ORIGINAL ARTICLE

Nano-amorphous composites of cilostazol–HP-b-CD inclusioncomplexes: physicochemical characterization, structureelucidation, thermodynamic studies and in vitro evaluation

Chirag Desai • Bala Prabhakar

Received: 2 July 2014 / Accepted: 27 August 2014

� Springer Science+Business Media Dordrecht 2014

Abstract The present research work aims to obtain nano-

amorphous composites of cilostazol and HP-b-CD using

combinatorial spray drying and inclusion complexation

technique. Physicochemical characterization of complexes

was performed in solution and solid state to understand the

mode of inclusion of cilostazol in the host cavity. Phase

solubility studies revealed formation of first-order soluble

complex with stability constant of 892.13 M-1, while Job’s

plot confirmed 2:1 (HP-b-CD:CLZ) stoichiometry. Struc-

ture elucidation of the complex was performed using UV,

FTIR and NMR spectroscopy. 1H NMR spectra revealed

only marginal chemical shifts for both CLZ and HP-b-CD

suggesting shallow penetration and presence of van der

Waals forces. Proximity relationships between CLZ and

HP-b-CD obtained from 2D (ROESY) NMR assisted

computer simulations to compute the binding energy of this

complex. Circular dichroism gave an estimate of the ori-

entation of CLZ in the HP-b-CD cavity. Solid state com-

plexes were evaluated using FTIR, DSC and hot stage

microscopy, all of which confirmed inclusion complexa-

tion. The amorphous nature of the complex was established

using XRD and atomic force microscopy. Thermodynamic

studies indicate exothermic nature of complexation while

signifying the role of steric interactions in complex for-

mation. The spray dried amorphous nanocomposites

exhibited lyotropic phase transitions with varying relative

humidity as suggested by dynamic vapor sorption analysis.

In vitro biorelevant dissolution media studies exhibited

negligible food effects as indicated by the fed/fasted ratio.

Keywords Nanocomposites � Spray drying � Inclusion

complex � Circular dichroism � 2D ROESY NMR �Thermodynamics � Biorelevant media

Introduction

Cilostazol (CLZ) is widely used in the treatment of inter-

mittent claudication which is the primary symptom of

peripheral arterial disease. It is a selective inhibitor of type-

3 phosphodiesterase (PDE3) with therapeutic focus on

increasing cAMP [1, 2]. Clinical applications of CLZ also

include glaucoma and erectile dysfunction [3, 4]. Inher-

ently a BCS class II molecule it exhibits low solubility and

is predisposed to low and variable oral bioavailability. Like

most other lipophilic drugs, co-administration of high fat

meal has shown significant increase in the rate and extent

of CLZ absorption. Its absorption in the GIT is slow,

variable, and incomplete. The absolute bioavailability of

CLZ is not known and relative bioavailability is unpre-

dictable [5, 6]. This limits the clinical efficacy of CLZ, but

opens new avenues for dosage form design to surmount

solubility and biovariability issues. One such approach is to

explore the synergism produced by combination of two

distinct solubility enhancement techniques, viz. spray

drying and cyclodextrin inclusion complexation to aug-

ment the solubility, wettability and in vitro dissolution

characteristics of CLZ. The efficient optimization of spray

drying process to obtain nanocomposites of uniform size

distribution, together with the amorphization of CLZ in

HP-b-CD may prove to be an apt particle engineering

approach for modulating the solubility profile of CLZ. The

structure of CLZ is presented in Fig. 1.

Cyclodextrins (CDs) and its derivatives are termed as

molecular-containers that host molecules of different

C. Desai � B. Prabhakar (&)

Shobhaben Pratapbhai Patel School of Pharmacy and

Technology Management, SVKM’s NMIMS, Mumbai 400056,

India

e-mail: balaprabhakar2014@gmail.com

123

J Incl Phenom Macrocycl Chem

DOI 10.1007/s10847-014-0447-x

polarities and molecular weights within their cavity [7, 8].

The resulting non-covalent inclusions or host–guest com-

plexes are of current scientific and technological interest

for solubility and bioavailability enhancement of poorly

soluble drug molecules [9]. Various pharmaceutical pro-

ducts containing cyclodextrins now exist in the market. The

aqueous solubility of natural CDs is low due to the rela-

tively strong intramolecular hydrogen bonding in the

crystal lattice. HP-b-CD is an analog of b-CD modified

with 2-hydroxypropyl unit, and is widely studied in the

field of pharmaceuticals owing to its ability to enhance

solubility and provide stability to various drug molecules.

It is known to produce more wettable amorphous com-

pounds with increased water solubility and complexing

power compared to b-CD [10]. Toxicological studies have

revealed that HP-b-CD is well tolerated by humans both by

intravenous and oral administration and is regarded as safe

by the FDA [11, 12].

Inclusion complexes (ICs) of CLZ with HP-b-CD pre-

pared using conventional techniques like kneading, co-

precipitation and solvent evaporation has been reported

earlier [13]. However, we feel that detailed characterization

of these complexes is necessary to understand the solubi-

lization efficiency of HP-b-CD for CLZ. It is necessary to

recognize the molecular interactions between guests and

CDs to understand the underlying stoichiometry and

geometry of the complexes.

Here, we report the use of industrially feasible and

efficient spray drying technique for preparation of nano-

amorphous composites. This paper emphasizes on the use

of robust analytical techniques like circular dichroism,

DSC, FITR, P-XRD and AFM to investigate the formation

of complex. 1D and 2D (ROESY) 1H NMR have been used

to deduce the structure of the formed complex. We also

report the use of molecular modeling and molecular

dynamics to understand the orientation of CLZ in the HP-

b-CD cavity and to compute binding energy which is

indicative of the stability of the complex [14]. Thermo-

dynamic analysis was carried out to understand the

enthalpy and entropy changes governing complexation

[15]. DVSO reflects the change in crystal structure and the

change in micro-environment of the complex due to

sorption/desorption with an increase in humidity. In vitro

dissolution studies of CLZ have been performed in bio-

relevant media to mimic in vivo performance of the for-

mulation prepared using CLZ–HP-b-CD nanocomposites.

Therefore, the emphasis of the present work was to

augment the solubility and in vitro dissolution of CLZ by

forming nano-amorphous spray dried composites with HP-

b-CD. The paper mainly focuses on the use of different

analytical principles like microscopy, spectroscopy, dif-

fractometry and thermodynamics to characterize and elu-

cidate complex formation. Moreover, an effort has been

made to understand complexation mechanism and reveal

various factors that contribute profoundly to the overall

stability of the complex.

Materials and methods

Materials

CLZ and HP-b-CD were obtained as gift samples from

Ipca laboratories and Roquette Chemicals, Mumbai, India,

respectively. Analytical grade reagents and double distilled

water were used throughout the study.

Preparation and evaluation of solid binary systems

Preparation of physical mixtures of CLZ and HP-b-CD

Physical mixtures (PMs) of CLZ and HP-b-CD were pre-

pared in 1:2 molar ratios. Both the ingredients were

weighed separately, passed through 100 mesh sieve and

mixed manually.

Preparation of nano-amorphous composites by spray

drying technique

CLZ and HP-b-CD were weighed in terms of their molar

ratios (1:2) obtained from phase solubility studies and Job’s

plot. Hydro-alcoholic solution containing ethanol and

water in 75:25 ratio was used as solvent for spray drying.

75 ml ethanolic solution of CLZ was added to 25 ml

aqueous solution of HP-b-CD under continuous stirring to

obtain a clear solution. The resulting solution was soni-

cated for 10 min and then stirred for 3–4 h using magnetic

stirrer at 30 �C. The feed was sprayed in the spray dryer

(LabUltima-222) under continuous stirring. The process

parameters were so adjusted that maximum product con-

centrate was obtained in cyclone II of the spray dryer

resulting in production of nanoamorphous composites with

uniform size distribution. The yield of the spray-drying

Fig. 1 Structure of CLZ and labeling of protons

J Incl Phenom Macrocycl Chem

123

process was measured as the weight percentage of the

powder obtained in the final operation compared with the

amounts of solids (CLZ ? HP-b-CD) present in the

sprayed solution. The product thus obtained was collected,

packed in aluminum foil and stored under dessicator until

further use.

The optimized conditions for spray drying were as

follows:

Inlet temperature: 75 �C.

Outlet temperature: 50 �C.

Cool temperature: 45 �C.

Inlet high temperature: 80 �C.

Outlet high temperature: 55 �C.

Spray rate: 1.5 ml/min.

Atomization air pressure: 1.5 kg/cm2.

Aspirator flow rate: 50 Nm3/h.

Batch size: 10 % w/v.

Particle size distribution, polydispersity index and zeta

potential studies

The particle size distribution of optimized and reproducible

batch of CLZ–HP-b-CD nanocomposites was determined

by laser diffraction technique. The laser diffraction analysis

was carried out at real refractive index of 1.58 and imag-

inary part of 0.001. Polydispersity index (PI) of optimized

batch was determined with photon correlation spectroscopy

using N4 plus submicron particle size analyzer (Beckman

Coulter, USA). Zeta potential of optimized batch was

evaluated using Zetasizer (Malvern Instruments, Malvern,

UK) with appropriate dilution of samples at 25 ± 1 �C.

Solution state characterization of nanocomposites

Spectral pattern of drug/ligand in presence of cyclodextrins

exhibits two distinct effects, the change in the intensity of

the band and/or a shift of the maximum wavelength [16].

The first effect, i.e., variation of intensity in presence of

different amounts of CDs, is used for determining the

association constant (phase solubility studies) while the

second one shows the influence of the surrounding on the

electronic system implied in the transition (circular

dichroism).

Saturation solubility of CLZ and CLZ–HP-b-CD

nanocomposites in water and biorelevant media

Effect of complexation on the aqueous solubility of CLZ

was studied using HPLC technique. 10 mg of CLZ and its

equivalent amount of complex were taken in separate

volumetric flasks and dispersed in 10 ml of water, fasted

state simulated intestinal fluid (FeSSIF), fed state simulated

intestinal fluid (FeSSIF), fasted state simulated gastric fluid

(FaSSGF) and fed state simulated gastric fluid (FeSSGF)

respectively. The samples were equilibrated at 30 ± 0.5 �C

for 24 h by shaking on a rotary shaker, following which

5 ml aliquots were filtered through 0.2 l membrane filter.

1 ml of the filtrate was then appropriately diluted with

respective solvent and injected into HPLC system.

Inclusion efficiency

The ability of HP-b-CD to include/encapsulate CLZ can be

estimated by determining its inclusion efficiency. 10 mg of

CLZ and its equivalent amount of IC were taken in separate

volumetric flasks and dissolved/dispersed in 10 ml meth-

anol respectively. The suspension of IC in methanol was

sonicated for 10 min and filtered through 0.2 l whatman

filter paper. 1 ml aliquot of the filtrate was suitably diluted

with methanol and analyzed at 257 nm. The absorbance of

CLZ in complexed state was compared with that of CLZ in

the free state to determine inclusion efficiency.

Determination of stoichiometry

Phase solubility studies and complexation effi-

ciency Phase solubility studies of CLZ with HP-b-CD

were performed in water according to the method reported

by Higuchi and Connors [17, 18]. Excess amount of CLZ

was added to the volumetric flask containing solutions of

increasing concentrations of HP-b-CD (2–16 mM). Each

flask was capped and shaken on a rotary shaker for 24 h at

30 ± 0.5 �C to attain equilibrium, following which 5 ml

aliquots of supernatant were withdrawn and filtered

through 0.2 l whatman filter paper. 1 ml aliquot of this

filtrate was appropriately diluted with water and analyzed

at 257 nm using a UV spectrophotometer. Phase solubility

diagram was plotted with HP-b-CD concentration on X

axis and CLZ concentration on Y axis. The stability con-

stant (Ks) was calculated using the following formula,

Ks ¼ Slope=S0 1� slopeð Þ; ð1Þ

where S0 is the maximum solubility of drug in the absence

of HP-b-CD.

Complexation efficiency (CE) is defined as the solubi-

lizing efficiency of CDs for guest molecule (in this case

HP-b-CD and CLZ respectively). Based on the results of

the phase solubility studies, CE of HP-b-CD for CLZ was

determined using the following formula,

CE ¼ CLZ/HP-b-CD½ �= HP-b-CD½ � ¼ Slope/ 1� Slopeð Þ;ð2Þ

where [CLZ] and [HP-b-CD] are molar fractions of CLZ

and HP-b-CD.

J Incl Phenom Macrocycl Chem

123

Continuous variation method (Job’s plot) Stoichiometry

of the complex was determined by continuous variation

(Job’s) method [19]. Equimolar (0.05 mM) solutions of

CLZ and HP-b-CD were prepared in methanol and water

respectively. Varying quantities (ml) of CLZ and HP-b-CD

solutions were mixed (1:9, 2:8, 3:7, …, 9:1) keeping the

final total volume to 10 ml. The samples were analyzed at

257 nm using UV spectrophotometer (Perkin Elmer

Lambda 25). The difference in absorbance of CLZ in

presence, and absence of HP-b-CD was plotted against R.

R = CLZ½ ��

CLZ½ �þ HP-b-CD½ �f g� �

ð3Þ

where R = ratio of mole fractions of CLZ and HP-b-CD,

where [CLZ] is the mole fraction of CLZ and [HP-b-

CD] is the mole fraction of HP-b-CD.

Circular dichroism spectroscopy Circular dichroism

spectroscopy was used to understand the manner in which

CLZ formed complex with HP-b-CD. The signs and

intensities of the induced circular dichroism (ICD) spectra

are interpreted in terms of the solution structures of the CD

complexes. Circular dichroism spectrum of CLZ–HP-b-CD

IC was obtained in methanol:water (85:15) as solvent,

using Jasco J-600 spectropolarimeter. Absorbance of the

sample was kept below two in the wavelength range of

200–400 nm.

Nuclear magnetic resonance spectroscopy (NMR) Struc-

ture elucidation of the complex was achieved using 1D and

2D 1H NMR (ROESY). The experiments were performed

on 6 mM solution of CLZ, HP-b-CD and CLZ–HP-b-CD

(1:2) complex in deuteriated dimethyl sulfoxide (DMSO-

d6) with a Bruker FT-NMR spectrophotometer operating at

500 MHz at 300 K. The ROESY spectrum was recorded

using 250 ms mixing time and 512 experiments to enhance

the signal to noise ratio. The fid’s were apodized with a

sine window function and zero filled to a matrix size of

2 9 1 K prior to fourier transformation using Bruker

Topsin 2.0.

Solid state characterization

Contact angle study

Effect of complexation on reduction of surface tension was

studied using contact angle measurement (CAM). CAM

was performed by static sessile drop method using Kruss

Contact Angle Goniometer G10. A drop was placed using a

syringe on compressed pellet of test substance. The pellet

was prepared by compressing at approximately 4 tons

pressure using KBr pellet press. Contact angle was evalu-

ated directly by measuring the angle formed between the

solid pellet and the tangent to the drop surface.

Fourier transform infrared spectroscopy (FTIR)

FTIR is a very useful tool to prove the existence of both

guest and host molecules in their complexes. FTIR spectra

of CLZ, HP-b-CD, PM and IC were recorded in the range

of 400–4,000 cm-1 using Perkin Elmer R*I spectropho-

tometer by KBr disc method.

Differential scanning calorimetry (DSC)

Thermal analysis of CLZ and its complexes was performed

to confirm the polymorphic state of CLZ and to identify

interactions between CLZ and HP-b-CD respectively. DSC

thermograms of CLZ, HP-b-CD, PM and IC were recorded

using ZYDIS calorimeter. About 1–3 mg of samples were

sealed in flat bottomed aluminum cubicles and heated at a

scan rate of 10 �C/min from 30 to 300 �C.

Powder X-ray diffractometry (P-XRD)

X-ray diffractograms of CLZ, HP-b-CD, PM and IC were

recorded on a Philips Analytical X’pert Pro MPD with

copper as anode using a voltage of 40 kV and a current of

35 mA. The diffractograms were recorded in 2h angle

range between 5� and 508 at a scanning rate of 18/min.

Atomic force microscopy (AFM)

A further in-depth morphological analysis was performed

using a DME Dualscope Rasterscope C21 atomic force

microscope (alternate contact mode, Mikromash Si tips,

Au-backside, k = 15 N/m) with a scanner of 50 lm with

three piezo electrodes for three axes X, Y and Z in a

noncontact mode. Silicon tips had an elastic constant of

40 nm equipped with dual scope DS 95-50 camera. The

data was processed using scanning probe microscopy

(SPM) program. The sample suspensions (1 % w/v) were

prepared in distilled water and a drop was placed on an

aluminum sheet. It was allowed to dry in a HEPA filter

zone and the dried region was analyzed.

Thermodynamics of complexation

Effect of temperature

Phase solubility studies (as mentioned under determination

of stoichiometry) were performed at 30, 40, 50 and

60 ± 0.5 �C, until solubility equilibria were reached. The

purpose of this study was to determine stability constants at

different temperatures. After equilibration, 5 ml aliquots of

supernatant were withdrawn and filtered through 0.2 lwhatman filter paper. 1 ml aliquot of filtrate was appro-

priately diluted with water and analyzed using UV

J Incl Phenom Macrocycl Chem

123

spectrophotometer (Perkin Elmer Lambda 25) at 257 nm.

Aforementioned temperature conditions were maintained

throughout the filtration and dilution procedures for each

study.

Effect of humidity

Sorption analysis was performed using TA instrument-

Q5000, USA and the data was processed using universal

analysis 4.5 v. Approximately 2–3 mg of sample was taken

in a pre tared quartz crucible and spread evenly on the pan.

The sample was exposed to increasing humidity from 35 %

RH to 90 % RH at 5 % RH step size with a step equilib-

rium criterion of \0.1 % w/w weight change for 10 min

for a maximum time step of 60 min. This sample was then

subjected for desorption from 90 % RH to 0 % RH with the

same criterion as that of sorption. Samples were subjected

to two cycles of sorption–desorption i.e., 40 to 90 to 0 and

then to 90 to 0 to 40 % RH. After sorption analysis, these

samples were then analyzed for their crystallinity by

P-XRD.

P-XRD was performed using Rigaku-Dmax2200 (Japan)

and data was processed using Rint 2000. Approximately

3–20 mg of test sample was placed into the silicon zero-

background holder and pressed using clean glass slide to

ensure co-planarity of sample surface to that of sample

holder. The test sample was mounted on goniometer and

scanned from 3� to 45� (2h) at a scan rate of 3� (2h) per

min at a step size of 0.02� (2h).

Molecular modeling studies

The computational studies were performed on a high per-

formance computing cluster with Intel Xeon hexacore

processors backed with Rocks Cluster Suite 6.1 (San Diego

Supercomputing Centre). The 3D coordinates of b-CD

were obtained from protein data bank (PDB id: 3CGT) [20]

and modified by alkylating appropriate hydroxyl groups to

obtain HP-b-CD [21]. The structure of CLZ was sketched

using Maestro v9.3.5 (Schrodinger Suite 2013.2). The

structures were prepared for docking in Schrodinger Suite

2013.2, defining the atom types and partial charges using

OLPS 2005. CLZ was placed in orientation in HP-b-CD

cavity as dictated by ROESY interpretations. The complex

was minimized to relax any clashes and then solvated with

DMSO to form a solvent shell of 10 A thickness around

complex. The complex was energy minimized in vacuo,

and the lowest energy structure was used as the starting

geometry for MD simulations using Desmond v. 3.1

incorporated in Schrodinger Suite. Subsequently, the min-

imized systems were simulated for 10 ns duration under

NPT ensemble [15, 21]. In this period the system was

relaxed with the Langevin thermostat and barostat.

Isotropic scaling of the velocities and pressure was used to

maintain the pressure at 1 bar and; temperature at 300 K

using Langevin algorithm. High-frequency vibrations were

removed by applying the SHAKE algorithm which con-

strains all bonds to their equilibrium values. The trajecto-

ries and corresponding energies were sampled every 10 ps.

During the productive simulation no constraints were

applied to the complexes.

In vitro dissolution studies of complexes

In vitro dissolution profiles of nanocomposites were

determined in 900 ml of 0.3 % SLS in water (USP official),

FaSSGF, FeSSGF, FaSSIF and FeSSIF as dissolution

media at 37 ± 0.5 �C using USP paddle apparatus (Elec-

trolab) at 75 rpm. Five milliliters of samples were with-

drawn at 5, 10, 15, 30, 45, 60 and 120 min and replaced

with the same amount of fresh dissolution medium to

maintain sink conditions. The solutions were immediately

filtered through 0.2 l filter and analyzed using validated

HPLC method. The simulated intestinal and gastric fluids;

FaSSIF, FeSSIF, FaSSGF and FeSSGF [22] were utilized

as dissolution media in order to predict in vivo dissolution

and the food effect on CLZ absorption.

Results and discussion

Preparation and evaluation of spray dried complexes

Optimization of spray drying process

Spray drying produces smooth spherical particles with high

specific surface area and low particle size. It is one of the

most commonly used techniques for preparation of nano-

amorphous solids from solutions [23–25]. Different organic

solvents like DMSO, DMF and methanol were studied as

solvent systems for spray drying. But, taking into consid-

eration the solubility of individual components, yield of the

product and environmental concerns associated with the

use of organic solvent, the choice was zeroed down to

hydroalcoholic solution containing ethanol and water.

Ethanol:water (75:25) gave a clear solution containing

CLZ and HP-b-CD in 1:2 molar ratio. Effect of various

process parameters like inlet air temperature, outlet air

temperature, feed rate, atomization air pressure and aspi-

rator speed were studied with respect to the yield of

product. The inlet and outlet air temperatures were opti-

mized at 75 and 50 �C, respectively, based on boiling

points of the solvents used and stability of the drug to get

dry and stable product. Effect of feed rate on the yield of

the product was studied by increasing the feed rate from

1 ml/min up to 4 ml/min. There was marked decrease in

J Incl Phenom Macrocycl Chem

123

the yield of the product with increase in feed rate. Maxi-

mum yield (75 %) was obtained at feed rate of 1.5 ml/

minute. Atomization air pressure and aspirator flow rate

were optimized at 1.5 kg/cm2 and 50 Nm3/h respectively

to achieve maximum yield and decrease the processing

time. All the above parameters were so adjusted that

maximum product concentrate could be obtained in

cyclone II of the spray dryer. Spray drying produced free

flowing complexes with significantly less moisture content

(2–4 % as determined by Karl-Fischer technique) com-

pared to pure HP-b-CD (10 % w/w). The spray dried

complexes so obtained in cyclone II were in nanosized

range with narrow size distribution.

Particle size distribution, polydispersity index and zeta

potential studies

Particle size distribution of optimized and reproducible

batches of CLZ nanocomposites as evaluated with laser

diffraction method was 401.18 ± 36 nm. An overlay graph

of optimized and reproducible batches of spray dried

product is presented in Fig. 2. It was observed that particle

size of the nanocomposite was far less compared to indi-

vidual components. This could be achieved by fine tuning

atomization air pressure, temperature, feed rate and aspi-

rator rate. Polydispersity index value obtained from photon

correlation spectroscopic studies was 0.241 which indicates

good homogeneity in particle size distribution. Zeta

potential value of optimized batch of nanocomposites was

found to be (-) 34. The presence of hydroxyl groups in

HP-b-CD resulted in negative values of zeta potential. The

higher zeta potential indicated desired stability of nanon-

ized system. Thus, spray drying technique produced free

flowing nanoamorphous inclusion complexes.

Fig. 2 Overlay graph of

particle size distribution of two

different batches of spray dried

products

Fig. 3 Saturation solubility of plain CLZ and CLZ in complexed

form in water, FaSSIF, FeSSIF, FaSSGF and FeSSGF

J Incl Phenom Macrocycl Chem

123

Solution state characterization of nanocomposites

Saturation solubility of CLZ and CLZ–HP-b-CD

nanocomposites in water and biorelevant media

Equilibrium solubility values of plain CLZ and CLZ–HP-

b-CD nanocomposites are shown in Fig. 3. CLZ is a neu-

tral lipophilic compound and shows higher solubility of

CLZ in FeSSIF and FeSSGF compared to FaSSIF and

FaSSGF. This could be attributed to micellar solubilization

due to higher concentration of sodium taurocholate and

lecithin in intestinal fluids. Going with the trend, the spray

dried nanocomposites also showed a similar solubility

pattern with a drastic increase in solubility compared to

free CLZ.

Inclusion efficiency

Inclusion efficiency plays an important role in deciding the

final weight of the dosage form. The more the inclusion

efficiency, the less is the amount of complex (equivalent to

drug) to be taken in the formulation. The percent inclusion

efficiency for 1:2 IC was found to be 97.18 ± 1.28, which

suggests uniform distribution of CLZ in the complex.

Determination of stoichiometry

Phase solubility studies Phase-solubility diagrams are

generally used to calculate binding constants of drug/

cyclodextrin complexes. Phase solubility studies exhibited

an AL type curve indicating formation of soluble complexes

of first order with respect to HP-b-CD and first or higher

order with respect to CLZ. There was a linear increase in

solubility of CLZ with an increase in HP-b-CD concen-

tration as seen in Fig. 4. Binding strength of the complex

was determined using stability constant values and the

apparent 1:1 stability constant, Ks was found to be

892.13 M-1 (usual range 100–20,000 M-1). Higher value

of Ks indicates good complexation of CLZ with HP-b-CD.

Generally, it is observed that poorly soluble drugs show

non-linear trend in the phase solubility diagram. Com-

plexation efficiency is regarded as a more accurate method

for determination of the solubilizing efficiency of CDs

because it is independent of both the intrinsic solubility of

the drug and the intercept of phase solubility diagram [26].

The complexation efficiency of CLZ–HP-b-CD complex

was found to be 3.61 9 10-3.

Continuous variation method (Job’s plot) Stoichiometry

of drug/cyclodextrin complexes cannot be derived from

simple phase-solubility studies, especially for poorly sol-

uble drugs. Therefore, Job’s plot is preferred over phase

solubility studies for determining stoichiometry of

drug:cyclodextrin complexes [27]. According to the con-

tinuous variation Job’s method, change in absorbance is

directly related to the concentration of complex, and can be

measured for a set of samples with continuously varying

the molar fraction of the components. The maximum

concentration of the complex will be present in the sample

where the molar ratio R corresponds to the complexation

stoichiometry. The maximum absorbance of CLZ–HP-b-

CD was observed for R = 0.7 (Fig. 5), which indicates 2:1

(HP-b-CD:CLZ) stoichiometry.

CLZ is a comparatively large molecule with rigidity in

its structure. As a result it might not be able to enter the

inner cavity of HP-b-CD completely. A look at the struc-

ture of CLZ reveals that tetrazole moiety is the only

hydrogen accepting group, making it most apt to interact

with hydroxyl groups of HP-b-CD. The degree of chemical

shifts produced for protons of CLZ and H-30 and H-50

protons of HP-b-CD will reveal whether the penetration is

deep or shallow (surface interaction). Tetrazoles are

Fig. 4 Phase solubility diagram of CLZ–HP-b-CD system in water at

30 �C

Fig. 5 Job’s plot determining Stoichiometry of CLZ–HP-b-CD

complex

J Incl Phenom Macrocycl Chem

123

characterized by p- p* transitions which produces a posi-

tively signed induced circular dichroism. This indicates

axial inclusion where the long axis of CLZ will be parallel

to the molecular axis of HP-b-CD. We foresee the inclu-

sion of tetrazole moiety attached to the cyclohexyl ring of

CLZ into the HP-b-CD cavity. The docking study reveals

that this structure was found to be highly probable and

energetically favorable. However, a detailed characteriza-

tion and structure elucidation has been carried out in the

following sections.

Circular dichroism spectroscopy Additional information

on the mode of inclusion of CLZ in the HP-b-CD cavity was

obtained using circular dichroism spectroscopy. CLZ, being

an achiral molecule did not give a circular dichroism

spectrum. Inclusion of CLZ in the chiral cavity of HP-b-CD

produced an induced circular dichroism spectrum, charac-

terized by positive band at 260 nm (Fig. 6), at the same

position as in the electronic absorption spectrum. It is well

known that intrinsic Cotton effects of CDs are observed

below 220 nm. The inclusion of optically inactive com-

pounds within the CD cavity generates extrinsic Cotton

effects in the wavelength region of the drug chromophore

[28, 29]. According to the semiempirical rules of Harata and

Kodaka [30], positive sign of the dichroic signal showed

that CLZ was included in the HP-b-CD cavity in such a way

that the transition moment of the chromophore was parallel

to the long axis of HP-b-CD cavity. Positively signed ICD

bands are associated with p-p* transition of the tetrazole

moiety of CLZ. It reveals axial inclusion in which the long

axis of CLZ is parallel to the molecular axis of HP-b-CD.

Thus ICD exhibit long-axis-polarised transitions.

NMR spectroscopy 1D 1H NMR spectrum of CLZ

showed signals for protons of the cyclohexyl fragment

from 1.97 ppm to 1.67 ppm as complex multiplets, further

upfield at 1.47–1.27 ppm appeared the protons of two

methylene groups from butyl fragments. The NH proton of

the quinolinone fragment is seen at 9.9 ppm and the aro-

matic protons are observed at 6.78–6.71 ppm. The forma-

tion of complex was evident from the changes in the

chemical shift values of CLZ (modest downfield) and HP-

b-CD (modest upfield) in the complex (Fig. 7). The

changes in the chemical shift values of CLZ and HP-b-CD

in the complex are depicted in Table 1.

Table 1 shows that there is a marginal shift towards

downfield in the proton signal from cyclohexyl region of the

drug indicating that this region of the drug interacts with

cyclodextrin. Since, the shift is only marginal (Dd -

0.027 ppm), which may have risen from just surface inter-

action and not complete inclusion of the drug in the cavity.

The mode of inclusion of guest molecule into the host cavity

of CD involves the insertion of the less polar portion of the

guest into the CD cavity as reported in earlier papers [31,

32]. The H-30 and H-50 protons of the glucose units are seen

facing towards the interior of the HP-b-CD cavity, whereas

H-60 protons are located at the rim with the primary alco-

hols. H-20 and H-40 are on the opposite sides of the cavity.

Penetration of guest into the cavity of CD can be gauged by

the change in the H30 and H50 protons of CDs. Chemical

shifts of both H-30 and H-50 protons of CD indicate deep

penetration whereas the shift in only H-30 protons occurs

when the cavity penetration is shallow. In CLZ–HP-b-CD

complex, only marginal chemical shifts were observed for

H-30 and H-50 protons of HP-b-CD. Moreover, H30 had a

slightly higher chemical shift compared to H50. Moreover,

the ratio for the chemical shift changes for these protons,

DdH-50/DdH-30 was found to be 0.69, which indicate shal-

low penetration of CLZ into HP-b-CD cavity.

Additionally, 2D-ROESY experiments were used to

validate through space intermolecular interactions [33, 34]

between CLZ and HP-b-CD in the complex and are pre-

sented as contour plot in Fig. 8. The interaction of cyclo-

hexyl and the methylene protons attached to the tetrazole

ring with the 8-OH of the HP-b-CD was evident from cross

peaks 1–4 as shown in Fig. 8. As cross peaks correlate

protons with separation lower than 4.0 A in space (dipolar

coupling), the observed correlations indicate that the

cyclohexane ring and the tetrazole moiety are included in

the b-CD cavity.

Solid state characterization

Contact angle study

The contact angle for CLZ was found to be 608, indicating

extremely poorly wettability of CLZ in water. Complexa-

tion with HP-b-CD greatly reduced the contact angle to

Fig. 6 Circular dichroism spectrum of CLZ in presence of HP-b-CD

J Incl Phenom Macrocycl Chem

123

188, thereby increasing the wettability and reducing the

surface tension of CLZ. Reduction in contact angle directly

relates to the increase in saturation solubility and dissolu-

tion of drug from the complex [35, 36]. Complexation with

HP-b-CD greatly increased the saturation solubility of CLZ

from 5 to 120.19 lg/ml and also released more than 90 %

of CLZ within 10 min as compared to 10 % release in

uncomplexed state.

FTIR spectroscopy

FTIR spectrum of CLZ was characterized by aromatic C=O

stretching of the amide band at 1,668 cm-1, tetrazole

moiety at 1,504 cm-1, N=N stretching of the tetrazole

moiety at 1,295 cm-1, aromatic ether at 1,196 cm-1, NH

stretching of the quinolinone moiety from 3,330 to

3,060 cm-1 and NH bending of the quinolinone moiety

Fig. 7 1HNMR spectra of a CLZ, b HP-b-CD, c CLZ–HP-b-CD complex

Table 1 Chemical Shifts of

CLZ in CLZ–HP-b-CD IC

a See Fig. 8 for type of protonsb d in ppm

Type of

proton

aab bab cab d1–

d3abeab fab gab hab iab j,k,l,m,nab

CLZ 9.9 2.829 2.399 6.77 3.966 1.425 1.274 2.96 4.39 1.823

CLZ-

HP-b-

CD

9.9 2.826 2.397 6.76 3.963 1.438 1.262 2.973 4.399 1.850

Dd 0.0 ?0.003 ?0.002 ?0.01 ?0.003 -0.013 ?0.012 -0.013 -0.009 -0.027

Type of proton Ha10 Ha20 Ha30 Ha40 Ha50 Ha60

HP-b-CD 4.832 3.319 3.674 3.357 3.373 3.625

CLZ–HP-b-CD 4.821 3.311 3.648 3.341 3.355 3.606

Dd ?0.011 ?0.008 ?0.026 ?0.016 ?0.018 ?0.019

J Incl Phenom Macrocycl Chem

123

from 1,570–1,515 cm-1. FTIR spectrum of HP-b-CD

showed broad absorption bands at 3,383 cm-1 (symmetric

and anti-symmetric O–H stretching), 2,929 cm-1 (CH2

aliphatic stretch), 1,155 cm-1 (C–H stretching) and

1,034 cm-1 (C–O-C bending vibrations) [37, 38]. The

spectrum for PM of CLZ–HP-b-CD was superimposable to

those of the pure compounds with attenuation of the CLZ

peaks as shown in Fig. 9. The shifts in the IR bands of CLZ

and HP-b-CD in the CLZ–HP-b-CD IC are shown in

Table 2. The insertion of cyclohexane ring of CLZ into the

electron rich cavity of HP-b-CD increases the density of

electron cloud, resulting in high frequency shifts. On the

other hand, decrease in the frequency between IC and its

constituent molecules may be due to the formation of

hydrogen bonds and presence of van der Waals forces

between CLZ and HP-b-CD. Thus, FTIR spectra prove the

formation of CLZ–HP-b-CD IC.

Differential scanning calorimetry studies

DSC thermograms of CLZ, HP-b-CD, PM and IC are

shown in Fig. 10. The CLZ thermogram showed a sharp

endothermic peak at 159–162 �C corresponding to its

melting point. DSC thermogram of HP-b-CD showed a

broad endotherm in the range of 65–125 �C, which can be

attributed to desolvation of water molecules present in the

HP-b-CD cavity [34]. Thermal curves of PMs of CLZ with

HP-b-CD showed endothermic peak of CLZ with lower

area, lower Hfus and a shift in the endothermic peak to a

slightly lower temperature. The thermal data of CLZ, HP-

b-CD and CLZ–HP-b-CD IC are shown in Table 3. In PM

there is 46.27 % reduction in the height of CLZ peak

indicating slight interaction. The endothermic peak of CLZ

disappeared in the inclusion complex, indicating strong

interactions of CLZ and HP-b-CD. This could be attributed

to the formation of an amorphous solid, encapsulation of

CLZ inside the HP-b-CD cavity, or both [39, 40].

Powder X-Ray diffraction studies

The change in crystallinity of CLZ on complexation with

HP-b-CD was studied using P-XRD. Two important

Fig. 8 2D ROESY spectrum of CLZ–HP-b-CD complex

Fig. 9 FTIR spectra of a CLZ, b HP-b-CD, c CLZ–HP-b-CD PM

and d CLZ–HP-b-CD complex

J Incl Phenom Macrocycl Chem

123

parameters viz. relative degree of crystallinity (RDC) and

full width at half maxima (FWHM) were calculated to

determine crystallinity [41, 42].

RDC ¼ Isam=Iref ; ð4Þ

where Isam is the peak height of CLZ in ICs, and Iref is the

peak height at the same angle for plain CLZ. The shifts in

2h values and the intensity of the principle peaks are

reported in Table 4. The P-XRD diffractograms of CLZ,

HP-b-CD, PMs and ICs are shown in Fig. 11. The crys-

talline nature of CLZ was evident from the presence of

intense peaks in the diffractogram at 12.678, 12.988,15.358, 15.768, 17.988, 18.718, 19.598, 22.198 and 22.588.The P-XRD pattern of HP-b-CD presents only an amor-

phous halo due to its non- crystalline nature. The diffrac-

togram of PM exhibits most of the principle peaks of CLZ

along with the amorphous halo of HP-b-CD, indicating

slight or no interaction between the pure components. In

contrast to these observations, IC showed a spectrum

similar to that of amorphous HP-b-CD and the disappear-

ance of characteristic crystalline peaks of CLZ. The dif-

fraction pattern of the peaks of IC was more diffused as

compared to pure components. The decrease in intensity

and FWHM values for IC’s compared to plain CLZ indi-

cate the transformation of CLZ from crystalline to amor-

phous state in the complex.

Atomic force microscopy

Visualization of surface topography of CLZ and CLZ–HP-

b-CD IC revealed a significant change in the morphology

of the surface when CLZ formed complex with HP-b-CD

as depicted in Fig. 12. The peak-to-valley distance in AFM

images was used as an indicator of the surface roughness

[43]. When the distance between the peak and the valley is

more it indicates a rough surface and if the distance is less

it indicates the smoothness of surface. Based on this peak

and valley distance, the AFM image of pure CLZ shows a

rough surface and uneven distribution. But in the case of

solid complex the rough surface of CLZ is changed into a

smooth surface. The changes in surface morphology and

the modification of the crystals may be taken as a proof for

the interaction of CLZ with HP-b-CD.

Thermodynamics of complexation

Effect of temperature

The solubility of CLZ in presence of HP-b-CD increased

with an increase in temperature. This resulted in an

increase in the slope of solubility curve, which may be

attributed to the liberation of water molecules bound in the

HP-b-CD cavity at higher temperatures. The decrease in Ks

values with increasing temperature indicates the exother-

mic nature of inclusion complexation [44, 45]. Typical

van’t Hoff plot, shown in Fig. 13 confirmed the linear

behavior over temperature range of 30–60 �C. Thermody-

namic parameters showed that the complexation phenom-

enon was dominated by favorable enthalpy changes as

compared to entropy changes as shown in Table 5. The

negative enthalpy change value suggested that inclusion of

CLZ into HP-b-CD is an enthalpy driven process, whereas

a positive entropy change suggested the role of hydro-

phobic interactions, which involves breakdown and

removal of structured water molecules inside the HP-b-CD

cavity and around the non-polar substrate. The magnitude

of DS was little higher suggesting role of steric interactions

in stabilization of complex [46].

Fig. 10 DSC thermographs of a CLZ, b HP-b-CD, c CLZ–HP-b-CD

PM and d CLZ–HP-b-CD complex

Table 2 Shifts in IR frequencies of CLZ and HP-b-CD in IC

Group assignment Wave number (cm-1) D (cm21)

CLZ CLZ in complex

C=O (Amide) 1,668.11 1,668.43 -0.32

Tetrazole 1,505.35 1,504.51 ?0.84

N=N 1,295.62 1,295.43 ?0.19

Ar–O–C 1,196.86 1,196.45 ?0.41

HP-b-CD HP-b-CD in complex

–OH stretching 3,383.28 3,374.19 ?9.09

–CH stretching 2,929.80 2,931.85 -2.05

C–H stretching 1,155.12 1,155.45 -0.33

C–O stretching 1,083.09 1,080.09 ?3.00

C–O–C bending 1,032.16 1,037.75 -5.59

J Incl Phenom Macrocycl Chem

123

Effect of humidity

Gravimetric vapor sorption study illustrated water vapor

lyotropic effect on spray dried nanocomposites of inclusion

complexes [47, 48]. CLZ, HP-b-CD and CLZ–HP-b-CD IC

could be classified as non-hygroscopic, hygroscopic and

slightly hygroscopic respectively as per the general clas-

sification reported in the earlier paper [49]. For CLZ, the

moisture increase below 80 % RH is 0.14 % and for HP-b-

CD and CLZ–HP-b-CD IC it is 10.62 and 1.65 % respec-

tively. Also, the moisture increase above 80 % RH for HP-

b-CD and CLZ–HP-b-CD IC is 1.19 and 0.52 % respec-

tively. Figure 14 illustrates the moisture sorbed versus

%RH at 25 �C for CLZ, HP-b-CD and CLZ–HP-b-CD IC.

Both, HP-b-CD and CLZ–HP-b-CD IC showed a mono-

phasic rate profile over the entire RH range and exhibited

similar adsorption–desorption profiles, indicating that the

presence of CLZ in the IC does not affect moisture uptake.

This is expected for CLZ, as crystalline materials are in a

thermodynamically stable state. Abrupt increase in weight

was observed particularly at 85 % RH for CLZ–HP-b-CD

complex which may have resulted from the occurrence of

capillary condensation in the pores. This rapid sorption of

water, at 85 % RH first led to surface dissolution of IC that

increased the mobility of the encapsulated CLZ. With

increased RH, the sorption kinetics changed from surface

adsorption to bulk absorption. Critical relative humidity,

the point at which the curve intersects the x-axis of the

uptake rate versus %RH profile was observed at 40 % RH.

The first hysteresis loop (between 90 and 80 % RH) is

primarily due to drying of the saturated solution. Below

80 % RH, desorption branch of the isotherm starts to

slightly deviate from the adsorption branch and the dif-

ference persists until low RH values were reached. This

hysteresis is probably due to the formation of a hydro-

phobic layer around the sample with lower diffusivity for

water [50].

XRD spectrum of CLZ–HP-b-CD IC showed a distinct

halo, both before and after DVSO analysis because of the

Table 4 XRD parameters of CLZ and CLZ–HP-b-CD IC

2h Intensity FWHM (cm) I/FWHM

CLZ CLZ–

HP-b-

CD

CLZ CLZ--

HP-b-

CD

CLZ CLZ–

HP-b-

CD

9.4 1,982.75 – 0.25 – 3,931 –

10.3 1,982.75 – 0.4 – 4,956.87 –

12.9 20,862.06 – 0.2 – 104,310.3 –

15.3 5,689.65 – 0.3 – 18,965.5 –

15.8 6,637.93 550.32 0.3 0.2 22,126.43 2,751.6

18.8 2,672.41 – 0.25 – 10,689.64 –

19.4 2,068.96 – 0.2 – 10,344.8 –

20.4 3,620.68 – 0.3 – 12,068.93 –

20.8 2,327.58 – 0.2 – 11,637.9 –

23.5 3,448.27 650.61 0.2 0.2 17,241.35 3,203.51

31.7 2,743.14 – 0.2 – 13,715.7 –

Table 3 Thermal data of CLZ, PM and CLZ–HP-b-CD complex

Qty. of CLZ in sample

(mg)

Tonset

(�C)

Tpeak

(�C)

Tendset

(�C)

Area

(mJ)

Hfus (Obs.) (J/

g)

Hfus (Theory) (J/

g)

%

difference

CLZ 3 159.42 161.9 163.04 259.19 112.69 – –

CLZ–HP-b-CD

(PM)

1 159.51 160.9 163.34 27.59 20.18 37.565 46.27

CLZ–HP-b-CD

(IC)1 – – – – – 37.565 100

Fig. 11 P-XRD spectra of a CLZ, b HP-b-CD, c CLZ–HP-b-CD PM

and d CLZ–HP-b-CD complex

J Incl Phenom Macrocycl Chem

123

amorphous nature of HP-b-CD as seen in Fig. 15. Most of the

principle peaks of CLZ disappeared/decreased in intensity or

broadened suggesting amorphous nature of the CLZ–HP-b-

CD IC spectrum. A channel type packing structure of IC was

observed, which may be attributed to increased molecular

mobility because of sudden absorption of moisture [51].

These observations were indicative of the transformation of

CLZ from crystalline to amorphous state, which might be

due to the inclusion of CLZ into HP-b-CD cavity.

Molecular modeling studies

Proximity relationships between CLZ and HP-b-CD

obtained from 2D ROESY NMR assisted computer simu-

lations to compute binding energy of the complex. The

structure of IC deduced from NMR studies (ROESY)

indicated shallow penetration of the cyclohexane ring and

the tetrazole moiety of CLZ in the HP-b-CD cavity.

Accordingly, the input structure (Fig. 16a) for molecular

dynamics simulation was prepared in DMSO solvent (since

NMR experiments were performed in DMSO).

Fig. 12 Atomic force microscopic images of a CLZ and b CLZ–HP-b-CD complex

Fig. 13 vant Hoff plot of CLZ–HP-b-CD complex at increasing

temperatures

Fig. 14 DVSO analysis of CLZ, HP-b-CD and CLZ–HP-b-CD

complex at different relative humidities

Table 5 Energy values of CLZ–HP-b-CD inclusion complexation

Temperature

(�C)

Ks DG (Kcal/

mol)

DH (Kcal/

mol)

DS (cal/

mol)

30 892.13 -4.085 -0.214 ?12.769

40 874.61 -4.208 ?12.754

50 851.26 -4.329 ?12.734

60 829.15 -4.443 ?12.616

J Incl Phenom Macrocycl Chem

123

The free energy of binding for CLZ–HP-b-CD com-

plexes was computed from the energetics of MD simula-

tions, using the following equation.

DG ¼ KBT � lnXi

minimaexp

�Ei

KBT

� �� �ð5Þ

Subsequently the free energy of binding was calculated

as the difference between the bound and unbound forms of

CLZ and HP-b-CD.

DDGbinding ¼ �DGcomplex þ DGhost þ DGguest ð6Þ

The stability of complexes over the entire simulation

trajectory was judged by the RMSD computed for the

CLZ–HP-b-CD frames captured over the productive MD

simulation as shown in Fig. 16b.

Computer simulations indicate that strength of the

complex was not very strong as it breaks open and behaves

as 1:1 complex (CLZ:HP-b-CD), which is also evident

from the ROESY data. The modeled complex revealed that

dispersive van der Waals interaction energy was a major

contributor for stabilization of complex which is comple-

mentary with both, NMR and thermodynamic data. Ther-

modynamic data indicated the role of non-bonded and

steric interactions in the process of complex formation.

Significant decrease in the total hydrophobic surface area

Fig. 15 XRD spectra of CLZ–HP-b-CD complex before and after

sorption cycle

Fig. 16 a Most probable

structure of CLZ–HP-b-CD

complex deduced from 2D

ROESY NMR. b RMSD graph

of HP-b-CD and CLZ–HP-b-

CD inclusion complex

J Incl Phenom Macrocycl Chem

123

(72 %) of CLZ upon complexation with HP-b-CD might be

responsible for increased solubility and dissolution of CLZ

in the IC. There was a good degree of correlation between

the experiments and modeling data suggesting that

molecular modeling was successfully used as a comple-

mentary tool for characterizing the drug inclusion com-

plexes with CDs.

In vitro dissolution studies of nanocomposites in water

and biorelevant media

The nano-amorphous composites showed a significant

increase in the solubility and dissolution characteristics of

CLZ as compared to plain CLZ. In vitro dissolution studies

in biorelevant media help to forecast the in vivo perfor-

mance of a drug. Biorelevant media compositions closely

mimic the actual physiological fluid compositions at fed

and fasted states and thus can be effectively used for

qualitative determination of extent of in vivo absorption

and food effects on dissolution [52–54]. The dissolution

profile of CLZ in each of these media and also in 0.3 %

SLS in water (USP dissolution medium) is shown in

Fig. 17. In vitro biorelevant dissolution media studies

exhibited negligible food effects. The nanocomposites of

CLZ–HP-b-CD exhibited [85 % drug release in all bio-

relevant media compositions within 15 min. The FeSSIF/

FaSSIF and FeSSGF/FaSSGF ratio of T50 % for nano-

composites was around 0.96 and 0.87 respectively. The

minimal difference in the absolute value of T50 % between

FeSSIF/FaSSIF and FeSSGF/FaSSGF could be attributed

to rapid dissolution of CLZ due to synergistic effect of a

combination of factors, viz. nanometric size range of the

composites (confirmed by particle size distribution), for-

mation of soluble inclusion complexes, amorphization of

CLZ in the complex (confirmed by DSC and XRD),

reduction in surface tension and improved wettability

(confirmed by contact angle study). The increase in the

dissolution rate of CLZ when physically mixed with HP-b-

CD was possibly a result of surfactant like properties of

HP-b-CD, thus improving the wettability and dissolution of

CLZ or possibly in situ formation of readily soluble com-

plex [55]. This observation was well supported by molec-

ular modeling studies which confirmed instant dissociation

of one molecule of HP-b-CD from the complex (data not

shown). This free HP-b-CD molecule formed in situ

complex with CLZ released from the nanocomposites in

dissolution medium.

Conclusion

The amalgamation of two different solubility enhancement

strategies, viz. spray drying and inclusion complexation

produced amorphous solids with nanoscale dimensions

which significantly modulate the solubility and dissolution

profile of cilostazol. Phase solubility studies and Job’s plot

revealed formation of a 2:1 (HP-b-CD:CLZ) stoichiometric

inclusion complex with stability constant of 892.13 M-1.

The close association between CLZ and HP-b-CD resulted

in changes in some of the characteristic spectral, phase

transitional and morphological properties of CLZ. Solid

state characterization confirmed the formation of a supra-

molecular complex in which CLZ was entrapped inside the

HP-b-CD cavity. However, structural elucidation using 1D

and 2D NMR (ROESY) in solution state revealed partial

inclusion of the cyclohexane ring and tetrazole moiety of

CLZ in the HP-b-CD cavity. Binding energy obtained from

molecular modeling revealed stability of the said inclusion

complex. DVSO studies confirmed lyotropic effect of spray

dried nano-amorphous complex with increasing humidity

values. In vitro dissolution profile of cilostazol in biorele-

vant media predicts the in vivo performance of CLZ and

Fig. 17 In-vitro dissolution

profiles of CLZ, CLZ–HP-b-CD

PM and CLZ–HP-b-CD

complex in water and

biorelevant media

J Incl Phenom Macrocycl Chem

123

corroborates the ability of nano-amorphous composites as a

dosage form design to reduce the biovariability associated

with pristine CLZ. Thus, CLZ–HP-b-CD nano-amorphous

composites could be considered as a promising strategy in

oral and parenteral delivery of CLZ.

Acknowledgments The authors are grateful to Dr. Evans Coutinho,

Dr. Raghuvir Pissurlenkar and Mr. Elvis Martis, Molecular Simula-

tions Group, Bombay College of Pharmacy for their dedicated support

in modeling studies, Dr. Vaibhav Sihorkar, Aurigene Labs Ltd. for

Dynamic Vapor Sorption analysis, Dr. Sudha Srivastava, Mrs. Mamta

Joshi and Mr. Devidas Jadhav, Tata Institute of Fundamental

Research (TIFR), Mumbai for NMR analysis, Mrs. Sandhya Shenoy,

Cadila Healthcare, Thane for DSC studies, Dr. M.M.V Ramana and

Mr. Nilesh Vasave, Mumbai University, Mr. Sarvesh Tiwari (Mettler

Toledo, Mumbai) for Hot Stage Microscopy, Mr. Nilesh Kulkarni and

Mrs. Bhagyashree Chalke, Tata Institute of Fundamental Research

(TIFR), Mumbai for XRD and SEM studies, Dr. Shyamalava

Mazumdar and Mr. Bharat Kansara for circular dichroism studies, Dr.

Jayesh Bellare, Dr. S. S. Major and Mr. Prem Verma, Indian Institute

of Technology (IIT), Mumbai for Atomic Force Microscopy studies,

Dr. S. S. Bhagwat, Chemical Engineering Division, Institute of

Chemical Technology (ICT), Mumbai for providing facility for con-

tact angle study and Ms. Deepashri Dixit and Mr. Satyendra Mishra

for DoE studies.

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