Aqueous Polysaccharide Associations Mediated by Î²-Cyclodextrin

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Biomacromolecules 2008, 9, 1434–1442

Aqueous Polysaccharide Associations Mediated by β-Cyclodextrin Polymers Véronique Wintgens,† Samia Daoud-Mahammed,‡ Ruxandra Gref,‡ Laurent Bouteiller,§ and Catherine Amiel*,† Systèmes Polymères Complexes, ICMPE, UMR 7182, 2 Rue Henry Dunant, 94320 Thiais, France, Faculté de Pharmacie, UMR 8612, Avenue Jean-Baptiste Clément, 92296 Chatenay-Malabry, France, and Laboratoire de Chimie des Polymères, UMR 7610, Université Pierre et Marie Curie 4, place Jussieu, 75252 Paris cedex05 Received November 6, 2007; Revised Manuscript Received February 22, 2008

Macromolecular assemblies were elaborated by mixing in water hydrophobically modified dextrans (MDCn) and β-cyclodextrin polymers (pβCD) interacting by inclusion complexation between the hydrophobic moieties of MDCn and the β-cyclodextrin cavities of pβCD. Dextrans have been modified by grafting alkyl groups (Cn) of varying chain lengths (n ) 8-16) and grafting ratio (3-6 mol%). Different pβCD polymers were synthesized by polycondensation of β-cyclodextrin and epichlorohydrin. The polymer–polymer interactions have been studied by fluorimetry, isothermal titration microcalorimetry, phase diagrams, and viscosimetry. The viscoelastic properties of the temporary networks (in the semidilute range) have been studied by rheology. The interaction mechanisms between the MDCn and pβCD can be understood taking into account the strength of the interaction between the alkyl group and the β-cyclodextrin cavity (mainly controlled by the alkyl chain length), the density of junctions between the chains (depending on the alkyl grafting density and the pβCD molecular weight), and additional cooperative effect (arising for high alkyl grafting density).

Introduction Supramolecular architectures involving polymers constitute an area of current interest and development due to their practical applications in solutions (associative thickeners,1 stimuli responsive gels,2 nanoparticles,3 gene carriers,4 and so on) or at interfaces (biosensing devices,5,6 and so on). The driving interaction mechanisms leading to polymer–polymer associations can result from hydrophobic interactions of amphiphilic copolymers in water,1–3 electrostatic interactions between polymers of opposite charges, responsible of well-defined polyelectrolyte multilayers at interfaces,7 or interactions involving a molecular recognition process such as hydrogen bond interactions in proteins8,9 or inclusion complexes with cyclodextrin (CD) compounds.10–16 In the frame of biological and pharmaceutical applications of these macromolecular assemblies, compounds made of polysaccharides or oligosaccharides showing good biocompatibility, biodegradability, and low toxicity are particularly attractive. For instance, hydrophobized polysaccharides were shown to be potential drug carriers as solubilization and protectionofpoorlywater-solubledrugsweregreatlyimproved.3,17–19 Cyclic oligosaccharides such as CDs constitute also very attractive drug carriers because a wide variety of lipophilic drugs can be included into the CD cavities.20,21 In addition, CD compounds can be used as tools to build supramolecular assemblies in polymer systems. Well-organized structures (polyrotaxans) corresponding to CDs threaded along a polymer chain are obtained when CDs are able to make inclusion complexes with the backbone units of a polymer.22,23 More * To whom correspondence should be addressed. E-mail: amiel@ glvt-cnrs.fr. † Systèmes Polymères Complexes, ICMPE, UMR 7182. ‡ Faculté de Pharmacie, UMR 8612. § Laboratoire de Chimie des Polymères, UMR 7610.

recently, the specific recognition between β-CD and hydrophobic derivatives such as alkyl groups Cn (n being the number of carbons of the alkyl chain) has been used to build threedimensional structures. A guest polymer containing several β-cyclodextrin units (β-cyclodextrin polymer) is mixed in water to a host polymer containing several lipophilic groups like alkyl or adamantyl (amphiphilic copolymer). The resulting complex interactions between the CD cavities and the lipophilic groups constitute the temporary cross-links of a network of connected chains. The structural and dynamic solution properties of the macromolecular assemblies have been studied in several systems.13–15,24–31 In particular, neutral amphiphilic copolymers of different natures (hydrophobically modified dextran, PEO, and so on) and architectures (telechelic or comblike) have been used, and it was shown that these systems are controlled by a main key parameter, related to the strength of the interaction between the host and the guest polymer. When the number of hydrophobic groups per amphiphilic copolymer chain is higher than a critical value, which is close to 3, the polymer–polymer interactions are strong enough to lead to associative phase separation. In the monophasic regions of the ternary phase diagrams, the structure and size of the soluble polymer–polymer complexes may vary as a function of the stoichiometry of the mixtures and of the total concentration. Very promising results related to drug delivery applications have been obtained by mixing hydrophobically modified dextrans bearing dodecyl moieties to β-cyclodextrin polymer. It was shown that these polymers spontaneously associate in solution leading to well-defined dispersion of nanoparticles (200 nm) or to viscoelastic gels depending on the concentration conditions.32–34 Encapsulation of lipophilic drugs such as tamoxifen or benzophenone could be favored by their inclusion complexation with the free CD cavities of the system. However, most of the studies have been performed with a hydrophobically

10.1021/bm800019g CCC: $40.75  2008 American Chemical Society Published on Web 04/05/2008

Aqueous Polysaccharide Associations

Biomacromolecules, Vol. 9, No. 5, 2008

Table 1. Characterization of the pβCD Samples pβCD sample

βCD content g/g

Mw g/mol

PI

pβCD0 pβCD1 pβCD2 pβCD3 pβCD4

0.88 0.59 0.505 0.67 0.70

8.6 × 103 1.59 × 105 2.3 × 105 1.55 × 105 6.9 × 105

1.8 1.9 2.3 1.5

modified dextran bearing 4 mol% of dodecyl groups, and the question that needs to be answered is how the supramolecular architectures are influenced by changes in the parameters of the system such as hydrophobic/hydrophilic molar ratio. The aim of this paper is to study the influence of the alkyl chain length on the association properties of the system. Dextrans modified with alkyl C8 to C16 have been synthesized with different grafting ratios. Phase diagrams, viscosimetry, and rheology give evidence of the polymer–polymer associations in solution. The affinity of the cyclodextrin cavities for the alkyl moieties has been studied in solution by fluorescence measurements and titration microcalorimetry.

Experimental Section 1. Materials. β-Cyclodextrin (βCD), pyrene, octanoyl chloride, decanoyl chloride, lauroyl chloride, palmitoyl chloride, pyridine, and 4-(dimethylamino)pyridine (DMAP) were purchased from SigmaAldrich (BP701, Saint Quentin Fallavier, France) and were used as received. Lithium chloride (Sigma-Aldrich) and dextran (DT40, Mw 40000 g/mol, Amersham, Sweden) were dried overnight under vacuum at 80 °C. N,N-Dimethylformamide (DMF) was anhydrous grade from Aldrich Chemicals, other solvents were analytical grade, and water was deionized quality. The poly-β-cyclodextrin polymer (pβCD) was prepared by polycondensation of β-cyclodextrin with epichlorohydrin under strong alkaline conditions.35 The different polymer samples used in this study have been characterized by 1H NMR in deuterated water and by size exclusion chromatography (columns TSK-gel type SW 4000-3000 (Tosoh Bioscience, Interchim, BP1140, Montluçon, France), refractive index, and laser light scattering detectors). The determined cage contents (g/g), the weight average molecular weight Mw, and polydispersity indexes PI are reported in Table 1 for the five pβCD samples. 2. Synthesis. The different hydrophobically modified dextrans were obtained by modification of a small proportion of the hydroxyl groups with alkanoyl chloride.36 Typically, 1 g of LiCl was dissolved under stirring and argon atmosphere in 100 mL of anhydrous DMF and heated at 80 °C. Then 4 g of DT40 was added and, after dissolving, 0.5 g of DMAP, 30 µL of pyridine, and the necessary amount of alkanoyl chloride (molar ratio varying between 1 and 10%) were added. The mixture was left 3 h at 80 °C and 15 h at room temperature. The polymer was isolated by precipitation into 1 L of 2-propanol and filtered. After dissolving the polymer in the minimum amount of water, it was purified by dialysis against water, and it was freeze-dried. Yields of polymer modification varied between 75 and 90%. The degree of alkyl substitution was obtained by 1H NMR in deuterated dimethyl sulfoxide (DMSO) from the ratio of the integration of the protons of the alkyl chains (0.8–1.8 ppm) and of the integration of the anomeric and hydroxylic protons (4.4–5.0 ppm). The name of the hydrophobically modified dextrans, MDCn-x, contains the length of the hydrophobic group (n ) 8, 10, 12 or 16) and the hydrophobic molar ratio, x (x is the molar percentage of alkyl group per glucose unit). Name, composition, and physical properties are reported in Table 2. The different fluorescence probes, 4-amino-N-n-Pr-phthalimide, 4-amino-N-i-But-phthalimide, and 4-amino-N-tert-butylphthalimide, were synthesized in the laboratory as previously described.37 3. Samples Preparation. All polymer solutions were prepared one day before the different experiments to get equilibrated samples, and

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Table 2. Composition and Physical Properties of the Different Dextrans, Intrinsic Viscosity [η], Huggins Constant k′, and Critical Aggregation Concentration cac

dextrans

% alkyl

alkyl number per chain

DT40 MDC8-2.5 MDC8-8.2 MDC10-5.3 MDC12-2.9 MDC12-3.3 MDC12-4.2 MDC12-4.8 MDC12-6.0 MDC16-1.3 MDC16-4.0

0 2.5 8.2 5.3 2.9 3.3 4.2 4.8 6.0 1.3 4.0

6 20 13 7 8 10 12 15 3 10

[η] in H2O L/g

k′

0.0185

0.33

0.0183 0.0175 0.0146

0.52 0.28 1.29

cac in H2O g/L >200 ∼20 ∼12 2.2 1.0 0.7 0.3

0.0147

1.85 0.3

in the case of viscosity measurements, the solutions were filtered over a Minisart RC15 Sartorius with 0.45 µm porosity. Samples for critical aggregation concentration (cac) determination were prepared in a two-step procedure: at first, 10 µL of a stock solution of pyrene (1.4 × 10-4 M) in dichloromethane was deposited on the bottom of a flask and left to evaporate, and then 2 mL of a polymer solution with the appropriate concentration was added to the flask and shaken for two days before measurements (the final concentration of pyrene in the samples was 7 × 10-7 M). To obtain the associating phase diagrams of pβCD and hydrophobically modified dextrans, stock solutions of each polymer at equal weight concentration and at a fixed water concentration were prepared. Then 1 mL of the mixture of these solutions in different ratios was prepared, stirred, left at 25 °C in a water bath for 48 h, and a simple visual observation was done. When the analysis of the supernatant was carried out, the supernatant was carefully removed of the mixture, weighed to get the percentage of the supernatant’s volume, then freezedried, and again weighed to get the weight of polymers in the supernatant and in the rich polymer phase (the results are at least the average of two experiments). For the association constants determination by fluorimetry, a stock solution of the probe in water (around 5 × 10-5 M) has been prepared and used to make a 10-2 M βCD solution to have the same probe concentration; then the mixture of these two solutions was used to record the fluorescence at different βCD concentrations (with excitation wavelength located at the isosbestic point to avoid any optical density variation). This allowed the determination of Kprobe. For the competition experiments, a stock solution of the probe with appropriate βCD concentration ([βCD] ∼ 1/Kprobe) was prepared and used to make a concentrated solution of the competitor; again, the mixture of these two solutions were used to record the fluorescence at different competitor concentrations. This allowed the determination of K (complexation constant between competitor and βCD). Two probes were used: 4-amino-N-n-Pr-phthalimide with Kprobe ) 480 M-1 and 4-aminoN-i-But-phthalimide with Kprobe ) 1400 M-1. For the association constants determination by microcalorimetry, a concentrated βCD (10-2 M) solution was placed in a 0.295 mL continuously stirred (310 rpm) syringe. The sample cell (1.435 mL) was filled with a solution of hydrophobically modified dextran. In the case of MDC12-4.2, a solution at 10-4 M in C12 groups was used to work at c < cac (c ) 0.38 g/L and cac ∼ 1 g/L). In the case of MDC105.3 and MDC8-2.5, solutions at 10-3 M in alkyl groups were used (at such concentrations, they were both under the cac, 3.1 g/L (cac ) 12 g/L) and 6.5 g/L (cac >200 g/L) for MDC10-5.3 and MDC8-2.5, respectively). A first volume of 2 µL was injected, without taking into account the observed heat, to remove the effect of solute diffusion across the syringe tip during the equilibration period. Subsequent additions of the solution (2–10 µL) were automatically injected into the sample cell every 200 s until the syringe was empty. 4. Instrumentation. The NMR spectra were recorded in deuterated DMSO or in deuterated water with a Bruker 200 or 300 MHz. The

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Figure 1. Fluorescence intensity ratio I1/I3 of pyrene in aqueous solutions of the different dextrans as a function of polymers concentration: (, MDC16-4.0; •, MDC12-4.2; 2, MDC10-5.3; +, MDC8-8.2; and 1, MDC8-2.5; the arrow shows the cac determined for MDC164.0 from the dashed lines.

pyrene fluorescence was recorded at 23 °C with a SLM Aminco 8100 by excitation at 335 nm with slits fixed at 16 and 0.5 nm, respectively, at the excitation and emission. Solution viscosities were determined with an Ubbelohde suspended level viscometer settled in a water bath at 25 °C. Rheological measurements were performed with an AR1000 rheometer from TA Instrument, using cone and plate geometries. All the dynamic rheological data were checked as a function of stress amplitude to ensure that the measurements were done in the linear viscoelastic domain. Isothermal titration microcalorimetry (ITC) was made using a MicroCal VP-ITC microcalorimeter. The experimental data were fitted by a theoretical titration curve using the instrument software.

Results and Discussion 1. Self-Association of the Hydrophobically Modified Dextrans in Water. Fluorescence. Pyrene was used as a probe in fluorescence experiments to show the presence of hydrophobic microdomains in water solutions of the different modified dextrans. The ratio of its first and third emission bands, I1/I3, depends on the polarity and on the hydrophobicity of the domain in which it is solubilized. Therefore, it is largely used to study the formation and the properties of aggregated systems.38,39 The I1/I3 ratio has a value around 1.9 in polar media, such as water, and decreases to 1.2 when pyrene is solubilized in relatively unpolar media, such as sodium dodecyl sulfate core micelles. Figure 1 shows the I1/I3 ratio as a function of concentration for different polymers. The I1/I3 ratio has a sigmoid decrease from a high value (≈1.90) at low polymer concentration to a low value (≈1.2–1.3) at high polymer concentration, which indicates the change of pyrene location from an aqueous polar environment to hydrophobic microdomains. The decrease of the I1/I3 ratio expands over a range of concentrations of around two decades, as reported for aqueous solutions of different polymers.40–42 This behavior was attributed to a less cooperative association of the hydrophobic moieties (as compared to micellization of surfactants) and also to the polydispersity of the polymers (in size and in distribution of the hydrophobic groups). In this work, we defined the cac values as the intercept of the tangent of the curve at the inflection point and of the tangent of the curve at high polymer concentration (this is shown in Figure 1 in the case of MDC16-4.0). In the case of hydrophobically modified polyacrylates, it was shown that small preaggregates were formed, to which pyrene would bind, leading to a decrease of the I1/I3 ratio well before polymeric micelles

Wintgens et al.

were formed and to a decrease of the I1/I3 ratio expanded over a wide range of concentrations.40 On the other hand, the cac of MDC12-4.2, determined using another probe, 4-amino-N-tertbutylphthalimide, which is more soluble in water than pyrene, is in good agreement with the one determined as mentioned before.37 The cac data are reported in Table 2; they depend both on the alkyl length and on the degree of alkyl substitution. As expected, increasing the alkyl length decreases the cac values because the hydrophobicity is enhanced. For instance, the cac values vary by almost 3 orders of magnitude from MDC16-4.0 (cac ) 0.3 g/L) to MDC8-2.5 (cac > 200 g/L). A similar effect of alkyl length has been reported in the case of other polymers40,43 and surfactants.44 Additionally, increasing the substitution ratio decreases the cac values of the studied dextrans for the same alkyl group. For example, MDC12 shows cac varying from 0.3 g/L to 2.2 g/L with the substitution ratio decreasing from 6 to 2.9% (Table 2). Viscosimetry. Intrinsic viscosity measurements are informative regarding the compacity of the polymers. Due to the difficulty to carry out experiments in water with the hydrophobically modified dextrans (bubbles formation in the capillary viscometer), experiments were conducted only with the less lipophilic samples. Values of the intrinsic viscosity were obtained by double extrapolation of the reduced, ηred, and inherent, ηinh, viscosities to zero concentration in water and are reported in Table 2. MDC8-8.2 and MDC10-5.3 have almost the same intrinsic viscosities, around 0.018 L/g, a value similar to that of the precursor polymer DT40 ([η] ) 0.0185 g/L) but MDC122.9 and MDC16-1.3 present a lower intrinsic viscosity, around 0.015 L/g. This lower value can be attributed to more compact coils through the formation of hydrophobic microdomains. In addition, MDC12-2.9 and MDC16-1.3 displayed positive slopes of ηinh compared to the negative slopes of the less lipophilic dextrans and also showed high values of the slopes of ηred (leading to a Huggins constant k′, ηred ) [η] + k′ × [ η]2 × C, reported in Table 2, which is largely higher than 0.5). This indicates a tendency to aggregate in the case of MDC12 and MDC16. 2. Association between the Hydrophobically Modified Dextrans with β-Cyclodextrin Polymers in Water. Associating Phase Diagram. Generally, ternary mixtures of polymer 1-polymer 2-solvent are showing segregative phase properties due to the low entropy of mixing of the polymers. This is also the case for β-cyclodextrin polymers and dextran, although they have very similar polysaccharide backbones. Nevertheless, the two incompatible polymers can be compatibilized by the introduction of the hydrophobic moieties. Due to inclusion complexes between alkyl groups and β-cyclodextrin cavities, additional interactions were experienced by the two polymers, leading either to a one-phase system or to an associative phase separation. An important parameter influencing the mechanism of phase separation is the number of alkyl groups.25,45 When the number of hydrophobic moieties per chain is lower than 3, mixtures of the two polymers give homogeneous one-phase systems. This was reported in the case of dextrans modified by adamantyl groups45 and was also observed in this work in the case of MDC16-1.3, which carries only an average number of three hexadecyl groups per chain. All the other studied dextrans gave associative phase separations. Mixtures prepared in the two-phase domain showed a polymer-rich phase topped by a phase mainly containing water. Volume and polymer weight of the supernatant have been determined in the case of the samples used for the rheological experiments (Table 3). In most samples, the supernatant contained less than 20%



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Table 3. Analysis of the Two-Phase Mixtures: Percentage of the Volume, Percentage of the Polymer Weight in the Supernatant, and Polymer Concentration of the Gel Phase for the Different Polymers’ Mixtures mixtures of polymers MDC12-3.3/pβCD2 MDC12-4.8/pβCD3 MDC12-6.0/pβCD3 MDC12-4.2/pβCD4

MDC12-4.2/pβCD4

polymer weight ratio

total concentration (g/L)

volume of supernatant (%)

90/10 60/40 50/50 50/50 50/50 50/50 50/50 20/80 33/67 50/50 67/33 80/20

150 130 130 33 50 67 75 66.7 66.7 66.7 66.7 66.7

22 52 83 74 60 60 83 69 61 57 59

of the polymers weight, reflecting the strength of the interactions between the polymers.46,47 The viscoelastic properties of the lower phase, the “gel-like” phase, are described in the last part. The extension of the two-phase domain depends on both the substitution ratio and the alkyl length. Increasing the substitution ratio increases the size of the two-phase domain. For instance, MDC12-4.2/pβCD3 showed a closed two-phase domain limited at low water content (≈95% in weight by weight), and for a ratio (weight of pβCD/total polymers weight) varying between

polymer weight in supernatant (%)

one phase 4 9 18 11 12 8 57 28 12 4 11

polymer concentration of gel phase (g/L) 150 160 248 162 171 149 171 165 156 149 148 144

30 and 70%, as shown in Figure 2a. In the case of MDC12, containing 6% of alkyl groups, only a two-phase domain has been observed (Figure 2b) in the studied concentration range (water content up to 87%). Therefore, increasing the number of hydrophobic groups per dextran chain should induce a larger cooperativity of the pβCD and MD chain interactions, explaining the changes in these phase diagrams. On the other hand, increasing the alkyl length also increases the size of the twophase domain. MDC10-5.3 (13 decyl groups per chain) led to

Figure 2. Associative phase diagrams (one phase (-), two-phase (+)) of (a) MDC12-4.2/pβCD3, (b) MDC12-6.0/pβCD3, (c) MDC10-5.3/pβCD1, and (d) MDC16–4.0/pβCD2.

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Wintgens et al.

Table 4. Thermodynamic Parameters for the Association of Different Modified Dextrans Kass (M-1) compound fluorescence method at 23 °C microcalorimetric method at 25 °C

MDC8-2.5 a

830 850b 470

MDC10-5.3 a

MDC12-4.2 c

1220 1180b 1200

c

1950 -1

-9.6 19

∆H (kJ mol ) -16.7 ∆S (J K mol-1) 2

-16.3 4

a

With 4-amino-N-n-Pr-phthalimide. b With 4-amino-N-i-But-phthalimide. c Value not determined (see text).

a closed two-phase domain (Figure 2c), and only two-phase mixtures were observed (Figure 2d) with MDC16-4.0 (10 hexadecyl groups per chain). This is certainly related to an enhancement of the interactions between hexadecyl groups and β-cyclodextrin cavities compared to decyl groups and β-cyclodextrin cavities. The 1/1 association constant between βCD and DoTAB (decyltrimethyl ammonium bromide) is around 15–20 times lower than that between βCD and CTAB (hexadecyltrimethyl ammonium bromide).37,48 Association Constant. To determine the association constants of the modified dextrans with βCD, we used two different techniques, a fluorimetric and a microcalorimetric titration. These determinations cannot be done with βCD polymers due to phase separations of some of the mixtures. The fluorimetric titration is an indirect method based on the competition between a fluorescent probe and a nonfluorescent competitor, both compounds forming association complexes with β-cyclodextrin. The used probe should present a fluorescence intensity, which largely varies with the βCD complexation. In this type of experiment, one usually works at fixed concentrations of βCD (around 1/Kprobe, where Kprobe is the association constant of the probe with βCD) and of the probe (around 5 × 10-5 M). Adding the competitor (in this work, the modified dextran) into the solution of probe/βCD leads to a decrease of the fluorescence intensity of the probe. This decrease is due to the dissociation of the fluorescent (probe/βCD) complex by formation of a complex between the competitor and βCD. These fluorescence variations allow the determination of the competitor association constant, K.37 In the experiments, one assumes that each alkyl group acts independently from each other, as mentioned in the following eqs 1 and 2:

probe + βCD a (probe ⁄ βCD)

Kprobe )

[probe ⁄ βCD] [probe][βCD]

(1) alkyl + βCD a (alkyl ⁄ βCD)

K )

[alkyl ⁄ βCD] [alkyl][βCD]

(2)

The data are reported in Table 4. Measurements have been done with two different probes, 4-amino-N-n-Pr-phthalimide and 4-amino-N-i-But-phthalimide, for which Kprobe is of the order of 480 and 1400 M-1, respectively. The results were reproducible and did not depend on the nature of the probe used. This fluorimetric method cannot be used with MDC12 or MDC16, because these compounds form hydrophobic microdomains at concentrations used in the fluorescence experiments. For example, the range of concentrations needed to determine an association constant of the order of 1500 M-1 is between 0.1 and 1 g/L, too close to the cac value of MDC12. Therefore, the hydrophobic microdomains disturb the fluorescence measure-

Figure 3. Enthalpogram of MDC12-4.2 at 10-4 M of alkyl group as a function of the molar ratio βCD/MDC12-4.2; the solid line represents the best fit of the experimental data (2) for a 1:1 stoichiometry of the complex. In the inset, enthalpogram obtained with initial concentration of MDC12-4.2 (O) fixed at 10-3 M.

ments, the probe being also highly fluorescent in such nonpolar microenvironments. The range of concentrations used in the case of MDC10-5.3 and MDC8-2.5 are 0.2–2 g/L and 0.6–6 g/L, respectively, below the cac values of these MD. The increase of the alkyl length (C8 to C10) leads to an increase of the association constant (from 850 M-1 to 1200 M-1), as expected. However, these values are three to eight times lower than the ones obtained for surfactants.37,48,49 This should be partly related to the large entropy loss when linking a small compound (βCD) to a large MD chain and also to a lower accessibility of the alkyl group bonded to the polymeric chain. Isothermal titration calorimetry (ITC) is a thermodynamic technique that allows studying the interactions of two species. When these two species interact, heat is either generated or absorbed. By measuring these interaction heats, the enthalpy variation ∆H may be determined from the following equation, and the binding constant K and the reaction stoichiometry n are obtained from the fit of enthalpograms:

MD - alkyl + nβCD a (MD-alkyl ⁄ nβCD) [MD-alkyl ⁄ βCD] (3) K ) [MD-alkyl][βCD]n After the successive βCD additions in the MDCn solution, heat flow curves were obtained. The process was exothermic, as expected for a complex formation, and integration of the heat flow peaks yielded the enthalpograms. The ones obtained for MDC12-4.2 are shown in Figure 3 with dodecyl concentration of 10-4 M and 10-3 M in the inset. At 10-3 M, MDC12-4.2 is above its cac, and the enthalpogram presents a special feature after the first βCD additions. The endothermic process, which is superimposed to the exothermic one of the complexation, is certainly the demicellization process because it vanishes with a MDC12-4.2 concentration lower than the cac (10-4 M). Unfortunately, we were not able to fit the experimental curve, taking into account both processes (demicellization and complexation), and therefore, to determine the thermodynamic parameters of the micellization. The best fit obtained for a 1:1 stoichiometry (n ) 1) and, according to eq 3, is also shown in Figure 3 for MDC12-4.2 at 10-4 M. The obtained association constant and the enthalpy variation are reported in Table 4 for three different MDCn. There is good agreement between the association constant values obtained by fluorimetric and microcalorimetry titration. We noticed again the increase of the association constant with the alkyl length and a value ten times lower for MDC12-4.2 than for a surfactant such as SDS or



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Figure 4. Specific viscosities of mixtures of MDC12-2.9 and pβCD0 as a function of the polymers weight ratio (MDCn/pβCD) at a total concentration of 30 g/L.

Figure 5. Specific viscosities of MDC12-2.9 (2) and stoichiometric mixture of MDC12-2.9/pβCD0 (polymers weight ratio, MDCn/pβCD, 80/20) (•) as a function of the MDC12-2.9 concentration.

DTAC.37 This confirms the tendency observed in fluorimetry with the modified dextrans having alkyl lengths lower than 12. The obtained enthalpy variations are in the expected range, in the order of 9.6 to 16.7 kJ/mol. M.V. Rekharsky and Co. suggested from data compilation50 a unit increment of ∆H per methylene. Using the one for 1-alkanol (∆H/dN ) -1.7 kJ/ mol), one obtains –13.4, -17.1, and -20.5 kJ/mol for the enthalpy variation of C8, C10, and C12 alkyl group with βCD, respectively. These values are of the same order as the ones reported in Table 4. In this table are also reported the entropy changes ∆S. The obtained values vary between 2 and 19 J · K · mol-1, depending on the chain length, and they are low compared to the ones calculated from data compilation50 where one obtains 37, 47, and 56 J · K · mol-1 for the entropy variation of C8, C10, and C12 alkyl group with βCD, respectively. As suggested before, this large entropy loss is an important parameter in the decrease of the association constants observed between the MD and the βCD compared to the ones obtained for surfactants.37,48,49 For these studied MD, the free energy change ∆G is mainly governed by the enthalpy change. Viscosimetry. The interactions between the modified dextrans and the β-cyclodextrin polymers may be characterized by measurements of the specific viscosity of aqueous mixtures with different ratio MDCn/pβCD. To avoid associative phase separation, we chose a β-cyclodextrin polymer having a low molecular weight (pβCD0) in all the viscosimetric experiments. Figure 4 reports the example of mixtures of pβCD0 and MDC12-2.9, the total concentration being kept at 30 g/L. The specific viscosity of the mixtures increased with increasing the weight ratio MDCn/pβCD, reached a maximum at 0.25 and then decreased. The viscosity variations reflect change in size and number of the aggregates. When the two polymers are neutrals, as is the case here, the viscosity maximum should correspond to the larger interaction strength between the β-cyclodextrin polymer and the modified dextran that is to the larger number of possible links between the chains. This situation should happen in mixtures of 1:1 stoichiometry, one cavity for one hydrophobic group. The composition of the mixture at the viscosity maximum (6 g/L MDC12-2.9 and 24 g/L pβCD0) extracted from the results of Figure 4 indeed corresponds to the 1:1 stoichiometric ratio, validating the explanation of the viscosity variations. The same experiment done with a dextran having a higher substitution ratio (MDC12-5.0, results not shown) also led to the same behavior with a viscosity maximum corresponding to one cavity for one alkyl group. The viscosity variations as a function of the mixture’s composition thus reflect the inclusion complex interactions between the two polymers, which are involving one

βCD cavity with one alkyl group. Similar viscosity variations with the composition have been described for other associative polymers systems involving cyclodextrin polymers.15,28–30 However, in the case of charged polymers, the viscosity maximum occurs at a 1:1 ratio only when the ionic strength of the medium is high enough to screen the electrostatic interactions. One has to notice that the concentrations of the viscosity experiments are larger than the critical aggregation concentration of the alkyl groups. Addition of pβCD to hydrophobically modified dextran thus induces a dissociation of the hydrophobic microdomains in favor of inclusion complex interactions. To study the influence of the total concentration on the properties of the associative network, we also measured the viscosities of stoichiometric mixtures of MDC12-2.9/pβCD0 and compared them to the viscosities of MDC12-2.9. Figure 5 shows the obtained results. The specific viscosity of MDC12-2.9 alone largely increases with the concentration; this can be explained by the hydrophobic interactions between the alkyl groups belonging to the dextran chains. At low concentration (larger than the cac ) 2.2 g/L), aggregates of finite size and with a higher compacity than the precursor dextran chains are formed, as explained in the previous section. A temporary network is formed when the concentration is increased above the critical overlapping concentration (c* ≈ 1/[η] ) 68 g/L), the alkyl groups being able to link the chains together. Addition of pβCD0 to MDC12-2.9 results in an important increase of the specific viscosity compared to the one of MDC12-2.9. Because the specific viscosity of pβCD0 is very low in the range of the studied concentration (0.234, 0.115, and 0.062 at 60, 30, and 17.6 g/L, respectively), the observed effect is related to the mixture of the polymers. Two different behaviors are observed at low and large concentrations. At low concentration, the intrinsic viscosity of the stoichiometric mixture of MDC12-2.9/ pβCD0 has been determined: [η]mixt ) 0.009 L/g. If the mixture was ideal (that is without interactions between the two polymers), one should expect that its intrinsic viscosity would be identical to the ideal value: [η]ideal ) ω1[η]1 + ω2[η]2, where ω1 and ω2 are the weight proportion of the two polymers, [η]1 and [η]2 being their intrinsic viscosities. The intrinsic viscosity of pβCD0 has not been determined, but the product (ω1[η]1) is equal to 0.011 L/g for MDC12–2.9, already larger than [η]mixt. This proves that the MDC12-2.9/pβCD0 mixtures in aqueous solution are not ideal, and the interactions between the two polymers induce more compact structures. The mixtures show a sharp viscosity increase around 30 g/L, a lower concentration than the overlapping concentration of MDC12-2.9. This means that the inclusion complex interactions between the two

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Wintgens et al.

Figure 6. (a) Frequency dependence of storage and loss moduli, G′ (2) and G′′ (∆), and of dynamic viscosity at 298 K (•) for MDC12-3.3/pβCD2 (polymers weight ratio, MDCn/pβCD, 90/10) at 150 g/L; (b) Dynamic viscosity |η*| for the polymer-rich phase of mixtures at 130 g/L of (1) MDC12-4.8/pβCD3 and (9) MDC12-6.0/pβCD3 (with MDCn/pβCD 60/40 and 50/50, respectively).

polymers are at the origin of a temporary network, which has a higher connectivity than the one produced for MDC12-2.9. Samples with higher concentrations show more marked viscoelastic behaviors and have been studied using a rheometer as will be discussed in the following section. Rheology. The viscoelastic properties of different mixtures MDC12/pβCD have been studied. For most of the studied systems, the mixtures are in the two-phase domain and the rheological experiments were done with the polymer-rich phase. Analysis of these two phases (volume of the supernatant, polymer weight in the supernatant) showed that for most of the studied samples, we could consider that the polymer-rich phase had a final concentration almost constant, around 155 g/L (Table 3). It is interesting to note that increasing the total concentration of the sample at a given ratio 50/50 leads to a decrease of the supernatant’s volume and that more than 80% of the polymers weight are in the polymer-rich phase. Increasing the ratio of MD/pβCD of the sample at 67 g/L also leads to a decrease of supernatant’s volume and for ratios over 50/50, 80% of the polymers weight are again in the polymer-rich phase. Unfortunately, we were not able to determine the polymers’ ratio in the polymer-rich phase. Only the sample DMC12-6.0/pβCD3 leads to a polymer-rich phase with a higher concentration, around 250 g/L. In the case of MDC12-3.3/pβCD2 at 150 g/L in a 90/10 ratio, the mixture gives one homogeneous phase. Figure 6a shows the frequency ω/2π dependence of the storage and loss moduli G′ and G′′ for this mixture. In the low frequency range (0.628 < ω < 31.4 rad · s-1), G′ and G′′ are proportional to ω1.92 and ω0.97, respectively. This is characteristic of a viscoelastic liquid almost responding to a Maxwell model for which G′ and G′′ are theoretically proportional to ω2 and ω1, respectively. The best fit of the data (MDC12-3.3/pβCD2 at 150 g/L in a 90/10 ratio) with a Maxwell model leads to a relaxation time τ equal to 0.0044 s, which corresponds to the lifetime of reversible junctions between both polymers. From Figure 6a, the crossover of G′ and G′′ can be estimated in the range of 30–50 Hz, a range that agrees with the theoretical relaxation time, even if the G′ points are scattered. Similar viscoelastic properties have been observed in other systems involving cyclodextrin polymers.51 The alkyl substitution influences the viscoelastic properties. The dynamic viscosity |η*|0, determined at low frequency in the viscous domain, largely depends on the mixtures (Figure 6b). It could vary by more than 5 times from MDC12-4.8/pβCD3 (|η*|0 ) 42 Pa · s) to MDC12-6.0/pβCD3 (|η*|0 ) 230 Pa · s). Increasing the degree of alkyl substitution (4.8-6.0%) of the MDC12 enhances the interactions between both polymers by

the increase of the number density of junctions per polymer chain ν. Using a simple mechanical model, one can expect a linear relationship between |η*|0 and the product (ν × τ). It appears that τ is approximately constant in all the samples tested (around 0.004 s) and, thus, |η*|0 should simply vary proportionally to ν. The viscosity |η*|0 increases by a factor of 5.5, whereas ν should increase by a factor of 1.9 (ν should vary linearly with the product (degree of alkyl substitution) × (concentration of the polymer rich phase)). The large increase of viscosity observed in this case cannot be solely attributed to the increase of the number density of junctions. It seems that enhanced cooperative effects are at the origin of this phenomenon. Figure 7a reports the variation of G′ and G′′ with the total concentration of the samples MDC12-4.2/pβCD4 in a 50/50 ratio. The rheological experiments are done on the polymerrich phases, which had almost a constant concentration. Therefore, the observed exponential increase of G′ and G′′ is not related to an effect of concentration, but to an effect of the polymer ratio in the polymer-rich phase. Figure 7b reports at one concentration (67 g/L) the effect of the polymer weight ratio MDCn/pβCD observed for the polymer-rich phase. G′ and G′′ increase, go through a maximum, and then decrease. The maximum both for G′ and G′′ correspond to a weight ratio of 40/60 in the mixture, which means a molar ratio of 1/3. Even if the polymers molar ratio is slightly different in the polymerrich phase than in the starting mixtures (1/3), this result is quite different from the one observed in viscosimetry, where the maximum was observed for the 1/1 molar ratio in diluted solution (Figure 4). All the cyclodextrin cavities are certainly not accessible in β-cyclodextrin polymers of high molecular weight (pβCD4), because the polymers are branched and compact; therefore, an excess of cages is necessary to get the maximum of G′ and G′′. Indeed, the MD chains, which are of finite size (Rh ≈ 6 nm), cannot penetrate the inner structure of pβCD4 (Rh ≈ 20 nm) and, thus, cannot reach the CD cavities located inside the core of pβCD4. A flow experiment at fixed shear rate has been realized over a temperature range varying from 278 to 343 K for the polymerrich phase of the mixture MDC12-6.0/pβCD3 (ratio 50/50) at 130 g/L (Figure 8). As expected, the viscosity decreases due to the dissociation of the complexes in the network. From the viscosity values, one can get the activation energy of the system from an Arrhenius relation:

Ln

Ea η(T) ) +C ( ) ηwater T RT

(4)

where η(T) and ηwater(T) are the viscosity of the polymers mixture and water at the temperature T, respectively. The inset



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Figure 7. Storage and loss moduli, G′ (2) and G′′ (∆; determined at a frequency of 1 Hz and at a stress of 1 Pa) for the polymer-rich phase of the mixtures MDC12-4.2/pβCD4 (a) as a function of the total concentration of the mixtures (polymers weight ratio, MDCn/pβCD, 50/50) and (b) as a function of MDCn/pβCD (total concentration of the mixtures, 67 g/L).

Figure 8. Viscosity variation as a function of temperature for the polymer-rich phase of the mixture MDC12-6.0/pβCD3 (polymers weight ratio, MDCn/pβCD, 50/50, at 130 g/L; flow experiment with a shear rate of 0.5 s-1). In the inset: logarithm of the ratio η(T)/ηwater(T) as a function of 1/T (the straight line is the best fit by eq 4).

of Figure 8 shows the plot Ln(η(T)/ηwater(T)) as a function of 1/T, and the slope of the straight line leads to an activation energy Ea of the order of 31 kJ/mol. Values in the same range (18–38 kJ/mol) were obtained for five different mixtures of modified dextran and pβCD. These activation energies due to the complex dissociation can be compared to the enthalpy variation of the complex formation measured by microcalorimetry (Table 4). The obtained values are at least two times higher than the enthalpy variation. This difference can be attributed to the use of a cyclodextrin polymer in the rheological experiments versus the use of βCD in the ITC measurements, which should induce an additional cooperative effect.

Conclusion It has been shown that hydrophobically modified dextrans bearing alkyl groups (MDCn) and β-cyclodextrin polymers (pβCD) can give rise to supramolecular architectures due to the inclusion complex interactions between the alkyl groups Cn and the β-cyclodextrin cavities. Prior to studies of the mixtures, the behavior of MDCns in solution has been analyzed by viscosity and fluorescence measurements. The MDCns selfassociate when the concentration is larger than a cac, which depends on the alkyl length and the grafting ratio. In a second stage, the properties of the aqueous MDCn/pβCD mixtures have

been studied using three kinds of β-cyclodextrin model compounds: a β-cyclodextrin monomer, a β-cyclodextrin oligomer (pβCD0), and β-cyclodextrin polymers of different molecular weights, and varying the hydrophobic/hydrophilic molar ratio of MDCn. β-Cyclodextrin monomer has been used to study the affinities of the alkyl groups of MDCn to the cavities of β-cyclodextrin. Two different and complementary methods have been used: a fluorimetric method based on competitive complexation using a fluorescent probe and a nonspecific method of isothermal titration microcalorimetry. The enthalpies are in the expected range (10–20 kJ/mol) for the inclusion of alkyl groups Cn (n ) 8-12) in β-cyclodextrin cavity. The complexation constants Kass are much lower than the ones usually obtained for small amphiphiles bearing the same alkyl group, probably due to combined entropic and steric effects, as the alkyl groups are linked to a macromolecule. There is an increased affinity between MDCn and β-cyclodextrin as the alkyl length n is increased (a factor 2–3 from n ) 8-12), but the sensitivity is much lower than for small alkyl amphiphiles (a factor of 10 from n ) 8-12). When multivalent β-cyclodextrin compounds instead of the β-cyclodextrin monomer are used to build three-dimensional structures, soluble complexes are obtained for low molecular weight β-cyclodextrin polymer (pβCD0) bearing only a few cyclodextrins per chain. Viscosity measurements as a function of the mixtures composition allowed to prove that the stoichiometry of the complex was one β-cyclodextrin cavity for one alkyl C12 group. Associative phase separations may occur when using larger molecular weights β-cyclodextrin polymers. The extension of the two phases domain was depending both on the alkyl chain length n and the grafting ratio. For instance, at a given grafting ratio, the two phases domain was more extended as n was increased due to increased affinity between the alkyl moieties and the cyclodextrin cavities. Conversely, at a given n value, the two phases domain was increasingly extended as the grafting ratio was increased due to an enhancement of the cooperative effects. At large enough concentrations, temporary networks of connected chains through inclusion complex interactions are formed. Their viscoelastic properties have been studied through rheological measurements. The strength of the temporary network was highly sensitive to the grafting ratio and, therefore, to the density of links. It also depended on the polymer weight ratio as can be expected but was optimum for

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a MDCn/pβCD composition different from the stoichiometry one alkyl for one CD. This was attributed to a lack of accessibility of some of the cyclodextrins in the large molecular weights cyclodextrin polymers. In summary, the associating properties of the aqueous mixtures MDCn/pβCD are extremely sensitive to the amphiphilic character of MDCn. Increasing the alkyl chain length induces modification of the associating properties due to the higher affinities between the connecting chains of the mixtures. Rather similar changes in the properties of the mixtures are observed when increasing the grafting ratio, but enhanced cooperative effects may occur in this case. The properties of the macromolecular assemblies can thus be easily tuned, varying the polymer parameters linked to the hydrophobic/hydrophilic balance in view of biomedical applications such as drug delivery.

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