CHAPTER ndash 4

Inclusion complexation of procainamide hydrochloride and

propafenone hydrochloride with α- and β-cyclodextrinslowast

In this chapter the encapsulation behaviour of two potential cardiovascular drugs

namely procainamide hydrochloride (PCA) and propafenone hydrochloride (PFO) with

α-CD and β-CD nanocavities has been studied by absorption fluorescence and time-

resolved fluorescence techniques The solid inclusion complexes were prepared and

characterized by SEM TEM FTIR DSC XRD and 1H NMR techniques Further the

geometries of these inclusion complexes were proposed with the help of computational

calculations (PM3 method) The chemical structures of PCA and PFO are given below

Chemical structures of (a) PCA and (b) PFO

41 Absorption and fluorescence measurements

In general inclusion complex formation with CD often causes change in the

electronic spectra of the guest molecules [87-93] In this regard the absorption and

fluorescence spectra of PCA and PFO drugs were recorded in various concentrations of

CDs at pH ~7 and the spectral data are given in Table 41 Fig 41 displays the

absorption spectra of the above drug molecules in aqueous solution as a function of

α-CD and β-CD concentrations In water PCA exhibits a strong absorption maximum at

278 nm with a slight shoulder (at 221 nm) whereas PFO shows two absorption maxima

at 303 and 249 nm Addition of various concentrations of β-CD into drug solution caused

blue shift about ~4 nm in the absorption maxima of PCA with gradual increase in the

molar extinction coefficient whereas in PFO the absorbance decreased at the same

wavelength However no significant spectral shift was observed in the presence of α-CD

except an increase in absorbance The above behaviour may be attributed to the

lowast Spectrochimica Acta Part A 115 (2013) 559

(a) (b) HH22NN

NNHH

OONN

OOOO

HHOO HHNN

93

enhanced dissolution of the drugs through hydrophobic interactions between the drugs

and the cavity of CDs These results indicate that both drugs are entrapped in the CD

cavity to form stable inclusion complexes Further the blue shift in β-CD solutions

reveals the less polar environment experienced by the drug molecule which is offered by

β-CD cavity rather than that of α-CD

Fig 42 shows the typical fluorescence spectra of PCA and PFO in different

concentrations of α-CD and β-CD The effect produced by the addition of CD on the

emission spectra of drugs in aqueous solution is more pronounced than the

corresponding effect on the absorption spectra The changes in the emission intensities of

drugs with increasing concentrations of CDs are shown in the inset of Fig 42 In

aqueous solution a single fluorescence emission is observed for both PCA (at 357 nm)

and PFO (at 420 nm) when excited at ~280 nm and 305 nm respectively The

fluorescence characteristics of the drug molecules undergo drastic changes in the

presence of CD With the addition of different concentrations of CD lead to a

considerable enhancement in the emission intensity of PCA especially for β-CD

Moreover the presence of β-CD concentrations produced a slight blue shift (~5 nm) in

PCA In PFO upon the addition of β-CD the fluorescence maxima was significantly red

shifted from 420 nm to 440 nm with a concomitant increase in the fluorescence intensity

whereas no significant emission shift was observed in the α-CD (Fig 42) These

phenomena suggest that the drug molecule is moving more deeply into the nonpolar CD

cavity during the formation of stable inclusion complexes An increase in the

fluorescence intensity after the formation of inclusion complex was also observed in

earlier studies [87-93] Since the CD cavity provided an apolar environment for the

guest and the movement of guest in the cavity was largely confined Further the

enhanced rigidity of the drugs resulted in an increase in their fluorescence quantum

yield

The binding constant (K) and stoichiometric ratio of the inclusion complexes of

drugs with CDs can be determined using the Benesi-Hildebrand (B-H) relation [120]

The K values were obtained from the slope of the linear plots Figs 43 and 44 depict

B-H double-reciprocal plot of 1AndashA0 and 1IndashI0 versus 1[CD] and 1[CD]2 In the case

of PCA according to eqn (2) the plots of 1AndashA0 and 1IndashI0 versus 1[CD] give upward

or downward curves as shown in Fig 43 While the plot of 1AndashA0 or 1IndashI0 versus

1[CD]2 shows a linear correlation However in PFO a good-linear relationship is

obtained when 1AndashA0 andor 1IndashI0 is plotted against 1[CD] (Fig 43) indicating that

the stoichiometry of PFOCD inclusion complex is 11 Further the non-linearity of

94

Table 41

95

Fig 41

96

Fig 42

97

Fig 43

98

Fig 44

99

the plots of 1AndashA0 and 1IndashI0 versus 1[CD]2 (Fig 44) ruled out the possibility of

12 stoichiometry between PFO and CDs This analysis reflects that 12 inclusion

complexes are formed between PCA and CDs whereas PFO formed 11 inclusion

complexes with CDs The K values for the inclusion complexes are given in Table 41

The quantitative comparison of binding constants of the two complexes suggests that

β-CD provides a better site to accommodate a deep inclusion of drugs in the β-CD

cavity Further it has been observed that the binding constants for 12 PCACD inclusion

systems are almost similar to the previously studied systems For example the binding

constant values of abs ~13542 M-1 and flu ~15576 M-1 for dothiepineβ-CD 12 inclusion

system [92] and abs ~14230 M-1 and flu ~48923 M-1 for fast violet-Bβ-CD 12

inclusion system [87]

The thermodynamic parameter free energy changes (∆G) for the binding of

guests within α-CD and β-CD were determined The negative free energy change values

in Table 41 indicate that the binding process is spontaneous and thermodynamically

favoured in the experimental temperature range (303 K) The negative values of ∆G

arose from the van der Waals interaction and the steric barrier caused by molecular

geometrical shape and the limit of CD cavity to the freedom of shift and rotation of guest

molecule

42 Effect of solvents

In order to understand the polarity around the drugs the absorption and

fluorescence spectra of PCA and PFO have been recorded in various solvents of different

polarities and hydrogen bonding abilities The absorption and fluorescence spectral

maxima in different solvents are compiled in Table 42 It can be observed from the

Table 42 that PCA shows much more interesting spectral features in different solvents

of various polarities than that of PFO In all solvents the absorption spectra of PCA are

much less structured with a slight shoulder around ~220 nm while the absorption spectra

of PFO are featureless with two peaks appearing around ~304 and 279 nm respectively

Data in Table 42 clearly indicate that the absorption spectra of PCA are red

shifted from cyclohexane to methanol but when compared to methanol blue shift is

observed in water The molar extinction coefficient is very high (~10-4 cm-1) indicating

that the observed absorption band corresponds to allowed πrarrπlowast transitions of the

benzene ring with a considerable charge transfer (CT) character Such kind of CT

process mainly originates from the aromatic ring or amino group (ndashNH2) to the amide

group (COndashNH) which is characterized by a high electron accepting character This can

be explained by analyzing the atomic orbital compositions of frontier molecular orbitals

100

of PCA as shown in Fig 45 The HOMO and LUMO energy calculations for PCA were

performed at PM3 level of theory In the HOMO the charge density is mainly

accumulated on the benzene ring and amino group (Fig 45) However in the case of

LUMO more charge density shifts to the benzene ring and carbonyl group This picture

clearly illustrates πrarrπlowast transition occurs when an electron density transfers from NH2

group to CminusC bond of the benzene ring and carbonyl group Thus nrarrπlowast transition of the

carbonyl group is hidden by an extended π conjugation of the phenyl ring to the

neighbouring carbonyl group [154-156] Also the absorption spectra of PCA are red

shifted in comparison to that of benzamide (cyclohexane asymp λabs ~270 230 nm) [157] and

benzanilide (cyclohexane asymp λabs ~262 254 247 nm λflu ~300 480 nm methanol asymp λabs

~261 nm λflu ~330 440 nm water asymp λabs ~256 nm λflu ~330 445 nm) [158] which

indicates an apparent perturbation effect of the amino group on the energy levels of the

parent benzamide molecule Further the spectral shifts observed in the absorption

spectrum of the drug molecule in protic and aprotic solvents are consistent with the

characteristic behaviour of aromatic molecules containing the amino group as an

auxochrome [90]

Table 42 Absorption and fluorescence spectral data (nm) and Stokes shifts

(cm-1) of PCA and PFO in different solvents

Solvents PCA PFO

λabs log ε λflu Stokes shifts λabs log ε λflu

Stokes shifts

Cyclohexane 280 223

sat 303 2660 300 249

sat 355s 336

5142

14-Dioxane 280 223

401 333

321 4460 300 248

369 367

340 331

3832

Ethyl acetate 285 222

416 353

326 4340 301 248

372 395

353s 339

4872

Acetonitrile 286 221

431 395

344 5871 302 246

377 390

353s 340

4783

2-Propanol 295 429 350 5235 303 248

378 398

354s 339

4667

Methanol 290 421 350 5746 303 249

376 401

419 338

9228

Water (pH ~65)

278 221s

408 303

357 7857 304 249

344 380

420 9176

sat - saturated

101

Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations

The fluorescence emission spectra of PCA and PFO in various solvents are

shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is

more pronounced than that on the corresponding effect on the absorption spectra which

reveals the excited state properties of the above drugs differ much from those in the

ground state PCA exhibits a single emission in all of the solvents whereas PFO gives

dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted

with increasing the polarity of solvents from cyclohexane to water The shift in the

emission maxima over absorption maxima indicates that the emitting state of the drug is

more polar than the ground state In PFO both SW and LW fluorescence bands are

affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are

largely red shifted and the half width of the LW fluorescence is significantly increased

(b) (a) H

OM

O

LU

MO

102

As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in

the present work can also be explained as hydrogen bond formation between the protic

solvents and electron withdrawing carbonyl group facilitates the formation of the

intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen

bonding seems to make the migration of electron density from benzene ring to the

electron withdrawing group more facile Further if the LW maximum is due to ICT this

should be more red shifted in protic solvents because ICT is more pronounced in protic

solvents than non-polar and aprotic solvents The spectral changes revealed that the LW

emission of PFO appeared as a result of ICT [160 161] which is more pronounced in

protic solvents than in other solvents The large Stokes shift indicates the strong changes

in the geometry of above molecules in the excited state [160 161] Moreover the

decreased emission intensity of PCA in water is likely due to the radiationless decay of

the probe molecule because of hydrogen bonding

Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

700

350

0 290 370 450

1

2

3 4

5

6

PCA λexci = 280 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

400

200

0 315 4075 500

1

2

3 4

5

PFO λexci = 305 nm

6

103

43 Time-resolved fluorescence studies

In order to confirm the interpretation of steady-state fluorescence spectroscopic

measurement data time-resolved fluorescence spectra and fluorescence decay times of

PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very

sensitive indicators for exploring the local environment around the excited state

fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and

CD environments are found to be quite different The typical time-resolved fluorescence

decay profile in different media is presented in Fig 47 and the average lifetimes are

given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly

affected by increasing the concentration of CD The results were judged by the statistical

fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission

wavelength was 355 nm Single exponential decay was observed for PCA in aqueous

solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve

becomes biexponential This indicates that PCA is encapsulated within the hydrophobic

nanocavity of CDs The life time of the inclusion complexes are higher than free drug

molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the

complexation ability of β-CD is higher in other words β-CD has given the privileged

encapsulation The amplitude of the complexed component also increased due to

increase in the complex formation and that of the decreased free species The

enhancement of τ1 and τ2 values with increase in the CD concentration is due to the

encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD

and the nature of the process with regard to short-lived species The decay of PCA is

dependent of the CD This may be due to the vibrational restriction of PCA in the excited

state

The fluorescence decay of PFO in water obtained by monitoring the emission

wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =

343 ns This analysis supports the presence of two emitting species ie LE and ICT

species in water possessing different fluorescence lifetimes However in the presence of

CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in

α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A

consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches

our interpretation of the steady-state fluorescence data indicating the formation of

inclusion complex between PFO and CDs The enhanced lifetimes associated with the

concentration of CD leads to the restriction of rotational degrees of freedom with

consequent impact on depletion of non-radiative decay channels Further when compared

104

to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows

the stronger hydrophobic interaction between PFO and β-CD

Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions

Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)

Drugs Medium Lifetime (ns) Pre exponential

factor ltτgt τ1 τ2 τ3 a1 a2 a3

PCA λemis = 355 nm

Water 056 041

α-CD 017 192 038 013 156

β-CD 021 386 032 007 313

PFO λemis = 420 nm

Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486

(a)

(b)

105

44 Molecular modeling studies

To scrutinize further the mechanism of inclusion complexation between the drugs

and CDs semiempirical quantum mechanical calculations at PM3 level of theory were

performed for the most favourable 11 inclusion mode This method is a very useful tool

for investigating the CD inclusion complexes in order to calculate the binding energy as

well as the geometrical structure of the inclusion complexes It is already proved that this

method is more reproducible when compared with the experimental data [162]

PM3 calculations for the inclusion process of drugs into the CD cavities were

carried out from the wider rim of the cavity because generally the approach of the guest

molecule to the CD cavity is more favourable for the wider rim side In addition when the

guest molecule is inserted into the cavity of CD the guest may rotate much more freely than

that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is

inserted from the wider rim of the CD cavity The energetically most favourable complex

formation strongly depends on the size and shape of the host and guest molecules From

PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately

~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular

dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit

entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped

within the hydrophobic cavity of CD So the calculations were carried out by inserting the

aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in

11 stoichiometric ratio followed by energy minimization But there was a failure in an

attempt to optimize 12 inclusion complexes of PCACD Hence only the energy

minimized structure of 12 PCACD complexes is given Upon the energy minimization it

was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host

molecules adjust themselves to give the most stable complex with least energy The 11

inclusion complex structure leading to minimum heat of formation shows the aniline moiety

or phenyl ring along with carbonyl group is encapsulated inside the CD cavity

The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD

PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for

12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in

Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the

hydrophobic CD cavity from the wider side and the binding geometries of both complexes

are almost similar Most prominently the simulation results are in good agreement with

the experimental data When compared to β-CD α-CD is closely associated with the drug

molecules consistent with the reduced diameter of the cavity

106

Figs 48

107

Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method

Fig 49 shows the structures of inclusion complexes of PCACDs in which both the

amino and methyl group protons along with phenyl ring are positioned nearly at narrow

rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and

methyl group protons of the drug in the process of complexation is huge compared with

other protons Further we could observe in the structures of 12 complexes that there are

several hydrogen bonds between the two neighbouring CD molecules (shown as green

dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes

The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that

could get involved in hydrogen bonding interactions Although the binding energies

prove the fact that the drug molecules enters with the aniline group or phenyl ring

through the wider rim of the CD cavity In order to explore the thermodynamic

parameters of the complexation process the statistical thermodynamic calculations were

carried out at 1 atm pressure and 298 K temperature The energy features

thermodynamic characteristics and electronic properties for the most stable 11 inclusion

complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated

drugs Further to quantify the interaction between drugs and CDs in the optimized

geometries we have determined the binding energy (ΔE) using Eqn (26) As expected

the binding energies of the inclusion complexes were more negative than the energies of

the isolated drugs and CDs suggest that the complexation process is more

thermodynamically favourable one The difference in binding energies (ΔE) for the most

stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal

mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD

(b)

2612 Aring

2701 Aring

2878 Aring

2804 Aring

(a)

2851 Aring

2675 Aring

108

The negative ΔH and ΔS values revealed that the formation of both inclusion complexes

is exothermic and enthalpy driven process On the other hand more negative ΔH values

observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion

complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG

values for these complexes are positive suggesting that the inclusion reaction was a non-

spontaneous process in vacuum phase Regrettably due to limitations in the calculating

ability of computers available with us the calculations for such a huge molecular system

could not be performed for aqueous phase However the experimental data as stated

earlier indicate that the formation of inclusion complexes in aqueous solution was

spontaneous one These variations may be due to the reason that all the calculations were

carried out in vacuum phase It is also considerable that during the inclusion

complexation in aqueous solution a number of water molecules are released from the

CD cavities

It can be observed from the complex structures that there are several

intermolecular hydrogen bonds between host and guest molecules in the structures (Figs

48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and

OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results

indicate that the intermolecular hydrogen bonding plays a significant role for the binding

energies and stabilizing the drugCD inclusion complexes

The geometric parameters such as bond distances bond angles and dihedral

angles of guest molecules before and after complexation in α-CD and β-CD were

obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion

complexes the geometry of drugs was completely changed The dihedral angles were

significantly altered indicating that guest must adapt a specific conformation to form a

more stable inclusion complex Further after the drug inclusion the geometrical

parameters of optimized structure of free CD also changed from truncated cone to an

oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen

and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that

those atoms played a crucial role in binding the drug molecules

The electronic structure of the inclusion complexes was also investigated in

addition to that of the isolated drug molecules HOMO as an electron donor represents

the capability to donate an electron and LUMO represents the capability to gain an

electron In HOMO of PCA the charge density is mainly accumulated on the benzene

ring and amino group whereas in PFO the charge density is observed on the CndashO and

CndashN bonds However in both cases of LUMO more charge density shifts to the benzene

109

ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during

HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC

bond of the benzene ring and carbonyl group It was also found that the charge densities

on each atom of the drugs within the CDs cavity were slightly changed Moreover the

energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical

species [145] The energy gaps for PCA and PFO as well as those for the most stable

complexes with CD were estimated by PM3 method and are shown in Table 44

Evidently the much lower energy gap which explains the fact that ultimate charge

transfer interaction occurs within the drug molecule The energy gap of drugβ-CD

complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD

complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex

is more stable than that of drugα-CD complex which is in good agreement with the

above discussed energy results In addition the EHOMOndashELUMO gap of both complexes

just fall in the range that of the PCA molecule suggesting there will be a blue shift in the

electronic spectrum of the guest molecule during binding and molecular recognition

This agrees well with the experimentally determined results

The polarity of the CD cavity is also taken into consideration for the formation of

most stable inclusion complexes The polarity of CD cavity significantly changed after

the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the

α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex

with respect to that of the dipole moment of optimized isolated hosts Although the

dipole moment of both complexes is lower than the sum of the dipole moments of

optimized isolated host and guest molecules From the above results one can conclude

that the dipole moment values show a strong correlation with the complexation

behaviour of CD

For the most stable complexes the physical parameters such as electronic

chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are

determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)

of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is

evident that the micro values of complexes differ remarkably from the isolated guest and host

molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD

therefore they are expected to act as electron acceptor in the inclusion complexation

interaction while the higher μ values of PCA indicates the drug act as electron donor In

the case of both η and ω values significant differences are observed when compared

with the isolated host (CD) molecule but it lies within the range that of the guest

110

Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method

Properties PCA PFO PCA α-CD

PCA β-CD

PFO α-CD

PFO β-CD

E -3427 -9462 -129371 -150783 -135294 -156835

∆E -1182 -1581 -1070 -1598

H 18209 19684 -40016 -49895 -37996 -48526

∆H -1141 -1349 -596 -1455

G 13929 14576 -53942 -65604 -51887 -63619

∆G -234 -581 1174 757

S 0143 0171 0449 0479 0456 0506

∆S -0047 -0073 -0068 -0074

EHOMO (eV) -886 -930 -829 -840 -946 -939

ELUMO (eV) -016 -024 009 004 -033 -019

EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920

Dipole moment (D) 439 168 583 995 784 1366

∆D -99 -673 -518 -031

micro -451 -477 -418 -410 -489 -479

η 435 453 422 419 456 460

S 022 022 023 024 021 021

ω 233 251 207 200 262 249

Unit =kcal mol-1 =kcalmol-Kelvin

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

93

enhanced dissolution of the drugs through hydrophobic interactions between the drugs

and the cavity of CDs These results indicate that both drugs are entrapped in the CD

cavity to form stable inclusion complexes Further the blue shift in β-CD solutions

reveals the less polar environment experienced by the drug molecule which is offered by

β-CD cavity rather than that of α-CD

Fig 42 shows the typical fluorescence spectra of PCA and PFO in different

concentrations of α-CD and β-CD The effect produced by the addition of CD on the

emission spectra of drugs in aqueous solution is more pronounced than the

corresponding effect on the absorption spectra The changes in the emission intensities of

drugs with increasing concentrations of CDs are shown in the inset of Fig 42 In

aqueous solution a single fluorescence emission is observed for both PCA (at 357 nm)

and PFO (at 420 nm) when excited at ~280 nm and 305 nm respectively The

fluorescence characteristics of the drug molecules undergo drastic changes in the

presence of CD With the addition of different concentrations of CD lead to a

considerable enhancement in the emission intensity of PCA especially for β-CD

Moreover the presence of β-CD concentrations produced a slight blue shift (~5 nm) in

PCA In PFO upon the addition of β-CD the fluorescence maxima was significantly red

shifted from 420 nm to 440 nm with a concomitant increase in the fluorescence intensity

whereas no significant emission shift was observed in the α-CD (Fig 42) These

phenomena suggest that the drug molecule is moving more deeply into the nonpolar CD

cavity during the formation of stable inclusion complexes An increase in the

fluorescence intensity after the formation of inclusion complex was also observed in

earlier studies [87-93] Since the CD cavity provided an apolar environment for the

guest and the movement of guest in the cavity was largely confined Further the

enhanced rigidity of the drugs resulted in an increase in their fluorescence quantum

yield

The binding constant (K) and stoichiometric ratio of the inclusion complexes of

drugs with CDs can be determined using the Benesi-Hildebrand (B-H) relation [120]

The K values were obtained from the slope of the linear plots Figs 43 and 44 depict

B-H double-reciprocal plot of 1AndashA0 and 1IndashI0 versus 1[CD] and 1[CD]2 In the case

of PCA according to eqn (2) the plots of 1AndashA0 and 1IndashI0 versus 1[CD] give upward

or downward curves as shown in Fig 43 While the plot of 1AndashA0 or 1IndashI0 versus

1[CD]2 shows a linear correlation However in PFO a good-linear relationship is

obtained when 1AndashA0 andor 1IndashI0 is plotted against 1[CD] (Fig 43) indicating that

the stoichiometry of PFOCD inclusion complex is 11 Further the non-linearity of

94

Table 41

95

Fig 41

96

Fig 42

97

Fig 43

98

Fig 44

99

the plots of 1AndashA0 and 1IndashI0 versus 1[CD]2 (Fig 44) ruled out the possibility of

12 stoichiometry between PFO and CDs This analysis reflects that 12 inclusion

complexes are formed between PCA and CDs whereas PFO formed 11 inclusion

complexes with CDs The K values for the inclusion complexes are given in Table 41

The quantitative comparison of binding constants of the two complexes suggests that

β-CD provides a better site to accommodate a deep inclusion of drugs in the β-CD

cavity Further it has been observed that the binding constants for 12 PCACD inclusion

systems are almost similar to the previously studied systems For example the binding

constant values of abs ~13542 M-1 and flu ~15576 M-1 for dothiepineβ-CD 12 inclusion

system [92] and abs ~14230 M-1 and flu ~48923 M-1 for fast violet-Bβ-CD 12

inclusion system [87]

The thermodynamic parameter free energy changes (∆G) for the binding of

guests within α-CD and β-CD were determined The negative free energy change values

in Table 41 indicate that the binding process is spontaneous and thermodynamically

favoured in the experimental temperature range (303 K) The negative values of ∆G

arose from the van der Waals interaction and the steric barrier caused by molecular

geometrical shape and the limit of CD cavity to the freedom of shift and rotation of guest

molecule

42 Effect of solvents

In order to understand the polarity around the drugs the absorption and

fluorescence spectra of PCA and PFO have been recorded in various solvents of different

polarities and hydrogen bonding abilities The absorption and fluorescence spectral

maxima in different solvents are compiled in Table 42 It can be observed from the

Table 42 that PCA shows much more interesting spectral features in different solvents

of various polarities than that of PFO In all solvents the absorption spectra of PCA are

much less structured with a slight shoulder around ~220 nm while the absorption spectra

of PFO are featureless with two peaks appearing around ~304 and 279 nm respectively

Data in Table 42 clearly indicate that the absorption spectra of PCA are red

shifted from cyclohexane to methanol but when compared to methanol blue shift is

observed in water The molar extinction coefficient is very high (~10-4 cm-1) indicating

that the observed absorption band corresponds to allowed πrarrπlowast transitions of the

benzene ring with a considerable charge transfer (CT) character Such kind of CT

process mainly originates from the aromatic ring or amino group (ndashNH2) to the amide

group (COndashNH) which is characterized by a high electron accepting character This can

be explained by analyzing the atomic orbital compositions of frontier molecular orbitals

100

of PCA as shown in Fig 45 The HOMO and LUMO energy calculations for PCA were

performed at PM3 level of theory In the HOMO the charge density is mainly

accumulated on the benzene ring and amino group (Fig 45) However in the case of

LUMO more charge density shifts to the benzene ring and carbonyl group This picture

clearly illustrates πrarrπlowast transition occurs when an electron density transfers from NH2

group to CminusC bond of the benzene ring and carbonyl group Thus nrarrπlowast transition of the

carbonyl group is hidden by an extended π conjugation of the phenyl ring to the

neighbouring carbonyl group [154-156] Also the absorption spectra of PCA are red

shifted in comparison to that of benzamide (cyclohexane asymp λabs ~270 230 nm) [157] and

benzanilide (cyclohexane asymp λabs ~262 254 247 nm λflu ~300 480 nm methanol asymp λabs

~261 nm λflu ~330 440 nm water asymp λabs ~256 nm λflu ~330 445 nm) [158] which

indicates an apparent perturbation effect of the amino group on the energy levels of the

parent benzamide molecule Further the spectral shifts observed in the absorption

spectrum of the drug molecule in protic and aprotic solvents are consistent with the

characteristic behaviour of aromatic molecules containing the amino group as an

auxochrome [90]

Table 42 Absorption and fluorescence spectral data (nm) and Stokes shifts

(cm-1) of PCA and PFO in different solvents

Solvents PCA PFO

λabs log ε λflu Stokes shifts λabs log ε λflu

Stokes shifts

Cyclohexane 280 223

sat 303 2660 300 249

sat 355s 336

5142

14-Dioxane 280 223

401 333

321 4460 300 248

369 367

340 331

3832

Ethyl acetate 285 222

416 353

326 4340 301 248

372 395

353s 339

4872

Acetonitrile 286 221

431 395

344 5871 302 246

377 390

353s 340

4783

2-Propanol 295 429 350 5235 303 248

378 398

354s 339

4667

Methanol 290 421 350 5746 303 249

376 401

419 338

9228

Water (pH ~65)

278 221s

408 303

357 7857 304 249

344 380

420 9176

sat - saturated

101

Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations

The fluorescence emission spectra of PCA and PFO in various solvents are

shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is

more pronounced than that on the corresponding effect on the absorption spectra which

reveals the excited state properties of the above drugs differ much from those in the

ground state PCA exhibits a single emission in all of the solvents whereas PFO gives

dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted

with increasing the polarity of solvents from cyclohexane to water The shift in the

emission maxima over absorption maxima indicates that the emitting state of the drug is

more polar than the ground state In PFO both SW and LW fluorescence bands are

affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are

largely red shifted and the half width of the LW fluorescence is significantly increased

(b) (a) H

OM

O

LU

MO

102

As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in

the present work can also be explained as hydrogen bond formation between the protic

solvents and electron withdrawing carbonyl group facilitates the formation of the

intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen

bonding seems to make the migration of electron density from benzene ring to the

electron withdrawing group more facile Further if the LW maximum is due to ICT this

should be more red shifted in protic solvents because ICT is more pronounced in protic

solvents than non-polar and aprotic solvents The spectral changes revealed that the LW

emission of PFO appeared as a result of ICT [160 161] which is more pronounced in

protic solvents than in other solvents The large Stokes shift indicates the strong changes

in the geometry of above molecules in the excited state [160 161] Moreover the

decreased emission intensity of PCA in water is likely due to the radiationless decay of

the probe molecule because of hydrogen bonding

Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

700

350

0 290 370 450

1

2

3 4

5

6

PCA λexci = 280 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

400

200

0 315 4075 500

1

2

3 4

5

PFO λexci = 305 nm

6

103

43 Time-resolved fluorescence studies

In order to confirm the interpretation of steady-state fluorescence spectroscopic

measurement data time-resolved fluorescence spectra and fluorescence decay times of

PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very

sensitive indicators for exploring the local environment around the excited state

fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and

CD environments are found to be quite different The typical time-resolved fluorescence

decay profile in different media is presented in Fig 47 and the average lifetimes are

given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly

affected by increasing the concentration of CD The results were judged by the statistical

fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission

wavelength was 355 nm Single exponential decay was observed for PCA in aqueous

solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve

becomes biexponential This indicates that PCA is encapsulated within the hydrophobic

nanocavity of CDs The life time of the inclusion complexes are higher than free drug

molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the

complexation ability of β-CD is higher in other words β-CD has given the privileged

encapsulation The amplitude of the complexed component also increased due to

increase in the complex formation and that of the decreased free species The

enhancement of τ1 and τ2 values with increase in the CD concentration is due to the

encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD

and the nature of the process with regard to short-lived species The decay of PCA is

dependent of the CD This may be due to the vibrational restriction of PCA in the excited

state

The fluorescence decay of PFO in water obtained by monitoring the emission

wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =

343 ns This analysis supports the presence of two emitting species ie LE and ICT

species in water possessing different fluorescence lifetimes However in the presence of

CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in

α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A

consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches

our interpretation of the steady-state fluorescence data indicating the formation of

inclusion complex between PFO and CDs The enhanced lifetimes associated with the

concentration of CD leads to the restriction of rotational degrees of freedom with

consequent impact on depletion of non-radiative decay channels Further when compared

104

to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows

the stronger hydrophobic interaction between PFO and β-CD

Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions

Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)

Drugs Medium Lifetime (ns) Pre exponential

factor ltτgt τ1 τ2 τ3 a1 a2 a3

PCA λemis = 355 nm

Water 056 041

α-CD 017 192 038 013 156

β-CD 021 386 032 007 313

PFO λemis = 420 nm

Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486

(a)

(b)

105

44 Molecular modeling studies

To scrutinize further the mechanism of inclusion complexation between the drugs

and CDs semiempirical quantum mechanical calculations at PM3 level of theory were

performed for the most favourable 11 inclusion mode This method is a very useful tool

for investigating the CD inclusion complexes in order to calculate the binding energy as

well as the geometrical structure of the inclusion complexes It is already proved that this

method is more reproducible when compared with the experimental data [162]

PM3 calculations for the inclusion process of drugs into the CD cavities were

carried out from the wider rim of the cavity because generally the approach of the guest

molecule to the CD cavity is more favourable for the wider rim side In addition when the

guest molecule is inserted into the cavity of CD the guest may rotate much more freely than

that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is

inserted from the wider rim of the CD cavity The energetically most favourable complex

formation strongly depends on the size and shape of the host and guest molecules From

PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately

~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular

dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit

entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped

within the hydrophobic cavity of CD So the calculations were carried out by inserting the

aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in

11 stoichiometric ratio followed by energy minimization But there was a failure in an

attempt to optimize 12 inclusion complexes of PCACD Hence only the energy

minimized structure of 12 PCACD complexes is given Upon the energy minimization it

was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host

molecules adjust themselves to give the most stable complex with least energy The 11

inclusion complex structure leading to minimum heat of formation shows the aniline moiety

or phenyl ring along with carbonyl group is encapsulated inside the CD cavity

The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD

PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for

12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in

Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the

hydrophobic CD cavity from the wider side and the binding geometries of both complexes

are almost similar Most prominently the simulation results are in good agreement with

the experimental data When compared to β-CD α-CD is closely associated with the drug

molecules consistent with the reduced diameter of the cavity

106

Figs 48

107

Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method

Fig 49 shows the structures of inclusion complexes of PCACDs in which both the

amino and methyl group protons along with phenyl ring are positioned nearly at narrow

rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and

methyl group protons of the drug in the process of complexation is huge compared with

other protons Further we could observe in the structures of 12 complexes that there are

several hydrogen bonds between the two neighbouring CD molecules (shown as green

dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes

The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that

could get involved in hydrogen bonding interactions Although the binding energies

prove the fact that the drug molecules enters with the aniline group or phenyl ring

through the wider rim of the CD cavity In order to explore the thermodynamic

parameters of the complexation process the statistical thermodynamic calculations were

carried out at 1 atm pressure and 298 K temperature The energy features

thermodynamic characteristics and electronic properties for the most stable 11 inclusion

complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated

drugs Further to quantify the interaction between drugs and CDs in the optimized

geometries we have determined the binding energy (ΔE) using Eqn (26) As expected

the binding energies of the inclusion complexes were more negative than the energies of

the isolated drugs and CDs suggest that the complexation process is more

thermodynamically favourable one The difference in binding energies (ΔE) for the most

stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal

mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD

(b)

2612 Aring

2701 Aring

2878 Aring

2804 Aring

(a)

2851 Aring

2675 Aring

108

The negative ΔH and ΔS values revealed that the formation of both inclusion complexes

is exothermic and enthalpy driven process On the other hand more negative ΔH values

observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion

complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG

values for these complexes are positive suggesting that the inclusion reaction was a non-

spontaneous process in vacuum phase Regrettably due to limitations in the calculating

ability of computers available with us the calculations for such a huge molecular system

could not be performed for aqueous phase However the experimental data as stated

earlier indicate that the formation of inclusion complexes in aqueous solution was

spontaneous one These variations may be due to the reason that all the calculations were

carried out in vacuum phase It is also considerable that during the inclusion

complexation in aqueous solution a number of water molecules are released from the

CD cavities

It can be observed from the complex structures that there are several

intermolecular hydrogen bonds between host and guest molecules in the structures (Figs

48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and

OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results

indicate that the intermolecular hydrogen bonding plays a significant role for the binding

energies and stabilizing the drugCD inclusion complexes

The geometric parameters such as bond distances bond angles and dihedral

angles of guest molecules before and after complexation in α-CD and β-CD were

obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion

complexes the geometry of drugs was completely changed The dihedral angles were

significantly altered indicating that guest must adapt a specific conformation to form a

more stable inclusion complex Further after the drug inclusion the geometrical

parameters of optimized structure of free CD also changed from truncated cone to an

oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen

and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that

those atoms played a crucial role in binding the drug molecules

The electronic structure of the inclusion complexes was also investigated in

addition to that of the isolated drug molecules HOMO as an electron donor represents

the capability to donate an electron and LUMO represents the capability to gain an

electron In HOMO of PCA the charge density is mainly accumulated on the benzene

ring and amino group whereas in PFO the charge density is observed on the CndashO and

CndashN bonds However in both cases of LUMO more charge density shifts to the benzene

109

ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during

HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC

bond of the benzene ring and carbonyl group It was also found that the charge densities

on each atom of the drugs within the CDs cavity were slightly changed Moreover the

energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical

species [145] The energy gaps for PCA and PFO as well as those for the most stable

complexes with CD were estimated by PM3 method and are shown in Table 44

Evidently the much lower energy gap which explains the fact that ultimate charge

transfer interaction occurs within the drug molecule The energy gap of drugβ-CD

complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD

complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex

is more stable than that of drugα-CD complex which is in good agreement with the

above discussed energy results In addition the EHOMOndashELUMO gap of both complexes

just fall in the range that of the PCA molecule suggesting there will be a blue shift in the

electronic spectrum of the guest molecule during binding and molecular recognition

This agrees well with the experimentally determined results

The polarity of the CD cavity is also taken into consideration for the formation of

most stable inclusion complexes The polarity of CD cavity significantly changed after

the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the

α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex

with respect to that of the dipole moment of optimized isolated hosts Although the

dipole moment of both complexes is lower than the sum of the dipole moments of

optimized isolated host and guest molecules From the above results one can conclude

that the dipole moment values show a strong correlation with the complexation

behaviour of CD

For the most stable complexes the physical parameters such as electronic

chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are

determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)

of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is

evident that the micro values of complexes differ remarkably from the isolated guest and host

molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD

therefore they are expected to act as electron acceptor in the inclusion complexation

interaction while the higher μ values of PCA indicates the drug act as electron donor In

the case of both η and ω values significant differences are observed when compared

with the isolated host (CD) molecule but it lies within the range that of the guest

110

Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method

Properties PCA PFO PCA α-CD

PCA β-CD

PFO α-CD

PFO β-CD

E -3427 -9462 -129371 -150783 -135294 -156835

∆E -1182 -1581 -1070 -1598

H 18209 19684 -40016 -49895 -37996 -48526

∆H -1141 -1349 -596 -1455

G 13929 14576 -53942 -65604 -51887 -63619

∆G -234 -581 1174 757

S 0143 0171 0449 0479 0456 0506

∆S -0047 -0073 -0068 -0074

EHOMO (eV) -886 -930 -829 -840 -946 -939

ELUMO (eV) -016 -024 009 004 -033 -019

EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920

Dipole moment (D) 439 168 583 995 784 1366

∆D -99 -673 -518 -031

micro -451 -477 -418 -410 -489 -479

η 435 453 422 419 456 460

S 022 022 023 024 021 021

ω 233 251 207 200 262 249

Unit =kcal mol-1 =kcalmol-Kelvin

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

94

Table 41

95

Fig 41

96

Fig 42

97

Fig 43

98

Fig 44

99

the plots of 1AndashA0 and 1IndashI0 versus 1[CD]2 (Fig 44) ruled out the possibility of

12 stoichiometry between PFO and CDs This analysis reflects that 12 inclusion

complexes are formed between PCA and CDs whereas PFO formed 11 inclusion

complexes with CDs The K values for the inclusion complexes are given in Table 41

The quantitative comparison of binding constants of the two complexes suggests that

β-CD provides a better site to accommodate a deep inclusion of drugs in the β-CD

cavity Further it has been observed that the binding constants for 12 PCACD inclusion

systems are almost similar to the previously studied systems For example the binding

constant values of abs ~13542 M-1 and flu ~15576 M-1 for dothiepineβ-CD 12 inclusion

system [92] and abs ~14230 M-1 and flu ~48923 M-1 for fast violet-Bβ-CD 12

inclusion system [87]

The thermodynamic parameter free energy changes (∆G) for the binding of

guests within α-CD and β-CD were determined The negative free energy change values

in Table 41 indicate that the binding process is spontaneous and thermodynamically

favoured in the experimental temperature range (303 K) The negative values of ∆G

arose from the van der Waals interaction and the steric barrier caused by molecular

geometrical shape and the limit of CD cavity to the freedom of shift and rotation of guest

molecule

42 Effect of solvents

In order to understand the polarity around the drugs the absorption and

fluorescence spectra of PCA and PFO have been recorded in various solvents of different

polarities and hydrogen bonding abilities The absorption and fluorescence spectral

maxima in different solvents are compiled in Table 42 It can be observed from the

Table 42 that PCA shows much more interesting spectral features in different solvents

of various polarities than that of PFO In all solvents the absorption spectra of PCA are

much less structured with a slight shoulder around ~220 nm while the absorption spectra

of PFO are featureless with two peaks appearing around ~304 and 279 nm respectively

Data in Table 42 clearly indicate that the absorption spectra of PCA are red

shifted from cyclohexane to methanol but when compared to methanol blue shift is

observed in water The molar extinction coefficient is very high (~10-4 cm-1) indicating

that the observed absorption band corresponds to allowed πrarrπlowast transitions of the

benzene ring with a considerable charge transfer (CT) character Such kind of CT

process mainly originates from the aromatic ring or amino group (ndashNH2) to the amide

group (COndashNH) which is characterized by a high electron accepting character This can

be explained by analyzing the atomic orbital compositions of frontier molecular orbitals

100

of PCA as shown in Fig 45 The HOMO and LUMO energy calculations for PCA were

performed at PM3 level of theory In the HOMO the charge density is mainly

accumulated on the benzene ring and amino group (Fig 45) However in the case of

LUMO more charge density shifts to the benzene ring and carbonyl group This picture

clearly illustrates πrarrπlowast transition occurs when an electron density transfers from NH2

group to CminusC bond of the benzene ring and carbonyl group Thus nrarrπlowast transition of the

carbonyl group is hidden by an extended π conjugation of the phenyl ring to the

neighbouring carbonyl group [154-156] Also the absorption spectra of PCA are red

shifted in comparison to that of benzamide (cyclohexane asymp λabs ~270 230 nm) [157] and

benzanilide (cyclohexane asymp λabs ~262 254 247 nm λflu ~300 480 nm methanol asymp λabs

~261 nm λflu ~330 440 nm water asymp λabs ~256 nm λflu ~330 445 nm) [158] which

indicates an apparent perturbation effect of the amino group on the energy levels of the

parent benzamide molecule Further the spectral shifts observed in the absorption

spectrum of the drug molecule in protic and aprotic solvents are consistent with the

characteristic behaviour of aromatic molecules containing the amino group as an

auxochrome [90]

Table 42 Absorption and fluorescence spectral data (nm) and Stokes shifts

(cm-1) of PCA and PFO in different solvents

Solvents PCA PFO

λabs log ε λflu Stokes shifts λabs log ε λflu

Stokes shifts

Cyclohexane 280 223

sat 303 2660 300 249

sat 355s 336

5142

14-Dioxane 280 223

401 333

321 4460 300 248

369 367

340 331

3832

Ethyl acetate 285 222

416 353

326 4340 301 248

372 395

353s 339

4872

Acetonitrile 286 221

431 395

344 5871 302 246

377 390

353s 340

4783

2-Propanol 295 429 350 5235 303 248

378 398

354s 339

4667

Methanol 290 421 350 5746 303 249

376 401

419 338

9228

Water (pH ~65)

278 221s

408 303

357 7857 304 249

344 380

420 9176

sat - saturated

101

Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations

The fluorescence emission spectra of PCA and PFO in various solvents are

shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is

more pronounced than that on the corresponding effect on the absorption spectra which

reveals the excited state properties of the above drugs differ much from those in the

ground state PCA exhibits a single emission in all of the solvents whereas PFO gives

dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted

with increasing the polarity of solvents from cyclohexane to water The shift in the

emission maxima over absorption maxima indicates that the emitting state of the drug is

more polar than the ground state In PFO both SW and LW fluorescence bands are

affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are

largely red shifted and the half width of the LW fluorescence is significantly increased

(b) (a) H

OM

O

LU

MO

102

As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in

the present work can also be explained as hydrogen bond formation between the protic

solvents and electron withdrawing carbonyl group facilitates the formation of the

intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen

bonding seems to make the migration of electron density from benzene ring to the

electron withdrawing group more facile Further if the LW maximum is due to ICT this

should be more red shifted in protic solvents because ICT is more pronounced in protic

solvents than non-polar and aprotic solvents The spectral changes revealed that the LW

emission of PFO appeared as a result of ICT [160 161] which is more pronounced in

protic solvents than in other solvents The large Stokes shift indicates the strong changes

in the geometry of above molecules in the excited state [160 161] Moreover the

decreased emission intensity of PCA in water is likely due to the radiationless decay of

the probe molecule because of hydrogen bonding

Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

700

350

0 290 370 450

1

2

3 4

5

6

PCA λexci = 280 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

400

200

0 315 4075 500

1

2

3 4

5

PFO λexci = 305 nm

6

103

43 Time-resolved fluorescence studies

In order to confirm the interpretation of steady-state fluorescence spectroscopic

measurement data time-resolved fluorescence spectra and fluorescence decay times of

PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very

sensitive indicators for exploring the local environment around the excited state

fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and

CD environments are found to be quite different The typical time-resolved fluorescence

decay profile in different media is presented in Fig 47 and the average lifetimes are

given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly

affected by increasing the concentration of CD The results were judged by the statistical

fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission

wavelength was 355 nm Single exponential decay was observed for PCA in aqueous

solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve

becomes biexponential This indicates that PCA is encapsulated within the hydrophobic

nanocavity of CDs The life time of the inclusion complexes are higher than free drug

molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the

complexation ability of β-CD is higher in other words β-CD has given the privileged

encapsulation The amplitude of the complexed component also increased due to

increase in the complex formation and that of the decreased free species The

enhancement of τ1 and τ2 values with increase in the CD concentration is due to the

encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD

and the nature of the process with regard to short-lived species The decay of PCA is

dependent of the CD This may be due to the vibrational restriction of PCA in the excited

state

The fluorescence decay of PFO in water obtained by monitoring the emission

wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =

343 ns This analysis supports the presence of two emitting species ie LE and ICT

species in water possessing different fluorescence lifetimes However in the presence of

CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in

α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A

consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches

our interpretation of the steady-state fluorescence data indicating the formation of

inclusion complex between PFO and CDs The enhanced lifetimes associated with the

concentration of CD leads to the restriction of rotational degrees of freedom with

consequent impact on depletion of non-radiative decay channels Further when compared

104

to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows

the stronger hydrophobic interaction between PFO and β-CD

Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions

Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)

Drugs Medium Lifetime (ns) Pre exponential

factor ltτgt τ1 τ2 τ3 a1 a2 a3

PCA λemis = 355 nm

Water 056 041

α-CD 017 192 038 013 156

β-CD 021 386 032 007 313

PFO λemis = 420 nm

Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486

(a)

(b)

105

44 Molecular modeling studies

To scrutinize further the mechanism of inclusion complexation between the drugs

and CDs semiempirical quantum mechanical calculations at PM3 level of theory were

performed for the most favourable 11 inclusion mode This method is a very useful tool

for investigating the CD inclusion complexes in order to calculate the binding energy as

well as the geometrical structure of the inclusion complexes It is already proved that this

method is more reproducible when compared with the experimental data [162]

PM3 calculations for the inclusion process of drugs into the CD cavities were

carried out from the wider rim of the cavity because generally the approach of the guest

molecule to the CD cavity is more favourable for the wider rim side In addition when the

guest molecule is inserted into the cavity of CD the guest may rotate much more freely than

that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is

inserted from the wider rim of the CD cavity The energetically most favourable complex

formation strongly depends on the size and shape of the host and guest molecules From

PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately

~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular

dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit

entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped

within the hydrophobic cavity of CD So the calculations were carried out by inserting the

aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in

11 stoichiometric ratio followed by energy minimization But there was a failure in an

attempt to optimize 12 inclusion complexes of PCACD Hence only the energy

minimized structure of 12 PCACD complexes is given Upon the energy minimization it

was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host

molecules adjust themselves to give the most stable complex with least energy The 11

inclusion complex structure leading to minimum heat of formation shows the aniline moiety

or phenyl ring along with carbonyl group is encapsulated inside the CD cavity

The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD

PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for

12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in

Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the

hydrophobic CD cavity from the wider side and the binding geometries of both complexes

are almost similar Most prominently the simulation results are in good agreement with

the experimental data When compared to β-CD α-CD is closely associated with the drug

molecules consistent with the reduced diameter of the cavity

106

Figs 48

107

Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method

Fig 49 shows the structures of inclusion complexes of PCACDs in which both the

amino and methyl group protons along with phenyl ring are positioned nearly at narrow

rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and

methyl group protons of the drug in the process of complexation is huge compared with

other protons Further we could observe in the structures of 12 complexes that there are

several hydrogen bonds between the two neighbouring CD molecules (shown as green

dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes

The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that

could get involved in hydrogen bonding interactions Although the binding energies

prove the fact that the drug molecules enters with the aniline group or phenyl ring

through the wider rim of the CD cavity In order to explore the thermodynamic

parameters of the complexation process the statistical thermodynamic calculations were

carried out at 1 atm pressure and 298 K temperature The energy features

thermodynamic characteristics and electronic properties for the most stable 11 inclusion

complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated

drugs Further to quantify the interaction between drugs and CDs in the optimized

geometries we have determined the binding energy (ΔE) using Eqn (26) As expected

the binding energies of the inclusion complexes were more negative than the energies of

the isolated drugs and CDs suggest that the complexation process is more

thermodynamically favourable one The difference in binding energies (ΔE) for the most

stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal

mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD

(b)

2612 Aring

2701 Aring

2878 Aring

2804 Aring

(a)

2851 Aring

2675 Aring

108

The negative ΔH and ΔS values revealed that the formation of both inclusion complexes

is exothermic and enthalpy driven process On the other hand more negative ΔH values

observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion

complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG

values for these complexes are positive suggesting that the inclusion reaction was a non-

spontaneous process in vacuum phase Regrettably due to limitations in the calculating

ability of computers available with us the calculations for such a huge molecular system

could not be performed for aqueous phase However the experimental data as stated

earlier indicate that the formation of inclusion complexes in aqueous solution was

spontaneous one These variations may be due to the reason that all the calculations were

carried out in vacuum phase It is also considerable that during the inclusion

complexation in aqueous solution a number of water molecules are released from the

CD cavities

It can be observed from the complex structures that there are several

intermolecular hydrogen bonds between host and guest molecules in the structures (Figs

48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and

OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results

indicate that the intermolecular hydrogen bonding plays a significant role for the binding

energies and stabilizing the drugCD inclusion complexes

The geometric parameters such as bond distances bond angles and dihedral

angles of guest molecules before and after complexation in α-CD and β-CD were

obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion

complexes the geometry of drugs was completely changed The dihedral angles were

significantly altered indicating that guest must adapt a specific conformation to form a

more stable inclusion complex Further after the drug inclusion the geometrical

parameters of optimized structure of free CD also changed from truncated cone to an

oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen

and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that

those atoms played a crucial role in binding the drug molecules

The electronic structure of the inclusion complexes was also investigated in

addition to that of the isolated drug molecules HOMO as an electron donor represents

the capability to donate an electron and LUMO represents the capability to gain an

electron In HOMO of PCA the charge density is mainly accumulated on the benzene

ring and amino group whereas in PFO the charge density is observed on the CndashO and

CndashN bonds However in both cases of LUMO more charge density shifts to the benzene

109

ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during

HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC

bond of the benzene ring and carbonyl group It was also found that the charge densities

on each atom of the drugs within the CDs cavity were slightly changed Moreover the

energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical

species [145] The energy gaps for PCA and PFO as well as those for the most stable

complexes with CD were estimated by PM3 method and are shown in Table 44

Evidently the much lower energy gap which explains the fact that ultimate charge

transfer interaction occurs within the drug molecule The energy gap of drugβ-CD

complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD

complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex

is more stable than that of drugα-CD complex which is in good agreement with the

above discussed energy results In addition the EHOMOndashELUMO gap of both complexes

just fall in the range that of the PCA molecule suggesting there will be a blue shift in the

electronic spectrum of the guest molecule during binding and molecular recognition

This agrees well with the experimentally determined results

The polarity of the CD cavity is also taken into consideration for the formation of

most stable inclusion complexes The polarity of CD cavity significantly changed after

the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the

α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex

with respect to that of the dipole moment of optimized isolated hosts Although the

dipole moment of both complexes is lower than the sum of the dipole moments of

optimized isolated host and guest molecules From the above results one can conclude

that the dipole moment values show a strong correlation with the complexation

behaviour of CD

For the most stable complexes the physical parameters such as electronic

chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are

determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)

of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is

evident that the micro values of complexes differ remarkably from the isolated guest and host

molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD

therefore they are expected to act as electron acceptor in the inclusion complexation

interaction while the higher μ values of PCA indicates the drug act as electron donor In

the case of both η and ω values significant differences are observed when compared

with the isolated host (CD) molecule but it lies within the range that of the guest

110

Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method

Properties PCA PFO PCA α-CD

PCA β-CD

PFO α-CD

PFO β-CD

E -3427 -9462 -129371 -150783 -135294 -156835

∆E -1182 -1581 -1070 -1598

H 18209 19684 -40016 -49895 -37996 -48526

∆H -1141 -1349 -596 -1455

G 13929 14576 -53942 -65604 -51887 -63619

∆G -234 -581 1174 757

S 0143 0171 0449 0479 0456 0506

∆S -0047 -0073 -0068 -0074

EHOMO (eV) -886 -930 -829 -840 -946 -939

ELUMO (eV) -016 -024 009 004 -033 -019

EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920

Dipole moment (D) 439 168 583 995 784 1366

∆D -99 -673 -518 -031

micro -451 -477 -418 -410 -489 -479

η 435 453 422 419 456 460

S 022 022 023 024 021 021

ω 233 251 207 200 262 249

Unit =kcal mol-1 =kcalmol-Kelvin

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

95

Fig 41

96

Fig 42

97

Fig 43

98

Fig 44

99

the plots of 1AndashA0 and 1IndashI0 versus 1[CD]2 (Fig 44) ruled out the possibility of

12 stoichiometry between PFO and CDs This analysis reflects that 12 inclusion

complexes are formed between PCA and CDs whereas PFO formed 11 inclusion

complexes with CDs The K values for the inclusion complexes are given in Table 41

The quantitative comparison of binding constants of the two complexes suggests that

β-CD provides a better site to accommodate a deep inclusion of drugs in the β-CD

cavity Further it has been observed that the binding constants for 12 PCACD inclusion

systems are almost similar to the previously studied systems For example the binding

constant values of abs ~13542 M-1 and flu ~15576 M-1 for dothiepineβ-CD 12 inclusion

system [92] and abs ~14230 M-1 and flu ~48923 M-1 for fast violet-Bβ-CD 12

inclusion system [87]

The thermodynamic parameter free energy changes (∆G) for the binding of

guests within α-CD and β-CD were determined The negative free energy change values

in Table 41 indicate that the binding process is spontaneous and thermodynamically

favoured in the experimental temperature range (303 K) The negative values of ∆G

arose from the van der Waals interaction and the steric barrier caused by molecular

geometrical shape and the limit of CD cavity to the freedom of shift and rotation of guest

molecule

42 Effect of solvents

In order to understand the polarity around the drugs the absorption and

fluorescence spectra of PCA and PFO have been recorded in various solvents of different

polarities and hydrogen bonding abilities The absorption and fluorescence spectral

maxima in different solvents are compiled in Table 42 It can be observed from the

Table 42 that PCA shows much more interesting spectral features in different solvents

of various polarities than that of PFO In all solvents the absorption spectra of PCA are

much less structured with a slight shoulder around ~220 nm while the absorption spectra

of PFO are featureless with two peaks appearing around ~304 and 279 nm respectively

Data in Table 42 clearly indicate that the absorption spectra of PCA are red

shifted from cyclohexane to methanol but when compared to methanol blue shift is

observed in water The molar extinction coefficient is very high (~10-4 cm-1) indicating

that the observed absorption band corresponds to allowed πrarrπlowast transitions of the

benzene ring with a considerable charge transfer (CT) character Such kind of CT

process mainly originates from the aromatic ring or amino group (ndashNH2) to the amide

group (COndashNH) which is characterized by a high electron accepting character This can

be explained by analyzing the atomic orbital compositions of frontier molecular orbitals

100

of PCA as shown in Fig 45 The HOMO and LUMO energy calculations for PCA were

performed at PM3 level of theory In the HOMO the charge density is mainly

accumulated on the benzene ring and amino group (Fig 45) However in the case of

LUMO more charge density shifts to the benzene ring and carbonyl group This picture

clearly illustrates πrarrπlowast transition occurs when an electron density transfers from NH2

group to CminusC bond of the benzene ring and carbonyl group Thus nrarrπlowast transition of the

carbonyl group is hidden by an extended π conjugation of the phenyl ring to the

neighbouring carbonyl group [154-156] Also the absorption spectra of PCA are red

shifted in comparison to that of benzamide (cyclohexane asymp λabs ~270 230 nm) [157] and

benzanilide (cyclohexane asymp λabs ~262 254 247 nm λflu ~300 480 nm methanol asymp λabs

~261 nm λflu ~330 440 nm water asymp λabs ~256 nm λflu ~330 445 nm) [158] which

indicates an apparent perturbation effect of the amino group on the energy levels of the

parent benzamide molecule Further the spectral shifts observed in the absorption

spectrum of the drug molecule in protic and aprotic solvents are consistent with the

characteristic behaviour of aromatic molecules containing the amino group as an

auxochrome [90]

Table 42 Absorption and fluorescence spectral data (nm) and Stokes shifts

(cm-1) of PCA and PFO in different solvents

Solvents PCA PFO

λabs log ε λflu Stokes shifts λabs log ε λflu

Stokes shifts

Cyclohexane 280 223

sat 303 2660 300 249

sat 355s 336

5142

14-Dioxane 280 223

401 333

321 4460 300 248

369 367

340 331

3832

Ethyl acetate 285 222

416 353

326 4340 301 248

372 395

353s 339

4872

Acetonitrile 286 221

431 395

344 5871 302 246

377 390

353s 340

4783

2-Propanol 295 429 350 5235 303 248

378 398

354s 339

4667

Methanol 290 421 350 5746 303 249

376 401

419 338

9228

Water (pH ~65)

278 221s

408 303

357 7857 304 249

344 380

420 9176

sat - saturated

101

Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations

The fluorescence emission spectra of PCA and PFO in various solvents are

shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is

more pronounced than that on the corresponding effect on the absorption spectra which

reveals the excited state properties of the above drugs differ much from those in the

ground state PCA exhibits a single emission in all of the solvents whereas PFO gives

dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted

with increasing the polarity of solvents from cyclohexane to water The shift in the

emission maxima over absorption maxima indicates that the emitting state of the drug is

more polar than the ground state In PFO both SW and LW fluorescence bands are

affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are

largely red shifted and the half width of the LW fluorescence is significantly increased

(b) (a) H

OM

O

LU

MO

102

As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in

the present work can also be explained as hydrogen bond formation between the protic

solvents and electron withdrawing carbonyl group facilitates the formation of the

intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen

bonding seems to make the migration of electron density from benzene ring to the

electron withdrawing group more facile Further if the LW maximum is due to ICT this

should be more red shifted in protic solvents because ICT is more pronounced in protic

solvents than non-polar and aprotic solvents The spectral changes revealed that the LW

emission of PFO appeared as a result of ICT [160 161] which is more pronounced in

protic solvents than in other solvents The large Stokes shift indicates the strong changes

in the geometry of above molecules in the excited state [160 161] Moreover the

decreased emission intensity of PCA in water is likely due to the radiationless decay of

the probe molecule because of hydrogen bonding

Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

700

350

0 290 370 450

1

2

3 4

5

6

PCA λexci = 280 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

400

200

0 315 4075 500

1

2

3 4

5

PFO λexci = 305 nm

6

103

43 Time-resolved fluorescence studies

In order to confirm the interpretation of steady-state fluorescence spectroscopic

measurement data time-resolved fluorescence spectra and fluorescence decay times of

PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very

sensitive indicators for exploring the local environment around the excited state

fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and

CD environments are found to be quite different The typical time-resolved fluorescence

decay profile in different media is presented in Fig 47 and the average lifetimes are

given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly

affected by increasing the concentration of CD The results were judged by the statistical

fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission

wavelength was 355 nm Single exponential decay was observed for PCA in aqueous

solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve

becomes biexponential This indicates that PCA is encapsulated within the hydrophobic

nanocavity of CDs The life time of the inclusion complexes are higher than free drug

molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the

complexation ability of β-CD is higher in other words β-CD has given the privileged

encapsulation The amplitude of the complexed component also increased due to

increase in the complex formation and that of the decreased free species The

enhancement of τ1 and τ2 values with increase in the CD concentration is due to the

encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD

and the nature of the process with regard to short-lived species The decay of PCA is

dependent of the CD This may be due to the vibrational restriction of PCA in the excited

state

The fluorescence decay of PFO in water obtained by monitoring the emission

wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =

343 ns This analysis supports the presence of two emitting species ie LE and ICT

species in water possessing different fluorescence lifetimes However in the presence of

CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in

α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A

consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches

our interpretation of the steady-state fluorescence data indicating the formation of

inclusion complex between PFO and CDs The enhanced lifetimes associated with the

concentration of CD leads to the restriction of rotational degrees of freedom with

consequent impact on depletion of non-radiative decay channels Further when compared

104

to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows

the stronger hydrophobic interaction between PFO and β-CD

Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions

Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)

Drugs Medium Lifetime (ns) Pre exponential

factor ltτgt τ1 τ2 τ3 a1 a2 a3

PCA λemis = 355 nm

Water 056 041

α-CD 017 192 038 013 156

β-CD 021 386 032 007 313

PFO λemis = 420 nm

Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486

(a)

(b)

105

44 Molecular modeling studies

To scrutinize further the mechanism of inclusion complexation between the drugs

and CDs semiempirical quantum mechanical calculations at PM3 level of theory were

performed for the most favourable 11 inclusion mode This method is a very useful tool

for investigating the CD inclusion complexes in order to calculate the binding energy as

well as the geometrical structure of the inclusion complexes It is already proved that this

method is more reproducible when compared with the experimental data [162]

PM3 calculations for the inclusion process of drugs into the CD cavities were

carried out from the wider rim of the cavity because generally the approach of the guest

molecule to the CD cavity is more favourable for the wider rim side In addition when the

guest molecule is inserted into the cavity of CD the guest may rotate much more freely than

that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is

inserted from the wider rim of the CD cavity The energetically most favourable complex

formation strongly depends on the size and shape of the host and guest molecules From

PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately

~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular

dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit

entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped

within the hydrophobic cavity of CD So the calculations were carried out by inserting the

aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in

11 stoichiometric ratio followed by energy minimization But there was a failure in an

attempt to optimize 12 inclusion complexes of PCACD Hence only the energy

minimized structure of 12 PCACD complexes is given Upon the energy minimization it

was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host

molecules adjust themselves to give the most stable complex with least energy The 11

inclusion complex structure leading to minimum heat of formation shows the aniline moiety

or phenyl ring along with carbonyl group is encapsulated inside the CD cavity

The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD

PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for

12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in

Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the

hydrophobic CD cavity from the wider side and the binding geometries of both complexes

are almost similar Most prominently the simulation results are in good agreement with

the experimental data When compared to β-CD α-CD is closely associated with the drug

molecules consistent with the reduced diameter of the cavity

106

Figs 48

107

Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method

Fig 49 shows the structures of inclusion complexes of PCACDs in which both the

amino and methyl group protons along with phenyl ring are positioned nearly at narrow

rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and

methyl group protons of the drug in the process of complexation is huge compared with

other protons Further we could observe in the structures of 12 complexes that there are

several hydrogen bonds between the two neighbouring CD molecules (shown as green

dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes

The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that

could get involved in hydrogen bonding interactions Although the binding energies

prove the fact that the drug molecules enters with the aniline group or phenyl ring

through the wider rim of the CD cavity In order to explore the thermodynamic

parameters of the complexation process the statistical thermodynamic calculations were

carried out at 1 atm pressure and 298 K temperature The energy features

thermodynamic characteristics and electronic properties for the most stable 11 inclusion

complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated

drugs Further to quantify the interaction between drugs and CDs in the optimized

geometries we have determined the binding energy (ΔE) using Eqn (26) As expected

the binding energies of the inclusion complexes were more negative than the energies of

the isolated drugs and CDs suggest that the complexation process is more

thermodynamically favourable one The difference in binding energies (ΔE) for the most

stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal

mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD

(b)

2612 Aring

2701 Aring

2878 Aring

2804 Aring

(a)

2851 Aring

2675 Aring

108

The negative ΔH and ΔS values revealed that the formation of both inclusion complexes

is exothermic and enthalpy driven process On the other hand more negative ΔH values

observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion

complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG

values for these complexes are positive suggesting that the inclusion reaction was a non-

spontaneous process in vacuum phase Regrettably due to limitations in the calculating

ability of computers available with us the calculations for such a huge molecular system

could not be performed for aqueous phase However the experimental data as stated

earlier indicate that the formation of inclusion complexes in aqueous solution was

spontaneous one These variations may be due to the reason that all the calculations were

carried out in vacuum phase It is also considerable that during the inclusion

complexation in aqueous solution a number of water molecules are released from the

CD cavities

It can be observed from the complex structures that there are several

intermolecular hydrogen bonds between host and guest molecules in the structures (Figs

48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and

OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results

indicate that the intermolecular hydrogen bonding plays a significant role for the binding

energies and stabilizing the drugCD inclusion complexes

The geometric parameters such as bond distances bond angles and dihedral

angles of guest molecules before and after complexation in α-CD and β-CD were

obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion

complexes the geometry of drugs was completely changed The dihedral angles were

significantly altered indicating that guest must adapt a specific conformation to form a

more stable inclusion complex Further after the drug inclusion the geometrical

parameters of optimized structure of free CD also changed from truncated cone to an

oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen

and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that

those atoms played a crucial role in binding the drug molecules

The electronic structure of the inclusion complexes was also investigated in

addition to that of the isolated drug molecules HOMO as an electron donor represents

the capability to donate an electron and LUMO represents the capability to gain an

electron In HOMO of PCA the charge density is mainly accumulated on the benzene

ring and amino group whereas in PFO the charge density is observed on the CndashO and

CndashN bonds However in both cases of LUMO more charge density shifts to the benzene

109

ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during

HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC

bond of the benzene ring and carbonyl group It was also found that the charge densities

on each atom of the drugs within the CDs cavity were slightly changed Moreover the

energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical

species [145] The energy gaps for PCA and PFO as well as those for the most stable

complexes with CD were estimated by PM3 method and are shown in Table 44

Evidently the much lower energy gap which explains the fact that ultimate charge

transfer interaction occurs within the drug molecule The energy gap of drugβ-CD

complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD

complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex

is more stable than that of drugα-CD complex which is in good agreement with the

above discussed energy results In addition the EHOMOndashELUMO gap of both complexes

just fall in the range that of the PCA molecule suggesting there will be a blue shift in the

electronic spectrum of the guest molecule during binding and molecular recognition

This agrees well with the experimentally determined results

The polarity of the CD cavity is also taken into consideration for the formation of

most stable inclusion complexes The polarity of CD cavity significantly changed after

the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the

α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex

with respect to that of the dipole moment of optimized isolated hosts Although the

dipole moment of both complexes is lower than the sum of the dipole moments of

optimized isolated host and guest molecules From the above results one can conclude

that the dipole moment values show a strong correlation with the complexation

behaviour of CD

For the most stable complexes the physical parameters such as electronic

chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are

determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)

of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is

evident that the micro values of complexes differ remarkably from the isolated guest and host

molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD

therefore they are expected to act as electron acceptor in the inclusion complexation

interaction while the higher μ values of PCA indicates the drug act as electron donor In

the case of both η and ω values significant differences are observed when compared

with the isolated host (CD) molecule but it lies within the range that of the guest

110

Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method

Properties PCA PFO PCA α-CD

PCA β-CD

PFO α-CD

PFO β-CD

E -3427 -9462 -129371 -150783 -135294 -156835

∆E -1182 -1581 -1070 -1598

H 18209 19684 -40016 -49895 -37996 -48526

∆H -1141 -1349 -596 -1455

G 13929 14576 -53942 -65604 -51887 -63619

∆G -234 -581 1174 757

S 0143 0171 0449 0479 0456 0506

∆S -0047 -0073 -0068 -0074

EHOMO (eV) -886 -930 -829 -840 -946 -939

ELUMO (eV) -016 -024 009 004 -033 -019

EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920

Dipole moment (D) 439 168 583 995 784 1366

∆D -99 -673 -518 -031

micro -451 -477 -418 -410 -489 -479

η 435 453 422 419 456 460

S 022 022 023 024 021 021

ω 233 251 207 200 262 249

Unit =kcal mol-1 =kcalmol-Kelvin

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

96

Fig 42

97

Fig 43

98

Fig 44

99

the plots of 1AndashA0 and 1IndashI0 versus 1[CD]2 (Fig 44) ruled out the possibility of

12 stoichiometry between PFO and CDs This analysis reflects that 12 inclusion

complexes are formed between PCA and CDs whereas PFO formed 11 inclusion

complexes with CDs The K values for the inclusion complexes are given in Table 41

The quantitative comparison of binding constants of the two complexes suggests that

β-CD provides a better site to accommodate a deep inclusion of drugs in the β-CD

cavity Further it has been observed that the binding constants for 12 PCACD inclusion

systems are almost similar to the previously studied systems For example the binding

constant values of abs ~13542 M-1 and flu ~15576 M-1 for dothiepineβ-CD 12 inclusion

system [92] and abs ~14230 M-1 and flu ~48923 M-1 for fast violet-Bβ-CD 12

inclusion system [87]

The thermodynamic parameter free energy changes (∆G) for the binding of

guests within α-CD and β-CD were determined The negative free energy change values

in Table 41 indicate that the binding process is spontaneous and thermodynamically

favoured in the experimental temperature range (303 K) The negative values of ∆G

arose from the van der Waals interaction and the steric barrier caused by molecular

geometrical shape and the limit of CD cavity to the freedom of shift and rotation of guest

molecule

42 Effect of solvents

In order to understand the polarity around the drugs the absorption and

fluorescence spectra of PCA and PFO have been recorded in various solvents of different

polarities and hydrogen bonding abilities The absorption and fluorescence spectral

maxima in different solvents are compiled in Table 42 It can be observed from the

Table 42 that PCA shows much more interesting spectral features in different solvents

of various polarities than that of PFO In all solvents the absorption spectra of PCA are

much less structured with a slight shoulder around ~220 nm while the absorption spectra

of PFO are featureless with two peaks appearing around ~304 and 279 nm respectively

Data in Table 42 clearly indicate that the absorption spectra of PCA are red

shifted from cyclohexane to methanol but when compared to methanol blue shift is

observed in water The molar extinction coefficient is very high (~10-4 cm-1) indicating

that the observed absorption band corresponds to allowed πrarrπlowast transitions of the

benzene ring with a considerable charge transfer (CT) character Such kind of CT

process mainly originates from the aromatic ring or amino group (ndashNH2) to the amide

group (COndashNH) which is characterized by a high electron accepting character This can

be explained by analyzing the atomic orbital compositions of frontier molecular orbitals

100

of PCA as shown in Fig 45 The HOMO and LUMO energy calculations for PCA were

performed at PM3 level of theory In the HOMO the charge density is mainly

accumulated on the benzene ring and amino group (Fig 45) However in the case of

LUMO more charge density shifts to the benzene ring and carbonyl group This picture

clearly illustrates πrarrπlowast transition occurs when an electron density transfers from NH2

group to CminusC bond of the benzene ring and carbonyl group Thus nrarrπlowast transition of the

carbonyl group is hidden by an extended π conjugation of the phenyl ring to the

neighbouring carbonyl group [154-156] Also the absorption spectra of PCA are red

shifted in comparison to that of benzamide (cyclohexane asymp λabs ~270 230 nm) [157] and

benzanilide (cyclohexane asymp λabs ~262 254 247 nm λflu ~300 480 nm methanol asymp λabs

~261 nm λflu ~330 440 nm water asymp λabs ~256 nm λflu ~330 445 nm) [158] which

indicates an apparent perturbation effect of the amino group on the energy levels of the

parent benzamide molecule Further the spectral shifts observed in the absorption

spectrum of the drug molecule in protic and aprotic solvents are consistent with the

characteristic behaviour of aromatic molecules containing the amino group as an

auxochrome [90]

Table 42 Absorption and fluorescence spectral data (nm) and Stokes shifts

(cm-1) of PCA and PFO in different solvents

Solvents PCA PFO

λabs log ε λflu Stokes shifts λabs log ε λflu

Stokes shifts

Cyclohexane 280 223

sat 303 2660 300 249

sat 355s 336

5142

14-Dioxane 280 223

401 333

321 4460 300 248

369 367

340 331

3832

Ethyl acetate 285 222

416 353

326 4340 301 248

372 395

353s 339

4872

Acetonitrile 286 221

431 395

344 5871 302 246

377 390

353s 340

4783

2-Propanol 295 429 350 5235 303 248

378 398

354s 339

4667

Methanol 290 421 350 5746 303 249

376 401

419 338

9228

Water (pH ~65)

278 221s

408 303

357 7857 304 249

344 380

420 9176

sat - saturated

101

Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations

The fluorescence emission spectra of PCA and PFO in various solvents are

shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is

more pronounced than that on the corresponding effect on the absorption spectra which

reveals the excited state properties of the above drugs differ much from those in the

ground state PCA exhibits a single emission in all of the solvents whereas PFO gives

dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted

with increasing the polarity of solvents from cyclohexane to water The shift in the

emission maxima over absorption maxima indicates that the emitting state of the drug is

more polar than the ground state In PFO both SW and LW fluorescence bands are

affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are

largely red shifted and the half width of the LW fluorescence is significantly increased

(b) (a) H

OM

O

LU

MO

102

As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in

the present work can also be explained as hydrogen bond formation between the protic

solvents and electron withdrawing carbonyl group facilitates the formation of the

intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen

bonding seems to make the migration of electron density from benzene ring to the

electron withdrawing group more facile Further if the LW maximum is due to ICT this

should be more red shifted in protic solvents because ICT is more pronounced in protic

solvents than non-polar and aprotic solvents The spectral changes revealed that the LW

emission of PFO appeared as a result of ICT [160 161] which is more pronounced in

protic solvents than in other solvents The large Stokes shift indicates the strong changes

in the geometry of above molecules in the excited state [160 161] Moreover the

decreased emission intensity of PCA in water is likely due to the radiationless decay of

the probe molecule because of hydrogen bonding

Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

700

350

0 290 370 450

1

2

3 4

5

6

PCA λexci = 280 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

400

200

0 315 4075 500

1

2

3 4

5

PFO λexci = 305 nm

6

103

43 Time-resolved fluorescence studies

In order to confirm the interpretation of steady-state fluorescence spectroscopic

measurement data time-resolved fluorescence spectra and fluorescence decay times of

PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very

sensitive indicators for exploring the local environment around the excited state

fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and

CD environments are found to be quite different The typical time-resolved fluorescence

decay profile in different media is presented in Fig 47 and the average lifetimes are

given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly

affected by increasing the concentration of CD The results were judged by the statistical

fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission

wavelength was 355 nm Single exponential decay was observed for PCA in aqueous

solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve

becomes biexponential This indicates that PCA is encapsulated within the hydrophobic

nanocavity of CDs The life time of the inclusion complexes are higher than free drug

molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the

complexation ability of β-CD is higher in other words β-CD has given the privileged

encapsulation The amplitude of the complexed component also increased due to

increase in the complex formation and that of the decreased free species The

enhancement of τ1 and τ2 values with increase in the CD concentration is due to the

encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD

and the nature of the process with regard to short-lived species The decay of PCA is

dependent of the CD This may be due to the vibrational restriction of PCA in the excited

state

The fluorescence decay of PFO in water obtained by monitoring the emission

wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =

343 ns This analysis supports the presence of two emitting species ie LE and ICT

species in water possessing different fluorescence lifetimes However in the presence of

CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in

α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A

consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches

our interpretation of the steady-state fluorescence data indicating the formation of

inclusion complex between PFO and CDs The enhanced lifetimes associated with the

concentration of CD leads to the restriction of rotational degrees of freedom with

consequent impact on depletion of non-radiative decay channels Further when compared

104

to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows

the stronger hydrophobic interaction between PFO and β-CD

Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions

Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)

Drugs Medium Lifetime (ns) Pre exponential

factor ltτgt τ1 τ2 τ3 a1 a2 a3

PCA λemis = 355 nm

Water 056 041

α-CD 017 192 038 013 156

β-CD 021 386 032 007 313

PFO λemis = 420 nm

Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486

(a)

(b)

105

44 Molecular modeling studies

To scrutinize further the mechanism of inclusion complexation between the drugs

and CDs semiempirical quantum mechanical calculations at PM3 level of theory were

performed for the most favourable 11 inclusion mode This method is a very useful tool

for investigating the CD inclusion complexes in order to calculate the binding energy as

well as the geometrical structure of the inclusion complexes It is already proved that this

method is more reproducible when compared with the experimental data [162]

PM3 calculations for the inclusion process of drugs into the CD cavities were

carried out from the wider rim of the cavity because generally the approach of the guest

molecule to the CD cavity is more favourable for the wider rim side In addition when the

guest molecule is inserted into the cavity of CD the guest may rotate much more freely than

that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is

inserted from the wider rim of the CD cavity The energetically most favourable complex

formation strongly depends on the size and shape of the host and guest molecules From

PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately

~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular

dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit

entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped

within the hydrophobic cavity of CD So the calculations were carried out by inserting the

aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in

11 stoichiometric ratio followed by energy minimization But there was a failure in an

attempt to optimize 12 inclusion complexes of PCACD Hence only the energy

minimized structure of 12 PCACD complexes is given Upon the energy minimization it

was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host

molecules adjust themselves to give the most stable complex with least energy The 11

inclusion complex structure leading to minimum heat of formation shows the aniline moiety

or phenyl ring along with carbonyl group is encapsulated inside the CD cavity

The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD

PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for

12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in

Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the

hydrophobic CD cavity from the wider side and the binding geometries of both complexes

are almost similar Most prominently the simulation results are in good agreement with

the experimental data When compared to β-CD α-CD is closely associated with the drug

molecules consistent with the reduced diameter of the cavity

106

Figs 48

107

Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method

Fig 49 shows the structures of inclusion complexes of PCACDs in which both the

amino and methyl group protons along with phenyl ring are positioned nearly at narrow

rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and

methyl group protons of the drug in the process of complexation is huge compared with

other protons Further we could observe in the structures of 12 complexes that there are

several hydrogen bonds between the two neighbouring CD molecules (shown as green

dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes

The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that

could get involved in hydrogen bonding interactions Although the binding energies

prove the fact that the drug molecules enters with the aniline group or phenyl ring

through the wider rim of the CD cavity In order to explore the thermodynamic

parameters of the complexation process the statistical thermodynamic calculations were

carried out at 1 atm pressure and 298 K temperature The energy features

thermodynamic characteristics and electronic properties for the most stable 11 inclusion

complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated

drugs Further to quantify the interaction between drugs and CDs in the optimized

geometries we have determined the binding energy (ΔE) using Eqn (26) As expected

the binding energies of the inclusion complexes were more negative than the energies of

the isolated drugs and CDs suggest that the complexation process is more

thermodynamically favourable one The difference in binding energies (ΔE) for the most

stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal

mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD

(b)

2612 Aring

2701 Aring

2878 Aring

2804 Aring

(a)

2851 Aring

2675 Aring

108

The negative ΔH and ΔS values revealed that the formation of both inclusion complexes

is exothermic and enthalpy driven process On the other hand more negative ΔH values

observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion

complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG

values for these complexes are positive suggesting that the inclusion reaction was a non-

spontaneous process in vacuum phase Regrettably due to limitations in the calculating

ability of computers available with us the calculations for such a huge molecular system

could not be performed for aqueous phase However the experimental data as stated

earlier indicate that the formation of inclusion complexes in aqueous solution was

spontaneous one These variations may be due to the reason that all the calculations were

carried out in vacuum phase It is also considerable that during the inclusion

complexation in aqueous solution a number of water molecules are released from the

CD cavities

It can be observed from the complex structures that there are several

intermolecular hydrogen bonds between host and guest molecules in the structures (Figs

48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and

OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results

indicate that the intermolecular hydrogen bonding plays a significant role for the binding

energies and stabilizing the drugCD inclusion complexes

The geometric parameters such as bond distances bond angles and dihedral

angles of guest molecules before and after complexation in α-CD and β-CD were

obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion

complexes the geometry of drugs was completely changed The dihedral angles were

significantly altered indicating that guest must adapt a specific conformation to form a

more stable inclusion complex Further after the drug inclusion the geometrical

parameters of optimized structure of free CD also changed from truncated cone to an

oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen

and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that

those atoms played a crucial role in binding the drug molecules

The electronic structure of the inclusion complexes was also investigated in

addition to that of the isolated drug molecules HOMO as an electron donor represents

the capability to donate an electron and LUMO represents the capability to gain an

electron In HOMO of PCA the charge density is mainly accumulated on the benzene

ring and amino group whereas in PFO the charge density is observed on the CndashO and

CndashN bonds However in both cases of LUMO more charge density shifts to the benzene

109

ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during

HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC

bond of the benzene ring and carbonyl group It was also found that the charge densities

on each atom of the drugs within the CDs cavity were slightly changed Moreover the

energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical

species [145] The energy gaps for PCA and PFO as well as those for the most stable

complexes with CD were estimated by PM3 method and are shown in Table 44

Evidently the much lower energy gap which explains the fact that ultimate charge

transfer interaction occurs within the drug molecule The energy gap of drugβ-CD

complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD

complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex

is more stable than that of drugα-CD complex which is in good agreement with the

above discussed energy results In addition the EHOMOndashELUMO gap of both complexes

just fall in the range that of the PCA molecule suggesting there will be a blue shift in the

electronic spectrum of the guest molecule during binding and molecular recognition

This agrees well with the experimentally determined results

The polarity of the CD cavity is also taken into consideration for the formation of

most stable inclusion complexes The polarity of CD cavity significantly changed after

the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the

α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex

with respect to that of the dipole moment of optimized isolated hosts Although the

dipole moment of both complexes is lower than the sum of the dipole moments of

optimized isolated host and guest molecules From the above results one can conclude

that the dipole moment values show a strong correlation with the complexation

behaviour of CD

For the most stable complexes the physical parameters such as electronic

chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are

determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)

of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is

evident that the micro values of complexes differ remarkably from the isolated guest and host

molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD

therefore they are expected to act as electron acceptor in the inclusion complexation

interaction while the higher μ values of PCA indicates the drug act as electron donor In

the case of both η and ω values significant differences are observed when compared

with the isolated host (CD) molecule but it lies within the range that of the guest

110

Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method

Properties PCA PFO PCA α-CD

PCA β-CD

PFO α-CD

PFO β-CD

E -3427 -9462 -129371 -150783 -135294 -156835

∆E -1182 -1581 -1070 -1598

H 18209 19684 -40016 -49895 -37996 -48526

∆H -1141 -1349 -596 -1455

G 13929 14576 -53942 -65604 -51887 -63619

∆G -234 -581 1174 757

S 0143 0171 0449 0479 0456 0506

∆S -0047 -0073 -0068 -0074

EHOMO (eV) -886 -930 -829 -840 -946 -939

ELUMO (eV) -016 -024 009 004 -033 -019

EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920

Dipole moment (D) 439 168 583 995 784 1366

∆D -99 -673 -518 -031

micro -451 -477 -418 -410 -489 -479

η 435 453 422 419 456 460

S 022 022 023 024 021 021

ω 233 251 207 200 262 249

Unit =kcal mol-1 =kcalmol-Kelvin

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

97

Fig 43

98

Fig 44

99

the plots of 1AndashA0 and 1IndashI0 versus 1[CD]2 (Fig 44) ruled out the possibility of

12 stoichiometry between PFO and CDs This analysis reflects that 12 inclusion

complexes are formed between PCA and CDs whereas PFO formed 11 inclusion

complexes with CDs The K values for the inclusion complexes are given in Table 41

The quantitative comparison of binding constants of the two complexes suggests that

β-CD provides a better site to accommodate a deep inclusion of drugs in the β-CD

cavity Further it has been observed that the binding constants for 12 PCACD inclusion

systems are almost similar to the previously studied systems For example the binding

constant values of abs ~13542 M-1 and flu ~15576 M-1 for dothiepineβ-CD 12 inclusion

system [92] and abs ~14230 M-1 and flu ~48923 M-1 for fast violet-Bβ-CD 12

inclusion system [87]

The thermodynamic parameter free energy changes (∆G) for the binding of

guests within α-CD and β-CD were determined The negative free energy change values

in Table 41 indicate that the binding process is spontaneous and thermodynamically

favoured in the experimental temperature range (303 K) The negative values of ∆G

arose from the van der Waals interaction and the steric barrier caused by molecular

geometrical shape and the limit of CD cavity to the freedom of shift and rotation of guest

molecule

42 Effect of solvents

In order to understand the polarity around the drugs the absorption and

fluorescence spectra of PCA and PFO have been recorded in various solvents of different

polarities and hydrogen bonding abilities The absorption and fluorescence spectral

maxima in different solvents are compiled in Table 42 It can be observed from the

Table 42 that PCA shows much more interesting spectral features in different solvents

of various polarities than that of PFO In all solvents the absorption spectra of PCA are

much less structured with a slight shoulder around ~220 nm while the absorption spectra

of PFO are featureless with two peaks appearing around ~304 and 279 nm respectively

Data in Table 42 clearly indicate that the absorption spectra of PCA are red

shifted from cyclohexane to methanol but when compared to methanol blue shift is

observed in water The molar extinction coefficient is very high (~10-4 cm-1) indicating

that the observed absorption band corresponds to allowed πrarrπlowast transitions of the

benzene ring with a considerable charge transfer (CT) character Such kind of CT

process mainly originates from the aromatic ring or amino group (ndashNH2) to the amide

group (COndashNH) which is characterized by a high electron accepting character This can

be explained by analyzing the atomic orbital compositions of frontier molecular orbitals

100

of PCA as shown in Fig 45 The HOMO and LUMO energy calculations for PCA were

performed at PM3 level of theory In the HOMO the charge density is mainly

accumulated on the benzene ring and amino group (Fig 45) However in the case of

LUMO more charge density shifts to the benzene ring and carbonyl group This picture

clearly illustrates πrarrπlowast transition occurs when an electron density transfers from NH2

group to CminusC bond of the benzene ring and carbonyl group Thus nrarrπlowast transition of the

carbonyl group is hidden by an extended π conjugation of the phenyl ring to the

neighbouring carbonyl group [154-156] Also the absorption spectra of PCA are red

shifted in comparison to that of benzamide (cyclohexane asymp λabs ~270 230 nm) [157] and

benzanilide (cyclohexane asymp λabs ~262 254 247 nm λflu ~300 480 nm methanol asymp λabs

~261 nm λflu ~330 440 nm water asymp λabs ~256 nm λflu ~330 445 nm) [158] which

indicates an apparent perturbation effect of the amino group on the energy levels of the

parent benzamide molecule Further the spectral shifts observed in the absorption

spectrum of the drug molecule in protic and aprotic solvents are consistent with the

characteristic behaviour of aromatic molecules containing the amino group as an

auxochrome [90]

Table 42 Absorption and fluorescence spectral data (nm) and Stokes shifts

(cm-1) of PCA and PFO in different solvents

Solvents PCA PFO

λabs log ε λflu Stokes shifts λabs log ε λflu

Stokes shifts

Cyclohexane 280 223

sat 303 2660 300 249

sat 355s 336

5142

14-Dioxane 280 223

401 333

321 4460 300 248

369 367

340 331

3832

Ethyl acetate 285 222

416 353

326 4340 301 248

372 395

353s 339

4872

Acetonitrile 286 221

431 395

344 5871 302 246

377 390

353s 340

4783

2-Propanol 295 429 350 5235 303 248

378 398

354s 339

4667

Methanol 290 421 350 5746 303 249

376 401

419 338

9228

Water (pH ~65)

278 221s

408 303

357 7857 304 249

344 380

420 9176

sat - saturated

101

Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations

The fluorescence emission spectra of PCA and PFO in various solvents are

shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is

more pronounced than that on the corresponding effect on the absorption spectra which

reveals the excited state properties of the above drugs differ much from those in the

ground state PCA exhibits a single emission in all of the solvents whereas PFO gives

dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted

with increasing the polarity of solvents from cyclohexane to water The shift in the

emission maxima over absorption maxima indicates that the emitting state of the drug is

more polar than the ground state In PFO both SW and LW fluorescence bands are

affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are

largely red shifted and the half width of the LW fluorescence is significantly increased

(b) (a) H

OM

O

LU

MO

102

As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in

the present work can also be explained as hydrogen bond formation between the protic

solvents and electron withdrawing carbonyl group facilitates the formation of the

intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen

bonding seems to make the migration of electron density from benzene ring to the

electron withdrawing group more facile Further if the LW maximum is due to ICT this

should be more red shifted in protic solvents because ICT is more pronounced in protic

solvents than non-polar and aprotic solvents The spectral changes revealed that the LW

emission of PFO appeared as a result of ICT [160 161] which is more pronounced in

protic solvents than in other solvents The large Stokes shift indicates the strong changes

in the geometry of above molecules in the excited state [160 161] Moreover the

decreased emission intensity of PCA in water is likely due to the radiationless decay of

the probe molecule because of hydrogen bonding

Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

700

350

0 290 370 450

1

2

3 4

5

6

PCA λexci = 280 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

400

200

0 315 4075 500

1

2

3 4

5

PFO λexci = 305 nm

6

103

43 Time-resolved fluorescence studies

In order to confirm the interpretation of steady-state fluorescence spectroscopic

measurement data time-resolved fluorescence spectra and fluorescence decay times of

PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very

sensitive indicators for exploring the local environment around the excited state

fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and

CD environments are found to be quite different The typical time-resolved fluorescence

decay profile in different media is presented in Fig 47 and the average lifetimes are

given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly

affected by increasing the concentration of CD The results were judged by the statistical

fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission

wavelength was 355 nm Single exponential decay was observed for PCA in aqueous

solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve

becomes biexponential This indicates that PCA is encapsulated within the hydrophobic

nanocavity of CDs The life time of the inclusion complexes are higher than free drug

molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the

complexation ability of β-CD is higher in other words β-CD has given the privileged

encapsulation The amplitude of the complexed component also increased due to

increase in the complex formation and that of the decreased free species The

enhancement of τ1 and τ2 values with increase in the CD concentration is due to the

encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD

and the nature of the process with regard to short-lived species The decay of PCA is

dependent of the CD This may be due to the vibrational restriction of PCA in the excited

state

The fluorescence decay of PFO in water obtained by monitoring the emission

wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =

343 ns This analysis supports the presence of two emitting species ie LE and ICT

species in water possessing different fluorescence lifetimes However in the presence of

CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in

α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A

consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches

our interpretation of the steady-state fluorescence data indicating the formation of

inclusion complex between PFO and CDs The enhanced lifetimes associated with the

concentration of CD leads to the restriction of rotational degrees of freedom with

consequent impact on depletion of non-radiative decay channels Further when compared

104

to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows

the stronger hydrophobic interaction between PFO and β-CD

Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions

Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)

Drugs Medium Lifetime (ns) Pre exponential

factor ltτgt τ1 τ2 τ3 a1 a2 a3

PCA λemis = 355 nm

Water 056 041

α-CD 017 192 038 013 156

β-CD 021 386 032 007 313

PFO λemis = 420 nm

Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486

(a)

(b)

105

44 Molecular modeling studies

To scrutinize further the mechanism of inclusion complexation between the drugs

and CDs semiempirical quantum mechanical calculations at PM3 level of theory were

performed for the most favourable 11 inclusion mode This method is a very useful tool

for investigating the CD inclusion complexes in order to calculate the binding energy as

well as the geometrical structure of the inclusion complexes It is already proved that this

method is more reproducible when compared with the experimental data [162]

PM3 calculations for the inclusion process of drugs into the CD cavities were

carried out from the wider rim of the cavity because generally the approach of the guest

molecule to the CD cavity is more favourable for the wider rim side In addition when the

guest molecule is inserted into the cavity of CD the guest may rotate much more freely than

that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is

inserted from the wider rim of the CD cavity The energetically most favourable complex

formation strongly depends on the size and shape of the host and guest molecules From

PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately

~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular

dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit

entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped

within the hydrophobic cavity of CD So the calculations were carried out by inserting the

aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in

11 stoichiometric ratio followed by energy minimization But there was a failure in an

attempt to optimize 12 inclusion complexes of PCACD Hence only the energy

minimized structure of 12 PCACD complexes is given Upon the energy minimization it

was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host

molecules adjust themselves to give the most stable complex with least energy The 11

inclusion complex structure leading to minimum heat of formation shows the aniline moiety

or phenyl ring along with carbonyl group is encapsulated inside the CD cavity

The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD

PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for

12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in

Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the

hydrophobic CD cavity from the wider side and the binding geometries of both complexes

are almost similar Most prominently the simulation results are in good agreement with

the experimental data When compared to β-CD α-CD is closely associated with the drug

molecules consistent with the reduced diameter of the cavity

106

Figs 48

107

Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method

Fig 49 shows the structures of inclusion complexes of PCACDs in which both the

amino and methyl group protons along with phenyl ring are positioned nearly at narrow

rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and

methyl group protons of the drug in the process of complexation is huge compared with

other protons Further we could observe in the structures of 12 complexes that there are

several hydrogen bonds between the two neighbouring CD molecules (shown as green

dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes

The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that

could get involved in hydrogen bonding interactions Although the binding energies

prove the fact that the drug molecules enters with the aniline group or phenyl ring

through the wider rim of the CD cavity In order to explore the thermodynamic

parameters of the complexation process the statistical thermodynamic calculations were

carried out at 1 atm pressure and 298 K temperature The energy features

thermodynamic characteristics and electronic properties for the most stable 11 inclusion

complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated

drugs Further to quantify the interaction between drugs and CDs in the optimized

geometries we have determined the binding energy (ΔE) using Eqn (26) As expected

the binding energies of the inclusion complexes were more negative than the energies of

the isolated drugs and CDs suggest that the complexation process is more

thermodynamically favourable one The difference in binding energies (ΔE) for the most

stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal

mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD

(b)

2612 Aring

2701 Aring

2878 Aring

2804 Aring

(a)

2851 Aring

2675 Aring

108

The negative ΔH and ΔS values revealed that the formation of both inclusion complexes

is exothermic and enthalpy driven process On the other hand more negative ΔH values

observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion

complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG

values for these complexes are positive suggesting that the inclusion reaction was a non-

spontaneous process in vacuum phase Regrettably due to limitations in the calculating

ability of computers available with us the calculations for such a huge molecular system

could not be performed for aqueous phase However the experimental data as stated

earlier indicate that the formation of inclusion complexes in aqueous solution was

spontaneous one These variations may be due to the reason that all the calculations were

carried out in vacuum phase It is also considerable that during the inclusion

complexation in aqueous solution a number of water molecules are released from the

CD cavities

It can be observed from the complex structures that there are several

intermolecular hydrogen bonds between host and guest molecules in the structures (Figs

48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and

OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results

indicate that the intermolecular hydrogen bonding plays a significant role for the binding

energies and stabilizing the drugCD inclusion complexes

The geometric parameters such as bond distances bond angles and dihedral

angles of guest molecules before and after complexation in α-CD and β-CD were

obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion

complexes the geometry of drugs was completely changed The dihedral angles were

significantly altered indicating that guest must adapt a specific conformation to form a

more stable inclusion complex Further after the drug inclusion the geometrical

parameters of optimized structure of free CD also changed from truncated cone to an

oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen

and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that

those atoms played a crucial role in binding the drug molecules

The electronic structure of the inclusion complexes was also investigated in

addition to that of the isolated drug molecules HOMO as an electron donor represents

the capability to donate an electron and LUMO represents the capability to gain an

electron In HOMO of PCA the charge density is mainly accumulated on the benzene

ring and amino group whereas in PFO the charge density is observed on the CndashO and

CndashN bonds However in both cases of LUMO more charge density shifts to the benzene

109

ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during

HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC

bond of the benzene ring and carbonyl group It was also found that the charge densities

on each atom of the drugs within the CDs cavity were slightly changed Moreover the

energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical

species [145] The energy gaps for PCA and PFO as well as those for the most stable

complexes with CD were estimated by PM3 method and are shown in Table 44

Evidently the much lower energy gap which explains the fact that ultimate charge

transfer interaction occurs within the drug molecule The energy gap of drugβ-CD

complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD

complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex

is more stable than that of drugα-CD complex which is in good agreement with the

above discussed energy results In addition the EHOMOndashELUMO gap of both complexes

just fall in the range that of the PCA molecule suggesting there will be a blue shift in the

electronic spectrum of the guest molecule during binding and molecular recognition

This agrees well with the experimentally determined results

The polarity of the CD cavity is also taken into consideration for the formation of

most stable inclusion complexes The polarity of CD cavity significantly changed after

the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the

α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex

with respect to that of the dipole moment of optimized isolated hosts Although the

dipole moment of both complexes is lower than the sum of the dipole moments of

optimized isolated host and guest molecules From the above results one can conclude

that the dipole moment values show a strong correlation with the complexation

behaviour of CD

For the most stable complexes the physical parameters such as electronic

chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are

determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)

of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is

evident that the micro values of complexes differ remarkably from the isolated guest and host

molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD

therefore they are expected to act as electron acceptor in the inclusion complexation

interaction while the higher μ values of PCA indicates the drug act as electron donor In

the case of both η and ω values significant differences are observed when compared

with the isolated host (CD) molecule but it lies within the range that of the guest

110

Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method

Properties PCA PFO PCA α-CD

PCA β-CD

PFO α-CD

PFO β-CD

E -3427 -9462 -129371 -150783 -135294 -156835

∆E -1182 -1581 -1070 -1598

H 18209 19684 -40016 -49895 -37996 -48526

∆H -1141 -1349 -596 -1455

G 13929 14576 -53942 -65604 -51887 -63619

∆G -234 -581 1174 757

S 0143 0171 0449 0479 0456 0506

∆S -0047 -0073 -0068 -0074

EHOMO (eV) -886 -930 -829 -840 -946 -939

ELUMO (eV) -016 -024 009 004 -033 -019

EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920

Dipole moment (D) 439 168 583 995 784 1366

∆D -99 -673 -518 -031

micro -451 -477 -418 -410 -489 -479

η 435 453 422 419 456 460

S 022 022 023 024 021 021

ω 233 251 207 200 262 249

Unit =kcal mol-1 =kcalmol-Kelvin

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

98

Fig 44

99

the plots of 1AndashA0 and 1IndashI0 versus 1[CD]2 (Fig 44) ruled out the possibility of

12 stoichiometry between PFO and CDs This analysis reflects that 12 inclusion

complexes are formed between PCA and CDs whereas PFO formed 11 inclusion

complexes with CDs The K values for the inclusion complexes are given in Table 41

The quantitative comparison of binding constants of the two complexes suggests that

β-CD provides a better site to accommodate a deep inclusion of drugs in the β-CD

cavity Further it has been observed that the binding constants for 12 PCACD inclusion

systems are almost similar to the previously studied systems For example the binding

constant values of abs ~13542 M-1 and flu ~15576 M-1 for dothiepineβ-CD 12 inclusion

system [92] and abs ~14230 M-1 and flu ~48923 M-1 for fast violet-Bβ-CD 12

inclusion system [87]

The thermodynamic parameter free energy changes (∆G) for the binding of

guests within α-CD and β-CD were determined The negative free energy change values

in Table 41 indicate that the binding process is spontaneous and thermodynamically

favoured in the experimental temperature range (303 K) The negative values of ∆G

arose from the van der Waals interaction and the steric barrier caused by molecular

geometrical shape and the limit of CD cavity to the freedom of shift and rotation of guest

molecule

42 Effect of solvents

In order to understand the polarity around the drugs the absorption and

fluorescence spectra of PCA and PFO have been recorded in various solvents of different

polarities and hydrogen bonding abilities The absorption and fluorescence spectral

maxima in different solvents are compiled in Table 42 It can be observed from the

Table 42 that PCA shows much more interesting spectral features in different solvents

of various polarities than that of PFO In all solvents the absorption spectra of PCA are

much less structured with a slight shoulder around ~220 nm while the absorption spectra

of PFO are featureless with two peaks appearing around ~304 and 279 nm respectively

Data in Table 42 clearly indicate that the absorption spectra of PCA are red

shifted from cyclohexane to methanol but when compared to methanol blue shift is

observed in water The molar extinction coefficient is very high (~10-4 cm-1) indicating

that the observed absorption band corresponds to allowed πrarrπlowast transitions of the

benzene ring with a considerable charge transfer (CT) character Such kind of CT

process mainly originates from the aromatic ring or amino group (ndashNH2) to the amide

group (COndashNH) which is characterized by a high electron accepting character This can

be explained by analyzing the atomic orbital compositions of frontier molecular orbitals

100

of PCA as shown in Fig 45 The HOMO and LUMO energy calculations for PCA were

performed at PM3 level of theory In the HOMO the charge density is mainly

accumulated on the benzene ring and amino group (Fig 45) However in the case of

LUMO more charge density shifts to the benzene ring and carbonyl group This picture

clearly illustrates πrarrπlowast transition occurs when an electron density transfers from NH2

group to CminusC bond of the benzene ring and carbonyl group Thus nrarrπlowast transition of the

carbonyl group is hidden by an extended π conjugation of the phenyl ring to the

neighbouring carbonyl group [154-156] Also the absorption spectra of PCA are red

shifted in comparison to that of benzamide (cyclohexane asymp λabs ~270 230 nm) [157] and

benzanilide (cyclohexane asymp λabs ~262 254 247 nm λflu ~300 480 nm methanol asymp λabs

~261 nm λflu ~330 440 nm water asymp λabs ~256 nm λflu ~330 445 nm) [158] which

indicates an apparent perturbation effect of the amino group on the energy levels of the

parent benzamide molecule Further the spectral shifts observed in the absorption

spectrum of the drug molecule in protic and aprotic solvents are consistent with the

characteristic behaviour of aromatic molecules containing the amino group as an

auxochrome [90]

Table 42 Absorption and fluorescence spectral data (nm) and Stokes shifts

(cm-1) of PCA and PFO in different solvents

Solvents PCA PFO

λabs log ε λflu Stokes shifts λabs log ε λflu

Stokes shifts

Cyclohexane 280 223

sat 303 2660 300 249

sat 355s 336

5142

14-Dioxane 280 223

401 333

321 4460 300 248

369 367

340 331

3832

Ethyl acetate 285 222

416 353

326 4340 301 248

372 395

353s 339

4872

Acetonitrile 286 221

431 395

344 5871 302 246

377 390

353s 340

4783

2-Propanol 295 429 350 5235 303 248

378 398

354s 339

4667

Methanol 290 421 350 5746 303 249

376 401

419 338

9228

Water (pH ~65)

278 221s

408 303

357 7857 304 249

344 380

420 9176

sat - saturated

101

Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations

The fluorescence emission spectra of PCA and PFO in various solvents are

shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is

more pronounced than that on the corresponding effect on the absorption spectra which

reveals the excited state properties of the above drugs differ much from those in the

ground state PCA exhibits a single emission in all of the solvents whereas PFO gives

dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted

with increasing the polarity of solvents from cyclohexane to water The shift in the

emission maxima over absorption maxima indicates that the emitting state of the drug is

more polar than the ground state In PFO both SW and LW fluorescence bands are

affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are

largely red shifted and the half width of the LW fluorescence is significantly increased

(b) (a) H

OM

O

LU

MO

102

As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in

the present work can also be explained as hydrogen bond formation between the protic

solvents and electron withdrawing carbonyl group facilitates the formation of the

intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen

bonding seems to make the migration of electron density from benzene ring to the

electron withdrawing group more facile Further if the LW maximum is due to ICT this

should be more red shifted in protic solvents because ICT is more pronounced in protic

solvents than non-polar and aprotic solvents The spectral changes revealed that the LW

emission of PFO appeared as a result of ICT [160 161] which is more pronounced in

protic solvents than in other solvents The large Stokes shift indicates the strong changes

in the geometry of above molecules in the excited state [160 161] Moreover the

decreased emission intensity of PCA in water is likely due to the radiationless decay of

the probe molecule because of hydrogen bonding

Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

700

350

0 290 370 450

1

2

3 4

5

6

PCA λexci = 280 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

400

200

0 315 4075 500

1

2

3 4

5

PFO λexci = 305 nm

6

103

43 Time-resolved fluorescence studies

In order to confirm the interpretation of steady-state fluorescence spectroscopic

measurement data time-resolved fluorescence spectra and fluorescence decay times of

PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very

sensitive indicators for exploring the local environment around the excited state

fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and

CD environments are found to be quite different The typical time-resolved fluorescence

decay profile in different media is presented in Fig 47 and the average lifetimes are

given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly

affected by increasing the concentration of CD The results were judged by the statistical

fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission

wavelength was 355 nm Single exponential decay was observed for PCA in aqueous

solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve

becomes biexponential This indicates that PCA is encapsulated within the hydrophobic

nanocavity of CDs The life time of the inclusion complexes are higher than free drug

molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the

complexation ability of β-CD is higher in other words β-CD has given the privileged

encapsulation The amplitude of the complexed component also increased due to

increase in the complex formation and that of the decreased free species The

enhancement of τ1 and τ2 values with increase in the CD concentration is due to the

encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD

and the nature of the process with regard to short-lived species The decay of PCA is

dependent of the CD This may be due to the vibrational restriction of PCA in the excited

state

The fluorescence decay of PFO in water obtained by monitoring the emission

wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =

343 ns This analysis supports the presence of two emitting species ie LE and ICT

species in water possessing different fluorescence lifetimes However in the presence of

CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in

α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A

consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches

our interpretation of the steady-state fluorescence data indicating the formation of

inclusion complex between PFO and CDs The enhanced lifetimes associated with the

concentration of CD leads to the restriction of rotational degrees of freedom with

consequent impact on depletion of non-radiative decay channels Further when compared

104

to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows

the stronger hydrophobic interaction between PFO and β-CD

Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions

Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)

Drugs Medium Lifetime (ns) Pre exponential

factor ltτgt τ1 τ2 τ3 a1 a2 a3

PCA λemis = 355 nm

Water 056 041

α-CD 017 192 038 013 156

β-CD 021 386 032 007 313

PFO λemis = 420 nm

Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486

(a)

(b)

105

44 Molecular modeling studies

To scrutinize further the mechanism of inclusion complexation between the drugs

and CDs semiempirical quantum mechanical calculations at PM3 level of theory were

performed for the most favourable 11 inclusion mode This method is a very useful tool

for investigating the CD inclusion complexes in order to calculate the binding energy as

well as the geometrical structure of the inclusion complexes It is already proved that this

method is more reproducible when compared with the experimental data [162]

PM3 calculations for the inclusion process of drugs into the CD cavities were

carried out from the wider rim of the cavity because generally the approach of the guest

molecule to the CD cavity is more favourable for the wider rim side In addition when the

guest molecule is inserted into the cavity of CD the guest may rotate much more freely than

that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is

inserted from the wider rim of the CD cavity The energetically most favourable complex

formation strongly depends on the size and shape of the host and guest molecules From

PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately

~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular

dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit

entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped

within the hydrophobic cavity of CD So the calculations were carried out by inserting the

aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in

11 stoichiometric ratio followed by energy minimization But there was a failure in an

attempt to optimize 12 inclusion complexes of PCACD Hence only the energy

minimized structure of 12 PCACD complexes is given Upon the energy minimization it

was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host

molecules adjust themselves to give the most stable complex with least energy The 11

inclusion complex structure leading to minimum heat of formation shows the aniline moiety

or phenyl ring along with carbonyl group is encapsulated inside the CD cavity

The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD

PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for

12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in

Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the

hydrophobic CD cavity from the wider side and the binding geometries of both complexes

are almost similar Most prominently the simulation results are in good agreement with

the experimental data When compared to β-CD α-CD is closely associated with the drug

molecules consistent with the reduced diameter of the cavity

106

Figs 48

107

Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method

Fig 49 shows the structures of inclusion complexes of PCACDs in which both the

amino and methyl group protons along with phenyl ring are positioned nearly at narrow

rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and

methyl group protons of the drug in the process of complexation is huge compared with

other protons Further we could observe in the structures of 12 complexes that there are

several hydrogen bonds between the two neighbouring CD molecules (shown as green

dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes

The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that

could get involved in hydrogen bonding interactions Although the binding energies

prove the fact that the drug molecules enters with the aniline group or phenyl ring

through the wider rim of the CD cavity In order to explore the thermodynamic

parameters of the complexation process the statistical thermodynamic calculations were

carried out at 1 atm pressure and 298 K temperature The energy features

thermodynamic characteristics and electronic properties for the most stable 11 inclusion

complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated

drugs Further to quantify the interaction between drugs and CDs in the optimized

geometries we have determined the binding energy (ΔE) using Eqn (26) As expected

the binding energies of the inclusion complexes were more negative than the energies of

the isolated drugs and CDs suggest that the complexation process is more

thermodynamically favourable one The difference in binding energies (ΔE) for the most

stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal

mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD

(b)

2612 Aring

2701 Aring

2878 Aring

2804 Aring

(a)

2851 Aring

2675 Aring

108

The negative ΔH and ΔS values revealed that the formation of both inclusion complexes

is exothermic and enthalpy driven process On the other hand more negative ΔH values

observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion

complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG

values for these complexes are positive suggesting that the inclusion reaction was a non-

spontaneous process in vacuum phase Regrettably due to limitations in the calculating

ability of computers available with us the calculations for such a huge molecular system

could not be performed for aqueous phase However the experimental data as stated

earlier indicate that the formation of inclusion complexes in aqueous solution was

spontaneous one These variations may be due to the reason that all the calculations were

carried out in vacuum phase It is also considerable that during the inclusion

complexation in aqueous solution a number of water molecules are released from the

CD cavities

It can be observed from the complex structures that there are several

intermolecular hydrogen bonds between host and guest molecules in the structures (Figs

48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and

OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results

indicate that the intermolecular hydrogen bonding plays a significant role for the binding

energies and stabilizing the drugCD inclusion complexes

The geometric parameters such as bond distances bond angles and dihedral

angles of guest molecules before and after complexation in α-CD and β-CD were

obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion

complexes the geometry of drugs was completely changed The dihedral angles were

significantly altered indicating that guest must adapt a specific conformation to form a

more stable inclusion complex Further after the drug inclusion the geometrical

parameters of optimized structure of free CD also changed from truncated cone to an

oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen

and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that

those atoms played a crucial role in binding the drug molecules

The electronic structure of the inclusion complexes was also investigated in

addition to that of the isolated drug molecules HOMO as an electron donor represents

the capability to donate an electron and LUMO represents the capability to gain an

electron In HOMO of PCA the charge density is mainly accumulated on the benzene

ring and amino group whereas in PFO the charge density is observed on the CndashO and

CndashN bonds However in both cases of LUMO more charge density shifts to the benzene

109

ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during

HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC

bond of the benzene ring and carbonyl group It was also found that the charge densities

on each atom of the drugs within the CDs cavity were slightly changed Moreover the

energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical

species [145] The energy gaps for PCA and PFO as well as those for the most stable

complexes with CD were estimated by PM3 method and are shown in Table 44

Evidently the much lower energy gap which explains the fact that ultimate charge

transfer interaction occurs within the drug molecule The energy gap of drugβ-CD

complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD

complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex

is more stable than that of drugα-CD complex which is in good agreement with the

above discussed energy results In addition the EHOMOndashELUMO gap of both complexes

just fall in the range that of the PCA molecule suggesting there will be a blue shift in the

electronic spectrum of the guest molecule during binding and molecular recognition

This agrees well with the experimentally determined results

The polarity of the CD cavity is also taken into consideration for the formation of

most stable inclusion complexes The polarity of CD cavity significantly changed after

the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the

α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex

with respect to that of the dipole moment of optimized isolated hosts Although the

dipole moment of both complexes is lower than the sum of the dipole moments of

optimized isolated host and guest molecules From the above results one can conclude

that the dipole moment values show a strong correlation with the complexation

behaviour of CD

For the most stable complexes the physical parameters such as electronic

chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are

determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)

of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is

evident that the micro values of complexes differ remarkably from the isolated guest and host

molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD

therefore they are expected to act as electron acceptor in the inclusion complexation

interaction while the higher μ values of PCA indicates the drug act as electron donor In

the case of both η and ω values significant differences are observed when compared

with the isolated host (CD) molecule but it lies within the range that of the guest

110

Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method

Properties PCA PFO PCA α-CD

PCA β-CD

PFO α-CD

PFO β-CD

E -3427 -9462 -129371 -150783 -135294 -156835

∆E -1182 -1581 -1070 -1598

H 18209 19684 -40016 -49895 -37996 -48526

∆H -1141 -1349 -596 -1455

G 13929 14576 -53942 -65604 -51887 -63619

∆G -234 -581 1174 757

S 0143 0171 0449 0479 0456 0506

∆S -0047 -0073 -0068 -0074

EHOMO (eV) -886 -930 -829 -840 -946 -939

ELUMO (eV) -016 -024 009 004 -033 -019

EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920

Dipole moment (D) 439 168 583 995 784 1366

∆D -99 -673 -518 -031

micro -451 -477 -418 -410 -489 -479

η 435 453 422 419 456 460

S 022 022 023 024 021 021

ω 233 251 207 200 262 249

Unit =kcal mol-1 =kcalmol-Kelvin

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

99

the plots of 1AndashA0 and 1IndashI0 versus 1[CD]2 (Fig 44) ruled out the possibility of

12 stoichiometry between PFO and CDs This analysis reflects that 12 inclusion

complexes are formed between PCA and CDs whereas PFO formed 11 inclusion

complexes with CDs The K values for the inclusion complexes are given in Table 41

The quantitative comparison of binding constants of the two complexes suggests that

β-CD provides a better site to accommodate a deep inclusion of drugs in the β-CD

cavity Further it has been observed that the binding constants for 12 PCACD inclusion

systems are almost similar to the previously studied systems For example the binding

constant values of abs ~13542 M-1 and flu ~15576 M-1 for dothiepineβ-CD 12 inclusion

system [92] and abs ~14230 M-1 and flu ~48923 M-1 for fast violet-Bβ-CD 12

inclusion system [87]

The thermodynamic parameter free energy changes (∆G) for the binding of

guests within α-CD and β-CD were determined The negative free energy change values

in Table 41 indicate that the binding process is spontaneous and thermodynamically

favoured in the experimental temperature range (303 K) The negative values of ∆G

arose from the van der Waals interaction and the steric barrier caused by molecular

geometrical shape and the limit of CD cavity to the freedom of shift and rotation of guest

molecule

42 Effect of solvents

In order to understand the polarity around the drugs the absorption and

fluorescence spectra of PCA and PFO have been recorded in various solvents of different

polarities and hydrogen bonding abilities The absorption and fluorescence spectral

maxima in different solvents are compiled in Table 42 It can be observed from the

Table 42 that PCA shows much more interesting spectral features in different solvents

of various polarities than that of PFO In all solvents the absorption spectra of PCA are

much less structured with a slight shoulder around ~220 nm while the absorption spectra

of PFO are featureless with two peaks appearing around ~304 and 279 nm respectively

Data in Table 42 clearly indicate that the absorption spectra of PCA are red

shifted from cyclohexane to methanol but when compared to methanol blue shift is

observed in water The molar extinction coefficient is very high (~10-4 cm-1) indicating

that the observed absorption band corresponds to allowed πrarrπlowast transitions of the

benzene ring with a considerable charge transfer (CT) character Such kind of CT

process mainly originates from the aromatic ring or amino group (ndashNH2) to the amide

group (COndashNH) which is characterized by a high electron accepting character This can

be explained by analyzing the atomic orbital compositions of frontier molecular orbitals

100

of PCA as shown in Fig 45 The HOMO and LUMO energy calculations for PCA were

performed at PM3 level of theory In the HOMO the charge density is mainly

accumulated on the benzene ring and amino group (Fig 45) However in the case of

LUMO more charge density shifts to the benzene ring and carbonyl group This picture

clearly illustrates πrarrπlowast transition occurs when an electron density transfers from NH2

group to CminusC bond of the benzene ring and carbonyl group Thus nrarrπlowast transition of the

carbonyl group is hidden by an extended π conjugation of the phenyl ring to the

neighbouring carbonyl group [154-156] Also the absorption spectra of PCA are red

shifted in comparison to that of benzamide (cyclohexane asymp λabs ~270 230 nm) [157] and

benzanilide (cyclohexane asymp λabs ~262 254 247 nm λflu ~300 480 nm methanol asymp λabs

~261 nm λflu ~330 440 nm water asymp λabs ~256 nm λflu ~330 445 nm) [158] which

indicates an apparent perturbation effect of the amino group on the energy levels of the

parent benzamide molecule Further the spectral shifts observed in the absorption

spectrum of the drug molecule in protic and aprotic solvents are consistent with the

characteristic behaviour of aromatic molecules containing the amino group as an

auxochrome [90]

Table 42 Absorption and fluorescence spectral data (nm) and Stokes shifts

(cm-1) of PCA and PFO in different solvents

Solvents PCA PFO

λabs log ε λflu Stokes shifts λabs log ε λflu

Stokes shifts

Cyclohexane 280 223

sat 303 2660 300 249

sat 355s 336

5142

14-Dioxane 280 223

401 333

321 4460 300 248

369 367

340 331

3832

Ethyl acetate 285 222

416 353

326 4340 301 248

372 395

353s 339

4872

Acetonitrile 286 221

431 395

344 5871 302 246

377 390

353s 340

4783

2-Propanol 295 429 350 5235 303 248

378 398

354s 339

4667

Methanol 290 421 350 5746 303 249

376 401

419 338

9228

Water (pH ~65)

278 221s

408 303

357 7857 304 249

344 380

420 9176

sat - saturated

101

Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations

The fluorescence emission spectra of PCA and PFO in various solvents are

shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is

more pronounced than that on the corresponding effect on the absorption spectra which

reveals the excited state properties of the above drugs differ much from those in the

ground state PCA exhibits a single emission in all of the solvents whereas PFO gives

dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted

with increasing the polarity of solvents from cyclohexane to water The shift in the

emission maxima over absorption maxima indicates that the emitting state of the drug is

more polar than the ground state In PFO both SW and LW fluorescence bands are

affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are

largely red shifted and the half width of the LW fluorescence is significantly increased

(b) (a) H

OM

O

LU

MO

102

As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in

the present work can also be explained as hydrogen bond formation between the protic

solvents and electron withdrawing carbonyl group facilitates the formation of the

intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen

bonding seems to make the migration of electron density from benzene ring to the

electron withdrawing group more facile Further if the LW maximum is due to ICT this

should be more red shifted in protic solvents because ICT is more pronounced in protic

solvents than non-polar and aprotic solvents The spectral changes revealed that the LW

emission of PFO appeared as a result of ICT [160 161] which is more pronounced in

protic solvents than in other solvents The large Stokes shift indicates the strong changes

in the geometry of above molecules in the excited state [160 161] Moreover the

decreased emission intensity of PCA in water is likely due to the radiationless decay of

the probe molecule because of hydrogen bonding

Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

700

350

0 290 370 450

1

2

3 4

5

6

PCA λexci = 280 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

400

200

0 315 4075 500

1

2

3 4

5

PFO λexci = 305 nm

6

103

43 Time-resolved fluorescence studies

In order to confirm the interpretation of steady-state fluorescence spectroscopic

measurement data time-resolved fluorescence spectra and fluorescence decay times of

PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very

sensitive indicators for exploring the local environment around the excited state

fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and

CD environments are found to be quite different The typical time-resolved fluorescence

decay profile in different media is presented in Fig 47 and the average lifetimes are

given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly

affected by increasing the concentration of CD The results were judged by the statistical

fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission

wavelength was 355 nm Single exponential decay was observed for PCA in aqueous

solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve

becomes biexponential This indicates that PCA is encapsulated within the hydrophobic

nanocavity of CDs The life time of the inclusion complexes are higher than free drug

molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the

complexation ability of β-CD is higher in other words β-CD has given the privileged

encapsulation The amplitude of the complexed component also increased due to

increase in the complex formation and that of the decreased free species The

enhancement of τ1 and τ2 values with increase in the CD concentration is due to the

encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD

and the nature of the process with regard to short-lived species The decay of PCA is

dependent of the CD This may be due to the vibrational restriction of PCA in the excited

state

The fluorescence decay of PFO in water obtained by monitoring the emission

wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =

343 ns This analysis supports the presence of two emitting species ie LE and ICT

species in water possessing different fluorescence lifetimes However in the presence of

CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in

α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A

consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches

our interpretation of the steady-state fluorescence data indicating the formation of

inclusion complex between PFO and CDs The enhanced lifetimes associated with the

concentration of CD leads to the restriction of rotational degrees of freedom with

consequent impact on depletion of non-radiative decay channels Further when compared

104

to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows

the stronger hydrophobic interaction between PFO and β-CD

Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions

Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)

Drugs Medium Lifetime (ns) Pre exponential

factor ltτgt τ1 τ2 τ3 a1 a2 a3

PCA λemis = 355 nm

Water 056 041

α-CD 017 192 038 013 156

β-CD 021 386 032 007 313

PFO λemis = 420 nm

Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486

(a)

(b)

105

44 Molecular modeling studies

To scrutinize further the mechanism of inclusion complexation between the drugs

and CDs semiempirical quantum mechanical calculations at PM3 level of theory were

performed for the most favourable 11 inclusion mode This method is a very useful tool

for investigating the CD inclusion complexes in order to calculate the binding energy as

well as the geometrical structure of the inclusion complexes It is already proved that this

method is more reproducible when compared with the experimental data [162]

PM3 calculations for the inclusion process of drugs into the CD cavities were

carried out from the wider rim of the cavity because generally the approach of the guest

molecule to the CD cavity is more favourable for the wider rim side In addition when the

guest molecule is inserted into the cavity of CD the guest may rotate much more freely than

that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is

inserted from the wider rim of the CD cavity The energetically most favourable complex

formation strongly depends on the size and shape of the host and guest molecules From

PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately

~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular

dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit

entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped

within the hydrophobic cavity of CD So the calculations were carried out by inserting the

aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in

11 stoichiometric ratio followed by energy minimization But there was a failure in an

attempt to optimize 12 inclusion complexes of PCACD Hence only the energy

minimized structure of 12 PCACD complexes is given Upon the energy minimization it

was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host

molecules adjust themselves to give the most stable complex with least energy The 11

inclusion complex structure leading to minimum heat of formation shows the aniline moiety

or phenyl ring along with carbonyl group is encapsulated inside the CD cavity

The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD

PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for

12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in

Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the

hydrophobic CD cavity from the wider side and the binding geometries of both complexes

are almost similar Most prominently the simulation results are in good agreement with

the experimental data When compared to β-CD α-CD is closely associated with the drug

molecules consistent with the reduced diameter of the cavity

106

Figs 48

107

Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method

Fig 49 shows the structures of inclusion complexes of PCACDs in which both the

amino and methyl group protons along with phenyl ring are positioned nearly at narrow

rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and

methyl group protons of the drug in the process of complexation is huge compared with

other protons Further we could observe in the structures of 12 complexes that there are

several hydrogen bonds between the two neighbouring CD molecules (shown as green

dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes

The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that

could get involved in hydrogen bonding interactions Although the binding energies

prove the fact that the drug molecules enters with the aniline group or phenyl ring

through the wider rim of the CD cavity In order to explore the thermodynamic

parameters of the complexation process the statistical thermodynamic calculations were

carried out at 1 atm pressure and 298 K temperature The energy features

thermodynamic characteristics and electronic properties for the most stable 11 inclusion

complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated

drugs Further to quantify the interaction between drugs and CDs in the optimized

geometries we have determined the binding energy (ΔE) using Eqn (26) As expected

the binding energies of the inclusion complexes were more negative than the energies of

the isolated drugs and CDs suggest that the complexation process is more

thermodynamically favourable one The difference in binding energies (ΔE) for the most

stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal

mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD

(b)

2612 Aring

2701 Aring

2878 Aring

2804 Aring

(a)

2851 Aring

2675 Aring

108

The negative ΔH and ΔS values revealed that the formation of both inclusion complexes

is exothermic and enthalpy driven process On the other hand more negative ΔH values

observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion

complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG

values for these complexes are positive suggesting that the inclusion reaction was a non-

spontaneous process in vacuum phase Regrettably due to limitations in the calculating

ability of computers available with us the calculations for such a huge molecular system

could not be performed for aqueous phase However the experimental data as stated

earlier indicate that the formation of inclusion complexes in aqueous solution was

spontaneous one These variations may be due to the reason that all the calculations were

carried out in vacuum phase It is also considerable that during the inclusion

complexation in aqueous solution a number of water molecules are released from the

CD cavities

It can be observed from the complex structures that there are several

intermolecular hydrogen bonds between host and guest molecules in the structures (Figs

48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and

OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results

indicate that the intermolecular hydrogen bonding plays a significant role for the binding

energies and stabilizing the drugCD inclusion complexes

The geometric parameters such as bond distances bond angles and dihedral

angles of guest molecules before and after complexation in α-CD and β-CD were

obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion

complexes the geometry of drugs was completely changed The dihedral angles were

significantly altered indicating that guest must adapt a specific conformation to form a

more stable inclusion complex Further after the drug inclusion the geometrical

parameters of optimized structure of free CD also changed from truncated cone to an

oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen

and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that

those atoms played a crucial role in binding the drug molecules

The electronic structure of the inclusion complexes was also investigated in

addition to that of the isolated drug molecules HOMO as an electron donor represents

the capability to donate an electron and LUMO represents the capability to gain an

electron In HOMO of PCA the charge density is mainly accumulated on the benzene

ring and amino group whereas in PFO the charge density is observed on the CndashO and

CndashN bonds However in both cases of LUMO more charge density shifts to the benzene

109

ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during

HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC

bond of the benzene ring and carbonyl group It was also found that the charge densities

on each atom of the drugs within the CDs cavity were slightly changed Moreover the

energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical

species [145] The energy gaps for PCA and PFO as well as those for the most stable

complexes with CD were estimated by PM3 method and are shown in Table 44

Evidently the much lower energy gap which explains the fact that ultimate charge

transfer interaction occurs within the drug molecule The energy gap of drugβ-CD

complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD

complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex

is more stable than that of drugα-CD complex which is in good agreement with the

above discussed energy results In addition the EHOMOndashELUMO gap of both complexes

just fall in the range that of the PCA molecule suggesting there will be a blue shift in the

electronic spectrum of the guest molecule during binding and molecular recognition

This agrees well with the experimentally determined results

The polarity of the CD cavity is also taken into consideration for the formation of

most stable inclusion complexes The polarity of CD cavity significantly changed after

the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the

α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex

with respect to that of the dipole moment of optimized isolated hosts Although the

dipole moment of both complexes is lower than the sum of the dipole moments of

optimized isolated host and guest molecules From the above results one can conclude

that the dipole moment values show a strong correlation with the complexation

behaviour of CD

For the most stable complexes the physical parameters such as electronic

chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are

determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)

of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is

evident that the micro values of complexes differ remarkably from the isolated guest and host

molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD

therefore they are expected to act as electron acceptor in the inclusion complexation

interaction while the higher μ values of PCA indicates the drug act as electron donor In

the case of both η and ω values significant differences are observed when compared

with the isolated host (CD) molecule but it lies within the range that of the guest

110

Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method

Properties PCA PFO PCA α-CD

PCA β-CD

PFO α-CD

PFO β-CD

E -3427 -9462 -129371 -150783 -135294 -156835

∆E -1182 -1581 -1070 -1598

H 18209 19684 -40016 -49895 -37996 -48526

∆H -1141 -1349 -596 -1455

G 13929 14576 -53942 -65604 -51887 -63619

∆G -234 -581 1174 757

S 0143 0171 0449 0479 0456 0506

∆S -0047 -0073 -0068 -0074

EHOMO (eV) -886 -930 -829 -840 -946 -939

ELUMO (eV) -016 -024 009 004 -033 -019

EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920

Dipole moment (D) 439 168 583 995 784 1366

∆D -99 -673 -518 -031

micro -451 -477 -418 -410 -489 -479

η 435 453 422 419 456 460

S 022 022 023 024 021 021

ω 233 251 207 200 262 249

Unit =kcal mol-1 =kcalmol-Kelvin

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

100

of PCA as shown in Fig 45 The HOMO and LUMO energy calculations for PCA were

performed at PM3 level of theory In the HOMO the charge density is mainly

accumulated on the benzene ring and amino group (Fig 45) However in the case of

LUMO more charge density shifts to the benzene ring and carbonyl group This picture

clearly illustrates πrarrπlowast transition occurs when an electron density transfers from NH2

group to CminusC bond of the benzene ring and carbonyl group Thus nrarrπlowast transition of the

carbonyl group is hidden by an extended π conjugation of the phenyl ring to the

neighbouring carbonyl group [154-156] Also the absorption spectra of PCA are red

shifted in comparison to that of benzamide (cyclohexane asymp λabs ~270 230 nm) [157] and

benzanilide (cyclohexane asymp λabs ~262 254 247 nm λflu ~300 480 nm methanol asymp λabs

~261 nm λflu ~330 440 nm water asymp λabs ~256 nm λflu ~330 445 nm) [158] which

indicates an apparent perturbation effect of the amino group on the energy levels of the

parent benzamide molecule Further the spectral shifts observed in the absorption

spectrum of the drug molecule in protic and aprotic solvents are consistent with the

characteristic behaviour of aromatic molecules containing the amino group as an

auxochrome [90]

Table 42 Absorption and fluorescence spectral data (nm) and Stokes shifts

(cm-1) of PCA and PFO in different solvents

Solvents PCA PFO

λabs log ε λflu Stokes shifts λabs log ε λflu

Stokes shifts

Cyclohexane 280 223

sat 303 2660 300 249

sat 355s 336

5142

14-Dioxane 280 223

401 333

321 4460 300 248

369 367

340 331

3832

Ethyl acetate 285 222

416 353

326 4340 301 248

372 395

353s 339

4872

Acetonitrile 286 221

431 395

344 5871 302 246

377 390

353s 340

4783

2-Propanol 295 429 350 5235 303 248

378 398

354s 339

4667

Methanol 290 421 350 5746 303 249

376 401

419 338

9228

Water (pH ~65)

278 221s

408 303

357 7857 304 249

344 380

420 9176

sat - saturated

101

Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations

The fluorescence emission spectra of PCA and PFO in various solvents are

shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is

more pronounced than that on the corresponding effect on the absorption spectra which

reveals the excited state properties of the above drugs differ much from those in the

ground state PCA exhibits a single emission in all of the solvents whereas PFO gives

dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted

with increasing the polarity of solvents from cyclohexane to water The shift in the

emission maxima over absorption maxima indicates that the emitting state of the drug is

more polar than the ground state In PFO both SW and LW fluorescence bands are

affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are

largely red shifted and the half width of the LW fluorescence is significantly increased

(b) (a) H

OM

O

LU

MO

102

As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in

the present work can also be explained as hydrogen bond formation between the protic

solvents and electron withdrawing carbonyl group facilitates the formation of the

intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen

bonding seems to make the migration of electron density from benzene ring to the

electron withdrawing group more facile Further if the LW maximum is due to ICT this

should be more red shifted in protic solvents because ICT is more pronounced in protic

solvents than non-polar and aprotic solvents The spectral changes revealed that the LW

emission of PFO appeared as a result of ICT [160 161] which is more pronounced in

protic solvents than in other solvents The large Stokes shift indicates the strong changes

in the geometry of above molecules in the excited state [160 161] Moreover the

decreased emission intensity of PCA in water is likely due to the radiationless decay of

the probe molecule because of hydrogen bonding

Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

700

350

0 290 370 450

1

2

3 4

5

6

PCA λexci = 280 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

400

200

0 315 4075 500

1

2

3 4

5

PFO λexci = 305 nm

6

103

43 Time-resolved fluorescence studies

In order to confirm the interpretation of steady-state fluorescence spectroscopic

measurement data time-resolved fluorescence spectra and fluorescence decay times of

PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very

sensitive indicators for exploring the local environment around the excited state

fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and

CD environments are found to be quite different The typical time-resolved fluorescence

decay profile in different media is presented in Fig 47 and the average lifetimes are

given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly

affected by increasing the concentration of CD The results were judged by the statistical

fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission

wavelength was 355 nm Single exponential decay was observed for PCA in aqueous

solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve

becomes biexponential This indicates that PCA is encapsulated within the hydrophobic

nanocavity of CDs The life time of the inclusion complexes are higher than free drug

molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the

complexation ability of β-CD is higher in other words β-CD has given the privileged

encapsulation The amplitude of the complexed component also increased due to

increase in the complex formation and that of the decreased free species The

enhancement of τ1 and τ2 values with increase in the CD concentration is due to the

encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD

and the nature of the process with regard to short-lived species The decay of PCA is

dependent of the CD This may be due to the vibrational restriction of PCA in the excited

state

The fluorescence decay of PFO in water obtained by monitoring the emission

wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =

343 ns This analysis supports the presence of two emitting species ie LE and ICT

species in water possessing different fluorescence lifetimes However in the presence of

CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in

α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A

consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches

our interpretation of the steady-state fluorescence data indicating the formation of

inclusion complex between PFO and CDs The enhanced lifetimes associated with the

concentration of CD leads to the restriction of rotational degrees of freedom with

consequent impact on depletion of non-radiative decay channels Further when compared

104

to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows

the stronger hydrophobic interaction between PFO and β-CD

Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions

Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)

Drugs Medium Lifetime (ns) Pre exponential

factor ltτgt τ1 τ2 τ3 a1 a2 a3

PCA λemis = 355 nm

Water 056 041

α-CD 017 192 038 013 156

β-CD 021 386 032 007 313

PFO λemis = 420 nm

Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486

(a)

(b)

105

44 Molecular modeling studies

To scrutinize further the mechanism of inclusion complexation between the drugs

and CDs semiempirical quantum mechanical calculations at PM3 level of theory were

performed for the most favourable 11 inclusion mode This method is a very useful tool

for investigating the CD inclusion complexes in order to calculate the binding energy as

well as the geometrical structure of the inclusion complexes It is already proved that this

method is more reproducible when compared with the experimental data [162]

PM3 calculations for the inclusion process of drugs into the CD cavities were

carried out from the wider rim of the cavity because generally the approach of the guest

molecule to the CD cavity is more favourable for the wider rim side In addition when the

guest molecule is inserted into the cavity of CD the guest may rotate much more freely than

that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is

inserted from the wider rim of the CD cavity The energetically most favourable complex

formation strongly depends on the size and shape of the host and guest molecules From

PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately

~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular

dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit

entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped

within the hydrophobic cavity of CD So the calculations were carried out by inserting the

aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in

11 stoichiometric ratio followed by energy minimization But there was a failure in an

attempt to optimize 12 inclusion complexes of PCACD Hence only the energy

minimized structure of 12 PCACD complexes is given Upon the energy minimization it

was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host

molecules adjust themselves to give the most stable complex with least energy The 11

inclusion complex structure leading to minimum heat of formation shows the aniline moiety

or phenyl ring along with carbonyl group is encapsulated inside the CD cavity

The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD

PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for

12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in

Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the

hydrophobic CD cavity from the wider side and the binding geometries of both complexes

are almost similar Most prominently the simulation results are in good agreement with

the experimental data When compared to β-CD α-CD is closely associated with the drug

molecules consistent with the reduced diameter of the cavity

106

Figs 48

107

Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method

Fig 49 shows the structures of inclusion complexes of PCACDs in which both the

amino and methyl group protons along with phenyl ring are positioned nearly at narrow

rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and

methyl group protons of the drug in the process of complexation is huge compared with

other protons Further we could observe in the structures of 12 complexes that there are

several hydrogen bonds between the two neighbouring CD molecules (shown as green

dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes

The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that

could get involved in hydrogen bonding interactions Although the binding energies

prove the fact that the drug molecules enters with the aniline group or phenyl ring

through the wider rim of the CD cavity In order to explore the thermodynamic

parameters of the complexation process the statistical thermodynamic calculations were

carried out at 1 atm pressure and 298 K temperature The energy features

thermodynamic characteristics and electronic properties for the most stable 11 inclusion

complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated

drugs Further to quantify the interaction between drugs and CDs in the optimized

geometries we have determined the binding energy (ΔE) using Eqn (26) As expected

the binding energies of the inclusion complexes were more negative than the energies of

the isolated drugs and CDs suggest that the complexation process is more

thermodynamically favourable one The difference in binding energies (ΔE) for the most

stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal

mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD

(b)

2612 Aring

2701 Aring

2878 Aring

2804 Aring

(a)

2851 Aring

2675 Aring

108

The negative ΔH and ΔS values revealed that the formation of both inclusion complexes

is exothermic and enthalpy driven process On the other hand more negative ΔH values

observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion

complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG

values for these complexes are positive suggesting that the inclusion reaction was a non-

spontaneous process in vacuum phase Regrettably due to limitations in the calculating

ability of computers available with us the calculations for such a huge molecular system

could not be performed for aqueous phase However the experimental data as stated

earlier indicate that the formation of inclusion complexes in aqueous solution was

spontaneous one These variations may be due to the reason that all the calculations were

carried out in vacuum phase It is also considerable that during the inclusion

complexation in aqueous solution a number of water molecules are released from the

CD cavities

It can be observed from the complex structures that there are several

intermolecular hydrogen bonds between host and guest molecules in the structures (Figs

48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and

OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results

indicate that the intermolecular hydrogen bonding plays a significant role for the binding

energies and stabilizing the drugCD inclusion complexes

The geometric parameters such as bond distances bond angles and dihedral

angles of guest molecules before and after complexation in α-CD and β-CD were

obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion

complexes the geometry of drugs was completely changed The dihedral angles were

significantly altered indicating that guest must adapt a specific conformation to form a

more stable inclusion complex Further after the drug inclusion the geometrical

parameters of optimized structure of free CD also changed from truncated cone to an

oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen

and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that

those atoms played a crucial role in binding the drug molecules

The electronic structure of the inclusion complexes was also investigated in

addition to that of the isolated drug molecules HOMO as an electron donor represents

the capability to donate an electron and LUMO represents the capability to gain an

electron In HOMO of PCA the charge density is mainly accumulated on the benzene

ring and amino group whereas in PFO the charge density is observed on the CndashO and

CndashN bonds However in both cases of LUMO more charge density shifts to the benzene

109

ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during

HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC

bond of the benzene ring and carbonyl group It was also found that the charge densities

on each atom of the drugs within the CDs cavity were slightly changed Moreover the

energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical

species [145] The energy gaps for PCA and PFO as well as those for the most stable

complexes with CD were estimated by PM3 method and are shown in Table 44

Evidently the much lower energy gap which explains the fact that ultimate charge

transfer interaction occurs within the drug molecule The energy gap of drugβ-CD

complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD

complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex

is more stable than that of drugα-CD complex which is in good agreement with the

above discussed energy results In addition the EHOMOndashELUMO gap of both complexes

just fall in the range that of the PCA molecule suggesting there will be a blue shift in the

electronic spectrum of the guest molecule during binding and molecular recognition

This agrees well with the experimentally determined results

The polarity of the CD cavity is also taken into consideration for the formation of

most stable inclusion complexes The polarity of CD cavity significantly changed after

the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the

α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex

with respect to that of the dipole moment of optimized isolated hosts Although the

dipole moment of both complexes is lower than the sum of the dipole moments of

optimized isolated host and guest molecules From the above results one can conclude

that the dipole moment values show a strong correlation with the complexation

behaviour of CD

For the most stable complexes the physical parameters such as electronic

chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are

determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)

of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is

evident that the micro values of complexes differ remarkably from the isolated guest and host

molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD

therefore they are expected to act as electron acceptor in the inclusion complexation

interaction while the higher μ values of PCA indicates the drug act as electron donor In

the case of both η and ω values significant differences are observed when compared

with the isolated host (CD) molecule but it lies within the range that of the guest

110

Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method

Properties PCA PFO PCA α-CD

PCA β-CD

PFO α-CD

PFO β-CD

E -3427 -9462 -129371 -150783 -135294 -156835

∆E -1182 -1581 -1070 -1598

H 18209 19684 -40016 -49895 -37996 -48526

∆H -1141 -1349 -596 -1455

G 13929 14576 -53942 -65604 -51887 -63619

∆G -234 -581 1174 757

S 0143 0171 0449 0479 0456 0506

∆S -0047 -0073 -0068 -0074

EHOMO (eV) -886 -930 -829 -840 -946 -939

ELUMO (eV) -016 -024 009 004 -033 -019

EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920

Dipole moment (D) 439 168 583 995 784 1366

∆D -99 -673 -518 -031

micro -451 -477 -418 -410 -489 -479

η 435 453 422 419 456 460

S 022 022 023 024 021 021

ω 233 251 207 200 262 249

Unit =kcal mol-1 =kcalmol-Kelvin

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

101

Fig 45 The optimized ground state structures with numbering system and the HOMO and LUMO energy structures of (a) PCA and (b) PFO obtained by PM3 calculations

The fluorescence emission spectra of PCA and PFO in various solvents are

shown in Fig 46 The effect of the polarity of the medium on the fluorescence spectra is

more pronounced than that on the corresponding effect on the absorption spectra which

reveals the excited state properties of the above drugs differ much from those in the

ground state PCA exhibits a single emission in all of the solvents whereas PFO gives

dual emissions (SW and LW) In PCA the fluorescence maxima are regularly red shifted

with increasing the polarity of solvents from cyclohexane to water The shift in the

emission maxima over absorption maxima indicates that the emitting state of the drug is

more polar than the ground state In PFO both SW and LW fluorescence bands are

affected in nonpolar and polar solvents (Table 42) ie both SW and LW bands are

largely red shifted and the half width of the LW fluorescence is significantly increased

(b) (a) H

OM

O

LU

MO

102

As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in

the present work can also be explained as hydrogen bond formation between the protic

solvents and electron withdrawing carbonyl group facilitates the formation of the

intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen

bonding seems to make the migration of electron density from benzene ring to the

electron withdrawing group more facile Further if the LW maximum is due to ICT this

should be more red shifted in protic solvents because ICT is more pronounced in protic

solvents than non-polar and aprotic solvents The spectral changes revealed that the LW

emission of PFO appeared as a result of ICT [160 161] which is more pronounced in

protic solvents than in other solvents The large Stokes shift indicates the strong changes

in the geometry of above molecules in the excited state [160 161] Moreover the

decreased emission intensity of PCA in water is likely due to the radiationless decay of

the probe molecule because of hydrogen bonding

Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

700

350

0 290 370 450

1

2

3 4

5

6

PCA λexci = 280 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

400

200

0 315 4075 500

1

2

3 4

5

PFO λexci = 305 nm

6

103

43 Time-resolved fluorescence studies

In order to confirm the interpretation of steady-state fluorescence spectroscopic

measurement data time-resolved fluorescence spectra and fluorescence decay times of

PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very

sensitive indicators for exploring the local environment around the excited state

fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and

CD environments are found to be quite different The typical time-resolved fluorescence

decay profile in different media is presented in Fig 47 and the average lifetimes are

given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly

affected by increasing the concentration of CD The results were judged by the statistical

fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission

wavelength was 355 nm Single exponential decay was observed for PCA in aqueous

solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve

becomes biexponential This indicates that PCA is encapsulated within the hydrophobic

nanocavity of CDs The life time of the inclusion complexes are higher than free drug

molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the

complexation ability of β-CD is higher in other words β-CD has given the privileged

encapsulation The amplitude of the complexed component also increased due to

increase in the complex formation and that of the decreased free species The

enhancement of τ1 and τ2 values with increase in the CD concentration is due to the

encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD

and the nature of the process with regard to short-lived species The decay of PCA is

dependent of the CD This may be due to the vibrational restriction of PCA in the excited

state

The fluorescence decay of PFO in water obtained by monitoring the emission

wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =

343 ns This analysis supports the presence of two emitting species ie LE and ICT

species in water possessing different fluorescence lifetimes However in the presence of

CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in

α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A

consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches

our interpretation of the steady-state fluorescence data indicating the formation of

inclusion complex between PFO and CDs The enhanced lifetimes associated with the

concentration of CD leads to the restriction of rotational degrees of freedom with

consequent impact on depletion of non-radiative decay channels Further when compared

104

to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows

the stronger hydrophobic interaction between PFO and β-CD

Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions

Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)

Drugs Medium Lifetime (ns) Pre exponential

factor ltτgt τ1 τ2 τ3 a1 a2 a3

PCA λemis = 355 nm

Water 056 041

α-CD 017 192 038 013 156

β-CD 021 386 032 007 313

PFO λemis = 420 nm

Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486

(a)

(b)

105

44 Molecular modeling studies

To scrutinize further the mechanism of inclusion complexation between the drugs

and CDs semiempirical quantum mechanical calculations at PM3 level of theory were

performed for the most favourable 11 inclusion mode This method is a very useful tool

for investigating the CD inclusion complexes in order to calculate the binding energy as

well as the geometrical structure of the inclusion complexes It is already proved that this

method is more reproducible when compared with the experimental data [162]

PM3 calculations for the inclusion process of drugs into the CD cavities were

carried out from the wider rim of the cavity because generally the approach of the guest

molecule to the CD cavity is more favourable for the wider rim side In addition when the

guest molecule is inserted into the cavity of CD the guest may rotate much more freely than

that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is

inserted from the wider rim of the CD cavity The energetically most favourable complex

formation strongly depends on the size and shape of the host and guest molecules From

PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately

~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular

dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit

entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped

within the hydrophobic cavity of CD So the calculations were carried out by inserting the

aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in

11 stoichiometric ratio followed by energy minimization But there was a failure in an

attempt to optimize 12 inclusion complexes of PCACD Hence only the energy

minimized structure of 12 PCACD complexes is given Upon the energy minimization it

was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host

molecules adjust themselves to give the most stable complex with least energy The 11

inclusion complex structure leading to minimum heat of formation shows the aniline moiety

or phenyl ring along with carbonyl group is encapsulated inside the CD cavity

The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD

PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for

12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in

Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the

hydrophobic CD cavity from the wider side and the binding geometries of both complexes

are almost similar Most prominently the simulation results are in good agreement with

the experimental data When compared to β-CD α-CD is closely associated with the drug

molecules consistent with the reduced diameter of the cavity

106

Figs 48

107

Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method

Fig 49 shows the structures of inclusion complexes of PCACDs in which both the

amino and methyl group protons along with phenyl ring are positioned nearly at narrow

rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and

methyl group protons of the drug in the process of complexation is huge compared with

other protons Further we could observe in the structures of 12 complexes that there are

several hydrogen bonds between the two neighbouring CD molecules (shown as green

dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes

The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that

could get involved in hydrogen bonding interactions Although the binding energies

prove the fact that the drug molecules enters with the aniline group or phenyl ring

through the wider rim of the CD cavity In order to explore the thermodynamic

parameters of the complexation process the statistical thermodynamic calculations were

carried out at 1 atm pressure and 298 K temperature The energy features

thermodynamic characteristics and electronic properties for the most stable 11 inclusion

complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated

drugs Further to quantify the interaction between drugs and CDs in the optimized

geometries we have determined the binding energy (ΔE) using Eqn (26) As expected

the binding energies of the inclusion complexes were more negative than the energies of

the isolated drugs and CDs suggest that the complexation process is more

thermodynamically favourable one The difference in binding energies (ΔE) for the most

stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal

mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD

(b)

2612 Aring

2701 Aring

2878 Aring

2804 Aring

(a)

2851 Aring

2675 Aring

108

The negative ΔH and ΔS values revealed that the formation of both inclusion complexes

is exothermic and enthalpy driven process On the other hand more negative ΔH values

observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion

complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG

values for these complexes are positive suggesting that the inclusion reaction was a non-

spontaneous process in vacuum phase Regrettably due to limitations in the calculating

ability of computers available with us the calculations for such a huge molecular system

could not be performed for aqueous phase However the experimental data as stated

earlier indicate that the formation of inclusion complexes in aqueous solution was

spontaneous one These variations may be due to the reason that all the calculations were

carried out in vacuum phase It is also considerable that during the inclusion

complexation in aqueous solution a number of water molecules are released from the

CD cavities

It can be observed from the complex structures that there are several

intermolecular hydrogen bonds between host and guest molecules in the structures (Figs

48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and

OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results

indicate that the intermolecular hydrogen bonding plays a significant role for the binding

energies and stabilizing the drugCD inclusion complexes

The geometric parameters such as bond distances bond angles and dihedral

angles of guest molecules before and after complexation in α-CD and β-CD were

obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion

complexes the geometry of drugs was completely changed The dihedral angles were

significantly altered indicating that guest must adapt a specific conformation to form a

more stable inclusion complex Further after the drug inclusion the geometrical

parameters of optimized structure of free CD also changed from truncated cone to an

oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen

and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that

those atoms played a crucial role in binding the drug molecules

The electronic structure of the inclusion complexes was also investigated in

addition to that of the isolated drug molecules HOMO as an electron donor represents

the capability to donate an electron and LUMO represents the capability to gain an

electron In HOMO of PCA the charge density is mainly accumulated on the benzene

ring and amino group whereas in PFO the charge density is observed on the CndashO and

CndashN bonds However in both cases of LUMO more charge density shifts to the benzene

109

ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during

HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC

bond of the benzene ring and carbonyl group It was also found that the charge densities

on each atom of the drugs within the CDs cavity were slightly changed Moreover the

energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical

species [145] The energy gaps for PCA and PFO as well as those for the most stable

complexes with CD were estimated by PM3 method and are shown in Table 44

Evidently the much lower energy gap which explains the fact that ultimate charge

transfer interaction occurs within the drug molecule The energy gap of drugβ-CD

complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD

complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex

is more stable than that of drugα-CD complex which is in good agreement with the

above discussed energy results In addition the EHOMOndashELUMO gap of both complexes

just fall in the range that of the PCA molecule suggesting there will be a blue shift in the

electronic spectrum of the guest molecule during binding and molecular recognition

This agrees well with the experimentally determined results

The polarity of the CD cavity is also taken into consideration for the formation of

most stable inclusion complexes The polarity of CD cavity significantly changed after

the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the

α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex

with respect to that of the dipole moment of optimized isolated hosts Although the

dipole moment of both complexes is lower than the sum of the dipole moments of

optimized isolated host and guest molecules From the above results one can conclude

that the dipole moment values show a strong correlation with the complexation

behaviour of CD

For the most stable complexes the physical parameters such as electronic

chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are

determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)

of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is

evident that the micro values of complexes differ remarkably from the isolated guest and host

molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD

therefore they are expected to act as electron acceptor in the inclusion complexation

interaction while the higher μ values of PCA indicates the drug act as electron donor In

the case of both η and ω values significant differences are observed when compared

with the isolated host (CD) molecule but it lies within the range that of the guest

110

Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method

Properties PCA PFO PCA α-CD

PCA β-CD

PFO α-CD

PFO β-CD

E -3427 -9462 -129371 -150783 -135294 -156835

∆E -1182 -1581 -1070 -1598

H 18209 19684 -40016 -49895 -37996 -48526

∆H -1141 -1349 -596 -1455

G 13929 14576 -53942 -65604 -51887 -63619

∆G -234 -581 1174 757

S 0143 0171 0449 0479 0456 0506

∆S -0047 -0073 -0068 -0074

EHOMO (eV) -886 -930 -829 -840 -946 -939

ELUMO (eV) -016 -024 009 004 -033 -019

EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920

Dipole moment (D) 439 168 583 995 784 1366

∆D -99 -673 -518 -031

micro -451 -477 -418 -410 -489 -479

η 435 453 422 419 456 460

S 022 022 023 024 021 021

ω 233 251 207 200 262 249

Unit =kcal mol-1 =kcalmol-Kelvin

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

102

As demonstrated by Rettig [159] and our earlier studies [87-90] the results obtained in

the present work can also be explained as hydrogen bond formation between the protic

solvents and electron withdrawing carbonyl group facilitates the formation of the

intramolecular charge transfer (ICT) state in the S1 state In other words this hydrogen

bonding seems to make the migration of electron density from benzene ring to the

electron withdrawing group more facile Further if the LW maximum is due to ICT this

should be more red shifted in protic solvents because ICT is more pronounced in protic

solvents than non-polar and aprotic solvents The spectral changes revealed that the LW

emission of PFO appeared as a result of ICT [160 161] which is more pronounced in

protic solvents than in other solvents The large Stokes shift indicates the strong changes

in the geometry of above molecules in the excited state [160 161] Moreover the

decreased emission intensity of PCA in water is likely due to the radiationless decay of

the probe molecule because of hydrogen bonding

Fig 46 Fluorescence spectra of PCA and PFO in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethylacetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

700

350

0 290 370 450

1

2

3 4

5

6

PCA λexci = 280 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

400

200

0 315 4075 500

1

2

3 4

5

PFO λexci = 305 nm

6

103

43 Time-resolved fluorescence studies

In order to confirm the interpretation of steady-state fluorescence spectroscopic

measurement data time-resolved fluorescence spectra and fluorescence decay times of

PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very

sensitive indicators for exploring the local environment around the excited state

fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and

CD environments are found to be quite different The typical time-resolved fluorescence

decay profile in different media is presented in Fig 47 and the average lifetimes are

given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly

affected by increasing the concentration of CD The results were judged by the statistical

fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission

wavelength was 355 nm Single exponential decay was observed for PCA in aqueous

solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve

becomes biexponential This indicates that PCA is encapsulated within the hydrophobic

nanocavity of CDs The life time of the inclusion complexes are higher than free drug

molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the

complexation ability of β-CD is higher in other words β-CD has given the privileged

encapsulation The amplitude of the complexed component also increased due to

increase in the complex formation and that of the decreased free species The

enhancement of τ1 and τ2 values with increase in the CD concentration is due to the

encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD

and the nature of the process with regard to short-lived species The decay of PCA is

dependent of the CD This may be due to the vibrational restriction of PCA in the excited

state

The fluorescence decay of PFO in water obtained by monitoring the emission

wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =

343 ns This analysis supports the presence of two emitting species ie LE and ICT

species in water possessing different fluorescence lifetimes However in the presence of

CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in

α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A

consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches

our interpretation of the steady-state fluorescence data indicating the formation of

inclusion complex between PFO and CDs The enhanced lifetimes associated with the

concentration of CD leads to the restriction of rotational degrees of freedom with

consequent impact on depletion of non-radiative decay channels Further when compared

104

to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows

the stronger hydrophobic interaction between PFO and β-CD

Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions

Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)

Drugs Medium Lifetime (ns) Pre exponential

factor ltτgt τ1 τ2 τ3 a1 a2 a3

PCA λemis = 355 nm

Water 056 041

α-CD 017 192 038 013 156

β-CD 021 386 032 007 313

PFO λemis = 420 nm

Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486

(a)

(b)

105

44 Molecular modeling studies

To scrutinize further the mechanism of inclusion complexation between the drugs

and CDs semiempirical quantum mechanical calculations at PM3 level of theory were

performed for the most favourable 11 inclusion mode This method is a very useful tool

for investigating the CD inclusion complexes in order to calculate the binding energy as

well as the geometrical structure of the inclusion complexes It is already proved that this

method is more reproducible when compared with the experimental data [162]

PM3 calculations for the inclusion process of drugs into the CD cavities were

carried out from the wider rim of the cavity because generally the approach of the guest

molecule to the CD cavity is more favourable for the wider rim side In addition when the

guest molecule is inserted into the cavity of CD the guest may rotate much more freely than

that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is

inserted from the wider rim of the CD cavity The energetically most favourable complex

formation strongly depends on the size and shape of the host and guest molecules From

PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately

~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular

dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit

entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped

within the hydrophobic cavity of CD So the calculations were carried out by inserting the

aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in

11 stoichiometric ratio followed by energy minimization But there was a failure in an

attempt to optimize 12 inclusion complexes of PCACD Hence only the energy

minimized structure of 12 PCACD complexes is given Upon the energy minimization it

was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host

molecules adjust themselves to give the most stable complex with least energy The 11

inclusion complex structure leading to minimum heat of formation shows the aniline moiety

or phenyl ring along with carbonyl group is encapsulated inside the CD cavity

The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD

PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for

12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in

Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the

hydrophobic CD cavity from the wider side and the binding geometries of both complexes

are almost similar Most prominently the simulation results are in good agreement with

the experimental data When compared to β-CD α-CD is closely associated with the drug

molecules consistent with the reduced diameter of the cavity

106

Figs 48

107

Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method

Fig 49 shows the structures of inclusion complexes of PCACDs in which both the

amino and methyl group protons along with phenyl ring are positioned nearly at narrow

rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and

methyl group protons of the drug in the process of complexation is huge compared with

other protons Further we could observe in the structures of 12 complexes that there are

several hydrogen bonds between the two neighbouring CD molecules (shown as green

dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes

The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that

could get involved in hydrogen bonding interactions Although the binding energies

prove the fact that the drug molecules enters with the aniline group or phenyl ring

through the wider rim of the CD cavity In order to explore the thermodynamic

parameters of the complexation process the statistical thermodynamic calculations were

carried out at 1 atm pressure and 298 K temperature The energy features

thermodynamic characteristics and electronic properties for the most stable 11 inclusion

complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated

drugs Further to quantify the interaction between drugs and CDs in the optimized

geometries we have determined the binding energy (ΔE) using Eqn (26) As expected

the binding energies of the inclusion complexes were more negative than the energies of

the isolated drugs and CDs suggest that the complexation process is more

thermodynamically favourable one The difference in binding energies (ΔE) for the most

stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal

mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD

(b)

2612 Aring

2701 Aring

2878 Aring

2804 Aring

(a)

2851 Aring

2675 Aring

108

The negative ΔH and ΔS values revealed that the formation of both inclusion complexes

is exothermic and enthalpy driven process On the other hand more negative ΔH values

observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion

complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG

values for these complexes are positive suggesting that the inclusion reaction was a non-

spontaneous process in vacuum phase Regrettably due to limitations in the calculating

ability of computers available with us the calculations for such a huge molecular system

could not be performed for aqueous phase However the experimental data as stated

earlier indicate that the formation of inclusion complexes in aqueous solution was

spontaneous one These variations may be due to the reason that all the calculations were

carried out in vacuum phase It is also considerable that during the inclusion

complexation in aqueous solution a number of water molecules are released from the

CD cavities

It can be observed from the complex structures that there are several

intermolecular hydrogen bonds between host and guest molecules in the structures (Figs

48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and

OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results

indicate that the intermolecular hydrogen bonding plays a significant role for the binding

energies and stabilizing the drugCD inclusion complexes

The geometric parameters such as bond distances bond angles and dihedral

angles of guest molecules before and after complexation in α-CD and β-CD were

obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion

complexes the geometry of drugs was completely changed The dihedral angles were

significantly altered indicating that guest must adapt a specific conformation to form a

more stable inclusion complex Further after the drug inclusion the geometrical

parameters of optimized structure of free CD also changed from truncated cone to an

oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen

and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that

those atoms played a crucial role in binding the drug molecules

The electronic structure of the inclusion complexes was also investigated in

addition to that of the isolated drug molecules HOMO as an electron donor represents

the capability to donate an electron and LUMO represents the capability to gain an

electron In HOMO of PCA the charge density is mainly accumulated on the benzene

ring and amino group whereas in PFO the charge density is observed on the CndashO and

CndashN bonds However in both cases of LUMO more charge density shifts to the benzene

109

ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during

HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC

bond of the benzene ring and carbonyl group It was also found that the charge densities

on each atom of the drugs within the CDs cavity were slightly changed Moreover the

energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical

species [145] The energy gaps for PCA and PFO as well as those for the most stable

complexes with CD were estimated by PM3 method and are shown in Table 44

Evidently the much lower energy gap which explains the fact that ultimate charge

transfer interaction occurs within the drug molecule The energy gap of drugβ-CD

complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD

complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex

is more stable than that of drugα-CD complex which is in good agreement with the

above discussed energy results In addition the EHOMOndashELUMO gap of both complexes

just fall in the range that of the PCA molecule suggesting there will be a blue shift in the

electronic spectrum of the guest molecule during binding and molecular recognition

This agrees well with the experimentally determined results

The polarity of the CD cavity is also taken into consideration for the formation of

most stable inclusion complexes The polarity of CD cavity significantly changed after

the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the

α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex

with respect to that of the dipole moment of optimized isolated hosts Although the

dipole moment of both complexes is lower than the sum of the dipole moments of

optimized isolated host and guest molecules From the above results one can conclude

that the dipole moment values show a strong correlation with the complexation

behaviour of CD

For the most stable complexes the physical parameters such as electronic

chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are

determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)

of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is

evident that the micro values of complexes differ remarkably from the isolated guest and host

molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD

therefore they are expected to act as electron acceptor in the inclusion complexation

interaction while the higher μ values of PCA indicates the drug act as electron donor In

the case of both η and ω values significant differences are observed when compared

with the isolated host (CD) molecule but it lies within the range that of the guest

110

Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method

Properties PCA PFO PCA α-CD

PCA β-CD

PFO α-CD

PFO β-CD

E -3427 -9462 -129371 -150783 -135294 -156835

∆E -1182 -1581 -1070 -1598

H 18209 19684 -40016 -49895 -37996 -48526

∆H -1141 -1349 -596 -1455

G 13929 14576 -53942 -65604 -51887 -63619

∆G -234 -581 1174 757

S 0143 0171 0449 0479 0456 0506

∆S -0047 -0073 -0068 -0074

EHOMO (eV) -886 -930 -829 -840 -946 -939

ELUMO (eV) -016 -024 009 004 -033 -019

EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920

Dipole moment (D) 439 168 583 995 784 1366

∆D -99 -673 -518 -031

micro -451 -477 -418 -410 -489 -479

η 435 453 422 419 456 460

S 022 022 023 024 021 021

ω 233 251 207 200 262 249

Unit =kcal mol-1 =kcalmol-Kelvin

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

103

43 Time-resolved fluorescence studies

In order to confirm the interpretation of steady-state fluorescence spectroscopic

measurement data time-resolved fluorescence spectra and fluorescence decay times of

PCA and PFO in CD solutions were investigated Fluorescence lifetimes are often very

sensitive indicators for exploring the local environment around the excited state

fluorophores [138 139] The fluorescence decay behaviour of both drugs in water and

CD environments are found to be quite different The typical time-resolved fluorescence

decay profile in different media is presented in Fig 47 and the average lifetimes are

given in Table 43 Fig 47 demonstrates that the fluorescence decay is significantly

affected by increasing the concentration of CD The results were judged by the statistical

fitting parameter χ2 The excitation wavelength of PCA was 295 nm and emission

wavelength was 355 nm Single exponential decay was observed for PCA in aqueous

solution (056 ns) By the addition of CDs (10 times 10-3 M) single exponential curve

becomes biexponential This indicates that PCA is encapsulated within the hydrophobic

nanocavity of CDs The life time of the inclusion complexes are higher than free drug

molecule (PCA α-CD = 156 ns and β-CD = 313 ns) The above results reveal that the

complexation ability of β-CD is higher in other words β-CD has given the privileged

encapsulation The amplitude of the complexed component also increased due to

increase in the complex formation and that of the decreased free species The

enhancement of τ1 and τ2 values with increase in the CD concentration is due to the

encapsulation of PCA in the CD cavity The τ1 and τ2 values depend on the type of CD

and the nature of the process with regard to short-lived species The decay of PCA is

dependent of the CD This may be due to the vibrational restriction of PCA in the excited

state

The fluorescence decay of PFO in water obtained by monitoring the emission

wavelength at 420 nm is a bi-exponential plot with lifetime values τ1 = 038 ns and τ2 =

343 ns This analysis supports the presence of two emitting species ie LE and ICT

species in water possessing different fluorescence lifetimes However in the presence of

CD the fluorescence decays are fitted to tri-exponential pattern with three lifetimes (in

α-CDβ-CD) τ1 = 041154 ns τ2 = 289181 ns and τ3 = 411975 ns (Table 43) A

consistent enhancement of lifetimes of PFO with the addition of CD (001 M) matches

our interpretation of the steady-state fluorescence data indicating the formation of

inclusion complex between PFO and CDs The enhanced lifetimes associated with the

concentration of CD leads to the restriction of rotational degrees of freedom with

consequent impact on depletion of non-radiative decay channels Further when compared

104

to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows

the stronger hydrophobic interaction between PFO and β-CD

Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions

Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)

Drugs Medium Lifetime (ns) Pre exponential

factor ltτgt τ1 τ2 τ3 a1 a2 a3

PCA λemis = 355 nm

Water 056 041

α-CD 017 192 038 013 156

β-CD 021 386 032 007 313

PFO λemis = 420 nm

Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486

(a)

(b)

105

44 Molecular modeling studies

To scrutinize further the mechanism of inclusion complexation between the drugs

and CDs semiempirical quantum mechanical calculations at PM3 level of theory were

performed for the most favourable 11 inclusion mode This method is a very useful tool

for investigating the CD inclusion complexes in order to calculate the binding energy as

well as the geometrical structure of the inclusion complexes It is already proved that this

method is more reproducible when compared with the experimental data [162]

PM3 calculations for the inclusion process of drugs into the CD cavities were

carried out from the wider rim of the cavity because generally the approach of the guest

molecule to the CD cavity is more favourable for the wider rim side In addition when the

guest molecule is inserted into the cavity of CD the guest may rotate much more freely than

that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is

inserted from the wider rim of the CD cavity The energetically most favourable complex

formation strongly depends on the size and shape of the host and guest molecules From

PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately

~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular

dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit

entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped

within the hydrophobic cavity of CD So the calculations were carried out by inserting the

aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in

11 stoichiometric ratio followed by energy minimization But there was a failure in an

attempt to optimize 12 inclusion complexes of PCACD Hence only the energy

minimized structure of 12 PCACD complexes is given Upon the energy minimization it

was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host

molecules adjust themselves to give the most stable complex with least energy The 11

inclusion complex structure leading to minimum heat of formation shows the aniline moiety

or phenyl ring along with carbonyl group is encapsulated inside the CD cavity

The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD

PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for

12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in

Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the

hydrophobic CD cavity from the wider side and the binding geometries of both complexes

are almost similar Most prominently the simulation results are in good agreement with

the experimental data When compared to β-CD α-CD is closely associated with the drug

molecules consistent with the reduced diameter of the cavity

106

Figs 48

107

Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method

Fig 49 shows the structures of inclusion complexes of PCACDs in which both the

amino and methyl group protons along with phenyl ring are positioned nearly at narrow

rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and

methyl group protons of the drug in the process of complexation is huge compared with

other protons Further we could observe in the structures of 12 complexes that there are

several hydrogen bonds between the two neighbouring CD molecules (shown as green

dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes

The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that

could get involved in hydrogen bonding interactions Although the binding energies

prove the fact that the drug molecules enters with the aniline group or phenyl ring

through the wider rim of the CD cavity In order to explore the thermodynamic

parameters of the complexation process the statistical thermodynamic calculations were

carried out at 1 atm pressure and 298 K temperature The energy features

thermodynamic characteristics and electronic properties for the most stable 11 inclusion

complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated

drugs Further to quantify the interaction between drugs and CDs in the optimized

geometries we have determined the binding energy (ΔE) using Eqn (26) As expected

the binding energies of the inclusion complexes were more negative than the energies of

the isolated drugs and CDs suggest that the complexation process is more

thermodynamically favourable one The difference in binding energies (ΔE) for the most

stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal

mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD

(b)

2612 Aring

2701 Aring

2878 Aring

2804 Aring

(a)

2851 Aring

2675 Aring

108

The negative ΔH and ΔS values revealed that the formation of both inclusion complexes

is exothermic and enthalpy driven process On the other hand more negative ΔH values

observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion

complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG

values for these complexes are positive suggesting that the inclusion reaction was a non-

spontaneous process in vacuum phase Regrettably due to limitations in the calculating

ability of computers available with us the calculations for such a huge molecular system

could not be performed for aqueous phase However the experimental data as stated

earlier indicate that the formation of inclusion complexes in aqueous solution was

spontaneous one These variations may be due to the reason that all the calculations were

carried out in vacuum phase It is also considerable that during the inclusion

complexation in aqueous solution a number of water molecules are released from the

CD cavities

It can be observed from the complex structures that there are several

intermolecular hydrogen bonds between host and guest molecules in the structures (Figs

48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and

OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results

indicate that the intermolecular hydrogen bonding plays a significant role for the binding

energies and stabilizing the drugCD inclusion complexes

The geometric parameters such as bond distances bond angles and dihedral

angles of guest molecules before and after complexation in α-CD and β-CD were

obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion

complexes the geometry of drugs was completely changed The dihedral angles were

significantly altered indicating that guest must adapt a specific conformation to form a

more stable inclusion complex Further after the drug inclusion the geometrical

parameters of optimized structure of free CD also changed from truncated cone to an

oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen

and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that

those atoms played a crucial role in binding the drug molecules

The electronic structure of the inclusion complexes was also investigated in

addition to that of the isolated drug molecules HOMO as an electron donor represents

the capability to donate an electron and LUMO represents the capability to gain an

electron In HOMO of PCA the charge density is mainly accumulated on the benzene

ring and amino group whereas in PFO the charge density is observed on the CndashO and

CndashN bonds However in both cases of LUMO more charge density shifts to the benzene

109

ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during

HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC

bond of the benzene ring and carbonyl group It was also found that the charge densities

on each atom of the drugs within the CDs cavity were slightly changed Moreover the

energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical

species [145] The energy gaps for PCA and PFO as well as those for the most stable

complexes with CD were estimated by PM3 method and are shown in Table 44

Evidently the much lower energy gap which explains the fact that ultimate charge

transfer interaction occurs within the drug molecule The energy gap of drugβ-CD

complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD

complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex

is more stable than that of drugα-CD complex which is in good agreement with the

above discussed energy results In addition the EHOMOndashELUMO gap of both complexes

just fall in the range that of the PCA molecule suggesting there will be a blue shift in the

electronic spectrum of the guest molecule during binding and molecular recognition

This agrees well with the experimentally determined results

The polarity of the CD cavity is also taken into consideration for the formation of

most stable inclusion complexes The polarity of CD cavity significantly changed after

the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the

α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex

with respect to that of the dipole moment of optimized isolated hosts Although the

dipole moment of both complexes is lower than the sum of the dipole moments of

optimized isolated host and guest molecules From the above results one can conclude

that the dipole moment values show a strong correlation with the complexation

behaviour of CD

For the most stable complexes the physical parameters such as electronic

chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are

determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)

of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is

evident that the micro values of complexes differ remarkably from the isolated guest and host

molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD

therefore they are expected to act as electron acceptor in the inclusion complexation

interaction while the higher μ values of PCA indicates the drug act as electron donor In

the case of both η and ω values significant differences are observed when compared

with the isolated host (CD) molecule but it lies within the range that of the guest

110

Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method

Properties PCA PFO PCA α-CD

PCA β-CD

PFO α-CD

PFO β-CD

E -3427 -9462 -129371 -150783 -135294 -156835

∆E -1182 -1581 -1070 -1598

H 18209 19684 -40016 -49895 -37996 -48526

∆H -1141 -1349 -596 -1455

G 13929 14576 -53942 -65604 -51887 -63619

∆G -234 -581 1174 757

S 0143 0171 0449 0479 0456 0506

∆S -0047 -0073 -0068 -0074

EHOMO (eV) -886 -930 -829 -840 -946 -939

ELUMO (eV) -016 -024 009 004 -033 -019

EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920

Dipole moment (D) 439 168 583 995 784 1366

∆D -99 -673 -518 -031

micro -451 -477 -418 -410 -489 -479

η 435 453 422 419 456 460

S 022 022 023 024 021 021

ω 233 251 207 200 262 249

Unit =kcal mol-1 =kcalmol-Kelvin

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

104

to α-CD the increasing efficiency of lifetime is found to be higher at β-CD which shows

the stronger hydrophobic interaction between PFO and β-CD

Fig 47 Fluorescence decay curves of (a) PCA and (b) PFO in water and 001 M CD solutions

Table 43 Fluorescence decay parameters of PCA and PFO in water and 001 M CD solutions (λexcitation = 295 nm)

Drugs Medium Lifetime (ns) Pre exponential

factor ltτgt τ1 τ2 τ3 a1 a2 a3

PCA λemis = 355 nm

Water 056 041

α-CD 017 192 038 013 156

β-CD 021 386 032 007 313

PFO λemis = 420 nm

Water 038 343 041 004 180 α-CD 041 289 411 043 009 005 259 β-CD 154 181 975 016 011 003 486

(a)

(b)

105

44 Molecular modeling studies

To scrutinize further the mechanism of inclusion complexation between the drugs

and CDs semiempirical quantum mechanical calculations at PM3 level of theory were

performed for the most favourable 11 inclusion mode This method is a very useful tool

for investigating the CD inclusion complexes in order to calculate the binding energy as

well as the geometrical structure of the inclusion complexes It is already proved that this

method is more reproducible when compared with the experimental data [162]

PM3 calculations for the inclusion process of drugs into the CD cavities were

carried out from the wider rim of the cavity because generally the approach of the guest

molecule to the CD cavity is more favourable for the wider rim side In addition when the

guest molecule is inserted into the cavity of CD the guest may rotate much more freely than

that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is

inserted from the wider rim of the CD cavity The energetically most favourable complex

formation strongly depends on the size and shape of the host and guest molecules From

PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately

~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular

dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit

entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped

within the hydrophobic cavity of CD So the calculations were carried out by inserting the

aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in

11 stoichiometric ratio followed by energy minimization But there was a failure in an

attempt to optimize 12 inclusion complexes of PCACD Hence only the energy

minimized structure of 12 PCACD complexes is given Upon the energy minimization it

was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host

molecules adjust themselves to give the most stable complex with least energy The 11

inclusion complex structure leading to minimum heat of formation shows the aniline moiety

or phenyl ring along with carbonyl group is encapsulated inside the CD cavity

The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD

PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for

12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in

Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the

hydrophobic CD cavity from the wider side and the binding geometries of both complexes

are almost similar Most prominently the simulation results are in good agreement with

the experimental data When compared to β-CD α-CD is closely associated with the drug

molecules consistent with the reduced diameter of the cavity

106

Figs 48

107

Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method

Fig 49 shows the structures of inclusion complexes of PCACDs in which both the

amino and methyl group protons along with phenyl ring are positioned nearly at narrow

rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and

methyl group protons of the drug in the process of complexation is huge compared with

other protons Further we could observe in the structures of 12 complexes that there are

several hydrogen bonds between the two neighbouring CD molecules (shown as green

dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes

The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that

could get involved in hydrogen bonding interactions Although the binding energies

prove the fact that the drug molecules enters with the aniline group or phenyl ring

through the wider rim of the CD cavity In order to explore the thermodynamic

parameters of the complexation process the statistical thermodynamic calculations were

carried out at 1 atm pressure and 298 K temperature The energy features

thermodynamic characteristics and electronic properties for the most stable 11 inclusion

complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated

drugs Further to quantify the interaction between drugs and CDs in the optimized

geometries we have determined the binding energy (ΔE) using Eqn (26) As expected

the binding energies of the inclusion complexes were more negative than the energies of

the isolated drugs and CDs suggest that the complexation process is more

thermodynamically favourable one The difference in binding energies (ΔE) for the most

stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal

mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD

(b)

2612 Aring

2701 Aring

2878 Aring

2804 Aring

(a)

2851 Aring

2675 Aring

108

The negative ΔH and ΔS values revealed that the formation of both inclusion complexes

is exothermic and enthalpy driven process On the other hand more negative ΔH values

observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion

complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG

values for these complexes are positive suggesting that the inclusion reaction was a non-

spontaneous process in vacuum phase Regrettably due to limitations in the calculating

ability of computers available with us the calculations for such a huge molecular system

could not be performed for aqueous phase However the experimental data as stated

earlier indicate that the formation of inclusion complexes in aqueous solution was

spontaneous one These variations may be due to the reason that all the calculations were

carried out in vacuum phase It is also considerable that during the inclusion

complexation in aqueous solution a number of water molecules are released from the

CD cavities

It can be observed from the complex structures that there are several

intermolecular hydrogen bonds between host and guest molecules in the structures (Figs

48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and

OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results

indicate that the intermolecular hydrogen bonding plays a significant role for the binding

energies and stabilizing the drugCD inclusion complexes

The geometric parameters such as bond distances bond angles and dihedral

angles of guest molecules before and after complexation in α-CD and β-CD were

obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion

complexes the geometry of drugs was completely changed The dihedral angles were

significantly altered indicating that guest must adapt a specific conformation to form a

more stable inclusion complex Further after the drug inclusion the geometrical

parameters of optimized structure of free CD also changed from truncated cone to an

oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen

and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that

those atoms played a crucial role in binding the drug molecules

The electronic structure of the inclusion complexes was also investigated in

addition to that of the isolated drug molecules HOMO as an electron donor represents

the capability to donate an electron and LUMO represents the capability to gain an

electron In HOMO of PCA the charge density is mainly accumulated on the benzene

ring and amino group whereas in PFO the charge density is observed on the CndashO and

CndashN bonds However in both cases of LUMO more charge density shifts to the benzene

109

ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during

HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC

bond of the benzene ring and carbonyl group It was also found that the charge densities

on each atom of the drugs within the CDs cavity were slightly changed Moreover the

energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical

species [145] The energy gaps for PCA and PFO as well as those for the most stable

complexes with CD were estimated by PM3 method and are shown in Table 44

Evidently the much lower energy gap which explains the fact that ultimate charge

transfer interaction occurs within the drug molecule The energy gap of drugβ-CD

complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD

complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex

is more stable than that of drugα-CD complex which is in good agreement with the

above discussed energy results In addition the EHOMOndashELUMO gap of both complexes

just fall in the range that of the PCA molecule suggesting there will be a blue shift in the

electronic spectrum of the guest molecule during binding and molecular recognition

This agrees well with the experimentally determined results

The polarity of the CD cavity is also taken into consideration for the formation of

most stable inclusion complexes The polarity of CD cavity significantly changed after

the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the

α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex

with respect to that of the dipole moment of optimized isolated hosts Although the

dipole moment of both complexes is lower than the sum of the dipole moments of

optimized isolated host and guest molecules From the above results one can conclude

that the dipole moment values show a strong correlation with the complexation

behaviour of CD

For the most stable complexes the physical parameters such as electronic

chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are

determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)

of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is

evident that the micro values of complexes differ remarkably from the isolated guest and host

molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD

therefore they are expected to act as electron acceptor in the inclusion complexation

interaction while the higher μ values of PCA indicates the drug act as electron donor In

the case of both η and ω values significant differences are observed when compared

with the isolated host (CD) molecule but it lies within the range that of the guest

110

Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method

Properties PCA PFO PCA α-CD

PCA β-CD

PFO α-CD

PFO β-CD

E -3427 -9462 -129371 -150783 -135294 -156835

∆E -1182 -1581 -1070 -1598

H 18209 19684 -40016 -49895 -37996 -48526

∆H -1141 -1349 -596 -1455

G 13929 14576 -53942 -65604 -51887 -63619

∆G -234 -581 1174 757

S 0143 0171 0449 0479 0456 0506

∆S -0047 -0073 -0068 -0074

EHOMO (eV) -886 -930 -829 -840 -946 -939

ELUMO (eV) -016 -024 009 004 -033 -019

EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920

Dipole moment (D) 439 168 583 995 784 1366

∆D -99 -673 -518 -031

micro -451 -477 -418 -410 -489 -479

η 435 453 422 419 456 460

S 022 022 023 024 021 021

ω 233 251 207 200 262 249

Unit =kcal mol-1 =kcalmol-Kelvin

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

105

44 Molecular modeling studies

To scrutinize further the mechanism of inclusion complexation between the drugs

and CDs semiempirical quantum mechanical calculations at PM3 level of theory were

performed for the most favourable 11 inclusion mode This method is a very useful tool

for investigating the CD inclusion complexes in order to calculate the binding energy as

well as the geometrical structure of the inclusion complexes It is already proved that this

method is more reproducible when compared with the experimental data [162]

PM3 calculations for the inclusion process of drugs into the CD cavities were

carried out from the wider rim of the cavity because generally the approach of the guest

molecule to the CD cavity is more favourable for the wider rim side In addition when the

guest molecule is inserted into the cavity of CD the guest may rotate much more freely than

that from the narrower rim For both drugs the phenyl moiety along with carbonyl group is

inserted from the wider rim of the CD cavity The energetically most favourable complex

formation strongly depends on the size and shape of the host and guest molecules From

PM3 calculations the internal diameter of α-CD and β-CD was found to be approximately

~65 and 56 Aring respectively and their height is ~78 Aring According to the molecular

dimensions of drugs (long axis PCA = 126807 Aring and PFO = 12544 Aring) it is too large to fit

entirely into the CD cavity and therefore the guest molecule cannot be fully entrapped

within the hydrophobic cavity of CD So the calculations were carried out by inserting the

aniline moiety (for PCA) or phenyl ring with C=O group (for PFO) into the CDs cavity in

11 stoichiometric ratio followed by energy minimization But there was a failure in an

attempt to optimize 12 inclusion complexes of PCACD Hence only the energy

minimized structure of 12 PCACD complexes is given Upon the energy minimization it

was observed that the phenyl ring is pulled inside the cavity and the atoms of guest or host

molecules adjust themselves to give the most stable complex with least energy The 11

inclusion complex structure leading to minimum heat of formation shows the aniline moiety

or phenyl ring along with carbonyl group is encapsulated inside the CD cavity

The optimized geometries of 11 inclusion complexes of PCAα-CD PCAβ-CD

PFOα-CD and PFOβ-CD obtained by PM3 method and energy minimized structures for

12 complexes of PCACD are shown in Figs 48 and 49 respectively As shown in

Figs 48 and 49 the aniline moiety of phenyl ring of the drugs is encapsulated into the

hydrophobic CD cavity from the wider side and the binding geometries of both complexes

are almost similar Most prominently the simulation results are in good agreement with

the experimental data When compared to β-CD α-CD is closely associated with the drug

molecules consistent with the reduced diameter of the cavity

106

Figs 48

107

Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method

Fig 49 shows the structures of inclusion complexes of PCACDs in which both the

amino and methyl group protons along with phenyl ring are positioned nearly at narrow

rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and

methyl group protons of the drug in the process of complexation is huge compared with

other protons Further we could observe in the structures of 12 complexes that there are

several hydrogen bonds between the two neighbouring CD molecules (shown as green

dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes

The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that

could get involved in hydrogen bonding interactions Although the binding energies

prove the fact that the drug molecules enters with the aniline group or phenyl ring

through the wider rim of the CD cavity In order to explore the thermodynamic

parameters of the complexation process the statistical thermodynamic calculations were

carried out at 1 atm pressure and 298 K temperature The energy features

thermodynamic characteristics and electronic properties for the most stable 11 inclusion

complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated

drugs Further to quantify the interaction between drugs and CDs in the optimized

geometries we have determined the binding energy (ΔE) using Eqn (26) As expected

the binding energies of the inclusion complexes were more negative than the energies of

the isolated drugs and CDs suggest that the complexation process is more

thermodynamically favourable one The difference in binding energies (ΔE) for the most

stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal

mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD

(b)

2612 Aring

2701 Aring

2878 Aring

2804 Aring

(a)

2851 Aring

2675 Aring

108

The negative ΔH and ΔS values revealed that the formation of both inclusion complexes

is exothermic and enthalpy driven process On the other hand more negative ΔH values

observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion

complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG

values for these complexes are positive suggesting that the inclusion reaction was a non-

spontaneous process in vacuum phase Regrettably due to limitations in the calculating

ability of computers available with us the calculations for such a huge molecular system

could not be performed for aqueous phase However the experimental data as stated

earlier indicate that the formation of inclusion complexes in aqueous solution was

spontaneous one These variations may be due to the reason that all the calculations were

carried out in vacuum phase It is also considerable that during the inclusion

complexation in aqueous solution a number of water molecules are released from the

CD cavities

It can be observed from the complex structures that there are several

intermolecular hydrogen bonds between host and guest molecules in the structures (Figs

48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and

OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results

indicate that the intermolecular hydrogen bonding plays a significant role for the binding

energies and stabilizing the drugCD inclusion complexes

The geometric parameters such as bond distances bond angles and dihedral

angles of guest molecules before and after complexation in α-CD and β-CD were

obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion

complexes the geometry of drugs was completely changed The dihedral angles were

significantly altered indicating that guest must adapt a specific conformation to form a

more stable inclusion complex Further after the drug inclusion the geometrical

parameters of optimized structure of free CD also changed from truncated cone to an

oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen

and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that

those atoms played a crucial role in binding the drug molecules

The electronic structure of the inclusion complexes was also investigated in

addition to that of the isolated drug molecules HOMO as an electron donor represents

the capability to donate an electron and LUMO represents the capability to gain an

electron In HOMO of PCA the charge density is mainly accumulated on the benzene

ring and amino group whereas in PFO the charge density is observed on the CndashO and

CndashN bonds However in both cases of LUMO more charge density shifts to the benzene

109

ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during

HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC

bond of the benzene ring and carbonyl group It was also found that the charge densities

on each atom of the drugs within the CDs cavity were slightly changed Moreover the

energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical

species [145] The energy gaps for PCA and PFO as well as those for the most stable

complexes with CD were estimated by PM3 method and are shown in Table 44

Evidently the much lower energy gap which explains the fact that ultimate charge

transfer interaction occurs within the drug molecule The energy gap of drugβ-CD

complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD

complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex

is more stable than that of drugα-CD complex which is in good agreement with the

above discussed energy results In addition the EHOMOndashELUMO gap of both complexes

just fall in the range that of the PCA molecule suggesting there will be a blue shift in the

electronic spectrum of the guest molecule during binding and molecular recognition

This agrees well with the experimentally determined results

The polarity of the CD cavity is also taken into consideration for the formation of

most stable inclusion complexes The polarity of CD cavity significantly changed after

the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the

α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex

with respect to that of the dipole moment of optimized isolated hosts Although the

dipole moment of both complexes is lower than the sum of the dipole moments of

optimized isolated host and guest molecules From the above results one can conclude

that the dipole moment values show a strong correlation with the complexation

behaviour of CD

For the most stable complexes the physical parameters such as electronic

chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are

determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)

of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is

evident that the micro values of complexes differ remarkably from the isolated guest and host

molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD

therefore they are expected to act as electron acceptor in the inclusion complexation

interaction while the higher μ values of PCA indicates the drug act as electron donor In

the case of both η and ω values significant differences are observed when compared

with the isolated host (CD) molecule but it lies within the range that of the guest

110

Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method

Properties PCA PFO PCA α-CD

PCA β-CD

PFO α-CD

PFO β-CD

E -3427 -9462 -129371 -150783 -135294 -156835

∆E -1182 -1581 -1070 -1598

H 18209 19684 -40016 -49895 -37996 -48526

∆H -1141 -1349 -596 -1455

G 13929 14576 -53942 -65604 -51887 -63619

∆G -234 -581 1174 757

S 0143 0171 0449 0479 0456 0506

∆S -0047 -0073 -0068 -0074

EHOMO (eV) -886 -930 -829 -840 -946 -939

ELUMO (eV) -016 -024 009 004 -033 -019

EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920

Dipole moment (D) 439 168 583 995 784 1366

∆D -99 -673 -518 -031

micro -451 -477 -418 -410 -489 -479

η 435 453 422 419 456 460

S 022 022 023 024 021 021

ω 233 251 207 200 262 249

Unit =kcal mol-1 =kcalmol-Kelvin

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

106

Figs 48

107

Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method

Fig 49 shows the structures of inclusion complexes of PCACDs in which both the

amino and methyl group protons along with phenyl ring are positioned nearly at narrow

rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and

methyl group protons of the drug in the process of complexation is huge compared with

other protons Further we could observe in the structures of 12 complexes that there are

several hydrogen bonds between the two neighbouring CD molecules (shown as green

dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes

The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that

could get involved in hydrogen bonding interactions Although the binding energies

prove the fact that the drug molecules enters with the aniline group or phenyl ring

through the wider rim of the CD cavity In order to explore the thermodynamic

parameters of the complexation process the statistical thermodynamic calculations were

carried out at 1 atm pressure and 298 K temperature The energy features

thermodynamic characteristics and electronic properties for the most stable 11 inclusion

complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated

drugs Further to quantify the interaction between drugs and CDs in the optimized

geometries we have determined the binding energy (ΔE) using Eqn (26) As expected

the binding energies of the inclusion complexes were more negative than the energies of

the isolated drugs and CDs suggest that the complexation process is more

thermodynamically favourable one The difference in binding energies (ΔE) for the most

stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal

mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD

(b)

2612 Aring

2701 Aring

2878 Aring

2804 Aring

(a)

2851 Aring

2675 Aring

108

The negative ΔH and ΔS values revealed that the formation of both inclusion complexes

is exothermic and enthalpy driven process On the other hand more negative ΔH values

observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion

complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG

values for these complexes are positive suggesting that the inclusion reaction was a non-

spontaneous process in vacuum phase Regrettably due to limitations in the calculating

ability of computers available with us the calculations for such a huge molecular system

could not be performed for aqueous phase However the experimental data as stated

earlier indicate that the formation of inclusion complexes in aqueous solution was

spontaneous one These variations may be due to the reason that all the calculations were

carried out in vacuum phase It is also considerable that during the inclusion

complexation in aqueous solution a number of water molecules are released from the

CD cavities

It can be observed from the complex structures that there are several

intermolecular hydrogen bonds between host and guest molecules in the structures (Figs

48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and

OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results

indicate that the intermolecular hydrogen bonding plays a significant role for the binding

energies and stabilizing the drugCD inclusion complexes

The geometric parameters such as bond distances bond angles and dihedral

angles of guest molecules before and after complexation in α-CD and β-CD were

obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion

complexes the geometry of drugs was completely changed The dihedral angles were

significantly altered indicating that guest must adapt a specific conformation to form a

more stable inclusion complex Further after the drug inclusion the geometrical

parameters of optimized structure of free CD also changed from truncated cone to an

oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen

and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that

those atoms played a crucial role in binding the drug molecules

The electronic structure of the inclusion complexes was also investigated in

addition to that of the isolated drug molecules HOMO as an electron donor represents

the capability to donate an electron and LUMO represents the capability to gain an

electron In HOMO of PCA the charge density is mainly accumulated on the benzene

ring and amino group whereas in PFO the charge density is observed on the CndashO and

CndashN bonds However in both cases of LUMO more charge density shifts to the benzene

109

ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during

HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC

bond of the benzene ring and carbonyl group It was also found that the charge densities

on each atom of the drugs within the CDs cavity were slightly changed Moreover the

energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical

species [145] The energy gaps for PCA and PFO as well as those for the most stable

complexes with CD were estimated by PM3 method and are shown in Table 44

Evidently the much lower energy gap which explains the fact that ultimate charge

transfer interaction occurs within the drug molecule The energy gap of drugβ-CD

complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD

complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex

is more stable than that of drugα-CD complex which is in good agreement with the

above discussed energy results In addition the EHOMOndashELUMO gap of both complexes

just fall in the range that of the PCA molecule suggesting there will be a blue shift in the

electronic spectrum of the guest molecule during binding and molecular recognition

This agrees well with the experimentally determined results

The polarity of the CD cavity is also taken into consideration for the formation of

most stable inclusion complexes The polarity of CD cavity significantly changed after

the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the

α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex

with respect to that of the dipole moment of optimized isolated hosts Although the

dipole moment of both complexes is lower than the sum of the dipole moments of

optimized isolated host and guest molecules From the above results one can conclude

that the dipole moment values show a strong correlation with the complexation

behaviour of CD

For the most stable complexes the physical parameters such as electronic

chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are

determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)

of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is

evident that the micro values of complexes differ remarkably from the isolated guest and host

molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD

therefore they are expected to act as electron acceptor in the inclusion complexation

interaction while the higher μ values of PCA indicates the drug act as electron donor In

the case of both η and ω values significant differences are observed when compared

with the isolated host (CD) molecule but it lies within the range that of the guest

110

Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method

Properties PCA PFO PCA α-CD

PCA β-CD

PFO α-CD

PFO β-CD

E -3427 -9462 -129371 -150783 -135294 -156835

∆E -1182 -1581 -1070 -1598

H 18209 19684 -40016 -49895 -37996 -48526

∆H -1141 -1349 -596 -1455

G 13929 14576 -53942 -65604 -51887 -63619

∆G -234 -581 1174 757

S 0143 0171 0449 0479 0456 0506

∆S -0047 -0073 -0068 -0074

EHOMO (eV) -886 -930 -829 -840 -946 -939

ELUMO (eV) -016 -024 009 004 -033 -019

EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920

Dipole moment (D) 439 168 583 995 784 1366

∆D -99 -673 -518 -031

micro -451 -477 -418 -410 -489 -479

η 435 453 422 419 456 460

S 022 022 023 024 021 021

ω 233 251 207 200 262 249

Unit =kcal mol-1 =kcalmol-Kelvin

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

107

Fig 49 The energy minimized structures of 12 inclusion complexes of (a) PCAα-CD and (b) PCAβ-CD obtained by PM3 method

Fig 49 shows the structures of inclusion complexes of PCACDs in which both the

amino and methyl group protons along with phenyl ring are positioned nearly at narrow

rim of the CD cavity and so the effect on the 1H NMR chemical shifts of amino and

methyl group protons of the drug in the process of complexation is huge compared with

other protons Further we could observe in the structures of 12 complexes that there are

several hydrogen bonds between the two neighbouring CD molecules (shown as green

dotted line in Fig 49) which further stabilized the structure of 12 inclusion complexes

The drugs have two functional groups ie C=O and ndashNH2 or ndashOH groups that

could get involved in hydrogen bonding interactions Although the binding energies

prove the fact that the drug molecules enters with the aniline group or phenyl ring

through the wider rim of the CD cavity In order to explore the thermodynamic

parameters of the complexation process the statistical thermodynamic calculations were

carried out at 1 atm pressure and 298 K temperature The energy features

thermodynamic characteristics and electronic properties for the most stable 11 inclusion

complexes drugα-CD and drugβ-CD are listed in Table 44 along with the isolated

drugs Further to quantify the interaction between drugs and CDs in the optimized

geometries we have determined the binding energy (ΔE) using Eqn (26) As expected

the binding energies of the inclusion complexes were more negative than the energies of

the isolated drugs and CDs suggest that the complexation process is more

thermodynamically favourable one The difference in binding energies (ΔE) for the most

stable complexes of the two CDs with drugs are 399 kcal mol-1 (for PCA) and 528 kcal

mol-1 (for PFO) indicating that the drugβ-CD complex is more stable than drugα-CD

(b)

2612 Aring

2701 Aring

2878 Aring

2804 Aring

(a)

2851 Aring

2675 Aring

108

The negative ΔH and ΔS values revealed that the formation of both inclusion complexes

is exothermic and enthalpy driven process On the other hand more negative ΔH values

observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion

complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG

values for these complexes are positive suggesting that the inclusion reaction was a non-

spontaneous process in vacuum phase Regrettably due to limitations in the calculating

ability of computers available with us the calculations for such a huge molecular system

could not be performed for aqueous phase However the experimental data as stated

earlier indicate that the formation of inclusion complexes in aqueous solution was

spontaneous one These variations may be due to the reason that all the calculations were

carried out in vacuum phase It is also considerable that during the inclusion

complexation in aqueous solution a number of water molecules are released from the

CD cavities

It can be observed from the complex structures that there are several

intermolecular hydrogen bonds between host and guest molecules in the structures (Figs

48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and

OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results

indicate that the intermolecular hydrogen bonding plays a significant role for the binding

energies and stabilizing the drugCD inclusion complexes

The geometric parameters such as bond distances bond angles and dihedral

angles of guest molecules before and after complexation in α-CD and β-CD were

obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion

complexes the geometry of drugs was completely changed The dihedral angles were

significantly altered indicating that guest must adapt a specific conformation to form a

more stable inclusion complex Further after the drug inclusion the geometrical

parameters of optimized structure of free CD also changed from truncated cone to an

oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen

and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that

those atoms played a crucial role in binding the drug molecules

The electronic structure of the inclusion complexes was also investigated in

addition to that of the isolated drug molecules HOMO as an electron donor represents

the capability to donate an electron and LUMO represents the capability to gain an

electron In HOMO of PCA the charge density is mainly accumulated on the benzene

ring and amino group whereas in PFO the charge density is observed on the CndashO and

CndashN bonds However in both cases of LUMO more charge density shifts to the benzene

109

ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during

HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC

bond of the benzene ring and carbonyl group It was also found that the charge densities

on each atom of the drugs within the CDs cavity were slightly changed Moreover the

energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical

species [145] The energy gaps for PCA and PFO as well as those for the most stable

complexes with CD were estimated by PM3 method and are shown in Table 44

Evidently the much lower energy gap which explains the fact that ultimate charge

transfer interaction occurs within the drug molecule The energy gap of drugβ-CD

complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD

complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex

is more stable than that of drugα-CD complex which is in good agreement with the

above discussed energy results In addition the EHOMOndashELUMO gap of both complexes

just fall in the range that of the PCA molecule suggesting there will be a blue shift in the

electronic spectrum of the guest molecule during binding and molecular recognition

This agrees well with the experimentally determined results

The polarity of the CD cavity is also taken into consideration for the formation of

most stable inclusion complexes The polarity of CD cavity significantly changed after

the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the

α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex

with respect to that of the dipole moment of optimized isolated hosts Although the

dipole moment of both complexes is lower than the sum of the dipole moments of

optimized isolated host and guest molecules From the above results one can conclude

that the dipole moment values show a strong correlation with the complexation

behaviour of CD

For the most stable complexes the physical parameters such as electronic

chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are

determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)

of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is

evident that the micro values of complexes differ remarkably from the isolated guest and host

molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD

therefore they are expected to act as electron acceptor in the inclusion complexation

interaction while the higher μ values of PCA indicates the drug act as electron donor In

the case of both η and ω values significant differences are observed when compared

with the isolated host (CD) molecule but it lies within the range that of the guest

110

Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method

Properties PCA PFO PCA α-CD

PCA β-CD

PFO α-CD

PFO β-CD

E -3427 -9462 -129371 -150783 -135294 -156835

∆E -1182 -1581 -1070 -1598

H 18209 19684 -40016 -49895 -37996 -48526

∆H -1141 -1349 -596 -1455

G 13929 14576 -53942 -65604 -51887 -63619

∆G -234 -581 1174 757

S 0143 0171 0449 0479 0456 0506

∆S -0047 -0073 -0068 -0074

EHOMO (eV) -886 -930 -829 -840 -946 -939

ELUMO (eV) -016 -024 009 004 -033 -019

EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920

Dipole moment (D) 439 168 583 995 784 1366

∆D -99 -673 -518 -031

micro -451 -477 -418 -410 -489 -479

η 435 453 422 419 456 460

S 022 022 023 024 021 021

ω 233 251 207 200 262 249

Unit =kcal mol-1 =kcalmol-Kelvin

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

108

The negative ΔH and ΔS values revealed that the formation of both inclusion complexes

is exothermic and enthalpy driven process On the other hand more negative ΔH values

observed for the drugsβ-CD complexes compared to the drugsα-CD inclusion

complexes demonstrates the guest molecules are tightly bound to the β-CD cavity ΔG

values for these complexes are positive suggesting that the inclusion reaction was a non-

spontaneous process in vacuum phase Regrettably due to limitations in the calculating

ability of computers available with us the calculations for such a huge molecular system

could not be performed for aqueous phase However the experimental data as stated

earlier indicate that the formation of inclusion complexes in aqueous solution was

spontaneous one These variations may be due to the reason that all the calculations were

carried out in vacuum phase It is also considerable that during the inclusion

complexation in aqueous solution a number of water molecules are released from the

CD cavities

It can be observed from the complex structures that there are several

intermolecular hydrogen bonds between host and guest molecules in the structures (Figs

48 and 49) The hydrogen bonds are described as CminusHO C=OH NminusHO and

OminusHH with the lengths ranging from 256 to 296 Aring (lt30 Aring) [142] The above results

indicate that the intermolecular hydrogen bonding plays a significant role for the binding

energies and stabilizing the drugCD inclusion complexes

The geometric parameters such as bond distances bond angles and dihedral

angles of guest molecules before and after complexation in α-CD and β-CD were

obtained from PM3 optimized structures (Table 45) It is noticeable that in the inclusion

complexes the geometry of drugs was completely changed The dihedral angles were

significantly altered indicating that guest must adapt a specific conformation to form a

more stable inclusion complex Further after the drug inclusion the geometrical

parameters of optimized structure of free CD also changed from truncated cone to an

oval shape (Figs 48 and 49) Further the hydrogen bond formed between the oxygen

and hydrogen atoms of primarysecondary hydroxyl groups of CD molecules reveals that

those atoms played a crucial role in binding the drug molecules

The electronic structure of the inclusion complexes was also investigated in

addition to that of the isolated drug molecules HOMO as an electron donor represents

the capability to donate an electron and LUMO represents the capability to gain an

electron In HOMO of PCA the charge density is mainly accumulated on the benzene

ring and amino group whereas in PFO the charge density is observed on the CndashO and

CndashN bonds However in both cases of LUMO more charge density shifts to the benzene

109

ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during

HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC

bond of the benzene ring and carbonyl group It was also found that the charge densities

on each atom of the drugs within the CDs cavity were slightly changed Moreover the

energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical

species [145] The energy gaps for PCA and PFO as well as those for the most stable

complexes with CD were estimated by PM3 method and are shown in Table 44

Evidently the much lower energy gap which explains the fact that ultimate charge

transfer interaction occurs within the drug molecule The energy gap of drugβ-CD

complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD

complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex

is more stable than that of drugα-CD complex which is in good agreement with the

above discussed energy results In addition the EHOMOndashELUMO gap of both complexes

just fall in the range that of the PCA molecule suggesting there will be a blue shift in the

electronic spectrum of the guest molecule during binding and molecular recognition

This agrees well with the experimentally determined results

The polarity of the CD cavity is also taken into consideration for the formation of

most stable inclusion complexes The polarity of CD cavity significantly changed after

the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the

α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex

with respect to that of the dipole moment of optimized isolated hosts Although the

dipole moment of both complexes is lower than the sum of the dipole moments of

optimized isolated host and guest molecules From the above results one can conclude

that the dipole moment values show a strong correlation with the complexation

behaviour of CD

For the most stable complexes the physical parameters such as electronic

chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are

determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)

of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is

evident that the micro values of complexes differ remarkably from the isolated guest and host

molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD

therefore they are expected to act as electron acceptor in the inclusion complexation

interaction while the higher μ values of PCA indicates the drug act as electron donor In

the case of both η and ω values significant differences are observed when compared

with the isolated host (CD) molecule but it lies within the range that of the guest

110

Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method

Properties PCA PFO PCA α-CD

PCA β-CD

PFO α-CD

PFO β-CD

E -3427 -9462 -129371 -150783 -135294 -156835

∆E -1182 -1581 -1070 -1598

H 18209 19684 -40016 -49895 -37996 -48526

∆H -1141 -1349 -596 -1455

G 13929 14576 -53942 -65604 -51887 -63619

∆G -234 -581 1174 757

S 0143 0171 0449 0479 0456 0506

∆S -0047 -0073 -0068 -0074

EHOMO (eV) -886 -930 -829 -840 -946 -939

ELUMO (eV) -016 -024 009 004 -033 -019

EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920

Dipole moment (D) 439 168 583 995 784 1366

∆D -99 -673 -518 -031

micro -451 -477 -418 -410 -489 -479

η 435 453 422 419 456 460

S 022 022 023 024 021 021

ω 233 251 207 200 262 249

Unit =kcal mol-1 =kcalmol-Kelvin

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

109

ring and carbonyl group (Fig 45) This calculation clearly demonstrated that during

HOMOrarrLUMO transition transfer of electron density occurs from amino group to CndashC

bond of the benzene ring and carbonyl group It was also found that the charge densities

on each atom of the drugs within the CDs cavity were slightly changed Moreover the

energy gap (EHOMOndashELUMO) reflects the chemical activity and stability of the chemical

species [145] The energy gaps for PCA and PFO as well as those for the most stable

complexes with CD were estimated by PM3 method and are shown in Table 44

Evidently the much lower energy gap which explains the fact that ultimate charge

transfer interaction occurs within the drug molecule The energy gap of drugβ-CD

complex (-844 eV for PCA and -920 eV for PFO) is more than that of drugα-CD

complex (-838 eV for PCA and -913 eV for PFO) reveals that the drugβ-CD complex

is more stable than that of drugα-CD complex which is in good agreement with the

above discussed energy results In addition the EHOMOndashELUMO gap of both complexes

just fall in the range that of the PCA molecule suggesting there will be a blue shift in the

electronic spectrum of the guest molecule during binding and molecular recognition

This agrees well with the experimentally determined results

The polarity of the CD cavity is also taken into consideration for the formation of

most stable inclusion complexes The polarity of CD cavity significantly changed after

the guest is inserted into the cavity As depicted in Table 44 the dipole moment of the

α-CD in the inclusion complex is decreased whereas it is increased in β-CD complex

with respect to that of the dipole moment of optimized isolated hosts Although the

dipole moment of both complexes is lower than the sum of the dipole moments of

optimized isolated host and guest molecules From the above results one can conclude

that the dipole moment values show a strong correlation with the complexation

behaviour of CD

For the most stable complexes the physical parameters such as electronic

chemical potential (micro) hardness (η) softness (S) and electrophilicity (ω) values are

determined from HOMO as ionization energy (IE) and LUMO as electron affinity (EA)

of the systems using the Eqns (28 29 210 and 211) respectively On comparison it is

evident that the micro values of complexes differ remarkably from the isolated guest and host

molecules (Table 44) PFO molecule has lower micro value (-477 eV) than that of CD

therefore they are expected to act as electron acceptor in the inclusion complexation

interaction while the higher μ values of PCA indicates the drug act as electron donor In

the case of both η and ω values significant differences are observed when compared

with the isolated host (CD) molecule but it lies within the range that of the guest

110

Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method

Properties PCA PFO PCA α-CD

PCA β-CD

PFO α-CD

PFO β-CD

E -3427 -9462 -129371 -150783 -135294 -156835

∆E -1182 -1581 -1070 -1598

H 18209 19684 -40016 -49895 -37996 -48526

∆H -1141 -1349 -596 -1455

G 13929 14576 -53942 -65604 -51887 -63619

∆G -234 -581 1174 757

S 0143 0171 0449 0479 0456 0506

∆S -0047 -0073 -0068 -0074

EHOMO (eV) -886 -930 -829 -840 -946 -939

ELUMO (eV) -016 -024 009 004 -033 -019

EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920

Dipole moment (D) 439 168 583 995 784 1366

∆D -99 -673 -518 -031

micro -451 -477 -418 -410 -489 -479

η 435 453 422 419 456 460

S 022 022 023 024 021 021

ω 233 251 207 200 262 249

Unit =kcal mol-1 =kcalmol-Kelvin

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

110

Table 44 Energy features thermodynamic parameters and HOMO-LUMO energy calculations for PCA PFO and the 11 inclusion complexes by PM3 method

Properties PCA PFO PCA α-CD

PCA β-CD

PFO α-CD

PFO β-CD

E -3427 -9462 -129371 -150783 -135294 -156835

∆E -1182 -1581 -1070 -1598

H 18209 19684 -40016 -49895 -37996 -48526

∆H -1141 -1349 -596 -1455

G 13929 14576 -53942 -65604 -51887 -63619

∆G -234 -581 1174 757

S 0143 0171 0449 0479 0456 0506

∆S -0047 -0073 -0068 -0074

EHOMO (eV) -886 -930 -829 -840 -946 -939

ELUMO (eV) -016 -024 009 004 -033 -019

EHOMOndashELUMO (eV) -870 -906 -838 -844 -913 -920

Dipole moment (D) 439 168 583 995 784 1366

∆D -99 -673 -518 -031

micro -451 -477 -418 -410 -489 -479

η 435 453 422 419 456 460

S 022 022 023 024 021 021

ω 233 251 207 200 262 249

Unit =kcal mol-1 =kcalmol-Kelvin

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

111

Table 45

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

112

45 Solid inclusion complex studies

451 Scanning Electron Microscopy (SEM)

In order to inspect the influence of complexation on the surface morphology of

nanomaterials SEM photographs were taken for the isolated components ie free drugs

free CDs and their corresponding inclusion complexes The selected SEM photographs

of PCA PFO and the corresponding inclusion complexes are shown in Fig 410 A

drastic change in the morphology and shape of the inclusion complexes from the starting

materials was observed In Fig 410a typical crystal of PCA was found to be in irregular

shape with a smooth surface and obtuse corners The SEM images indicate that the PFO

particles appeared as micronised crystals of oval shaped stone-like structure in

agreement with the sharp peaks observed by powder XRD In contrast the solid

containing PCA in the complexed form with α-CD was in quite different size Further

PCAβ-CD complex was in irregular shape Likewise both the solid complexes of PFO

with CDs are quite distinct from those of pure materials in which the actual

morphologies of PFO and CDs disappeared The PFOα-CD and PFOβ-CD complexes

in Figs 410e and f showed agglomerated particles with irregular shape These changes

in the shape and size parameters of the solid samples are indicative of the formation of

inclusion complexes between the drugs and CDs

452 Transmission Electron Microscopy (TEM)

The morphologies of various kinds of supramolecular self-assemblies are

demonstrated in TEM analysis (Fig 411) Fig 411 illustrates that the nanoparticles

were observed for PCAα-CD PFOα-CD and PFOβ-CD with diameter size of 46 nm

150 nm and 210 nm respectively In the case of PCAβ-CD complex the different

structural features are obtained by supramolecular self-assembly in highly pure water and

characterized by TEM and micro-Raman imaging The TEM images in Fig 411c-f show

a well-ordered 2D microtube structures about 225-31 μm in width while the length

extends above 20 μm which is consistent with the result of the micro-Raman images

(Fig 412) Further the thickness of wall of the microtubes is approximately 220-275 nm

Also it can be observed in TEM images of microtubes hallow cavities with diameters

ranging approximately from 162 μm to 252 μm A proper matching between the

dimension of the guest and the β-CD cavity size is ascribed to be responsible for the

formation of extended supramolecular structure [163] Interestingly one important

observation was made from the TEM images of β-CD complex that there is a branch of

nanorod structure (Fig 411d) This rod-like structure is actually assembled by thousands

of smaller nanotubes in a stack way layer by layer The width of such rods is

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

113

Fig 410 SEM photographs of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(b)

(a)

(c)

(e)

(d)

(f)

50 microm

100 microm

10 microm

50 microm

20 microm

20 microm

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

114

Fig 411

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

115

approximately in the range of 570 nm Further the microtubes could be formed by the

secondary self-assembly of nanotubes of β-CD induced by the antiarrhythmic drug as

guest molecule (Fig 413)

The detailed structural characteristics of such microtubes formed from CD

nanotubes have been often reported [164] Recently some researchers reported that a

linear structure was obtained from β-CD inclusion complex by encapsulating linear guest

molecule through the intra and intermolecular hydrogen bonding as well as the

intradimer π-π interactions whereas the wave-type structure was observed other than the

linear structure due to the non-linear molecular structure of the guest molecule [164] For

example 4-hydroxyazobenzeneβ-CD complex showed a linear nanotube structure

evidently demonstrated by Liu et al [164] Moreover they have also reported that a

wave-type structure for a non-linear molecule 4-aminoazobenzene in complexed form

with β-CD acting as the host molecule Based on the literature it is proposed that the

single PCAβ-CD nanotube is likely to adopt a linear structure due to the linear

molecular structure of PCA The microtubes are formed by the secondary self-

aggregation of nanotubes of PCAβ-CD as shown in Figs 411c-f These secondary self-

assembly of nanotubes of 12 inclusion complexes for the formation of nanorod structure

were already reported by Wu et al [165] and Sowmiya et al [166] According to their

inference it can also be explained that even with low concentration of guest molecules

the lesser amount of nanotubes could be formed with β-CD Furthermore in order to

form nanotubes the presence of guest molecule is essential and the aggregation of

nanotubes is dependent on the concentration of guest molecules This reveals that

inclusion complex formation and their extensions to construct nanomicro structures are

induced by hydrophobic interactions between CD and guest molecules

Raman imaging was also performed for further confirmation of the micro-sized

tube structures Large number of such microtubes is found in the morphologies obtained

from micro-Raman images (Fig 412) which are exactly same in size as that in the TEM

images With these images herein reported the bunch of such micro-sized tubes formed

due to self-assembly of nanotubes of β-CD complexes and also their arrangement with a

regular fashion But a clear morphology for PCAα-CD complex in Raman imaging was

not obtained because such particles are present nano in size Two drops of the solution

was slowly evaporated on a clean glass surface at room temperature for Raman imaging

whereas the solution was directly deposited on a carbon coated TEM grid for electron

microscopic analysis Moreover when adjusting the pH value to 130 it was found that

the turbid solution becomes completely transparent since the pKa value of β-CD is 120

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

116

hydroxyl groups of CD will turn to negative oxygenic ions at higher pH value (~120)

Also under this condition the breakdown of hydrogen bonding between neighbouring

CD would lead to the collapse of the nanotubular structure [167] Consequently there

was no tubular structure except spherical ones observed by TEM images Therefore

besides hydrophobic interaction and van der Waals interaction hydrogen bonding

between the hydroxyl groups of neighbouring CDs is essential for the formation of the

nanotubular structure

The represented nanomicro tube structures are formed through the secondary

assembly of nanotubes The mechanism for the formation of microtube structures from

the secondary assembly of nanotubes of β-CD induced by PCA as guest molecule can be

explained as follows (Fig 414) The examination of prepared sample of micro tubular

structures by TEM indicated that rhombus-shaped nano sheets were formed Further a

closer investigation at the cleavage of the wall of the microtube confirmed the presence

of several thin supramolecular level layers which have thickness-dependent contrasts

and are stacked together to form multi-layered bulk nanosheets (Fig 411d) In addition

to that micro-Raman imaging was performed on the bulk aqueous solutions which were

already prepared for the TEM analysis as same Interestingly in some areas of the

sample a few multilayered bulk sheets were found along with the microtubes

Furthermore the observation of sheet like structure suggests that the microtubes are

formed from the nanosheets through a rolling mechanism Chandrasekhar and coworker

[168] have clearly demonstrated the shape-shifts of nanosheets into nanotubes through

the sheet rolling mechanism in the presence of water It is also monitored in the present

case the occurrence of the shape-shifts in the absence of any external forces A closer

examination of a tube in Fig 412e clearly showed the presence of a sheet-like structure

This inference is also supported by the closer investigation at the cleavage of the

microtube wall (Fig 411e)

The above results revealed that intermolecular hydrogen bonding plays an

important role in the supramolecular ordering and self-assembly besides the hydrophobic

interactions and van der Waals forces which contribute to the formation of the PCA

β-CD complex Through these intermolecular interactions 12 complexes of CD

aggregate can form a sheet like structure with a thickness of around 14-17 nm since the

width of CD is approximately 137-156 nm The nano rods and supramolecular level

layers in the wall of the microtubes probably originate from the hydrophobic surface

effect on the sheet like structures in the presence of water Further the hydrogen bonding

is the driving force for the secondary self-assembly Therefore it is expected that rod like

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

117

structures can also be formed in solution In addition to this B-H double reciprocal plots

did not show any linear relationship for 11 as like for 12 complexation even at lower

concentration where a good linear correlation was observed in the plot of 1(AndashA0)

andor 1(IndashI0) against 1[CD]2 This suggests nanotubes formation at higher

concentration of CDs (4 times 10-4 M) in solution

Fig 412 micro-Raman photographs of (a-e) micro tubes of PCA β-CD complex (e) The blue lined oval indicates the tube structure formed from the sheet like structure

Fig 413 Structure of nanotube formation of 12 PCAβ-CD complexes and their

secondary self-assembly

Sheet-like structure

Microtube structure

(a) (b)

(c) (d)

(e)

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

118

Fig 414 Possible formation mechanism of microtube from nanotube of 12 PCAβ-CD inclusion complex based on TEM and micro-Raman data (a) Upper and side view of a nanotube (176 nm) (bc) Upper and side view of secondary self-assembly of nanotubes to form a nanosheet (d) 1D molecular level layers are tightly stacked with another one molecular axis-b and form 2D nano sheet (ef) Formation of microtube from the nanosheets

12 complex

(a) Staking

(b)

Multilayer nanosheets

Single nanosheet

Microtube (f)

Rolling force

(c) (e)

(d)

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

119

453 FTIR spectral analysis

FTIR spectra of the inclusion complexes of PCAα-CD PCAβ-CD PFOα-CD

and PFOβ-CD as well as those for pure drugs are shown in Fig 415 The IR spectra of

both drugs showed its characteristic bands in the frequency range 400-4000 cm-1 Both

CD showed prominent peaks at 3300 1638 1035 and 564 cm-1 as related in the

literature In Fig 415a the peaks at 3215 3402 cm-1 are due to the primary amine and

1512 cm-1 for secondary amide bending vibrations of PCA Among those bands the

primary amine stretching vibrations disappeared in the inclusion complexes and the

secondary amide bending vibration moved to lower frequency (1509 cm-1) The CndashH

stretching and bending vibrations at 3319 2978 and 1392 cm-1 disappeared in the

inclusion complexes The sharp benzene ring skeleton stretching vibration at 1599 cm-1

diminished or shifted in the inclusion complexes (Figs 415b and c) Further the CndashH

stretching frequencies at 630 and 600 cm-1 also disappeared in the PCACD complexes

and the OH stretching peak of CD moved to higher frequency which might be due to the

existence of the intermolecular hydrogen bonding between the drug and CD These

obvious changes in the IR spectra confirmed that the inclusion complexes were formed

and the benzene ring with the secondary amine group of PCA was included into the CD

nanocavity

The C=O stretching vibration of pure PFO drug located at 1662 cm-1 (Fig 415d)

The CndashO stretching and CndashOndashC stretching vibrations bands are observed at 1165 cm-1

and 1031 cm-1 The OH stretching and NH stretching vibrations appeared at 3423 cm-1

and 3315 cm-1 respectively The symmetrical stretching vibration of =CH2 at 2972 cm-1

and ndashCH2 stretching vibration at 2926 cm-1 were observed The aromatic ring stretching

and CndashN stretching frequencies occurred at 1593 cm-1 and 1329 cm-1 The OH bending

vibration observed at 1304 cm-1 and the band at 732 cm-1 for out of plane deformation

The bands at 854-636 cm-1 are attributed to the CH bending vibrations However all

these bands shifted to lower frequency in the inclusion complexes (Figs 415e and f) in

concert with the disappearance of some of the characteristic bands of PFO For example

the frequency at 3423 cm-1 (for OH stretching) and 3315 cm-1 (for NH stretching)

completely disappeared in the inclusion complexes The C=O stretching vibration at

1662 cm-1 shifted to 1651 cm-1 in the inclusion complex Further the CH bending

vibrations shifted to 831-706 cm-1 The aromatic ring stretching shifted to 1586 cm-1 in

α-CD complex whereas it is completely disappeared in the β-CD complex The observed

changes in the IR spectra of PFOCD inclusion complexes are due to the restriction of

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

120

the vibration of free drug molecule upon encapsulation into CD cavity indicating the

formation of inclusion complexes between PFO and CD

Fig 415 FTIR spectra of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

400

Tra

nsm

ittan

ce

4000 3000 2000 1000 Wavenumber (cm-1)

(a)

(b)

(c)

3402

74

3215

63

1512

33

3319

79

2978

36

1392

73

1599

13

1545

12

630

33

601

73

2928

21

3377

66 16

396

4

1509

40

2941

71

2577

13

2467

18

1643

50

1294

35

1184

40

1028

15

964

50

767

74

841

05

1608

01

1157

39

1030

08

570

98

844

90

2928

55

3375

73

1640

84

1509

65

1608

78

1157

50

1031

15

578

70

846

83

1413

95

1294

35

1334

85

(d)

(e)

(f)

767

74

854

54

3315

93

1030

08

1662

79

3423

96 65

393

2972

57

1593

35

1452

53

1165

33

2781

60

1304

21

706

15

636

57

3396

95

2930

14

1452

53

1155

46

767

74

707

94

1652

20

1030

08

831

47

1586

76

3393

09

2930

29

1452

86

1155

46

758

57

706

47

1651

89

1031

55

858

40

1329

07

732

45

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

121

454 DSC analysis

The thermal analysis is a preliminary and widely used analytical tool for the

characterization of multicomponent systems such as host-guest inclusion complexes in

the solid state Fig 416 illustrates the DSC curves of PCA PFO PCAα-CD PCA

β-CD PFOα-CD and PFOβ-CD The DSC curve of PCA (Fig 416a) showed a sharp

endothermic peak at 1686 degC corresponding to the melting point of the drug As can be

seen from the analysis of the PCACD inclusion complexes in Figs 416b and c the

peaks of CD which represent water losses are strongly reduced in intensity and shifted to

lower temperature In addition the complete disappearance of PCA thermogram clearly

indicates a strong interaction between the PCA and CD molecules The DSC data

suggested the true inclusion complex formation and the drug is well dispersed in the

hydrophobic CD nanocavities

It can be seen in Fig 416d the DSC analysis of PFO curve was typical of a

crystalline anhydrous substance showed an initial flat profile followed by a sharp

endothermic peak at ~1722 ordmC which is characterized by the melting point of the drug

On the contrary the melting endothermic peaks corresponding to PFO was disappeared

in the DSC curves of PFOCD complexes Further α-CD dehydration peak at 792 ordmC was

completely disappeared and other endothermic peaks correspond to the dehydration of

free CDs were shifted to lower temperature in the inclusion complexes The comparative

analysis of DSC curves of inclusion complexes in Fig 416 with those of the pure drug

and both CDs reveals the strong interactions of PFO with CDs and the formation of

stable inclusion complexes The overall DSC results indicate that usual thermal

properties of the drugs as well as the isolated CDs were altered after inclusion complex

formation

455 XRD analysis

Powder XRD is a useful tool for the detection of CD complexation in powder or

microcrystalline states Fig 417 presents the XRD patterns of PCA PFO and their

corresponding solid inclusion complexes The diffractograms of PCA and PFO displayed

a series of intense and sharp peaks at diffraction angles (2θ) of 104ordm 116ordm 166ordm 181ordm

193ordm 203ordm 214ordm 224ordm 236ordm 254ordm 264ordm 281ordm 296ordm and 764ordm 1210ordm 1629ordm

2184ordm 2888ordm 3923ordm 4233ordm respectively demonstrating the crystalline nature of the

drugs The diffraction patterns obtained for both α-CD and β-CD also showed that those

are crystalline substances because of the presence of characteristics peaks at 2θ angles

range from 5ordm to 50ordm The pattern of the physical mixture is just a superimposition of the

crystalline drug in the CD patterns in which the characteristic peaks of drug were still

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

122

Fig 416 DSC thermograms of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex (25-185 oC at 10 oCmin)

(d)

(e)

1722degC

1313degC 1075degC

1188degC

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180

(f)

Exo

ther

mic

(mW

mg)

40 Temperature (oC)

(a)

(b)

(c)

1686degC

1258degC 712degC 1048degC

1166degC

60 80 100 120 140 160 180

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

123

Fig 417 Powder XRD patterns of (a) PCA (b) PCAα-CD complex (c) PCAβ-CD complex (d) PFO (e) PFOα-CD complex and (f) PFOβ-CD complex

(a)

(b)

(c)

(d)

(e)

(f)

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

124

detectable (Figure not shown) In contrast the XRD patterns of PCAα-CD and PCA

β-CD complexes are amorphous and display halo patterns which are evidently different

from the physical mixture suggesting the interaction between the drug and CD In

addition most of the crystalline diffraction peaks of CD diminished in Fig 417 and the

diffraction peaks for pure PCA in the 2θ values of 166ordm 235ordm 254ordm 281ordm and 296ordm

disappeared indicating that the formation of inclusion complex between the PCA and

CD However the diffraction patterns of the PFOCD inclusion complexes

(Figs 417e and f) showed complete disappearance or considerable reduction of

characteristic diffraction peaks apparent in the XRD pattern of PFO and CD which

suggest the formation of an inclusion complex The presence of new peaks at 2θ values

of 1319ordm 2521ordm and 3122ordm (for PFOα-CD complex) and 618ordm 1702ordm and 1957ordm (for

PFOβ-CD complex) indicate that the inclusion complexes were formed with a new solid

phase Further the inclusion complexes showed amorphous halo behaviour as evidenced

by broader diffraction peaks with lower intensity obtained for the solid samples

456 1H NMR spectral analysis

The mechanism of interaction between PCA or PFO and CD was investigated

using proton nuclear magnetic resonance (1H NMR) spectroscopy Figs 418 and 419

illustrate the qualitative features concerning the interaction of drugs with CDs 1H NMR

spectra of both CDs possessing six types of protons are very close to those already

reported by different authors H-1 H-2 H-3 H-4 H-5 and H-6 protons are located at

497 356 389 352 and 374-377 ppm respectively A detailed investigation of 1H

NMR spectra of inclusion complexes indicates that the addition of drugs influences the

chemical shifts of CD protons signals Especially H-3 (~014 ppm) and H-5 (~010 ppm)

protons located in the interior of the CD cavity are greatly shifted upfield rather than

those of other protons Furthermore H-5 and H-6 signals appeared somewhat resolved in

comparison to that of isolated CD suggesting the drug molecules enter into close

proximity with both H-5 and H-6 protons A negligible shift is observed for the signals

of H-1 H-2 and H-4 protons located on the exterior of CD cavity These chemical shift

changes suggest interaction of the drugs with the internal protons of CD

The chemical shift values of the different protons of PCA PFO and the changes

in chemical shift (Δδ) of those protons in the inclusion complexes are listed in Table 46

The proton signals of PCA (Fig 418a) are in agreement with the results reported by

Janik et al [169] Figs 418b and c clearly displayed the chemical shifts of PCA protons

in the presence of α-CDβ-CD It is noteworthy that all the chemical shifts of PCA

protons are influenced by the addition of CD The methyl protons (Hf and Hj) of PCA

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

125

only undergo a downfield shift while the other protons including the aromatic and amino

protons show an upfield shift The signals for all drug protons are shifted from 004 ppm

to 042 ppm Further the shifts are accompanied by a loss in resolution of Hg and Hi

protons The signals corresponding to amino protons (He) and amide protons (Hb) are

significantly affected upon inclusion complexation Therefore it can be concluded that

PCA is inserted inside the cavity with these protons closer to the interior protons of CD

(H-3 and H-5) Surprisingly the most significant changes have been observed on the Ha

and He protons which indicates the aromatic ring and the amino protons are included

into the CD cavity

Comparative analysis of the 1H NMR spectra of PFO in the presence of α-CD and

β-CD also revealed that the complex formation which evince by the significant changes

in the chemical shift values of drug molecules (Fig 419) As can be the 1H NMR

spectral data seen in Table 46 the aromatic protons (Hc Hd He and Hf) and methylene

protons (Hk and Hm) have shown significant chemical shift changes in the presence of

CDs However in the case of β-CD inclusion complex besides the above chemical shift

changes the amino protons (Ha) and hydroxyl protons (Hb) of the PFO molecule was

largely shifted to upfield andor downfield respectively The chemical shift changes of

these protons suggest the penetration of PFO involves insertion of the aromatic skeleton

and part of the aliphatic chain along with the carbonyl group This interpretation is

supported by strong frequency changes observed on H-3 and H-5 protons of the CD

Some of the most significant chemical shift changes on the spins of the CD protons come

from the diamagnetic shielding of the aromatic part of the drug In the structure of α-CD

or β-CD H-3 and H-5 protons are located inside the CD cavity Among these

H-3 protons are positioned at the wider rim of the cavity while H-5 protons placed at the

narrower rim of the methylene (H-6) bearing the primary hydroxyl groups All other

protons (H-1 H-2 and H-4) are located outside of the cavity Upon the addition of PFO

the significant upfield shifts was observed only for internal protons of CD These shifts

are indicative of the fact that the aromatic part of guest molecule entered closer to the

internal protons of CD and the stable inclusion complexes are formed between the drugs

and CDs

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

126

Fig 418 1H NMR spectra of (a) PCA (b) PCAα-CD complex and (c) PCAβ-CD complex Inset Fig Assignation of protons of PCA

(c)

(b)

(a) a b

c d e

i

g

j

f

a e b

N

C O

NH CH2

CH2 NH

H

H

CH2

CH2

CH3

CH3 c

c

d

d f

g

i

i

j

j Cl +

e

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

127

Fig 419 1H NMR spectra of (a) PFO (b) PFOα-CD complex and (c) PFOβ-CD complex Inset Fig Assignation of protons of PFO

(c)

(b)

(a) a

b cd ef

gi

j mn

l

p

q r

s

k

a

b

c

d

e

f

g i

j

g i k

l

m n

p q

r

s O

C

O

CH2 CH2

CH2

OH

CH CH2

HN CH2 CH2 CH3

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

128

Table 46 Chemical shift values (δ ppm) of PCA PFO and changes of chemical shift

(Δδ) after forming the inclusion complexes

Protons PCA (δ)

PCAα-CD (Δδ)

PCAβ-CD (Δδ)

PFO (δ)

PFOα-CD (Δδ)

PFOβ-CD (Δδ)

Ha 1034 -033 -042 915 -004 -037

Hb 846 -010 -013 595 -005 028

Hc 762 007 -004 754 -004 -016

Hd 653 -012 -015 751 -001 -010

He 568 -007 -009 716 -008 -009

Hf 357 006 004 702 -007 -010

Hg Hi 332-315 721-

727 0001 0002

Hj 122 013 014 715 0001 001

Hk 334

Hl 292 001 005

Hm 432 006 010

Hn 412 -002 -011

Hp 292 0001 004

Hq 273 0001 001

Hr 164 001 -001

Hs 087 0002 0006

-merged with CD protons

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes

129

46 Conclusion

The encapsulation behaviour of α-CD and β-CD with PCA and PFO was

investigated by absorption fluorescence time-resolved fluorescence SEM TEM FTIR

DSC XRD 1H NMR and PM3 methods The present study showed that the above drugs

did not show any significant spectral shifts in the solvents In aqueous solutions each

CD has been found to form inclusion complexes with PCA and PFO Addition of CD to

aqueous solutions of the above drugs has resulted in the observation of the enhancement

of fluorescence intensity The stoichiometry of PCA with CDs is found to be 12 whereas

PFO forms 11 complex with CDs The red shifted ICT emission of PFO in CD indicates

the fact that phenyl ring along with carbonyl group is present in the inner part of the

hydrophobic CD cavity SEM FTIR DSC XRD and 1H NMR results suggested that the

formation stable inclusion complexes of the drugs with both CDs in the solid state The

results of solid state studies showed the important modifications in the physicochemical

properties of free drug The supramolecular nanomicro structures were also fabricated in

aqueous solution by the self-assembly of α-β-CD nanocavity induced by drug

molecules The analysis of thermodynamics of inclusion process indicates that the drug-

CD complexation is spontaneous and hydrogen bonding plays an important role in the

complexation process Investigations of thermodynamic and electronic properties

confirmed the stability of the inclusion complexes