Journal of Colloid and Interface Science 354 (2011) 517–527

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Journal of Colloid and Interface Science

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A comprehensive study of the spontaneous formation of nanoassemblies in waterby a ‘‘lock-and-key’’ interaction between two associative polymers

Mohammad Othman a, Kawthar Bouchemal a, Patrick Couvreur a, Didier Desmaële b, Estelle Morvan b,Thierry Pouget c, Ruxandra Gref a,⇑a Univ. Paris-Sud XI, Faculté de Pharmacie, UMR CNRS 8612, 92296 Châtenay-Malabry Cedex, Franceb Univ. Paris-Sud XI, Faculté de Pharmacie, UMR CNRS 8076, BioCIS, 92296 Châtenay-Malabry Cedex, Francec LVMH Recherche Parfums et Cosmétiques, Département Innovation Matériaux et Technologies, 185 Avenue de Verdun, F-45804 Saint Jean de Braye, France

a r t i c l e i n f o

Article history:Received 31 August 2010Accepted 9 November 2010Available online 4 December 2010

Keywords:Hydrophobized dextranPoly beta cyclodextrinIsothermal titration microcalorimetryNMRAssociative polymerNanoassemblies

0021-9797/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.jcis.2010.11.015

Abbreviations: CAC, critical association concentraGibbs free energy; DH, enthalpy changes; DS, entropyscattering; FFEM, freeze fracture electron microscopy amicrocalorimetry; K, the constants of association;lauroyl side chains; mM, millimole; NMR, nuclear maometry of the interactions; NAs, nanoassemblies; Pbdextrin; PDI, polydispersity index; SD, standard devsulphate.⇑ Corresponding author. Address: Université Paris

UMR CNRS 8612, tour D5, 2ème étage, 5 rue JB ClémeFrance. Fax: +33 1 46 83 59 46.

E-mail address: ruxandra.gref@u-psud.fr (R. Gref).

a b s t r a c t

Nanoassemblies (NAs) with sizes ranging from 60 to 160 nm were spontaneously formed in water aftermixing a host polymer (polymerized cyclodextrin (pb-CD)) and a guest polymer (dextran grafted withlauroyl side chains (MD)). The combination of microscopy, dynamic light scattering (DLS), nuclear mag-netic resonance (1H NMR), isothermal titration calorimetry (ITC) and molecular modelling was used toinvestigate the parameters which govern the association between MD and pb-CD. Remarkably, whenpb-CD was progressively added to a solution of MD, NAs with a well-defined diameter were spontane-ously formed and their diameter was constant whatever the composition of the system. According toNMR data, almost all the alkyl chains of MD were included into CDs’ cavities of the polymer when themolar ratio lauroyl chain (C12)/CD was P1. The hydrophobic interaction between C12 and the hydropho-bic cavities of CDs appears as the main driving force for NAs’ formation, with a minor contribution arisingfrom van der Waals’ interactions. The inclusion of C12 into b-CD cavities is almost a completely enthalpy-driven process, whereas the MD-C12/pb-CD interaction was found to be an entropy-driven process. Majorconclusions which can be drawn from these studies are that the interactions between the two polymersare restricted neither by the MD substitution yield, nor by the micellization of MD. The simultaneouseffects of several CD linked together in pb-CD and of many alkyl chains grafted on dextran were necessaryto generate these stable NAs.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

Cyclodextrins (CDs) are cyclic oligosaccharides composed of(1 ? 4)-linked a-D-glucosyl units, among which the most com-monly investigated are the a-, b- and c-CDs, consisting of 6, 7 and8 glucosyl units, respectively [1]. CDs can be described as hollow,truncated cones with a hydrophilic surface and a hydrophobic pock-et, which form inclusion complexes with a variety of hydrophobic

ll rights reserved.

tion; CDs, cyclodextrins; DG,changes; DLS, dynamic lightfter; ITC, isothermal titrationMD, modified dextran withgnetic resonance; N, stoichi--CD, polymer of beta cyclo-iation; SDS, sodium dodecyl

Sud, Faculté de Pharmacie,nt, 92296 Châtenay Malabry,

molecules of interest, phenomenon known as host–guest interaction[2]. The inclusion of a guest drug can improve apparent solubility,physical and chemical stabilities, dissolution and bioavailability[3], thus making CDs very attractive drug carriers.

Hydrophobized polysaccharides, such dextran [4,5] or pullulan[6,7] are also particularly attractive in the biomedical field, dueto their biocompatibility and low toxicity, which are advantageousfor biological and pharmaceutical applications. For example, cho-lesteryl-bearing pullulans were proposed as potential drug carriersas they can self-assemble in water under the form of aggregatesable to solubilise small hydrophobic drugs or proteins [8,9].

The association of CDs and modified polysaccharides in drugdelivery devices, combining the advantageous properties of both ofthese materials, is of great interest. Huh et al. described systems inwhich CDs formed inclusion complexes with poly(ethylene glycol)grafted dextrans [10]. Supramolecular gel-like networks were ob-tained by mixing a CD-bearing host polysaccharides and ahydrophobically modified guest polymer [11,12]. More recently,studies were carried out in our group to design for the first time sta-ble dispersions of 200 nm-sized nanoassemblies (NAs), combining

518 M. Othman et al. / Journal of Colloid and Interface Science 354 (2011) 517–527

the properties of both polysaccharides and CDs [13]. We showed thatthese nanoassemblies spontaneously formed by mixing two aque-ous solutions of soluble polymers: a hydrophobically modified dex-tran obtained by grafting alkyl moieties onto the polysaccharidebackbone (MD) and a b-cyclodextrin polymer (pb-CD). The stabilityof the nanoassemblies was ensured by a ‘‘lock-and-key’’ mechanism:the CDs in the pb-CD polymer formed inclusion complexes with thependant alkyl side chains on MD (Fig. 1). Many CDs in the NAs re-mained empty and could be used to include compounds of interest,such as drugs (tamoxifen) [14], sunscreen agents (benzophenone)[14–16] or contrast agents (complex of gadolinium functionalizedwith adamantane) [17,18].

These results clearly established the potential of these NAs forthe entrapment of various molecules of interest, but also raisedsome issues that still need to be addressed. Indeed, the mechanismof formation of the loaded NAs has not been yet fully elucidated.There is clearly a competition between drugs and alkyl chains forthe CDs. However, surprisingly, loading molecules of interest inhigh amounts did not destabilize the NAs by displacing the alkylside chains of MD from the b-CD cavities [16,17].

The present study aims at gaining information on the mecha-nism of formation of the MD/pb-CD nanoassemblies and their sta-bility as a function of the MD substitution degree. The startingpoint to get answers is the knowledge of the thermodynamicparameters, the stoichiometry of the interactions (N) in the systemand the constants of association (K). The Gibbs free energy (DG) be-tween the CDs in pb-CD and the alkyl chains bound to MD can bederived from enthalpy (DH) and the entropy (DS) of the interaction[19,20]. In this study, isothermal titration microcalorimetry (ITC)was used as a powerful tool enabling to assess all these parametersduring the NAs’ formation. This technique has been used in con-junction with proton nuclear magnetic resonance (1H NMR),microscopic investigations, dynamic light scattering (DLS) andmolecular modelling. The interactions of MD with various alkylgrafting densities (substitution degrees from 2.7% to 7%) witheither CDs free in solution (monomer of b-CD) or pb-CD werestudied.

2. Materials and methods

2.1. Materials

b-CD (Cavamax� W7 Pharma was purchased from Wacker FineChemicals, Burghausen, Germany). Pyrene, lauroyl chloride,

++

MDP -CD

Nanoassembly

Fig. 1. Schematic representation of a complex (nanoassembly) from two associativepolymers: MD (which could be in molecular or micellar form) and pb-CD.

pyridine, and 4-(dimethylamino) pyridine (DMAP) were purchasedfrom Sigma–Aldrich (Saint Quentin Fallavier, France) and wereused as received without further purification. Lithium chloride(Acros organics, Belgium) and dextran (average molecular weight40.000 g/mol, Amersham – Sweden) were dried overnight undervacuum at 80 �C. Anhydrous grade N,N-Dimethylformamide(DMF) was from Aldrich Chemicals. The other solvents were ofanalytical grade. MilliQ� water (Millipore France) was used in allexperiments.

The polymer pb-CD was prepared as previously described, bypolycondensation of b-CD with epichlorhydrin under strong alka-line conditions [21]. The b-CD content in the polymer, as deter-mined by 1H NMR spectroscopy, was 70% w/w. The molecularweight of pb-CD was around 1.5 � 106 g/mol, as determined bygel permeation chromatography.

Dextran grafted with alkyl side chains was synthesized byadapting a previously published method [13]. Briefly, 4.0 g of dex-tran were solubilised in 100 mL of DMF containing 1.0 g of lithiumchloride. Then, 0.5 g of DMAP, 30 mg of pyridine, and variousamounts of lauroyl chloride (0.162, 0.286, 0.366, and 0.405 grespectively) were added to the dextran solution. The reactionwas carried out at 80 �C for 3 h. The obtained MD was isolatedby precipitation in isopropyl alcohol. It was further solubilised indeionised water, purified by dialysis for 48 h using membraneswith a cut-off of 6000–8000 g/mol (Spectra/Por�) and finallyfreeze-dried. The substitution degrees of MD, defined as the per-centage of glucose units substituted with alkyl chains, were deter-mined according to the 1H NMR spectra in DMSO-d6. They wererespectively equal to 2.7%, 5%, 6.5%, and 7% for the samples namedMD2.7, MD5, MD6.5 and MD7 corresponding to theoretical molarmasses ranging from 41,000 to 43,000 g/mole. With the assump-tion that the substitution was homogeneous on the dextran back-bone, one can calculate the average molar mass of one MDrepeating units (6182, 3422, 2674, and 2496 g/mol respectively).

The molar concentrations refer to the MD or pb-CD contents oflauroyl chains (C12) or b-CDs, respectively.

The residual amount of water in each product (b-CD, pb-CD andMD) used for the ITC experiments was accurately determined bythermogravimetric analysis. It was found 14 wt.% for b-CD, 11 wt.%for pb-CD, and about 9 wt.% for MD with the different ratios of sub-stitution. This residual amount of water was taken into account toaccurately determine the concentrations of each product.

2.2. Methods

2.2.1. Critical association concentration (CAC) of MD by using pyreneas a fluorescent probe

Samples for spectroscopic analysis were prepared as follows: apyrene-saturated solution in MilliQ� water was obtained by stirringovernight a suspension of pyrene in water, followed by filtration toremove excess of undissolved pyrene microcrystals. MD stocksolution (5–10 g/L) was prepared in pyrene-saturated water. Itwas left to equilibrate under agitation over 24 h protected fromlight. Subsequently, the stock solution was diluted with pyrene-saturated water to obtain solutions of varying concentrations(1 � 10�3–7.5 g/L), which were further equilibrated under agitationfor 24 h. An estimation of the CAC value was obtained by monitoringthe changes in the ratio of the pyrene excitation spectra intensities atk = 333 nm (I333) for pyrene in water and k = 336 nm (I336) for pyrenein the hydrophobic medium within the micelle core [22]. Excitationspectra were measured at emission wavelength kem = 390 nm. TheCAC values were determined at the intercept of the tangent of thecurve at the inflection point and of the tangent of the curve at highpolymer concentration [18]. Fluorescence spectra were measuredat 23 �C with a SPEX Fluorolog FL1T11 fluorimeter controlled bycomputer (Spex Industries, Edison, USA).

M. Othman et al. / Journal of Colloid and Interface Science 354 (2011) 517–527 519

2.2.2. Morphology of the nanoassembliesNanoassemblies were visualised by freeze fracture electron

microscopy (FFEM). For FFEM observations, 10 mg/mL water solu-tions of pb-CD and MD5 were mixed at room temperature to formNAs suspensions. Gels were prepared as previously described [14]by mixing pb-CD and MD solutions at concentrations of 50 mg/mL.Immediately after mixing, the gel phase sedimented and was easilyrecovered with a spatula.

Nanoassemblies’ suspensions and gels were further incubatedwith glycerol (30% v/v), used as a cryoprotectant. A drop of eachsample (50 lL) was then placed on a copper support and immedi-ately frozen in liquid propane, cooled with liquid nitrogen, andthen kept in liquid nitrogen. Fracturing and shadowing were per-formed in a Balzers BAF 400 freeze-etching unit. The replicas werewashed in THF and distilled water and placed on copper grids.Observations were made under a transmission electron microscopePhilips CM120 operating at 120 kV.

2.2.3. Yield of nanoassemblies’ formationNAs were obtained by injecting under stirring a solution of

pb-CD (10 mM with respect to the CDs in the polymer) to a solu-tion of MD (1 mM with respect to the alkyl chains) according tothe protocol 1 in ITC (see below). The molar ratios of pb-CD/MDat the end were 2CD/1C12. The freshly prepared nanoassemblies’suspensions were centrifuged at 20 �C, 147,000 g for 60 min.Supernatants were collected in glass vials, frozen at �20 �C in aconventional freezer, and freeze-dried for 48 h, in order to deter-mine the weight of the polymers which did not participate tonanoassemblies formation. The formation yields were calculatedfrom the mass ratio of the polymers forming nanoassemblies andthe polymers initially introduced in the preparation procedures.

2.2.4. Nanoassemblies’ preparation and size measurement to mimicITC experiments

Nanoassemblies were obtained at room temperature by twodifferent methods:

(i) Successive injections of 10 lL of 10 mM pb-CD solution inMilliQ� water, into 1.41 mL of MD solutions at concentra-tions between 0.05 and 1 mM.

(ii) Successive injections of a MD solution (5 mM C12) into1.41 mL of 1 mM pb-CD solution. The mean particle size andpolydispersity index were determined at 25 �C, by dynamiclight scattering (DLS) (Zetasizer Nano ZS, Malvern Instru-ments Ltd., UK). The selected angle was 90� and time intervalsof 10 min between two injections were respected. Both uni-modal and size distribution processor analysis were per-formed. All experiments were performed in triplicate.Turbidity measurements of the NAs suspensions wereassessed spectrophotometrically at 500 nm using a ShimadzuUV-2101/PC UV/VIS in the condition previously described.

2.2.5. 1H NMR studiesThe 1H NMR spectra were performed on an Avance 400 NMR

spectrometer from Bruker operating at 400 MHz and 300 K. Thepolymers were solubilised in deuterium oxide (D2O 99.9% D Sigma)and 256 scans were collected, with a delay between two scans of15 s. Five-hundreds microliters of each sample (the polymers so-lely, or a mixture of different molar ratios pb-CD/MD) were intro-duced into a 5 mm probe. The spectra were recorded with a flipangle of 90� and a spectral width of 8250 Hz. A solution of 3-tri-methylsilyl 2,20,3,30-tetradeuteropropionic acid (TSP-d4) (Euriso-top, Saint-Aubin, France) has been used, providing an externalfield-frequency lock and reference for proton chemical shifts(d = 0.00 ppm). Calibration was performed using the residual sig-nals of the solvent as an internal field-frequency lock for 1H

NMR. Chemical shifts were referenced to the solvent values(d = 4.79 ppm for HOD) while taking into account temperatureeffects.

2.2.6. Isothermal titration microcalorimetry experimentsITC (MicroCal Inc., USA) has been used for determining the asso-

ciation constant and the enthalpy of the interaction between theguest (C12 of MD) and the CDs (mono or poly b-CD) from a singletitration curve. The ITC instrument was periodically calibratedeither electrically using an internal electric heater, or chemicallyby measuring the dilution enthalpy of methanol in water [18,20].This standard reaction was in excellent agreement (1–2%) withMicroCal constructor data.

Two protocols were achieved to study the mechanism of theinteraction of MD with b-CD and pb-CD.

2.2.6.1. Protocol 1. Aliquots of 10 lL of titrant consisting of b-CD(10 mM) or pb-CD solutions (concentration of b-CD cavi-ties = 10 mM) placed in a syringe were used to titrate the guestsolutions of MD (concentrations of alkyl chains = 0.1, 0.2, 0.3, 0.6or 1 mM) placed in the calorimetric cell. In this case, the substitu-tion degrees of MD were varied (2.7%, 5%, 6.5% and 7%). A back-ground titration, consisting in injecting the same cyclodextrinsolution in solely MilliQ� water placed in the sample cell, was sub-tracted from each experimental titration to account for dilution ef-fects. Concentrations of MD lower than the critical associationconcentration (CAC) were used in order to estimate clearly thethermodynamic parameters. Solutions of pb-CD 2 or 5 mM concen-tration were used to titrate the diluted solutions of MD.

2.2.6.2. Protocol 2. Aliquots of 10 lL of titrant consisting of MD(concentration of alkyl chains = 5 or 10 mM) placed in a syringewere used to titrate a pb-CD solution (concentration of b-CD cavi-ties = 1 mM) placed in the calorimetric cell. A background titration,consisting in injecting the same MD solution in solely MilliQ�

water placed in the sample cell, was subtracted from each experi-mental titration to account for dilution effects.

For both protocols, solutions were degassed before each titra-tion. All polymer solutions were prepared one day before the dif-ferent experiments to get equilibrated samples. The titrantsolution was delivered over 25 s and the corresponding heat flowwas recorded. Intervals between injections were 600 s to allowcomplete equilibration and agitation speed was 394 rpm. Temper-ature was fixed at 298.15 K (25 �C).

The data were collected automatically and subsequently ana-lyzed with either a one-site or two site binding model by the Win-dows-based Origin software package supplied by Microcal. A heatflow was recorded as a function of time, which corresponds to anyprocesses able to produce or consume energy following each injec-tion. The concentration of the titrant and the sample were used tofit the heat flow per injection to an equilibrium binding equation,providing best fit values of the stoichiometry N, the binding con-stant K and the change in enthalpy DH [15,19,20,23]. From theseresults, the free energy DG and the entropy DS were deductedaccording:

DG ¼ �RT ln K ¼ DH � TDS ð1Þ

where R is the gas constant (8.314 J k�1 mol�1) and T is the absolutetemperature of the interaction in Kelvin.

2.2.7. Molecular modellingMolecular modelling was used for a better understanding of the

interaction of alkyl chains on MD with b-CD. The b-CD structurewas taken from Martin Chaplin web page from London South BankUniversity [24]. The structures of the different molecules havebeen drawn (ChemDraw Ultra 6.0, CambridgeSoft) and presented

520 M. Othman et al. / Journal of Colloid and Interface Science 354 (2011) 517–527

to the b-CD molecule. Further, the dreiding force field was mini-mized with the software DS ViewerPro 6.0 (Accelrys SoftwareInc.) leading to the more likely supramolecular assembly. Bumpmonitorization, minimization of the dreiding force field [25] andmolecular rendering (solvent accessible surface) were alsoachieved with DS Viewer Pro 6.0 in the case of these optimizedstructures [18,20].

3. Results

3.1. MD CAC determination

The CAC of the amphiphilic MD was estimated by fluorescencespectroscopy using pyrene, a hydrophobic fluorescence probe thatpreferentially partitions into the hydrophobic core of the micelle.Two methods were described in the literature for the determina-tion of the CAC of polymeric micelles using pyrene as fluorescentprobe [22]. The first method [26], takes advantage of the changesin the vibronic fine structure of the pyrene emission and monitorsthe changes in the ratio of the intensities I1 and I3 of the [0, 0] and[0, 2] bands, respectively. In the second method [27], which hasbeen suggested to be more accurate, determination of the CACcan be obtained by monitoring the changes in the ratio of the pyr-ene excitation spectra intensities at I333 nm for pyrene in water andI336 nm for pyrene in a hydrophobic medium. Therefore, the lattermethod was used here. The excitation spectrum undergoes a smallshift to longer wavelengths as the probe passes from a hydrophilicto a hydrophobic environment [27]. This shift is quantified in termsof the ratio, I336/I333, of the fluorescence intensities at 336 and333 nm. The intensities I336/I333 ratios of pyrene fluorescence wereplotted against the logarithm of MD concentration in aqueoussolutions (Fig. 2).

Sigmoidal curves were obtained for all substitution yields ofMD. The CAC values of the MD in water are presented in Table 1.

0,001 0,01 0,1 1 10C g/L

CAC

0,6

0,7

0,8

0,9

1,0

1,1

1,2

1,3

1,4

I 336

/I33

3

Fig. 2. Experimental determination of CAC of MD: ratio of pyrene fluorescenceintensities I336 nm/I333 nm as a function of logarithm of MD concentrations(1 � 10�3–7.5 g/L) for � MD2.7, j MD5, N MD6.5, and s MD7.

Table 1Determination of the nanoassemblies’ formation yields and CAC of MD withformation was obtained by means of ultracentrifugation then freeze-dnanoassemblies and the polymers initially introduced in the fabrication pr

Polymer Grafted C12 (Mol%) Average number of C12

per dextran chainNAs d(±SD)

MD2.7 2.7 6.7 63 (±5MD5 5 12.4 119 (±MD6.5 6.5 16.1 161 (±MD7 7 17.3 96 (±8

* Mean ± SD (n = 3).

When the substitution degree of MD was increased from 2.7% to7%, the CAC was shifted to lower values, from 0.4 to 0.11 mM,respectively (Table 1). For example CAC values of 2.5 and 0.7 g/Lwere obtained for MD2.7 and MD5, respectively. These values arein agreement with the previously reported ones, i.e. 2.2, 1, and0.7 g/L for MD2.9, MD4.2, and MD4.8, respectively [28].

3.2. Nanoassemblies’ morphology

Fig. 3A shows a typical picture of the cross section of theMD/pb-CD colloidal systems, showing spherically shaped nanopar-ticles composed of tiny spheres. The high molecular weightcross-linked pb-CD alone appeared as tiny spherical particles ofaround (20 nm) (Fig. 3B), No specific feature could be observed inthe MD solutions, whatever their concentration up to 10 g/L (resultsnot shown).

When highly concentrated solutions of MD and pb-CD (50 mg/mL) were mixed, a viscous homogeneous gel phase was obtainedinstead of nanoassemblies (Fig. 3C).

3.3. Nanoassemblies’ formation by directly mixing MD and Pb-CD

Nanoassemblies’ formation was also studied in identical exper-imental conditions like those employed in ITC and NMR experi-ments (Table 1, Fig. 4).

However, at the lowest MD substitution degrees (2.7%) polydis-persed nanoassemblies (PDI P0.3) with a mean diameter around63 nm were formed (Fig. 4). In this case, the yield of NAs’ formationwas lower (67 wt.%). In contrast, for the other substitution yields ofMD (5%, 6.5% and 7%) monodispersed nanoassemblies with poly-dispersity index (PDI 60.1) were formed, as revealed by DLS(Fig. 4). The obtained NAs diameters (63–161 nm) were higherthan those of the MD micelles (around 20 nm) and of the pb-CDmacromolecules (around 30 nm) as determined by the same tech-nique (DLS).

It was found that MD associated spontaneously with pb-CD toform NAs, whatever the substitution degree of MD and at concen-trations greater than the CAC (Table 1). Well formed NAs were ob-tained only for substitution degrees P5. The typical Tyndall effectwas observed in these cases, and satisfying yields (75–81%) wereobtained. The analysis by 1H NMR of the supernatant afterfreeze-drying (results not shown) confirmed the presence of bothassociative polymers.

3.4. Nanoassemblies’ formation by progressive mixing of MD andpb-CD

In the aim to understand at which pb-CD/MD ratio the nanoas-semblies are obtained, the ITC experiments were mimicked by pro-gressively injecting pb-CD into MD solution or the contrary. Themean diameter was measured after each injection and typical re-sults are presented in Fig 5. NAs were instantaneously formed in

different substitution degrees (2.7%–7%). The yield of nanoassemblies’rying of supernatants, from the mass ratio of polymers formingocedure.

iameternm

PDI Yield of NAsformation (%)

CAC

[MD] (g/L) [MD] (mM)

) 0.3 67 ± 4* 2.5 0.47) 0.1 75 ± 3 0.7 0.29) 0.1 79 ± 5 0.4 0.15) 0.1 81 ± 4 0.27 0.11

Fig. 3. Transmission Electron Microscopy images after freeze-fracture of: (A) pb-CD/MD5 nanoassemblies, at a concentration of 10 g/L, the arrows indicate toindividual particles were observed which could be pb-CD; (B) pb-CD aqueoussolution at a concentration of 10 g/L and (C) the gel obtained by mixing pb-CD andMD5 at concentrations of 50 g/L.

Size (d.nm)

Inte

nsit

y (%

)

P -CD/ MD

P -CD/ MD

Fig. 4. Size distributions by intensity of nanoassemblies made by mixing solutionsof pb-CD (10 mM) and MD2.7 or MD5. In the first case polydispersed (PDI = 0.3)nanoassemblies were formed compared to the second case (PDI = 0.06). Thevolumes and the concentrations were the same as at the end of ITC titration(protocol 1 (section ITC materials and methods)).

Fig. 5. Determination of the nanoassemblies’ mean diameter when the ITCexperiments were mimicked by: (A) injection of 5 mM solutions of MD2.7 (squarej) or MD7 (triangle N) in (1.41 mL) of 1 mM solution of pb-CD. (B) injection ofpb-CD 10 mM into (1.41 mL) of 1 mM of MD2.7 (square j) or MD7 (triangle N). Themean diameters of nanoassemblies were expressed as a function of the, injectedconcentration of MD or pb-CD respectively.

M. Othman et al. / Journal of Colloid and Interface Science 354 (2011) 517–527 521

the two cases, as soon as the aqueous solutions of the twopolymers were mixed together, either MD/pb-CD or pb-CD/MD.When the MD solution was injected into the pb-CD one, the meanNAs diameter increased progressively as a function of MD concen-tration (Fig. 5A).

This result was reproduced, whatever the substitution degree ofMD (results not shown). Remarkably, the successive injections ofpb-CD solution into the MD led to the formation of NAs with thesame size whatever the amount of pb-CD or the substitution de-gree of MD. For example, 63 nm and 96 nm-sized NAs were formedwith MD2.7 and MD7, respectively, immediately after the first injec-tions (Fig. 5B). The following introduction of pb-CD into the systemdid not change the NAs’ mean diameter, but only the count rate in-creased and the laser attenuation decreased as shown by DLS.Moreover, the turbidity of the NA suspensions, studied by monitor-ing the suspension absorbance at 500 nm, continuously increased,which is also an indication of the increase of the number of NAs.

Thus, when adding progressively the MD solution into thepb-CD one, NAs were immediately formed and their size increasedprogressively until reaching a characteristic mean diameter (dY)which appeared to be a function of the MD substitution degree.When adding progressively the pb-CD solution into the MD one,NAs were also immediately formed, and they instantaneouslyreached the dY.

3.4.1. 1H NMR studiesThe interaction between the MD alkyl chains and the CD cavi-

ties in pb-CD was studied by 1H NMR spectroscopy. Since the inter-action takes place by inclusion of the alkyl chains in CDs cavities,the analysis of the spectra was limited to the region comprised be-tween 2.6 and 0.7 ppm which characterizes the MD lauroyl moie-ties. More particularly, a special attention was given to the A andB peaks (Fig. 6), which correspond to the methyl end group (CH3)and the adjacent methylene (CH2) protons, respectively. Fig. 6shows the region from 1.8 to 0.7 ppm of the spectra of pb-CD(Fig. 6I), MD7 (Fig. 6II), and different pb-CD/ MD7 mixtures withmolar ratios CD/C12 from 0.4 to 10 (Fig. 6 (III–VI)).

The pb-CD polymer does not show any peak in this region (Fig. 6I).In the case of the uncomplexed MD (Fig. 6II), the A (0.93 ppm) and B(1.33 ppm) signals presented a narrow band-width at half intensityand were symmetric. However, for the molar ratios CD/C12 lowerthan one (0.4, Fig. 6III) the signals A and B were only broader thanin the case of MD, but they were not downfield shifted. In the caseof NAs formed with molar ratios CD/C12 higher than or equal toone, the band-width at half intensity of both A and B signals wasbroadened (Fig. 6 (IV–VI), and these peaks were downfield shifted(d = 0.97 ppm, and d = 1.37 ppm, respectively).

Fig. 6. Region of the 1H NMR spectra corresponding to alkyl chains of MD: study ofthe associative polymers alone: I (Pb-CD), II (MD7), and of the nanoassembliesprepared at different molar ratios of CD in pb-CD/C12 in MD7: III (NAs (0.4/1)), IV(NAs (1/1)), V (NAs (2/1)), VI (NAs (10/1)).

522 M. Othman et al. / Journal of Colloid and Interface Science 354 (2011) 517–527

The same observations were drawn whatever the substitutiondegrees of MD, in conditions mimicking the ITC experiments(280 lL of a 10 mM pb-CD solution in D2O was injected in1.41 mL of a 1 mM MD solution). The molar ratio CD/C12 was equalto two, and practically all the C12 chains were in the bound (com-plexed) form, as shown by the enlargement and downfield shift ofthe peaks A and B (results not shown).

3.5. Characterization of the interaction of MD with pb-CD by using ITC

A typical ITC experiment consisted in the injection of the pb-CDsolution (10 mM) contained in the syringe into a MD7 solution(1 mM) contained in the titration cell (see protocol 1 Section 2).During pb-CD addition, the two materials interacted and the exo-thermic heat released was measured (Fig. 7A). This heat was di-rectly proportional to the amount of pb-CD added within the MDsolution. As the population of interacting molecules in the cell be-came more and more saturated with pb-CD, the heat signal pro-gressively diminished during titration (Fig. 7A).

After integrating the heat as a function of the molar ratio betweenpb-CD and MD, it was possible to fit the integration curve to a stan-dard single site model, making possible to determine: K = 6500 M�1,N = 0.7 and the thermodynamic parameters of the interactionDH = �6.5 kJ M�1; TDS = 15.3 kJ M�1 and DG = �21.75 kJ M�1. Thena second series of ITC experiments were performed by injectingthis time the MD solution into the pb-CD solution contained inthe titration cell (see protocol 2 Section 2). From the results inFig. 7D, association constants were found in the same order ofmagnitude for pb-CD/MD7 and for MD7/pb-CD interactions (6500and 6100 M�1 respectively) (Fig. 7D).

However, as we can see from Table 2, the association constantsfor the interaction MD/pb-CD (6500 6 K6 12,000 M�1) were muchhigher than for the interaction MD/b-CD (1600 6 K6 6700 M�1), inthe same conditions of temperature, MD substitution degree andconcentration (Table 2). Indeed, these constants were 1.3–7.6times higher, depending on the substitution degree of MD.Noteworthy, our data are in agreement with previously published

results in the case of MD (substitution yield 4.2%) interacting withb-CD. The association constants of MD4.2/b-CD interaction deter-mined by ITC were in the range of 1950 M�1 [28].

Concerning the stoichiometry of the interaction between MDand b-CD, it was greater than 1 as shown in Table 2. However, inthe case of MD/pb-CD interaction, N was less than that with nativeb-CD.

These results were obtained with concentrations of MD of 1 mMabove the CAC. In the ITC experiments, for each MD substitutiondegree, we have progressively lowered the MD concentration from1 mM until we have reached the sensitivity limits of the method.Therefore, it was possible to investigate the effect of the aggrega-tion state of MD (presence of micelles and/or free chains in solu-tion) upon the parameters determined by ITC. As shown from theresults in Table 3, the association constant of MD/pb-CD interac-tion progressively increased when the substitution degree andthe concentration of MD were lowered.

Particularly, when MD was in the form of unimers, the interac-tion was reflected by gigantic association constant (K was equal to3.7 � 104 and 6.1 � 104 M�1 for MD2.7 and MD6.5 respectively)(Table 3) and (Fig. 8A).

3.6. Molecular modelling

To have a deeper understanding of the system, the interactionsof C12 free chains with b-CD have been further investigated. Fig. 9presents the molecular modelling results, showing that two CDscan interact with one sodium dodecyl sulphate (SDS) chain.

4. Discussion

It has been shown that in aqueous media MD and pb-CD asso-ciate whatever their concentrations to form supramolecular NAs[13,28]. The aim of the present study was to investigate the mech-anism of association of MD and pb-CD and to identify the keyparameters which led to the formation of stable NAs. For this,NAs were formed in various conditions, using a range of MD substi-tution degrees. Association constants of MD and pb-CD were deter-mined, as well as the thermodynamic parameters related to thisassociation.

MD is an amphiphilic polymer which associates in water toform ‘‘polymeric micelles’’ [29], consisting of a hydrophobic core(essentially alkyl chains) stabilized by a corona of hydrophilic dex-tran exposed to the aqueous environment. Depending on its con-centration and the substitution degree of MD, before mixing withpb-CD, MD could be under the form of micelles or/and free chainsin water (Fig. 1). Therefore, as a first approach, the polymer con-centration at which the association takes place, known as the crit-ical association concentration CAC, was determined for eachsynthesized MD polymer. As expected, the CAC of MD was foundto be dependent on its substitution degree (Table 1). The CAC val-ues decreased with increasing molecular content of lauroyl moie-ties linked to the polymer backbone, reflecting the increase inhydrophobicity of the polymer.

NAs of 63–161 nm were spontaneously formed by mixingpb-CD with MD, according to the substitution degree of MD (Table1). The morphology, formation yield, size and inclusion of alkylchains into the CDs have been studied.

The morphology of the NAs was investigated by FFEM, which isa technique of choice enabling to observe the surface as well as thecross-sections of colloidal systems, without the need to dry them.The high molecular weight pb-CD polymer could be observed astiny spherical particles of around 20 nm, whereas no specific fea-ture could be detected in the MD solutions (Fig. 3B). When assem-bled with MD, the spherically-shaped pb-CD polymer seems to be

Fig. 7. Typical ITC data corresponding to the binding interaction during the titration of MD7 (1 mM) with pb-CD (10 mM) (A and B) and the titration of pb-CD (1 mM) with MD(10 mM) (C and D) at 25 �C. Left panels show exothermic heat flows which are released upon successive injection of 10 lL aliquots of pb-CD into MD (A) and MD into pb-CD(C). The right panels show integrated heat data, giving a differential binding curve which was fitted to a standard single-site binding model yielding the following parametersN, K, and DH.

M. Othman et al. / Journal of Colloid and Interface Science 354 (2011) 517–527 523

‘‘concentrated’’ in the NAs’ core, with practically no/or little freepb-CD in the dispersion medium (Fig. 3A). In the gels obtained athigh polymer concentrations (Fig. 3C), the pb-CD polymer appearsto be highly concentrated into a globally homogeneous structure.These observations suggest that MD chains physically cross-linkthe pb-CD polymer under the form of gels or disperse NAs with agel-like (nanosponge) structure. This was consistent with our pre-viously reported data [17] indicating that the NAs have high watercontents of about 70 wt.%.

The NAs’ formation yield increased with the MD substitutiondegree (Table 1). It appears that the more substituted was theMD polymer, the more efficient was the formation of the NAs(Fig. 3A), since the simultaneous effect of many alkyl chains on

MD with several CDs’ cavities in pb-CD is physically efficient. How-ever, it should be noted that MD with substitution degrees >10% isnot soluble and cannot be used for NAs’ preparation. Therefore,there is an optimal substitution degree range (between 4% and7%) for NAs’ formation.

NAs formed also immediately when the pb-CD was progres-sively mixed with the MD, instead of a rapid mixing. Remarkably,whatever the substitution degree of MD and the ratio pb-CD/MD,the size of the NAs stays stable after the first few injections(Fig. 5B). A possible explanation is that the pb-CD macromoleculesin the injected concentrated solution were immediately physicallycross-linked by MD chains present in the receiver phase and thisresulted in regular well defined NAs.

Table 2Stoichiometries (N), association constants (K), and thermodynamic parameters of host–guest interactions. 10 mM solution of either b-CD or pb-CD was injected into a 1 mMsolution of MD (different substitution degrees). The temperature was fixed at 298 K (25 �C).

Host Guest Ka (M�1) N DHa (kJ M�1) TDS b (kJ M�1) DGb (kJ M�1)

b-CD MD2.7 6700 ± 200 1.2 �16.4 ± 0.4 5.4 �21.8MD5 2500 ± 300 1.3 �16.4 ± 0.3 3.0 �19.4MD6.5 1500 ± 200 1.4 �14.6 ± 0.7 3.6 �18.2MD7 1600 ± 250 1.0 �14.0 ± 0.6 4.4 �18.4

pb-CD MD2.7 9000 ± 500 1.1 �5.4 ± 0.5 17.2 �22.6MD5 12,000 ± 2500 0.95 �5.5 ± 0.2 17.8 �23.3MD6.5 11,460 ± 3200 0.9 �5.5 ± 0.9 17.7 �23.2MD7 6500 ± 400 0.7 �6.5 ± 0.8 15.25 �21.75

a Mean ± SD (n = 3).b DG ¼ �RT ln K ¼ DH � TDS.

Table 3Complex association constant (K) and thermodynamic parameters corresponding to inclusion complex of MD2.7 (I), MD5 (II), MD6.5 (III) and MD7 (IV) with pb-CD (10 mM) at 298 K(25 �C). The concentration of MD was changed in the aim to obtain thermodynamic parameters when the MD concentration was above and below the CAC.

[MD] mM K (M�1) N DH (kJ M�1) TDS (kJ M�1) DG (kJ M�1)

I. Effect of MD2.7 concentration on the thermodynamic parameters based on a 1:1 complexation of MD-C12 chains with pb-CDC > CAC 1 9000 ± 900 1.1 �5.4 ± 0.5 17.2 �22.6

0.6 12,900 ± 1400 1 �5.0 ± 0.4 18.5 �23.5C < CAC 0.3 25,000 ± 2100 1 �4.9 ± 0.6 20.2 �25.1

0.2 37,500 ± 3700 1 �4.0 ± 0.9 22.1 �26.1

II. Effect of MD5 concentration on the thermodynamic parameters based on a 1:1 complexation of MD-C12 chains with pb-CDC > CAC 1 12,000 ± 2500 0.95 �5.5 ± 0.2 17.8 �23.3

0.6 14,300 ± 1100 0.95 �5.7 ± 0.3 18.0 �23.70.3 22,100 ± 1900 0.94 �6.1 ± 1.1 18.7 �24.8

C < CAC 0.2 45,100 ± 3300 1 �6.3 ± 0.2 20.3 �26.6

III. Effect of MD6.5 concentration on the thermodynamic parameters based on a 1:1 complexation of MD-C12 chains with pb-CDC > CAC 1 11,460 ± 3200 0.9 �5.5 ± 0.9 17.7 �23.2

0.6 16,250 ± 1900 0.9 �5.5 ± 0.4 18.5 �24.00.3 32,750 ± 3500 0.9 �5.1 ± 0.1 20.6 �25.7

C < CAC 0.1 61,050 ± 5400 1 �4.1 ± 0.1 23.2 �27.3

IV. Effect of MD7 concentration on the thermodynamic parameters based on a 1:1 complexation of MD-C12 chains with pb-CDC > CAC 1 6500 ± 2400 0.7 �6.5 ± 0.8 15.3 �21.8

0.6 10,500 ± 360 0.7 �6.0 ± 0.2 16.9 �22.90.3 22,650 ± 590 0.7 �5.6 ± 0.2 19.3 �24.9

Fig. 8. (A) Effect of MD concentration and SD on association constants. (B) Enthalpy–entropy compensation plot corresponding to inclusion complex formation of pb-CD(10 mM) with MD (1 mM) with different substitution degrees ((j) DM2.7, (d) DM5, (M) DM6.5 and (s) DM7).

524 M. Othman et al. / Journal of Colloid and Interface Science 354 (2011) 517–527

NMR studies were performed to determine the proportion ofthe alkyl chains included inside the CDs in all the NAs. The NMRchemical shift method is widely employed to confirm the inclusionof a guest molecule into a CD cavity and to assess the configurationof the complex formed [13,14,30]. The broadening of the peaks Aand B according to Fig. 6 indicates that two species of alkyl chains,

bound and unbound, coexist in exchange [14,31,32]. The adjacentCH2 protons included into b-CD cavity were not surrounded bythe same atoms, and became consequently unequivalent, leadingto the appearance of an asymmetric B signal compared to that ob-tained for a single MD solution. It was estimated that alkyl chainswith a maximum of five or six carbon atoms could accommodate in

Fig. 9. Optimized structures and the most stable supramolecular assemblies ofsodium dodecyl sulphate SDS and b-CD yielded when using DS ViewerPro 6.0software. Atoms were represented in different colours: carbon (grey); oxygen (red);sulphur (yellow), hydrogen (white) and sodium (blue–violet). Lateral view on theleft and vertical view on the right. (For interpretation of the references to colour inthis figure legend, the reader is referred to the web version of this article.)

M. Othman et al. / Journal of Colloid and Interface Science 354 (2011) 517–527 525

the a-CD cavity [33–35]. Therefore, one would expect that, for ali-phatic guests with a number of carbon atoms P7, at least onemethylene group is forced to stay outside the hydrophobic cavityand will not be able to form effective contacts with the CD’s innerwall, as the length of the guest C12 is significantly longer than theformal depth (7.9 Å) [2] of CD’s geometrical cavity. This could ex-plain the broadening of (CH2) peak.

Moreover, deconvolution studies enabled to demonstrate thatat molar ratios CD/C12 greater than or equal to one, practically allthe MD alkyl chains were interacting with the CDs [13].

However, for the molar ratios CD/C12 lower than one (0.4) thesignals A and B were only broader than that in the case of MD,but were not downfield shifted, reflecting the existence of free al-kyl chains. To summarize these observations, when CD/C12 P1,both CH3 and CH2 groups of C12 in MD interacted with the CD cav-ities of pb-CD, leading to NAs formation by a ‘‘lock-and-key’’ mech-anism. The multivalent character of the interactions between thetwo polymers should ensure the stability of the NAs.

All these studies have shown that by a zip-fastening effect, pb-CD was able to de-micellize MD to form the NAs. For this reason,ITC has been used in complement to previous experiments to gaininformation on the force and thermodynamics of the interactionsbetween pb-CD and MD. ITC is considered as one of the most mod-ern and sensitive method available for the investigation of theinteraction of dilute species in solutions [2,19,36,37]. Advantagesof this methodology are that turbidity and colour of the solutionor suspension have no influence on the measurement and thatthe thermodynamic parameters can be measured without immobi-lisation, modification or labelling.

It has been previously shown that b-CD could accommodate upto 12 hydrogen-bonded water molecules within its so-calledhydrophobic core [38]. These water molecules might be displacedupon guest inclusion. Thus, taking into account the initially in-cluded or interacting water molecules, the 1:1 complexation inter-action of MD C12 chains with b-CD cavity may be written asfollows:

MD-C12 � gH2Oþ CD � hH2O $ MD-C12 � CD � ðg þ h� iÞH2O

þ iH2O ð2Þ

where g represents the number of water molecules interacting withthe free guest C12 of MD, h the number of tightly bound hydrationwater molecules inside the cyclodextrines’ cavity, and i the net dis-placement of water upon complexation [2]. The binding constantfor a 1:1 complexation of the cyclodextrin with the guest moleculeis expressed by:

K ¼ ½MD� C12 � CD�½MD� C12�½CD� ð3Þ

where [CD], [MD-C12] and [MD-C12�CD] are the concentrationsof the cyclodextrin, lauroyl of MD, and the inclusion complexrespectively.

However, we demonstrated from the results in Table 2 that theinteraction between MD and the polymer (pb-CD) was much stron-ger than the one between MD and the monomer (b-CD) (Table 2).This is in agreement with previously published data [28] in thecase of MD with a substitution degree = 4.2% interacting withb-CD. It seems reasonable to attribute these results to the simulta-neous effect of the cross-linked CDs in the pb-CD polymer.

Table 2 shows that the association constants, determined in con-ditions of NAs formation ‘‘MD concentration 1 mM’’, are higher forMD5 than other MD substitution degrees. This result is consistentwith the observed optimal stability of the NAs in this optimal narrowset of experimental conditions [13]. Indeed, for the lowest substitu-tion degree (2.7%) the alkyl chains are not dense enough to ensuresufficient physical cross-links between MD and pb-CD, in order tostabilize the supramolecular nanoassemblies. On the other hand,for the highest substitution degree (7%) the predominance of highamounts of micelles or aggregates might perturb the NAs formation.

Noteworthy, working with ITC requires special attention on theexperimental conditions in order to attribute unambiguously theobserved heat effects to the phenomena to be studied. In our case,contrastingly to pb-CD which did not show any surface activity[13], MD is an amphiphilic macromolecule which can self-associ-ate in water (see CAC section). Therefore, ITC experiments wereconducted at MD concentrations higher and at MD concentrationslower than the CAC (Table 3, Fig. 8A). Values of K very high as6.1 � 104 M�1 were obtained for the interaction between MD6.5

(<CAC) and pb-CD (Table 3). These constants are among the highestreported in the literature [39–46]. Examples of high K (103–106 M�1) mainly concern the interaction between adamantlyderivatives and b-CDs [40–43,45]. In contrast, the interaction be-tween alkyl chains (decanoic acid) and the native b-CD was lessstronger (K = 10.9 � 103 M�1) [43].

The remarkably high K observed here might be explained by aproximity effect between the interacting species, C12 and CDs. In-deed, as soon as one complex is formed, the remaining alkyl chainson MD become in proximity with other free CDs cages in pb-CDand the probability to form new inclusion complexes increases. Itis probably sufficient for a few alkyl chains to be captured by thepb-CD cavities to produce a chain reaction leading to the strongassociation of the two associative polymers, by a ‘‘gear-wheel’’-or ‘‘zip-fastening’’ process. This high affinity between MD andpb-CD could explain the strong interactions between these poly-mers resulting in an excellent NAs stability.

The variations of K with the substitution degree of MD were notsignificantly different. The association of MD in solution, micellesor free macromolecules, appears as the key factor governingK. Possibly, the MD under the form of micelles have a lower affinityfor the pb-CD cavities, since the hydrophobic parts (C12) areembedded inside the MD micelles. Presumably, the interactionsbetween the CDs and the MD chains in the form of unimers(c < CAC) do not suffer from these effects. Moreover, steric encum-brance effects of pb-CD might additionally occur impeding theinclusive interactions.

The calculated CD/C12 stoichiometries in the case of b-CD/MDinteraction were higher than one (Table 2). Probably, some C12 sidechains might form inclusion complexes with more than one b-CDunit, which indicates the coexistence of two stoichiometries 1:1CD/C12, and 2:1 CD/C12 as we confirmed by molecular modelling(Fig. 9). This is in agreement with reported data in the literaturefor the dodecyl chain inclusion in CDs [47,48].

526 M. Othman et al. / Journal of Colloid and Interface Science 354 (2011) 517–527

The calculated CD/C12 stoichiometries for the pb-CD/MD inter-action were lower than that with b-CD ones. This might be attrib-uted to the additive contributions of: (i) the micelles orhydrophobic microdomains resulting from interactions inter andintra MD molecules; (ii) the steric hindrance in pb-CD, renderingdifficult for the C12 to reach the embedded CDs inside the pb-CDand (iii) the size of the CD cavities in pb-CD which are large enoughto accommodate more than one alkyl chain at the same time. Thepresence of micelles or MD aggregates in solution was previouslyshown by CAC measurements. The steric hindrance effects seemobvious in the structure of the pb-CD polymer, showing tiny parti-cles (Fig. 2B).

From a thermodynamic point of view, complexation thermody-namic parameters have been shown to reflect the nature of thenon-covalent interactions occurring between the guest and cyclo-dextrin molecules [30]. The interaction is dominated by van derWaals’ forces when the process is enthalpy-driven with minorfavourable or unfavourable entropies of interaction (|DH|>|TDS|).However, hydrophobic interactions between two apolar moleculesat room temperature have been known as entropy-driven pro-cesses, where the entropy of interaction is large and positive whilethe enthalpy of the process is small (|DH|<|TDS|) [49]. For bothpb-CD/MD7 (10/1 mM) and MD7/pb-CD (10/1 mM) complexes(Fig 7), the interactions were exclusively exothermic DH <0 witha positive entropic contribution DS >0 and mostly entropy drivenð DHj j < TDSj jÞ. Furthermore, the formation processes of b-CD/MDand pb-CD/MD complexes were spontaneous in both cases asevidenced by negative values of the Gibbs free energy DG (Fig. 7and Table 2).

These thermodynamic behaviours were found to be similarwhatever the concentration of MD and its substitution degree(Table 3). The overall thermodynamic parameters were compara-ble whatever the substitution degree of MD (Tables 2 and 3). Theinteraction of MD with pb-CD is mainly governed by the entropychanges (Table 3), in agreement with the entropy increase. Theexpansion of the CD ring size, as it is the case when the naturalCDs are chemically modified, increases the entropy, these trendsare consistent with greater ring flexibility and a larger number ofassociated water molecules in and around the cavity of the CD asthe ring size increases [2]. Thus, large positive entropy changesusually arise from the significantly important translational andconformational freedoms of host and guest upon complexation[50,51]. Comparable negative and small enthalpies and positiveentropies, in water, were reported in the case of propranolol com-plexation by natural c-CD [52]. The positive entropies were attrib-uted to the large internal diameter of c-CD, leading to a looseadaptation of the naphthyl group of propranolol to the CD cavity.Thus, it could be suggested that the interactions of lauroyl chainswith pb-CD are predominantly mediated by hydrophobic interac-tions as explained previously. This does not exclude the possibili-ties of H bond formation. Indeed, it has been previously shownby molecular modelling that MD established a stable network ofhydrogen bonds with pb-CD due to the proximity between thetwo polysaccharide polymers [13].

To summarize, from a thermodynamic point of view, weobserved that:

(i) Both MD/pb-CD and MD/b-CD are exothermic and spontane-ous processes (DH <0 and DG <0). However, the inclusion ofC12 into b-CD cavities is an enthalpy-driven process|DH| > |TDS|, whereas the MD-C12/pb-CD interaction is anentropy-driven process |DH| < |TDS| whatever the substitu-tion degree of MD (Table 2).

(ii) For the same MD substitution degree, the decrease of theconcentration resulted in an increase of DS (Table 3). How-ever, little variation of DH was observed in this case.

(iii) Finally, the enthalpy changes DH were plotted against TDSchanges for all the results, the compensation plot onFig. 8B showed a linear relationship between DH and TDS,showing that in the aggregation process, the changes inDH were compensated by the changes in DS. Briefly, theentropic gain that arises from desolvation upon guest inclu-sion is quite significant.

In the case of the interaction of MD with the native b-CD, thefree energy changes DG was mainly governed by the enthalpychange, as it has been reported in the case of the inclusion of meth-ylene groups into both b- and a-CD cavities, which was an almostcompletely enthalpy-driven process [2].

Furthermore, the interactions of C12 free chains with b-CD havebeen further investigated, taking as a typical example the inclusionof dodecyl chains of SDS into b-CD. Indeed, SDS is known to formboth 1:1 and 2:1 b-CD/SDS complexes [53], according to the equa-tions Eqs. (4) and (5):

b-CDþ SDS $K1 b-CD—SDS ð4Þ

b-CDþ b-CD—SD $K2 ðb-CDÞ2—SDS ð5Þ

Thus, Molecular modelling showed that b-CD cavity size waslarge with respect to the alkyl chains to provide a significant con-tribution due to van der Waals-type interactions (Fig. 9). As a re-sult, the flexibility of the supramolecular complex formed is high,supporting the previously observed data (large gains in entropy)in the case of pb-CD.

5. Conclusions

This study was dedicated to a better comprehension of the nat-ure of the non-covalent interactions between a hydrophobizeddextran and mono- and poly b-CD. ITC was used as a principaltechnique for these investigations, as it allowed determining theassociation constants, as well as the stoichiometry and the thermo-dynamic parameters from a simple titration curve. It has beendemonstrated that the spontaneous formation of nanoassembliesafter mixing of MD with pb-CD resulted from the formation ofinclusion complexes with entropy-driven process, whereas theinclusion of C12 of MD into b-CD cavities was almost a completelyenthalpy-driven process. Possibly, because of chemical modifica-tion of the native cyclodextrin, the polymer of CD has larger hydro-phobic cavities than b-CD. Nanoassemblies formation was notrestricted neither by the hydrophobic dextran substitution de-grees, nor by the micellization of dextran. The simultaneous effectsof several cyclodextrins linked together in poly-b cyclodextrin andof many alkyl chains grafted on dextran were necessary to gener-ate these stable colloids. Thus, the cohesion of the nanoassembliesis presumably due to the establishment of numerous weak interac-tions between alkyl chains on MD and b-CD from pb-CD, acting asphysical cross-links between the chains of the two associativepolymers.

References

[1] J. Szejtli, Chem. Rev. 98 (1998) 1743.[2] M.V. Rekharsky, Y. Inoue, Chem. Rev. 98 (1998) 1875.[3] D. Duchene, F. Glomot, C. Vaution, Cyclodextrins and Their Industrial Uses,

Editions de Santé ed., Editions de Santé, Paris, 1987.[4] C. Rouzes, A. Durand, M. Leonard, E. Dellacherie, J. Colloid Interface Sci. 253

(2002) 217.[5] E. Rotureau, E. Dellacherie, A. Durand, Eur. Polym. J. 42 (2006) 1086.[6] C. Duval-Terrie, J. Huguet, G. Muller, Colloids Surf., A 220 (2003) 105.[7] W. Henni, M. Deyme, M. Stchakovsky, D. LeCerf, L. Picton, V. Rosilio, J. Colloid

Interface Sci. 281 (2005) 316.[8] K. Akiyoshi, S. Deguchi, N. Moriguchi, J. Yamaguchi, J. Sunamoto, Macro-

molecules 26 (1993) 3062.

M. Othman et al. / Journal of Colloid and Interface Science 354 (2011) 517–527 527

[9] S.W. Jung, Y.I. Jeong, S.H. Kim, Int. J. Pharm. 254 (2003) 109.[10] K.M. Huh, T. Ooya, W.K. Lee, S. Sasaki, I.C. Kwon, S.Y. Jeong, N. Yui,

Macromolecules 34 (2001) 8657.[11] C. Amiel, B. Sebille, Adv. Colloid Interface Sci. 79 (1999) 105.[12] R. Auzély-Velty, M. Rinaudo, Macromolecules 35 (2002) 7955.[13] R. Gref, C. Amiel, K. Molinard, S. Daoud-Mahammed, B. Sebille, B. Gillet, J.C.

Beloeil, C. Ringard, V. Rosilio, J. Poupaert, P. Couvreur, J. Control. Release 111(2006) 316.

[14] S. Daoud-Mahammed, C. Ringard-Lefebvre, N. Razzouq, V. Rosilio, B. Gillet, P.Couvreur, C. Amiel, R. Gref, J. Colloid Interface Sci. 307 (2007) 83.

[15] K. Bouchemal, P. Couvreur, S. Daoud-Mahammad, J. Poupaert, R. Gref, J. Therm.Anal. Calorim. 98 (2009) 57.

[16] S. Daoud-Mahammed, P. Couvreur, R. Gref, Int. J. Pharm. 332 (2007) 185.[17] E. Battistini, E. Gianolio, R. Gref, P. Couvreur, S. Fuzerova, M. Othman, S. Aime,

B. Badet, P. Durand, Chemistry 14 (2008) 4551.[18] M. Othman, K. Bouchemal, P. Couvreur, R. Gref, Int. J. Pharm. 379 (2009) 218.[19] K. Bouchemal, Drug Discovery Today 13 (2008) 960.[20] F. Segura-Sanchez, K. Bouchemal, G. Lebas, C. Vauthier, N.S. Santos-Magalhaes,

G. Ponchel, J. Mol. Recognit. 22 (2009) 232.[21] E. Renard, A. Deratani, G. Volet, B. Sebille, Eur. Polym. J. 33 (1997) 49.[22] M.F. Francis, L. Lavoie, F.M. Winnik, J.C. Leroux, Eur. J. Pharm. Biopharm. 56

(2003) 337.[23] S. Daoud-Mahammed, P. Couvreur, K. Bouchemal, M. Cheron, G. Lebas, C.

Amiel, R. Gref, Biomacromolecules (2009).[24] M. Chaplin, London South Bank University, 2008. Available from: <http://

www.lsbu.ac.uk/water/cycloh.html>.[25] S. Mayo, B.D. Olafson, W.A. Goddard, J. Phys. Chem. 94 (1990) 8897.[26] K. Kalyanasundaram, J.K. Thomas, J. Am. Chem. Soc. 99 (1977) 2039.[27] M. Wilhelm, C.L. Zhao, Y. Wang, R. Xu, M.A. Winnik, J.L. Mura, G. Riess, M.D.

Croucher, Macromolecules 24 (1991) 1033.[28] V. Wintgens, S. Daoud-Mahammed, R. Gref, L. Bouteiller, C. Amiel,

Biomacromolecules 9 (2008) 1434.[29] G.S. Kwon, T. Okano, Adv. Drug Delivery Rev. 21 (1996) 107.[30] Y. Inoue, Annu. Rep. NMR Spectrosc. 27 (1993) 59.

[31] A. Harada, Acc. Chem. Res. 34 (2001) 456.[32] A. Harada, H. Adachi, Y. Kawaguchi, M. Kamachi, Macromolecules 30 (1997)

5181.[33] I. Sanemasa, T. Osajima, T. Deguchi, Bull. Chem. Soc. Jpn. 63 (1990) 2814.[34] Y. Matsui, K. Mochida, Bull. Chem. Soc. Jpn. 52 (1979) 2808.[35] S. Augustat, Nahrung/Food 25 (2006) 701.[36] C. Roques, K. Bouchemal, G. Ponchel, Y. Fromes, E. Fattal, J. Control. Release 138

(2009) 71.[37] W.Q. Tong, J.L. Lach, T.F. Chin, J.K. Guillory, J. Pharm. Biomed. Anal. 9 (1991)

1139.[38] G. Crini, C. Cosentino, S. Bertini, A. Naggi, G. Torri, C. Vecchi, L. Janus, M.

Morcellet, Carbohydr. Res. 308 (1998) 37.[39] A. Bom, M. Bradley, K. Cameron, J.K. Clark, J. Van Egmond, H. Feilden, E.J.

MacLean, A.W. Muir, R. Palin, D.C. Rees, M.Q. Zhang, Angew. Chem. Int. Ed.Engl. 41 (2002) 266.

[40] F.P. Charbonnier, S. Penadés, Eur. J. Org. Chem. 2004 (2004) 3650.[41] W.C. Cromwell, K. Bystrom, M.R. Eftink, J. Phys. Chem. 89 (1985) 326.[42] M.R. Eftink, M.L. Andy, K. Bystrom, H.D. Perlmutter, D.S. Kristol, J. Am. Chem.

Soc. 111 (1989) 6765.[43] R. Gelb, L. Schwartz, J. Inclusion Phenom. 7 (1989) 465.[44] Y. Izutani, K. Kanaori, T. Imoto, M. Oda, FEBS J. 272 (2005) 6154.[45] V.H.S. Tellini, A. Jover, L. Galantini, F. Meijide, J.V. Tato, Acta Crystallogr. 60

(2004) 204.[46] B. Zhang, R. Breslow, J. Am. Chem. Soc. 115 (1993) 9353.[47] G. Castruonovo, V. Elia, D. Fessas, F. Velleca, G. Viscardi, Carbohydr. Res. 287

(1996) 127.[48] A.C.S. Lino, Y. Takahata, C. Jaime, J. Mol. Struct. (Theochem) 594 (2002) 207.[49] P.M. Wiggins, Physica A 238 (1997) 113.[50] M. Rekharsky, Y. Inoue, J. Am. Chem. Soc. 122 (2000) 10949.[51] M. Rekharsky, Y. Inoue, J. Am. Chem. Soc. 122 (2000) 4418.[52] G. Castronuovo, M. Niccoli, Bioorg. Med. Chem. 14 (2006) 3883.[53] H. Mwakibete, R. Cristantino, D.M. Bloor, E. Wyn-Jones, J.F. Holzwarth,

Langmuir 11 (1995) 57.