Associative Network Based on Cyclodextrin Polymer: A Model System

Oct 9, 2009 - Associative networks have been elaborated by mixing in aqueous media a cyclodextrin polymer to a dextran bearing adamantyl groups...
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Biomacromolecules 2009, 10, 3283–3289

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Associative Network Based on Cyclodextrin Polymer: A Model System for Drug Delivery Anne-Magali Layre, Gise`le Volet, Ve´ronique Wintgens, and Catherine Amiel* Syste`mes Polyme`res Complexes, ICMPE, UMR 7182, 2 rue Henri Dunant, 94320 Thiais, France Received July 31, 2009; Revised Manuscript Received September 23, 2009

Associative networks have been elaborated by mixing in aqueous media a cyclodextrin polymer to a dextran bearing adamantyl groups. The two polymers interact mainly via inclusion complexes between adamantyl groups and cyclodextrin cavities, as evidenced by the high complexation constants determined by isothermal titration microcalorimetry (∼104 L mol-1). Additional interaction mechanisms participating in the strength of the network, mainly hydrogen bonding and electrostatic interactions, are sensitive to the pH and ionic strength of the medium, as shown by pH-dependent rheological properties. The loading and release of an apolar model drug, benzophenone, has been studied at two pH values and different cyclodextrin polymer content. Slow releases have been obtained (10-12 days) with slower kinetics at pH 2 than at pH 7. Analysis of the experiments at pH 7 shows that drug release is controlled both by diffusion in the network and by inclusion complex interactions with cyclodextrin cavities.

Introduction Associative polymers have attracted increasing interest in the field of biotechnology due to their ability to build supramolecular assemblies in aqueous media.1,2 Moreover, the structures can be easily tuned to respond to external stimuli such as pH, ionic strength, wavelength, or temperature.2–4 Associative polymers are mostly made of hydrophilic units and contain a minority of hydrophobic moieties that are associating into hydrophobic microdomains. These systems are particularly attractive for drug delivery applications because apolar drugs can be solubilized into the hydrophobic microdomains and the hydrophilic surrounding can help to control the drug release. Compounds made of polysaccharides or oligosaccharides are specially attractive because of their good biocompatibility, biodegradability and low toxicity.5–8 Cyclic oligosaccharides such as cyclodextrins (CD) are also very attractive as drug carriers because a wide variety of lipophilic drugs can be included into the CD cavities.9,10 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.11,12 More recently, the specific recognition between βCD and hydrophobic derivatives such as alkyl groups Cn (n being the number of carbons of the alkyl chain) has been used to build three-dimensional structures. A guest polymer containing several CD units (βCD polymer) is mixed in water to a host polymer containing several lipophilic groups like alkyl or adamantyl groups (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.3,13–23 It has been shown that the properties of the temporary networks are depending on the architectures of the * To whom correspondence should be addressed. E-mail: amiel@ icmpe.cnrs.fr.

two polymers of the mixture (linear or branched), the density of key-locks related to the number of CD unit per CD polymer, the number of hydrophobic group per amphiphilic polymer, and the presence of charge on one or the other polymer. In particular, the use of βCD polymer bearing acid groups help to build assemblies responsive to pH and ionic strength.22 These systems are of great interest for drug delivery applications as they combine the low toxicity of the hydrophilic compounds, the easiness of preparation of the temporary networks that are formed by simple mixing of the two polymers in aqueous media, the specific affinity for apolar drugs, and the ability to control the drug release. Chemical hydrogels containing βCD units in the networks have been also studied by several authors.16,24–26 The drug release was shown to be controlled both by the network swelling and diffusion properties and by the affinity of the CD units for the drugs. In the study presented here, the supramolecular assemblies were obtained by mixing a polyacid bearing pendent βCD units, poly[(methyl vinyl ether)-alt-(maleic acid)]-g-βCD (P(MVEMA)-g-βCD), and a hydrophobically modified dextran (DextAda). The adamantyl groups of Dext-Ada induce associations between the two polymers by making inclusion complexes with the CD units. In a previous work, associating networks made of (P(MVE-MA)-g-βCD) and hydrophobically modified three arm star polyethylene oxide (PEO-Ad3)22 were shown to be strongly pH-dependent. The aim of the present work is to design a system for drug delivery having a higher density of links. Thus, Dext-Ada, with a dextran backbone bearing 10-20 adamantyl units per chain, was used instead of PEO-Ada3. The paper is divided into three parts. In the first part, the attractive inclusion complex interactions between the two polymers, P(MVE-MA)-g-βCD) and Dext-Ada, are evidenced and quantified by titration microcalorimetry. Then the rheological properties of the temporary networks are analyzed as a function of concentration, composition, and pH. In the last part, benzophenone, a hydrophobic compound widely used for sunscreens in the cosmetic field, is used as a model drug. The loading capacities and in vitro releases, studied at two pH conditions

10.1021/bm900866p CCC: $40.75  2009 American Chemical Society Published on Web 10/09/2009

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(pH 2 and pH 7.2), show the ability of the system to control the drug delivery.

Materials and Methods Materials. β-Cyclodextrin (βCD) was a gift from Roquette Company (France). 1-Adamantanecarbonyl chloride, pyridine, 4-(dimethylamino)pyridine (DMAP), poly[(methyl vinyl ether)-alt-(maleic anhydride)] (P(MVE-MA)), lithium hydride (LiH), and benzophenone (Bz) were purchased from Sigma-Aldrich (BP701, Saint Quentin Fallavier, France) and used as received. Poly(ethylene oxide) (PEO) was purchased from Fluka. Lithium chloride (Sigma-Aldrich) and dextran (DT40, Mw 40000 g mol-1, Amersham, Sweden) were dried overnight under vacuum at 80 °C. N,N-Dimethylformamide (DMF) was anhydrous grade from Sigma-Aldrich, other solvents were analytical grade, and water was deionized quality. Synthesis of Polymers. a. Dextran Modified by Adamantyl Group. Dextran (DT40) modified by adamantyl groups (Dext-Ada) was synthesized as previously described by esterification reaction with 1-adamantanecarbonyl chloride.21 Typically, 1 g of LiCl was dissolved under stirring and an 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 0.42 g 1-adamantanecarbonyl chloride was 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 filtration. After dissolving the polymer in the minimum amount of water, it was purified by dialysis against water and then it was freeze-dried. The degree of substitution of adamantyl groups was 4.6% per mole of glucose unit. It was determined by 1H NMR in deuterated dimethyl sulfoxide from the ratio of the integration of the protons of adamantyl groups and of the integration of anomeric (Ha) and hydroxylic protons. 1H NMR (DMSO-d6) of Dext-Ada: δ 4.93 ppm (OH), 4.87 ppm (Ha), 4.67 ppm (OH), 4.53 ppm (OH), 3.8-3.2 (CH), 1.96 ppm (3H, adamantyl), 1.84 ppm (6H, adamantyl), 1.66 ppm (6H, adamantyl). b. P(MVE-MA)-g-βCD. β-Cyclodextrin polymer (P(MVE-MA)-gβCD) was prepared as previously described27 by grafting βCD onto P(MVE-MA), which is an alternated copolymer with a number average molecular weight (Mn) of about 4.8 × 105 g mol-1 and a polydispersity index of 2.6. P(MVE-MA)-g-βCD was prepared by an esterification reaction between βCD and the anhydride bonds as follows: βCD (21.8 g) was dried overnight under vacuum at 110 °C and dissolved in dried DMF (389 mL) under nitrogen. After addition of LiH (153 mg) as a powder, the solution was stirred for 12 h at room temperature. P(MVEMA) (2 g) was dissolved in dried DMF and was added to the CD solution. The mixture was stirred overnight at room temperature. DMF was evaporated under vacuum and the solid obtained was dissolved in water. Unreacted βCDs and low molecular weight products were removed by centrifugation and ultrafiltration in an Amicon cell equipped with a cellulose membrane (molecular weight cutoff of 3 × 104 g mol-1). The ultrafiltration was stopped after addition of a large water excess (10 times the initial volume). P(MVE-MA)-g-βCD was isolated by freeze-drying. A degree of substitution of 84 mol %, corresponding to the grafting of βCD residues per maleic anhydride unit, was determined by FTIR quantitative analysis (Golden Gate ATR, Tensor 27 Bruker instrument).27 Two bands are characteristic of P(MVE-MA)g-βCD: σ 1713 cm-1 (νCdO of acid and ester groups) and σ 1574 cm-1 (νCOO-). The chemical structure of P(MVE-MA)-g-βCD is shown in Figure 1a. The obtained polymer has two kinds of units resulting from the modification of P(MVE-MA) in two steps: first grafting of alkoxide βCD on P(MVE-MA) and second hydrolysis of unreacted anhydride functions. c. Adamantyl End-Capped Poly(ethylene oxide). The poly(ethylene oxide) with a methoxy group at one end and a hydroxyl group at the other end has a number average molecular weight (Mn) of about 5 × 103 g mol-1. Hydrophobically modified PEO (PEO-Ada) was obtained by reaction of the terminal OH group of the polymer with 1-adamantyl isocyanate. The precursor polymer (5 g) was dried by heating under

Figure 1. Chemical structure of (A) P(MVE-MA)-g-βCD and (B) DextAda.

vacuum at 70 °C overnight and dissolved in dried 1,2-dichloroethane (200 mL). Dibutyltin dilaurate (25 µL) and triethylamine (25 µL) were added as catalysts. After introducing 1-adamantyl isocyanate in excess (498 mg), the mixture was heated at 65 °C for 3 h. The solvent was evaporated under vacuum and the obtained product was dissolved in water and stirred overnight to destroy the excess of 1-adamantyl isocyanate. A white precipitate was eliminated by filtration. Activated carbon was added to trap the remaining free adamantyl moieties. After filtration, PEO-Ada polymer was isolated by freeze-drying. The end group modification was close to 100%, as checked by 1H NMR spectroscopy. 1H NMR (DMSO-d6) of the polymer precursor (PEO-OH): δ 4.6 ppm (t, OH), 3.5 ppm (CH2). 1H NMR (DMSO-d6) of PEO-Ada: δ 3.5 ppm (CH2), 2.0 ppm (3H, adamantyl), 1.9 ppm (6H, adamantyl), 1.6 ppm (6H, adamantyl). Preparation of Dext-Ada/P(MVE-MA)-g-βCD Associative Networks. All polymer solutions at different concentrations were prepared one day before the different experiments to get equilibrated samples. The polymer concentration (Cp) was in the range of 40-145 g L-1. Associative networks were obtained by simply mixing, at room temperature, an aqueous solution of Dext-Ada and an aqueous solution of P(MVE-MA)-g-βCD, with different ratios (weight ratio of 25/75, 50/50, or 75/25, approximately corresponding to molar ratio Ada/CD of 1/1, 1/3, or 1/8). At pH 7 and 4, immediately after the two polymer solutions were put in contact, a one-phase viscous solution was obtained. Contrarily, at pH 2, a two-phase mixture was obtained: 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 sample used for the rheological and in vitro benzophenone release experiments, allowing the determination of the polymer concentration in the rich-phase. Supernatant always contained less than 10% of the total polymer weight. Isothermal Titration Microcalorimetry Experiments. Isothermal titration microcalorimetry (ITC) was measured using a MicroCal VPITC microcalorimeter. In each titration, 28 injections of 10 µL of concentrated PEO-Ada or Dext-Ada solutions (10-3 mol L-1 in adamantyl groups) were added from the computer controlled 295 µL microsyringe at an interval of 180 s into the cell (volume ) 1.4569 mL) containing the P(MVE-MA)-g-βCD solution (1 × 10-4 mol L-1 of CD) while stirring at 450 rpm. The experiments were done at 25 °C. The raw experimental data are obtained as the amount of heat produced per second following each injection as a function of time. Integration of the heat flow peaks by the instrument software (after taking into account heat of dilution) provides the amount of heat produced per injection. The experimental data were fitted with a

Associative Network Based on Cyclodextrin Polymer theoretical titration curve using the instrument software with a model assuming a 1:1 stoichiometry for the adamantyl/CD complex. The enthalpy change, ∆H, the apparent association constant, Kapp, and the overall stoichiometry, n ) [Ada]/[CD], were the adjustable parameters. Rheological Experiments. Oscillatory experiments were performed with a cone-plate rheometer (AR1000 from TA Instruments). All the dynamics rheological data were checked as a function of strain amplitude to ensure that the measurements were performed in the linear viscoelastic region. The cone used has a diameter of 40 mm and an angle of 1°. A solvent trap was used to prevent evaporation of solvent. The viscoelastic properties of the associative networks were determined by measuring changes of the storage modulus G′ and the loss modulus G′′, at 25 °C, applying a shear stress at fixed frequency. Frequency experiments were performed at a stress located in the range of linear viscoelasticity in the frequency range of 0.1-80 Hz. Preparation of Benzophenone-Loaded Associative Networks. The polymer solutions at Cp ) 80 g L-1 were prepared in phosphate buffer (pH ) 7.2) with an ionic strength of 0.154 mol L-1, to match the biological conditions. Loaded Dext-Ada/P(MVE-MA)-g-βCD associative networks were obtained by mixing at room temperature a DextAda solution and a P(MVE-MA)-g-βCD solution already loaded with Bz. The Bz-loaded P(MVE-MA)-g-βCD solution was prepared by adding an excess of Bz to a P(MVE-MA)-g-βCD solution. After 2 days of stirring, the insoluble fraction of Bz was removed by centrifugation (40 min, 13000 G, Sigma Labozentrifugen 3-15, Germany). The amount of Bz into the associative network was evaluated by UV spectrometry at 257 nm assuming a value of 16000 L mol-1 cm-1 for the molar extinction coefficient (Varian Cary 50Bio), leading to the determination of the drug loading (DL; Supporting Information) and the loading efficiency (LE; Supporting Information). In Vitro Release of Benzophenone Experiments. A membrane diffusion system (cutoff of 6-8000 g mol-1) was used for in vitro release studies. The loaded Dext-Ada/P(MVE-MA)-g-βCD associative network are prepared as described (at pH 7.2 in phosphate buffer with Cp ) 80 g L-1, Dext-Ada/P(MVE-MA)-g-βCD ratio 25/75 (w/w) or 50/50 (w/w), and at pH 2 with Cp ) 95 g L-1, Dext-Ada/P(MVEMA)-g-βCD ratio 50/50 (w/w)). It should be noted that, in the case of the sample at pH 2, Cp is the concentration of the bottom phase. A known weight of loaded associative network (typically 1 g) was then set into the membrane diffusion system (surface ) 2.27 cm2). Then, 4 mL of solution (pH 2 or phosphate buffer pH 7.2) was added as a receptor fluid and the system was stored at 37 °C. Release studies were done either without renewal of the release medium or, to be in sink conditions, with renewal of the supernatant two times by day (after 7 and 17 h, respectively) and replacement by fresh medium. The supernatant samples were subjected to UV analysis to determine Bz content as previously described. In equilibrium conditions, Bz is partitioning between the gel-like phase and the release medium and the released amount of Bz can be calculated, leading either to estimate KBz or to simulate a release experiment (Supporting Information).

Results and Discussion Chemical structures of Dext-Ada and P(MVE-MA)-g-βCD are shown in Figure 1. The synthesized Dext-Ada had a substitution yield of 4.6% per mole of glucose unit and the synthesized P(MVE-MA)-g-βCD had a degree of substitution of 84% per mole of maleic anhydride unit. Binding Properties of P(MVE-MA)-g-βCD. Isothermal titration calorimetry (ITC) is a thermodynamic technique that allows studying interactions of two species. When two species interact, heat is either generated or absorbed and by measuring these interaction heats, the enthalpy variation ∆H is determined. The apparent binding constant, Kapp, and the overall stoichiometry, n, are obtained from the fit of enthalpograms and the entropy variation ∆S is deduced from the following relation:

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∆G ) -RT lnK ) ∆H - T∆S

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(1)

where R and T are the gas constant and the temperature, respectively. In this work, complex formation between P(MVE-MA)-gβCD and PEO-Ada or Dext-Ada were investigated by ITC at pH 4 and 7 (without or with NaCl at 0.1 mol L-1). The PEOAda oligomer was chosen as a model for adamantyl group complexation with βCD in comparison of Dext-Ada, for which about 12 adamantyl groups are bound by one dextran chain. The process is exothermic as expected for inclusion complex formation. Integration of the heat flow peaks leads to enthalpograms and the one obtained for Dext-Ada at pH 7 is shown in Figure 2. The fit is done using a simple model where one assumes that each hydrophobic group acts independently from each other, according to the following equation:

Ada + βCD a (Ada/βCD)

K)

[Ada/βCD] [Ada][βCD]

(2)

In all cases, an overall stoichiometry around 0.4 was deduced from the fits, indicating that more than a half of the cavities are not accessible to the adamantyl groups (Supporting Information, Table S1). The n value is independent of the total chain charge because no effect of pH or salt addition is observed. The high degree of CD substitution probably induces a steric hindrance for the complexation with adamantyl groups leading to n values lower than 1. Additionally, K values around 2 × 104 L mol-1 were determined; they are 1 order of magnitude lower than the association constant determined for adamantyl derivatives with native βCD, again reflecting the lower accessibility of CD cavities. Such a decrease in the association constant between native βCD and βCD polymers has already been reported.16,28,29 For example, Weickenmeier et al.28 reported values of 3.3 × 105 L mol-1 and 2.9 × 103 L mol-1 for the association constant of 1-adamantanecarboxylic acid anion with native βCD and poly[(N-vinyl-2-pyrrolidone)-co-(maleic anhydride)]-g-βCD, respectively. The enthalpy variations ∆H are large (between -25 kJ mol-1 and -55 kJ mol-1), the complexation process being mainly enthalpy driven. The good matching in size and shape of the host (adamantyl group) and the guest (βCD cavity) leads to strong van der Waals interactions and, therefore, to large enthalpy changes.30,31 Rheological Properties of Associative Networks. Mixing of P(MVE-MA)-g-βCD and Dext-Ada solutions at high enough concentration (>40 g L-1) led immediately to gel-like solutions whose rheological properties were studied. Figure 3A,B reports the shear stress and the frequency dependence of the storage and loss moduli, G′ and G′′, for mixtures at different DextAda/P(MVE-MA)-g-βCD weight ratio (Cp ) 80 g L-1), respectively. Mixtures were characterized by very stable values of both G′ and G′′ under shear stress. At a fixed frequency of 1 Hz, G′′ was always higher than G′, showing the predominant viscous properties of the mixture. The highest values of G′ and G′′ were obtained for Dext-Ada/P(MVE-MA)-g-βCD weight ratio of 50/50, corresponding to a molar ratio of one adamantyl group to 3 CD cavities. On the other hand, G′ and G′′ increase with the oscillation frequency (Figure 3B); at 75/25 ratio and in the low frequency range, G′ and G′′ deviate from the Maxwell model. G′ and G′′ were found to be proportional to ω1.4 and ω0.9, respectively (following Maxwell G′ and G′′ should scale as ω2 and ω1). Polydispersity in the molecular weight distribution and in the copolymers composition may induce broad relaxation times distribution, leading to model deviation. Such

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Figure 2. ITC titration of P(MVE-MA)-g-βCD ([CD] ) 10-4 mol L-1) by Dext-Ada ([Ada] ) 10-3 mol L-1) at 25 °C: (A) raw data (heat flow vs time) obtained for 27 automatic injections of 10 µL at pH 7; (B) the enthalpogram with the best fit of the experimental points (integrated heat vs molar ratio [Ada]/[CD]).

deviations were already reported in the literature for other associating polymer systems.32,33 In the high frequency range, G′ and G′′ cross over at a frequency ωcross of 19 rad · s-1 and 160 rad · s-1 for Dext-Ada/P(MVE-MA)-g-βCD weight ratio of 50/50 and 25/75, respectively. No crossing point is observed for polymers weight ratio of 75/25. The corresponding terminal relaxation time of the systems (1/ωcross) strongly varies with the polymers weight ratio. The highest relaxation time is found for the strongest physical elastic network obtained for a ratio of one adamantyl group to 3 CD cavities. This ratio is quite close to the one determined by ITC (n ) 0.4, 1 Ada for 2.5 CD). In the following experiments, studies of other parameters that influence the rheological properties of the associative networks were done at Dext-Ada/P(MVE-MA)-g-βCD weight ratio of 50/50. Figure 4A reports the effect of the total polymers concentration. As expected, increasing the total concentration raises G′ and G′′ up. The crossing frequencies are only slightly influenced by the concentration increase, whereas the modulus at the crossing point G′cross ) G′′cross increases sharply with the concentration, from 110 Pa at 80 g L-1 to 600 Pa at 145 g L-1. In a first approximation, G′cross is of the order of G0/2, G0 being the theoretical plateau modulus of the network in the frame of a Maxwell model with a single relaxation time. Then the molar density of junction points ν between the chains can be estimated from the relationship G0 ∼ νRT. At the largest concentration, ν is of the order 4.9 × 10-4 mol L-1. This number is much lower than the molar concentration in adamantyl groups in the medium Cada ) 2 × 10-2 mol L-1. As the network formation is necessarily attributed to inclusion complexes between adamantyl

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groups and CD units, this means that the number of elastically active junction points is only a few percent of the total number of links between the chains. At pH 7, the CD polymer chains are highly charged in water solution; therefore, we also studied the salt effect on the rheological properties of the associating network Dext-Ada/P(MVE-MA)-g-βCD. Figure 4B reports on the obtained results for different NaCl concentrations. Only a moderate effect on G′ and G′′ is observed. The most charged associating network (pH 7 without NaCl) has slightly higher storage and loss moduli, indicating a weak change of the chains conformation by charges screening. Whatever their charge density, the CD polymer chains are probably quite rigid due to their high CD substitution level. Figure 4C reports on pH effect on the rheological properties; G′ and G′′ values do not follow the pH order. Three different interaction mechanisms can be at the origin of the mechanical properties. Inclusion complexes are attractive interactions between the adamantyl groups and the CD cavities and occur similarly in the pH range (see Table S1). Additional hydrogen bond interactions between the acidic functions and the hydroxyl groups present on CDs and dextran should occur mainly at low pH. At pH larger than the pKa, electrostatic repulsions between the charges borne by the P(MVE-MA)-g-βCD chains have to be taken into account. The scheme in Figure 5 is trying to rationalize the different effects. At pH 2, the strength of the associative networks comes both from the inclusion complex formation and the hydrogen bonds. At pH 4, close to the pKa value of the carboxylic groups, the inclusion complex formation is mainly responsible for the networks strength; therefore, the storage and loss moduli are lower at pH 4 than at pH 2. An increase of pH leads to an increase in the global charge of the chains, inducing a swelling of the associative networks at pH 7; therefore, the storage and loss moduli are higher at pH 7 than at pH 4, but still not as high as at pH 2. Drug Entrapment and Release from the Associative Networks. Benzophenone (Bz) was chosen as a model to evaluate the capacity of the associative networks to load and deliver a hydrophobic drug. Bz is known to form inclusion complex with βCD with an association constant of 1900 L mol-1.34 Loading the associative networks (at pH 2 and 7.2) was accomplished by simple mixing of Dext-Ada and Bz-loaded P(MVE-MA)-g-βCD solutions. Bz loading was determined by UV spectrometry. The drug loading, DL, was at least 2 times higher at pH 2 (2.5%) than at pH 7.2 (1.08%) for the associative network with 50/50 polymer weight ratio, and around 1.5 times higher at pH 7.2 for the associative network with 25/75 polymer weight ratio (1.55%) compared to 50/50 (Supporting Information, Table S2). It is expected that the amount of Bz incorporated into the associative networks increases with the amount of P(MVE-MA)-g-βCD, which is the case on going from 50/50 to 25/75 polymer weight ratio. It is more difficult to understand the difference between the drug loadings at pH 2 and pH 7.2. First of all, there could be an influence of the associative phase separation occurring at pH 2 and not at pH 7.2. Hydrogen bonds between acid functions of P(MVE-MA)-g-βCD and Bz can also develop more favorably at pH 2 than at pH 7.2 and explain the larger DL value at pH 2. Influence of Bz loading on the rheological properties was investigated at pH 7 for Dext-Ada/P(MVE-MA)-g-βCD weight ratio of 50/50. Figure 4D shows that Bz incorporation has no effect on the storage and loss moduli under frequency sweep, contrarily to previous results on hydrogels formed between dextran modified by dodecyl groups and a neutral βCD polymer where a decrease of G′ and G′′ was noticed.35 Higher loading

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Figure 3. Storage and loss moduli, G′ (filled markers) and G′′ (empty markers) as a function of shear stress (A) and as a function of frequency for Dext-Ada/P(MVE-MA)-g-βCD (B) weight ratio 25/75 (9), 50/50 (2), and 75/25 ((), at 25 °C, pH 7, Cp ) 80 g L-1 and [NaCl] ) 0.1 mol L-1.

Figure 4. Storage and loss moduli, G′ (filled markers) and G′′ (empty markers) as a function of frequency for Dext-Ada/P(MVE-MA)-g-βCD weight ratio of 50/50 at 25 °C (A) with Cp equal to 80 ((), 100 (2), and 145 g L-1 (9) at pH 7 and [NaCl] ) 0.1 mol L-1 (B) with NaCl concentration equal to 0 ((), 0.2 (2), and 0.4 mol L-1 (9) at pH 7 and Cp ) 80 g L-1 (C) with pH equal to 7 ((), 4 (2), and 2 (9) at Cp ) 80 g L-1 (D) with Cp ) 80 g L-1 at pH 7 in phosphate buffer without (2) and with (() Bz.

efficiency in this previous work (89% compared to 26%) could explain these different rheological properties. The in vitro Bz release studies for Dext-Ada/P(MVE-MA)g-βCD weight ratio of 50/50 were carried out at 37 °C, using a membrane diffusion system both at pH 2 and 7.2. In a first step, the drug release was investigated without renewal of the release medium. Figure 6A presents the Bz release for Dext-Ada/P(MVE-MA)-g-βCD weight ratio of 50/ 50 in buffer solution at pH 7.2. The first part of the release has been fitted with a kt0.5 function, where k and t are a rate constant and the release time, respectively. This is characteristic of a

Fickian transport, where k is proportional to D0.5, D being the diffusion coefficient of Bz in the sample. The fit is shown on Figure 6A at pH 7.2. The obtained k values were 14.9 and 25.7 s-0.5 at pH 2 and 7.2, respectively. This allows to calculate the ratio of the diffusion coefficients DpH2/DpH7 ) 0.34. The lower diffusion coefficient at pH 2 can be linked to the higher moduli of the network at this pH compared to pH 7.2 (see Figure 4C). In equilibrium conditions, Bz is partitioned between the gellike phase and the release medium, the process of inclusion complex with the CD cavities ensuring its retention in the gellike phase. One can notice in Figure 6A that the medium is

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Figure 5. Representative scheme of the pH effect on the associative network.

saturated before 20-24 h at a value of 20%. Therefore, an apparent complexation constant KBz around 1000 L mol-1 can be estimated at pH 7.2 (Supporting Information). This value is twice as small as the one estimated from the literature data for Bz complexation with native βCD (2050 L mol-1 at 37 °C).36 The embedding of the CD units into the associative network can be at the origin of the lower stability of the complexes. In another set of experiments, the release medium was completely refreshed two times per day to obtain a complete release. Figure 6B shows the Bz cumulative release as a function of time for Dext-Ada/P(MVE-MA)-g-βCD weight ratio of 50/ 50 at pH 2 and pH 7.2. The Bz release took several days and the whole of Bz was almost released after 10 days. The release was faster at pH 7.2 than at pH 2, in accordance with the rheological studies (see Figure 4C) and with the kinetics release (see above). The Bz release at pH 7.2 with a polymer weight ratio of 25/75 showed no influence of the polymer weight ratio on the release rate (results not shown). We did not notice any loss of integrity of the associative network during the release. It seems that no structure modification of the associative networks occurred during the 11 days of observation. These experiments clearly show that the incorporation of Bz into the associative network allowed its sustained release. The origin of the sustained release can be attributed to the CD units in the network. Several authors have studied the release of drugs from βCD hydrogels and they have shown that the loading and the release where strongly dependent on the CD content in the hydrogel, the release being slower when the CD content is increased.24–26 As previously reported, Bz loading

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and release have been studied on similar associative networks made of neutral βCD polymer and dextran modified by dodecyl groups.35 In the same concentration range, the release profile was comparable to the one reported here. In all these CD-based systems, the drug release can be controlled by the network swelling, the diffusion of the drug and the drug affinity toward the CD units. In our experimental conditions at pH 7.2, the swelling is limited because there is a membrane diffusion system and the diffusion equilibrium is reached before each measurement. Thus, the release can only be inferred to the partition of the drug between the gel-like phase and the release medium. The amount of Bz released can be calculated at each renewal of the medium. To be able to simulate the experiment of Figure 6B, we assumed in a first approximation that the total Bz molar number total total ) is low compared to the total CD molar number (nCD ). (nBz f Therefore, the free CD molar number (nCD) is constant and equal to ntotal CD - nAda, where nAda is the total molar number of adamantyl groups. The ratio, R, defined as the ratio of released Bz molar total , will depend only number into the released volume over nBz on the experimental conditions and on the complexation constant (Supporting Information). R will thus be constant at each step. The amount of released Bz before the first renewal is equal total total , before the second renewal R(1 - R)nBz , before the to RnBz th i-1 total i renewal R(1 - R) nBz . The cumulative release of Bz before i total R(1 - R)j-1nBz . the ith renewal is thus ∑j)1 The experiment has been simulated using this simple model for a renewal of the release medium each 12 h. The model nicely fits the experimental points within the error bars (Figure 6B) using an R value of 0.23, which is close to the experimental value deduced at the first renewal. At least 6 days are necessary to release more than 95% of the loaded Bz. This calculation clearly shows that release experiments with renewal of the medium at sufficiently long time intervals are completely controlled by the partition of the drug between the CD compartment and the release medium, the drug partition being controlled by the affinity of the drug for the CD cavities.

Conclusion Mixing in aqueous media a CD polymer to a dextran bearing adamantyl groups leads to formation of associative networks. The main interactions between the two polymers are due to the inclusion complexes formation between adamantyl groups and CD cavities: high affinity constants (∼104 L mol-1) were determined by ITC. Additional interaction mechanisms participate in the cohesion of the networks: hydrogen bonds and electrostatic repulsions. These interactions are sensitive to the

Figure 6. (A) In vitro release (with the kt0.5 function fit) and (B) in vitro cumulative release of Bz from Dext-Ada/P(MVE-MA)-g-βCD weight ratio of 50/50 at (0) pH 7.2 in phosphate buffer (Cp 80 g L-1) (with the simulated experiment) and (() pH 2 (Cp 95 g L-1) at T ) 37 °C.

Associative Network Based on Cyclodextrin Polymer

pH and ionic strength of the medium. The largest storage and loss moduli are obtained at pH 2 where hydrogen bonds between carboxylic acid groups of the CD polymer and OH groups of the dextran backbone should form and increase the number of junctions between the chains. At the highest studied pH (pH 7.2), the P(MVE-MA)-g-βCD chains are fully charged and hydrogen bonds have a lower probability to occur. However, the electrostatic repulsions between the carboxylic acid groups participate to the increase of the storage and loss moduli by inducing a swelling of the network. At an intermediate pH close to the pKa of the polyacid (pH 4), the P(MVE-MA)-g-βCD chains are only partly charged and the additional interactions provide minimum contributions to the network cohesion. In this paper we have demonstrated that pH-sensitive associative networks are of great interest for the delivery of apolar drugs. The loading and release of an apolar model drug, benzophenone, was studied at two pH and different P(MVEMA)-g-βCD content. The loading (between 1 and 2%) was shown to increase with the CD content and to decrease with the pH. Sustained releases of Bz have been obtained at the two pHs, a slower kinetic being observed at pH 2 in good correlation with the rheological properties. The drug release is controlled both by diffusion in the network and by inclusion complex interactions with CD cavities. These two mechanisms can be decoupled in two kinds of experiments. First, in release experiments with no renewal of the release medium, the kinetic is mainly controlled by Fickian transport of the drug. Second, in experiments where the medium is renewed at sufficiently long time period, the equilibrium is reached before each renewal and the release, only due to the partition of the drug between the associative network and the release medium, can be calculated at each step knowing the complexation constant of the drug. This new drug delivery system combines several advantages compared to previously described ones that are the easiness of preparation of the temporary networks, the pH responsive properties, and the ability to control the release of apolar drugs due to their specific inclusion complex interactions with βCD cavities. Supporting Information Available. Determination of the drug loading (DL) and the drug efficiency (LE), determination of the released amount of Bz, and Tables S1 and S2. This material is available free of charge via the Internet at http:// pubs.acs.org.

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