J. Chem. Thermodynamics 67 (2013) 112–119

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J. Chem. Thermodynamics

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Testing the effect of the cavity size and the number of molecularsubstitutions in host–guest complexes formed by 2-hydroxypropyl-cyclodextrins and n-octyl-b-D-glucopyranoside

0021-9614/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jct.2013.08.003

⇑ Corresponding author at : Departamento de Física Aplicada, Facultad de Física,Universidad de Santiago de Compostela, Campus Vida, E-15782 Santiago deCompostela, Spain.

E-mail addresses: silpeca@unam.mx (S. Pérez-Casas), Pilar.Brocos@usc.es(P. Brocos), Angel.Pineiro@usc.es (Á. Piñeiro).

Sibila Concha-Santos a, Silvia Pérez-Casas a, Pilar Brocos b, Ángel Piñeiro a,b,⇑a Departamento de Fisicoquímica, Facultad de Química, Universidad Nacional Autónoma de México, Cd. Universitaria, México, D.F. 04510, Mexicob Departamento de Física Aplicada, Facultad de Física, Universidad de Santiago de Compostela, Campus Vida, E-15782 Santiago de Compostela, Spain

a r t i c l e i n f o a b s t r a c t

Article history:Received 20 July 2013Accepted 7 August 2013Available online 17 August 2013

Keywords:2-Hydroxypropyl cyclodextrinn-Octyl-b-D-glucopyranosideHost–guest complexIsothermal titration calorimetryDemicellization heatNumber of molecular substitutions

The thermodynamic characterization of host–guest complex formation processes based on modifiedcyclodextrins (CDs) presents several difficulties that are absent when using native CDs. They are mainlydue to the relatively wide distribution of the degree of molecular substitution and also to the indetermi-nacy in the location of the modified groups. A more common trouble would arise from the coexistence ofcoupled processes, such as CD-surfactant complexation and surfactant demicellization. In the presentwork a reasonable approach based on the analysis of isothermal titration calorimetric measurementsis employed to assess the effect of the number of molecular substitutions as well as of the CD cavity sizein supra-molecular complexes formed by 2-hydroxypropyl-CDs and n-octyl-b-D-glucopyranoside. Theemployed method considers both first- (1:1) and second-order (1:2 and 2:1) stoichiometries. The resultswere significantly different from those previously observed for native CDs. The substituted groupsincrease entropically the stability of 1:1 species based on a-CD, while compete with the surfactant forthe CD cavity in the case of 1:1 and 1:2 species based on b- and c-CD, respectively. Also, the modifiedb-CD seems to enhance the formation of non-inclusion second-order complexes.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Native cyclodextrins (CDs) are cyclic oligosaccharides formedby 6 (a), 7 (b), or 8 (c) d-glucopyranoside units linked by a(1–4)glycosidic bonds. Their molecular geometry is usually describedas a truncated cone with a hydrophobic cavity able to capturenon-polar groups in water solution. Such ability has been exploitedfor decades, leading to a huge amount of applications including thesolubilization and transport of drugs [1,2], the design of new mate-rials [3–7], and the optimization of catalysis processes [8,9].Although the amount of scientific work based on cyclodextrins isvast, most of it is addressed to direct applications and much lessfundamental work has been published. Thus, basic properties ofnative CDs such as the difference in the solubility between a-, b-,and c-CD have not been still clearly explained in terms of theirmolecular structure and interactions with other molecules, or withthe solvent, although several attempts have been performed[10,11]. The advance in the fundamental knowledge of CD-based

systems is expected to facilitate the rational design of new pro-cesses and materials, as well as the optimization of current appli-cations and products.

The cavity size of native CDs is the most important factor todefine their affinity for other molecules. For instance, a-CD bindsefficiently alkyl chains while cyclic groups are better encapsulatedby b-CD [12]. The c-CD often hosts more than one molecule in itscavity [13–15]. A number of chemical modifications have beenintroduced in native CDs to overcome their limited range ofsolubility, to optimize their ability for specific applications, or toprovide them with new functionalities [1,7,9,16,17]. The substitu-tion of hydroxyl H atoms by 2-hydroxypropyl (HP) groups repre-sents perhaps the most widely employed chemical modificationin cyclodextrins. The main feature of these modified CDs, whencompared to the native ones, is their much higher solubility. As re-gards pharmaceutical applications, their advantages are: (a) in-creased drug solubility; (b) increased drug dissolution speed; (c)increased drug bioavailability; (d) reduced drug side-effects; and(e) increased stability of drugs [17,18]. A major difficulty for thecharacterization of systems based on modified cyclodextrins is thatthe location and number of chemical substitutions are not entirelyunder control. Thus, commercially available modified CDs consistof a distribution of species. Typically, any measurement of systems

SCHEME 2. Structure of 2-hydroxypropyl-cyclodextrins, with n = 6, 7, and 8 for a-,b-, and c-cyclodextrin, respectively. The substitution of hydrogen by a 2-hydroxy-propyl group (R in the scheme) can be made in the positions 2, 3, and 6 of each ring(O2–O3 and O6 are called the secondary and primary oxygen atoms, respectively).The average number of molecular substitutions per glucopyranoside ring in theemployed cyclodextrins is indicated in table 1.

TABLE 1List of chemicals employed in this work including their molar mass in g �mol�1 (M).The average number of molecular substitutions (MS) per glucopyranoside unit (bothAldrich specification and certified value) and the average number of crystallizedwater molecules per CD (#H2O) are indicated for the modified cyclodextrins.

S. Concha-Santos et al. / J. Chem. Thermodynamics 67 (2013) 112–119 113

based on those molecules will result from the contribution of eachof those species weighted by its concentration in the distribution.

During the last years, we have been trying to further the under-standing of CD complexes by using model systems and a variety oftechniques. This allowed us to provide quantitative structural, dy-namic, and energetic information on several supra-molecular com-plexes and their aggregates [13,15,19–22]. Based on fundamentalinformation at atomic level obtained from molecular dynamicssimulations and from the analysis of isothermal titration calorim-etry (ITC) experiments, together with microscopy images and sur-face tension measurements, we have been able to show that highlyviscoelastic films are expected to be formed for CD systems andconditions where 2:1 complexes (one guest hosted by two CDs)largely dominate [20–22]. It is our aim to continue studying differ-ent systems based on cyclodextrins in order to get informationuseful for the rational design of functional materials. In the presentwork we perform a thermodynamic characterization of the supra-molecular complexes that result from the interaction between(2-hydroxypropyl)-a-, -b-, or -c-cyclodextrins (HP-a-CD, CAS RN:128446-33-3; HP-b-CD, CAS RN: 128446-35-5; HP-c-CD, CAS RN:128446-34-4) and n-octyl-b-D-glucopyranoside (OGP, CAS RN: 29836-26-8) in water solution, based on the analysis of isothermaltitration calorimetry (ITC) measurements. The results are discussedin terms of the differences between native and HP-modified cyclo-dextrins and also as a function of the CD size. The effect of thenumber of molecular substitutions (MS) per glucopyranoside unitexhibited by the HP-b-CD is also discussed.

2. Experimental

2.1. Materials

Both the n-octyl-b-D-glucopyranoside (scheme 1) and the mod-ified cyclodextrins (scheme 2) were purchased with the highestpurity available from Aldrich and used without further purifica-tion. As regards HP-b-CD, three samples with different MS specifi-cation were used (table 1), so that the present comparative studydeals in fact with five ternary systems. The certificates of analysisprovided by Aldrich for HP-a-CD and HP-b-CD give the averagevalue of the corresponding MS distribution (we will always referto these certified values throughout this work), but this is notthe case for HP-c-CD, whose representative MS value was takenat the middle of the specified distribution range. The solutionswere carefully prepared by mass (balance model AT250, Mettler,Switzerland) taking into account the water content of the corre-sponding CD, which was determined by the Karl-Fisher method(701 KF Titrino, Metrohm, Swiss) using the Hydranal Composite5 reagent from Sigma–Aldrich (table 1). The uncertainty in the con-centration of the final solutions was less than 10�4 M in the case ofCDs (its main contribution being the uncertainty in the CD watercontent) and less than 10�5 M in the case of OGP. Water was dis-tilled and deionised using a Barnstead Nanopure Infinity equip-ment, its resistivity being 18.0 ± 0.2 MX � cm�1. Water was alsodegassed by ultrasound before adding the corresponding solute.

SCHEME 1. Structure of n-octyl-b-D-glucopyranoside.

2.2. Calorimetry

All the calorimetric measurements were performed at T =(298.15 ± 0.01) K using a Thermal Activity Monitor (TAM) model2277 from Thermometric (Sweden) equipped with a 2201 highperformance calorimetric unit. Under optimal conditions, thisinstrument is capable of detecting ±1 lW. Approximately 2 mL ofa cyclodextrin aqueous solution were introduced into the sampleand reference cells, each with a total volume of 4 mL. An OGP solu-tion with a concentration of either �(223 or 270) mM, approxi-mately ten times its critical micelle concentration (cmc), wastitrated to the sample cell employing a syringe pump (model 612Lund from Thermometric, Sweden) controlled by a computer. Inall experiments, between 20 and 50 titrations of 16 lL were per-formed. The experiments were designed to achieve an OGP concen-tration in the sample cell higher than its apparent cmc (cmcap) forthat particular CD solution. Pure water was simultaneously titratedinto the reference cell to subtract the heat evolved due to the dilu-tion of the cyclodextrin from the total signal. For each of the fivecyclodextrins, experiments using the above described methodol-ogy were done at several CD concentrations, ranging from (5 to30) mM. An OGP dilution experiment into pure water, using anOGP concentration similar to that employed with the CD solutionswas also performed. Prior to start and end each experiment, anelectrical calibration of the calorimetric unit was carried out in or-der to adjust the power sensor in the cells. It is worth noting that

Ma MS #H2O

Spec. Certified

OGP 292.37HP-a-CD 1180 0.52 5.27 ± 0.08HP-b-CD 1380 0.5-0.7 0.64 4.49 ± 0.21

1460 0.8-1.0 0.8 4.45 ± 0.221540 0.86-1.28 1.19 4.47 ± 0.09

HP-c-CD b 1580 0.5-0.7 5.39 ± 0.08

a Average molar mass for the anhydrous modified CDs.b Since the average value of the MS distribution is not furnished in the Aldrichcertificate, we regard this cyclodextrin as bearing MS = 0.6, which is at the middle ofthe specified MS range.

114 S. Concha-Santos et al. / J. Chem. Thermodynamics 67 (2013) 112–119

this calorimeter furnishes positive and negative signals for exo-thermic and endothermic processes, respectively.

3. Thermodynamic characterization

As stated above, in all calorimetric experiments an aqueoussolution of the surfactant at a concentration approximately tentimes its cmc was titrated over the cyclodextrin solution locatedin the sample cell. These experiments are equivalent to those pre-viously reported with native cyclodextrins and the same surfactant[15]. The calorimetric signal arises from several contributions: (i)formation of complexes between the surfactant and the cyclodex-trin molecules; (ii) rupture of micelles when a tiny volume of ahighly concentrated surfactant solution is added to the much morediluted CD solution; (iii) dilution of the free surfactant molecules,i.e. those that are not part of a supra-molecular complex nor of amicelle; and (iv) dilution of micelles. The dilution of the cyclodex-trin solution does not contribute to the total signal because it iscounterbalanced by the titrations performed in the reference cell.

Since the concentration of cyclodextrin in the cell was muchlower than that of OGP in the syringe, after a number of titrationsthe CD molecules are saturated, i.e. most of them form part of com-plexes with surfactant molecules. The saturation concentration de-pends on the initial CD concentration, on the presence ofcomplexes with different stoichiometry and on their respectiveconcentration that can be determined from the correspondingequilibrium constants. It is worth noting that the contribution (i)described above is highly reduced once the saturation of the CDmolecules is reached. Beyond that concentration, contributions(ii) and (iii) dominate the total heat signal while the cmcap valueis not achieved. At higher OGP concentration only the dilution ofmicelles and perhaps their interaction with CD:OGP complexesare expected to contribute significantly, but the detected heat be-comes negligible. A representation of a raw calorimetric curvereflecting such evolution is shown in scheme 3, together with theexpected signal for the dilution of the surfactant in the absenceof cyclodextrin (inset). Thus, by subtracting the heat of demicelli-zation (Qm in scheme 3) from the total heat detected at a giventitration i (prior to the CD saturation), the contribution correspond-ing to the formation of CD:OGP complexes (Q com

i ) is isolated. Therelevant magnitude for the treatment of data is the accumulatedheat after j titrations (Q acc

j ):

Q accj ¼

Xj

i¼1

Q comi : ð1Þ

This experimental Q accj can be written in terms of the molar

enthalpies of formation DHij of the complex species present inthe solution as well as of their concentrations [CDiOGPj]:

OGP concentra�on

OGP concentra�on

Q

Calo

rim

etri

c Sig

nal (Q

)D

etec

ted

Hea

t /Ti

tra�

on

cmcap

cmc

Qm

Qm

(i)-(iv)

Satura�onof CDs

OGP Dilu�on in the absence of CD

Titra�on of OGP on a CD solu�on

(ii)-(iv)

(iv)

SCHEME 3. Schematic representation of the calorimetric signal from an experimentof OGP titrated over a CD solution. The inset corresponds to the titration of thesurfactant over pure water. The dominant contributions (see Section 3) areindicated in the boxed text. Qm stands for the heat of demicellization.

Qaccj ¼Vj DH11½CD1OGP1� þ ðDH11 þ DH12Þ½CD1OGP2�f

þ DH11 þ DH21Þ½CD2OGP1�ð g; ð2Þ

where Vj is the volume of the solution in the calorimetric cell after jtitrations. In equation (2), the concentrations of the complex speciesdepend on the equilibrium constants for their formation:

K11 ¼½CD1OGP1�½CDf � � ½OGPf �

; K21 ¼½CD2OGP1�

½CD1OGP1� � ½CDf �;

K12 ¼½CD1OGP2�

½CD1OGP1� � ½OGPf �; ð3Þ

where [Xf] is the concentration of molecules of the species X that arefree, i.e. the concentration of CD or OGP that are not forming com-plexes, micelles nor any other type of supra-molecular aggregate.Only complexes with stoichiometry 1:1, 1:2, and 2:1 were consid-ered in this work since higher-order structures are unlikely for thistype of molecules [13,15]. Thus, by combining the latter equationswith those obtained from the mass balance for both the CD andthe surfactant molecules (see equations (4) and (5)), we get 6 fittingparameters, namely DH11, K11, DH12, K12, DH21, and K21. They werefitted using a home-made code and the objective function given byequation (6). More details are given in previous works [15,22].

½CDf � ¼ ½CDT� � ½CD1OGP1� � 2½CD2OGP1� � ½CD1OGP2�; ð4Þ

½OGPf � ¼ ½OGPT� � ½CD1OGP1� � ½CD2OGP1� � 2½CD1OGP2�; ð5Þ

v2 ¼ 1n

Xn

j¼1

Q accj ðcalcÞ � Qacc

j ðexpÞh i2

: ð6Þ

4. Results and discussion

4.1. Analysis of the calorimetric signals

As illustrated in scheme 3, the raw calorimetric signal corre-sponding to the experiments performed in the present work hasseveral contributions. Since we are interested only in the thermo-dynamic characterization of the CD:OGP supra-molecular complexformation processes, the signal corresponding to the demicelliza-tion of OGP in the absence of cyclodextrin (Qm in the inset ofscheme 3) would require to be subtracted from each experiment.Then the resulting data could be directly analyzed in terms ofthe model described in the previous section. Three or four experi-ments were performed for each system using different initial con-centrations of the corresponding CD in the sample cell. Thetitration volume of OGP was the same in all cases (16 lL) whilethe surfactant concentration was either �223 mM or, only in twoexperiments, �270 mM. The profile of the calorimetric signals asa function of the OGP concentration exhibits a large dependenceon the cyclodextrin structure (figure 1). Although the interactionslabeled as (i)–(iv) contribute to all these experimental profiles, onlysome of the curves obtained for HP-a-CD look like the graph shownin scheme 3, which illustrates the case where both supra-molecu-lar complexation and demicellization processes are exothermic.However, it is clear that all the experimental curves have a flat re-gion preceding a relatively sharp drop of the signal. (Only in theexperiment corresponding to 30 mM of HP-a-CD the final regionafter the plateau was not attained, which lacks importance sinceit is not needed for the above-described method of data treatment.)For a given cyclodextrin structure and OGP concentration, thatplateau takes similar or even practically identical values (see forinstance the experiments with (5, 10, and 15) mM of HP-a-CD,those with (10 and 15) mM of HP-b-CD (MS = 0.64), or those with(15 and 20) mM of HP-b-CD (MS = 0.8)). In each experimental

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40

−Q/

mJ

[OGP] / mM

HP-α-CD (MS=0.52)

0

5

10

15

20

25

0 10 20 30 40 50 60 70

−Q/

mJ

[OGP] / mM

HP-β-CD (MS=0.64)

0

5

10

15

20

25

0 10 20 30 40 50 60

−Q/

mJ

[OGP] / mM

HP-β-CD (MS=0.8)

-15

-10

-5

0

5

10

15

20

25

0 10 20 30 40 50 60

−Q/

mJ

[OGP] / mM

HP-β-CD (MS=1.19)

0

4

8

12

16

20

0 10 20 30 40 50 60

−Q/

mJ

[OGP] / mM

HP-γ-CD (MS=0.6)

FIGURE 1. Raw calorimetric signals obtained by ITC for the working systems. The labels within the plots indicate the size and MS specification of the cyclodextrin involved ineach set of experiments. The employed CD concentrations are 5 mM (s), 10 mM (D), 15 mM (h), 20 mM (e), and 30 mM (+). The OGP concentration in the syringe was�223 mM for all the systems except for HP-a-CD and HP-b-CD (MS = 0.8) at 30 mM, where it was �270 mM.

S. Concha-Santos et al. / J. Chem. Thermodynamics 67 (2013) 112–119 115

curve, the average value of this plateau was identified as Qm inorder to get the values of Q com

i and Q accj . Note that, for the aim of

this work, the calculation of both quantities does not make sensebeyond the OGP concentration corresponding to the saturation ofthe CD. Thus, the middle region of the plateau was typically takenas the maximum OGP concentration for our analysis. The signal ob-tained upon the subtraction of Qm and the application of equation(1) was exothermic in all the experiments using HP-a-CD andendothermic for those using HP-b-CD and HP-c-CD (figure 2).The weakest signals were observed for HP-c-CD. Actually, theraw calorimetric curve for 5 mM of this cyclodextrin in the samplecell (figure 1) is very similar to that obtained from the single dem-icellization experiment, i.e. titration of the surfactant solution onpure water; hence that curve was not kept for analysis and the cor-responding data series is missing in figure 2. The magnitude of Qacc

j

grows significantly, in absolute value, when increasing the averagenumber of MS for HP-b-CD. The limiting absolute value of Qacc in-volved in the saturation of HP-b-CD with MS = 1.19 is twice and�1.3 larger than those obtained with MS = 0.64 and MS = 0.8,respectively. For a similar degree of HP-substitutions (MS � 0.6),

the final value of Qacc for HP-a-CD and HP-b-CD is one order ofmagnitude and twice larger, respectively, than that determinedfor HP-c-CD. It is also worth noting that both the plateau and thesubsequent drop of the total calorimetric signal (figure 1) takeplace at higher concentrations when increasing the concentrationof the cyclodextrin, which indicates a corresponding increase ofthe cmcap of the surfactant. All these dependences are related tothe presence and concentration of supra-molecular complexeswith different stoichiometry in the solution, as well as to the en-ergy involved in the formation of each chemical species.

4.2. Data treatment and thermodynamic parameters

The thermodynamic parameters resulting from the simulta-neous fit of all the data recorded for each cyclodextrin to the modeldescribed above are listed in table 2. It is worth mentioning thatthe parameters obtained from the simultaneous fit of severalcurves corresponding to the same system are expected to be muchmore reliable than those obtained from the fitting of a single curve,even if the standard deviation is larger in the former case. As it can

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

−Qac

c/

J

[OGP] / [CD]

HP-α-CD (MS=0.52)

-0.18

-0.15

-0.12

-0.09

-0.06

-0.03

0.00

0.0 0.5 1.0 1.5 2.0 2.5

−Qac

c/

J

[OGP] / [CD]

HP-β-CD (MS=0.64)

-0.26

-0.23

-0.20

-0.17

-0.14

-0.11

-0.08

-0.05

-0.02

0.0 0.5 1.0 1.5 2.0

−Qac

c/

J

[OGP] / [CD]

HP-β-CD (MS=0.8)

-0.35

-0.30

-0.25

-0.20

-0.15

-0.10

-0.05

0.00

0.0 0.5 1.0 1.5 2.0 2.5

−Qac

c/

J

[OGP] / [CD]

HP-β-CD (MS=1.19)

-0.09

-0.08

-0.07

-0.06

-0.05

-0.04

-0.03

-0.02

-0.01

0.00

0.0 0.5 1.0 1.5 2.0 2.5

−Qac

c/

J

[OGP] / [CD]

HP-γ-CD (MS=0.6)

FIGURE 2. Accumulated heat due to the CD:OGP complex formation, obtained after subtraction of the demicellization heat (Qm in scheme 3) from the raw calorimetric signal(in the region prior to the CD saturation) and subsequently application of equation (1). The labels within the plots indicate the size and MS specification of the cyclodextrininvolved in each set of experiments. The symbols and concentrations are the same as in figure 1. The curves correspond to the simultaneous fitting of all the datasets of eachplot to the equation (2).

116 S. Concha-Santos et al. / J. Chem. Thermodynamics 67 (2013) 112–119

be seen in figure 2, the employed model is able to describe per-fectly most of the data sets. The poorest fitting was obtained forHP-b-CD (MS = 0.8) and, to a lesser extent, for HP-c-CD, which pro-duced the smallest calorimetric signal. The modest fittings ob-tained for these systems could be connected to a combination ofdifferent factors, including the unavoidable uncertainties in thepreparation of mixtures as well as in the water content estimationand further correction, the accumulated errors due to the subtrac-tion of the demicellization signal of the surfactant, and the hetero-geneity of the employed chemicals. As stated in the experimentalsection, the compounds were purchased from Aldrich with thehighest available purity. For native cyclodextrins this would beP98%, but modified cyclodextrins are supplied as a mixture of ran-dom substitutions with a distribution centered on the certified MSvalue (table 1). Moreover, two cyclodextrin molecules exhibitingan identical number of HP-substitutions in different positions(scheme 2) might behave very differently as regards the formationof inclusion complexes [23,24]. Hence the heterogeneity of thechemicals arises from combining position isomerism with a MSdistribution of unknown width. This can clearly be a source of

uncertainty because the range of species that are present in thedistribution are expected to bear different affinities for the surfac-tant molecule and these affinities are expected to contribute withdifferent weight to the thermodynamic parameters resulting fromthe corresponding fitting. Additionally, an important error arisesfrom the subtraction of Qm from the total calorimetric signal. Asnoted in the previous section, the contribution to the total signaldue to the surfactant demicellization is usually easy to identifyas the average value of the plateau upon saturation of the cyclo-dextrin, but in some cases the binding and demicellization signalsseem to be coupled with each other and the plateau is not easy toidentify. This is the case of (10 and 20) mM of HP-c-CD, and that of30 mM of HP-b-CD (MS = 0.8). Being aware of these uncertaintysources, we take the employed data treatment and the resultingparameters as a reasonable approach to characterize the formationprocess of the obtained complexes.

The values listed in table 2 clearly indicate that the presence of1:2 complexes using HP-a-CD is negligible, although such speciesmight be formed with HP-b-CD and HP-c-CD. In fact, in the caseof HP-c-CD the formation of the 1:2 complexes displays a slight

TABLE 2Thermodynamic parameters for the binding of native (MS = 0, earlier work) and HP-modified cyclodextrins with OGP at T = 298.15 K, obtained from the simultaneous fitting of thedata series measured for each system to the model described by equation (2). The number of fitted curves and the value attained in each fit by the objective function v2, given byequation (6), are noted too.a

a-CD b-CD c-CD

MSb 0 0.52 0 0.64 0.8 1.19 0 0.6

K11/mol � dm�3 1700c 2800 762c 430 380 240 7c 40K12/mol � dm�3 0c 3 0c 20 22 41 509c 17K21/mol � dm�3 64c 45 38c 45 44 7 46c

DH11/kJ �mol�1 �25.8c �14 �6.4c 5.7 6.7 14 6.4DH12/kJ �mol�1 6.0 �7.8 �6.8 �15 �11DH21/kJ �mol�1 �7.3c �0.1 2.9c �6.5 �6.0 �32106�v2/J2 15 1.6 54 25 4.8# Curves 4 4 3 4 3

a The estimated errors for Kij and DHij are less than 10%.b Average values certified by Aldrich for HP-a-CD and HP-b-CD. For HP-c-CD, the representative value was taken at the middle of the specified MS range (see table 1).c Ref. [15].

S. Concha-Santos et al. / J. Chem. Thermodynamics 67 (2013) 112–119 117

positive cooperativity, as suggested by the relation K11 < 4K12 [25]and by the (slightly greater than unity) Hill slope at half saturation[26], which is calculated as 2/[1 + (K11/4K12)0.5] and amounts to 1.1.A significant presence of 2:1 complexes, i.e. with two cyclodextrinsand one surfactant molecule, is predicted for HP-a-CD and HP-b-CD. For HP-c-CD, the inclusion of the two additional fitting param-eters required to consider this third stoichiometry in the modelwas not statistically justified. Globally, the relative weight of thesecond-order species, including 1:2 and 2:1 complexes, increaseswith the number of glucopyranoside units in the cyclodextrin,which is mainly due to the sharp decrease of K11 in the same direc-tion, with values of (2800, 430, and 40) M�1 for similar degrees ofHP-substitution in HP-a-CD, HP-b-CD, and HP-c-CD, respectively.A less pronounced diminution of K11 is observed with the increasein the number of molecular substitutions in HP-b-CD, with valuesof (430, 380, and 240) M�1 for MS = 0.64, 0.8, and 1.19,respectively.

As stated above, the overall Qacc signal is exothermic for the sys-tems with HP-a-CD and endothermic for those involving the othertwo cyclodextrins, regardless of the number of molecular substitu-tions. This is reflected in the enthalpy values, DH11 being negativefor HP-a-CD and positive for HP-b-CD and HP-c-CD. The increasein the number of molecular substitutions for HP-b-CD produces aclear increment in DH11, which is (5.7, 6.7, and 14) kJ �mol�1 forMS = 0.64, 0.8, and 1.19, respectively. The enthalpy values corre-sponding to the formation of second-order complexes (1:2 and2:1) have the opposite sign to those of the 1:1 species (the onlyexception isDH21 for HP-a-CD, which is practically zero). The forma-tion of 1:2 species is enthalpically unfavorable for HP-a-CD, as ex-pected due to its cavity size. The variation of the second-orderthermodynamic parameters with the number of molecular substitu-tions for HP-b-CD is not monotonic. Both Kij and DHij (i – j) areinsensitive within error to the increase from MS = (0.64 to 0.8), butsuffer a dramatic change when going from 0.8 to 1.19. As regardsthe formation of 2:1 species, it should be remembered that the mostefficient arrangement for the native cyclodextrins is that namedhead-to-head [3,13,15], where a tight CD dimer is formed by intra-and intermolecular H-bonds between the secondary hydroxyls (O2

and O3 oxygens in scheme 2). Since the O6 oxygen is the most reac-tive site [24], high degrees of HP-substitution (MS > 1) are morelikely to hinder the formation of head-to-head dimers, hence caus-ing a dramatic decrease of K21, whose values are (45, 44, and 7)M-1 for MS = 0.64, 0.8, and 1.19, respectively. On the other hand, hav-ing MS = 1.19 means that every HP-b-CD molecule (with seven D-glucopyranoside units) has 8.33 HP-substitutions in average. Inpractice this entails a lengthening of the cyclodextrin cavity and awidening of the secondary O cavity entrance [23], so that the forma-tion of noninclusion complexes of the type CD:OPG/OGP (where one

of the guests is threading the CD while the other is sitting on theother end of the cavity [13,15]) becomes more likely and moreenthalpically favorable. Although recent investigations haverecognized the significant contribution of non-inclusion-based phe-nomena to drug solubilization [27–29], the thermodynamic charac-terization of this kind of arrays has not been addressed yet, since itwould require their explicit consideration in the applied models.In the present case, the presence of noninclusion complexes is justinferred by indirect evidence, namely, the comparison with thebinding of native b-CD with OGP, for which K12 = 0 (seeSection 4.3.2).

Although no structural measurements were performed in thepresent work, the thermodynamic parameters obtained for thepredicted stoichiometries, together with results previously pub-lished for similar systems, can shed light on some topological fea-tures of the different supra-molecular complexes formed by HP-CDs and OGP.

4.3. Comparison with previous results and structural interpretation

The interaction between OGP and the three native cyclodex-trins has been studied in detail several years ago by usingmolecular dynamics simulations as well as calorimetric and sur-face tension measurements, taking advantage of the negligiblesurface activity of the native CDs [15] (see thermodynamicparameters for MS = 0 in table 2). A similar study involving mod-ified cyclodextrins would present much more difficulties becausetheir surface activity is significant [8], hence requiring a more in-volved model to get thermodynamic parameters from surfacetension measurements, and also because topology parametersfor a variety of substitutions are required prior to performmolecular dynamics simulations of these systems (we are cur-rently working on this). A few articles based on moleculardynamics simulations of HP-CDs [23] or HP-CD based complexes[30–32] have been published at the moment. Amongst the latter,either they just give an outline of the inclusion complex [30], orare mainly focused on free energy calculations [31], or thelength of the trajectories (<1 ns) is too short to take any signif-icant conclusion on the structure and dynamics of the simulatedmolecules [32]. Simulations of native cyclodextrins performed inour lab show that at least 1 ns is required to stabilize the rootmean square deviation of the studied structures in the mostfavorable case (preformed inclusion complexes). Thus, the diffi-culties to study supra-molecular complexes based on modifiedCDs are then numerous and important, but ITC measurementsare accessible and highly informative upon the data treatmentproposed to subtract the micellization contribution to the totalsignal.

118 S. Concha-Santos et al. / J. Chem. Thermodynamics 67 (2013) 112–119

4.3.1. Native a-CD vs. HP-a-CDOur results indicate that HP-a-CD forms complexes with 1:1

stoichiometry, while the presence of 1:2 complexes is negligibleand that of 2:1 species might be significant. In qualitative terms, thisis equivalent to what was observed for the interaction between na-tive a-CD and the same surfactant molecule, the main differencebeing the higher stability of HP-a-CD1OGP1 species(K11 = 2800 M�1) as compared to a-CD1OGP1 (K11 = 1700 M�1). Suchincreased stability does not lie on a more favorable binding enthalpy,as DH11 becomes more positive when going from the native(�25.8 kJ �mol�1) to the modified cyclodextrin (�14 kJ �mol�1).Rather, it could be related to the release of ‘‘low entropy’’ water mol-ecules upon the inclusion of OGP, since the presence of 2-hydroxy-propyl groups reduces the water density near the cavity entrances[23]. The structure of 2:1 complexes based on native cyclodextrinswith different surfactants is mainly stabilized by two contributions:the introduction of alkyl chains threading the hydrophobic cavitiesof the CDs (the larger the surfactant chain the more important thiscontribution), and a network of hydrogen bonds between the twocyclodextrins in the supra-molecular aggregate. Such network ofH-bonds is practically saturated when the complexes are based onnative a-CDs [13]. The substitution of hydrogen atoms by 2-hydroxypropyl groups hampers the formation of CD–CD H-bondsand might explain the relatively lower stability of the 2:1 complexesbased on HP-a-CD as compared to those based ona-CD. Accordingly,in the first case the binding process is solely entropically driven(DH21 being nearly zero), while in the second case the binding en-thalpy is favorable too.

4.3.2. Native b-CD vs. HP-b-CDFor the cyclodextrins with seven glucopyranoside units, it is

remarkable that while DH11 is negative in the case of the nativemolecule, it becomes positive (and increasing with MS) for theHP-modified CDs. Moreover, K11 decreases when going fromMS = (0 to 1.19). This fact points to a competition between thesubstituted groups and the surfactant molecule for the cavity ofthe cyclodextrin, whose effect would be enhanced for growingMS. In fact, it has been shown by molecular dynamics simulationthat the 2-hydroxypropyl groups placed at the O6 positions movetowards the HP-b-CD cavity [23]. As regards 1:2 species, it isnoticeable that they apparently exist with HP-b-CD but not withb-CD, for which K12 = 0. This would make no sense if the 1:2 com-plex was visualized as a CD cavity threaded by two alkyl chains.However, the observed difference becomes reasonable when theformation of non-inclusion complexes is considered. Moleculardynamics simulations suggest that 1:1/1 complexes involving na-tive b-CDs could consist of the interaction of an inclusion 1:1 com-plex with another surfactant molecule lying on the widest entranceof the cavity [13,15]. This kind of interaction would be favoured inthe modified CD, since the 2-hydroxypropyl groups placed at theO2 and O3 positions adopt a more spread-out configuration ascompared to that of the corresponding hydroxyl groups in the na-tive b-CD, making the secondary O cavity entrance still wider [23].The value of DH21 is significantly more negative for HP-b-CD thanfor the native b-CD. (This behavior is opposite to that observed forthe cyclodextrins with six glucopyranoside units.) Concurrently,the corresponding equilibrium constants take similar values for0 6MS < 1 and much lower for MS = 1.19, meaning that the enthal-pic decrease on going from b-CD2OGP1 to HP-b-CD2OGP1 is practi-cally counterbalanced by an important entropic decrease of thesystem upon formation of this 2:1 complex. Further studies are re-quired to provide more structural information.

4.3.3. Native c-CD vs. HP-c-CDThe substitution of H atoms by HP groups in c-cyclodextrins has

serious consequences in the thermodynamic parameters corre-

sponding to the formation of CD:OGP complexes. The relationK11� 4K12 and a value of 1.9 for the Hill slope at half saturation[26] indicate that the formation of 1:2 species based on the nativecyclodextrin displays a marked positive cooperativity [25]. Thecavity formed by eight glucopyranoside rings is large enough tohost two surfactant molecules and the introduction of the firstmolecule facilitates the capture of the second one. For the modifiedCD the formation of the complexes with 1:2 stoichiometry can alsobe considered a positively cooperative process but to a much lesserextent, as suggested by the important decrease of the Hill slope athalf saturation, from 1.9 to 1.1. As a consequence, K11 is signifi-cantly higher for HP-c-CD than for c-CD: (40 vs. 7) M�1. As in Sec-tion 4.3.2, we propose that the differences between bothcyclodextrins are due to the competition between the OGP mole-cules and the HP groups for the cavities. In the present case thecompetition would appear once the first-order complex is formedand its effects would be enhanced by the larger flexibility of c-CD as compared to b-CD. As explained in Section 4.2, the value ofK21 for this system could not be estimated since the simultaneousconsideration of the three species resulted in a too large number offitting parameters that could not be statistically justified. This wasdue, at least in part, to the weak calorimetric signal produced bythis system (see figure 2). The same problem was observed in thepast for mixtures of the native c-CD with OGP, whose thermody-namic characterization had been performed by using surface ten-sion measurements because the calorimetric signal was too lowto be properly analyzed [15]. Consequently, no enthalpies were re-ported in that earlier work for mixtures of the native c-CD withOGP although, given the very small calorimetric signal obtainedfor such systems, they are expected to be low.

5. Conclusions and perspectives

The thermodynamic parameters corresponding to the forma-tion of CD:OGP complexes with (2-hydroxypropyl)-a-, -b-, and -c-cyclodextrins are significantly different from those obtainedwith the native CDs. The effect of the perturbation induced bythe substitution of H atoms by 2-hydroxypropyl groups is differentfor each of the cyclodextrins. While for a-CD there is an entropi-cally driven increase of K11, for b-CD K11 decreases as MS increases,probably due to the competition between the surfactant moleculesand the 2-hydroxypropyl groups for the cyclodextrin cavity. Thisexplanation would also account for the dramatic decrease of thehigh positive cooperativity associated to the formation of c-CD1-

OGP2 complexes. The formation of non-inclusion second-ordercomplexes detected in the past for native b-CD seems to be rein-forced by the presence of the HP groups.

The precise study of these systems is difficult due to several fac-tors, including the distribution of molecular substitutions and struc-tures in the employed cyclodextrins and the coupling betweendifferent processes in the solution, but the methodology employedin this work allows providing reasonable thermodynamic parame-ters for the main supra-molecular aggregates that are present inthe target systems. More specifically, the decoupling of the demicel-lization and complex formation processes by subtracting the Qm val-ues is key to perform the binding analysis. The uncertaintiesintroduced by this approach are counterbalanced, at least in part,by the simultaneous fitting of several data series obtained for thesame systems by using different CD concentrations. For the future,a better alternative would be to implement a thermodynamic modelfor the demicellization of the surfactant and to propose a globalmodel that considers both processes together. We are currentlyworking in this kind of models and implementations in our codefor the fitting of binding experiments. This code is expected to bemade publicly available in the near future. The study of this and sim-

S. Concha-Santos et al. / J. Chem. Thermodynamics 67 (2013) 112–119 119

ilar systems as a function of temperature is expected to be also extre-mely interesting since the chemical species present in the solution,as well as the corresponding thermodynamic parameters, haveproved to exhibit important differences by changing this variable[22]. Last, we are already working in the topologic parameterizationof 2-hydroxypropyl and other modified cyclodextrins in order tomake available detailed structural, dynamic, and energeticinformation at atomic level of this kind of systems by moleculardynamics simulation. The combination of different methodologieswill improve the fundamental knowledge of CDs and CD-basedcomplexes.

Acknowledgements

This work was supported by grants from the MINECO-Spain(MAT2011-25501), CONACyT-Mexico (49811), and PAPIIT-UNAM(IN105107 and IN 118511).

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JCT 13-438