Cyclodextrin Polymer Nanoassemblies: Strategies for Stability

Jan 11, 2012 - The main goal of this work was to develop two strategies for stabilization of nanoassemblies made of β-cyclodextrin polymer and amphip...
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Cyclodextrin Polymer Nanoassemblies: Strategies for Stability Improvement Véronique Wintgens,† Anne-Magali Layre,‡ Dominique Hourdet,‡ and Catherine Amiel*,† †

Systèmes Polymères Complexes, ICMPE (UMR 7182 CNRS-UPEC), 2 rue Henri Dunant, 94320 Thiais, France Soft Matter Science and Engineering (UMR 7615 UPMC−CNRS-ESPCI), ESCPI, 10 rue Vauquelin, 75231 Paris Cedex 05, France

‡

S Supporting Information *

ABSTRACT: The main goal of this work was to develop two strategies for stabilization of nanoassemblies made of βcyclodextrin polymer and amphiphilic dextran associated through host−guest complexes. The first strategy was to coat the nanoassemblies with a dextran derivative bearing adamantyl anchoring groups and hydrophilic poly(ethylene oxide-co-propylene oxide) side chains to increase the steric repulsion between the nanoassemblies. The second strategy developed was to post-reticulate the nanoassemblies upon UV irradiation. Photo-cross-linkable nanoassemblies have been prepared from new host or guest polymers bearing allylether or methacrylate groups. The modified nanoassemblies have been characterized by dynamic light scattering as a function of time and for various salt and competitor concentrations. The results of the first strategy show an improvement of shelf stability and resistance at relatively low concentrations of competitors. The second strategy is the most efficient in providing good shelf stability, much larger than with the first strategy, together with a large resistance to dissociation in presence of competitors.

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INTRODUCTION Recently, numerous works have been devoted to the design of nanoscale polymer assemblies due to their potential applications in the biomedical field.1−4 Methods using self-assembly of amphiphilic copolymers are most commonly described in the literature and generally lead to core/shell nanostructures able to carry apolar drugs in their apolar core.1,2 One of the major drawbacks of these systems is related to their production processes, which imply the use of organic solvent or surfactant, not easy to remove and limiting the use of the nanoassemblies in health industry. Polymers interacting via host−guest interactions can spontaneously self-assemble in aqueous solution without the use of organic solvents or surfactants. Cyclodextrin-based polymeric systems have been proposed. Cyclodextrins (CDs) are cyclic oligosaccharides, the most common ones being α, β, and γCD, which contain six, seven, and eight glucose units, respectively. CDs are truncated coneshaped molecules having a relatively hydrophobic interior cavity that can accommodate a wide range of apolar guests. For example, adamantyl groups (Ada) match quite perfectly the cavity size of βCD cavities leading to high association constants. 5 Recent reviews report host−guest polymer assemblies originated from CD polymers mixed with guest polymers and their biomedical applications.3,4,6 Recently, we reported that neutral host and guest polymers can directly form nanoassemblies by simple mixing in pure water.7−11 The strong host guest interactions between the two polymers (bearing βCD and hydrophobic groups (Ada or © 2012 American Chemical Society

dodecyl (C12)), respectively) lead to nanoassemblies of tailorable sizes, depending not only on the βCD and guest substitution level but also on the total concentration and composition of the mixtures. Moreover, neither the nature of the host polymer, linear dextran chains with βCD pending groups or branched copolymers βCD/epichlorohydrin (Poly(βCD-Ep)), nor the nature of the guest groups, Ada or C12 groups, did affect strongly the nanoassemblies properties. The conjectured mechanism for the formation of nanoscale assemblies should result from an associative phase separation of the two polymers in solution. The growing nanosized polymer rich phases should be kinetically stabilized by an external shell made of guest polymer and containing ions adsorbed from the solution. Regarding the good properties of biocompatibility and low toxicity of these nanoassemblies, promising drug delivery applications were considered.7−9 Encapsulation of lipophilic drugs such as tamoxifen or benzophenone could be achieved by their inclusion complex formation with the free βCD cavities of the system. Despite these interesting properties, these systems present a major drawback that is their strong sensitivity to environmental conditions. Indeed, they combine the characteristics of colloidal systems that can be destabilized by additives promoting aggregation (salt or non adsorbing polymers) and the Received: November 15, 2011 Revised: January 10, 2012 Published: January 11, 2012 528

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characteristics of a supramolecular system that can be dissociated by the addition of a competitor. This problem encompasses the case of CD nanoassemblies and is a general feature of nanoscale polymer self assemblies. Therefore, the main goal of this work is to develop strategies aiming at stabilizing the βCD nanoassemblies in complex media. Two methods are proposed. The first one is to provide a shell containing poly(ethylene oxide-co-propylene oxide) (PEPO) grafts that will improve the steric stabilization of nanoassemblies. Grafting PEPO chains onto dextran bearing Ada groups (Dext-Ada-PEPO) will allow a direct anchoring of a steric shell on the nanoassemblies, taking advantage of host− guest interactions between Ada groups and βCD cavities of the nanoassemblies. The second method is to post-reticulate the nanoassemblies to prevent them from dissociation and to keep their integrity in complex media. New host and guest polymers bearing reactive groups, allylether (AE) or methacrylate moieties, will be prepared to form reactive nanoassemblies that will be cross-linked subsequently upon UV irradiation. The Article uses reference CD nanoassemblies made of associated Poly(βCD-Ep) and dextrans bearing Ada groups (Dext-Ada) or C12 groups (Dext-C12) to study the efficiency of stabilization strategies. The first part is devoted to the synthesis of the different compounds. Disubstituted dextran (Dext-Ada-PEPO) was synthesized for the stabilizing shell method. For the photo-cross-linking method, either host and guest polymers were post-modified with reactive groups allowing photo-cross-linking: Dext-C12 bearing methacryloyl groups (Dext-C12-MA) and Poly(βCD-Ep) bearing AE groups (Poly(βCD-Ep)-AE). The second part of this Article deals with the characterization of the nanoassemblies and their shelf stability before post-modification. Post-modified nanoassemblies using the PEPO corona or the photo-cross-linking strategies are analyzed in the last two parts in terms of shelf stability, sensibility to salt, or addition of host competitors.

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Figure 1. Structure of Poly(βCD-Ep). substitution was obtained by 1H NMR in deuterated DMSO from the ratio between the protons of alkyl chains (0.8 to 1.8 ppm for DextC12 and 1.6 to 2.0 ppm for Dext-Ada) and the anomeric and hydroxylic protons of dextran (4.4 to 5.0 ppm). Dext-MA was prepared by modification of DT40 with GMA as already reported.15,16 DT40 (5 g) was dissolved in 40 mL of anhydrous DMSO under stirring and nitrogen atmosphere. Then, 1 g of DMAP and 0.73 mL of GMA were added, and the mixture was left 24 h at room temperature. Dext-MA was isolated by precipitation in 200 mL of ethanol, filtration, and drying. After dissolving the polymer in 100 mL of water, it was purified by dialysis against water and then freeze-dried. We obtained 4.9 g of Dext-MA. The degree of substitution of Dext-MA, 11.2 mol %, was determined by 1H NMR in deuterated DMSO from the integration of (CH2) protons of MA (5.65 and 6.10 ppm) and anomeric and hydroxylic protons of dextran (4.4 to 5.0 ppm). Dext-C12-MA was prepared by a two-step synthesis. After modification of a small proportion of the hydroxyl groups by lauroyl chloride, the obtained Dext-C12 was further modified by reacting with GMA as previously: we used 3.5 g of Dext-C12, 30 mL of anhydrous DMSO, 0.7 g of DMAP, and 0.52 mL of GMA. Dext-C12-MA was isolated by precipitation into 300 mL of propan-2-ol and purified by dialysis against water. After freeze-drying, 3.3 g of Dext-C12-MA was obtained. The substitution degree of Dext-C12-MA was also obtained by 1H NMR in deuterated DMSO. Dext-Ada-PEPO was prepared by a three-step synthesis. After modification of a small proportion of hydroxyl groups by 1adamantanecarbonyl chloride, the obtained Dext-Ada was further modified by esterification reaction with succinic anhydride (Dext-AdaCOOH) and finally grafted with amino-terminated PEPO (Jeffamine M-2005) by peptide coupling (Dext-Ada-PEPO). Typically, 2.68 g of LiCl was initially dissolved under stirring and nitrogen atmosphere in 200 mL of anhydrous DMF and heated to 85 °C. Then, 10.17 g of DT40 was added, and after solubilization, 1.23 g of 1-adamantanecarbonyl chloride, 1.44 g of DMAP, and 86 μL of pyridine were introduced. The reaction was maintained for 3 h under these conditions, and 10 mL of the mixture was sampled out and treated with propan-2-ol to recover the Dext-Ada sample. In the remaining 190 mL of the reaction medium left under nitrogen at 85 °C, 0.885 g of succinic anhydride, 0.109 g of DMAP, and 10 μL of pyridine were added. The mixture was left 18 h at 85 °C and 3 h at room temperature. The disubstituted dextran, Dext-Ada-CO2H, was isolated by precipitation in 2 L of propan-2-ol, filtration, and drying. After the polymers (Dext-Ada or Dext-Ada-CO2H) were dissolved in the minimum amount of water, the samples were purified by dialysis against water and then freeze-dried. We obtained 0.5 g of Dext-Ada and 5.92 g of Dext-Ada-CO2H, respectively. The degree of substitution of Dext-Ada-CO2H was obtained by 1H NMR in deuterated DMSO from the integration of −(CH2)2−CO protons (2.45 and 2.60 ppm) and anomeric and hydroxylic protons (4.4 to 5.0 ppm). In a last step, 5 g of Dext-Ada-CO2H was dissolved in 100 mL of water, and the solution was cooled in an ice bath (T ≅ 2 to 3 °C). At the same time, the same amount of PEPO (m = 5 g) was dissolved under acidic form in 50 mL of cold water (T = 2 to 3 °C). Once the polymers dissolved, the two solutions were mixed, and the pH was adjusted to 5.4 by adding HCl 1 mol L−1. Then, 0.347 g of NHS and 1.832 g of EDC

MATERIALS AND METHODS

Materials. Lauroyl chloride, 1-adamantanecarbonyl chloride, succinic anhydride, glycidyl methacrylate (GMA), allyl glycidyl ether (AGE), tetrabutylammonium hydroxide (water solution, 40%), 4(dimethylamino)pyridine (DMAP), N-hydroxysuccinimide (NHS, 98%), and 2,2′-dimethoxy-2-phenyl acetophenone (DMPAP) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France) and used as received. 1-[3-Dimethylaminopropyl]-3-ethyl carbodiimide hydrochloride (EDC, 98%) was purchased from Acros and used as received. Jeffamine M-2005 (PEPONH2), an amino-terminated poly(ethylene oxide-co-propylene oxide) with 29 PO and 6 EO units (Mn = 2000 g/mol), was kindly supplied by Huntsman (Belgium) and used as received. Lithium chloride (Sigma-Aldrich) and dextran (DT40, Mw 4.3 × 104 g mol−1, Amersham Sweden) were dried overnight under vacuum at 100 °C before use. Pyridine, N,Ndimethylformamide (DMF) and dimethylsulfoxide (DMSO) were anhydrous grade from Sigma-Aldrich; other solvents were analytical grade, and water was deionized quality. The synthesis of Poly(βCD-Ep) has been described12 and a schematic structure of the branched polymer is given in Figure 1. The two Poly(βCD-Ep) samples used in this work were characterized by 1 H NMR in deuterated water (Bruker 400 MHz) and by size exclusion chromatography (columns TSK-gel type SW 4000−3000, refractive index, and laser light scattering detectors). The βCD content, the weight-average molar mass, Mw, the number-average molar mass, Mn, and the dispersity of molar masses, (D = Mw/Mn), can be found as Supporting Information (Table S1). Synthesis. The synthesis of monosubstituted dextrans, Dext-C12 and Dext-Ada, has already been described.13,14 The degree of 529

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Figure 2. Reaction scheme of dextran modification. were added to the mixture, and the reaction was left to proceed at T = 2 to 3 °C during 24 h. After dilution in water, 4.22 g of Dext-AdaPEPO was recovered by dialysis against pure water. The substitution degree of the different synthesized dextrans can be found as Supporting Information in Table S2. Poly(βCD-Ep)-AE was obtained by modification of Poly(βCD-Ep) by AGE. The synthesis was adapted from dextran modification by 1,2epoxy-alkane.17 We dissolved 1 g of Poly(βCD-Ep) in 15 mL of DMSO, and 7 mL of tetrabutylammonium hydroxide (1 mol L−1) in water was added. The mixture was left under stirring for 3 days. Poly(βCD-Ep)-AE was isolated by precipitation in 300 mL of acetone, filtration, and drying. After dissolving the polymer in the minimum amount of water, it was purified by dialysis against water and then freeze-dried. We obtained 0.8 g of Poly(βCD-Ep)-AE. The degree of substitution of Poly(βCD-Ep)-AE was obtained by 1H NMR in deuterated D2O from the integrations of the (CH) proton (5.85 ppm), the (CH2) protons, and the anomeric protons (4.9 to 5.3 ppm). Nanoassemblies Preparation. All polymer solutions were prepared at least 1 day before the different experiments to get equilibrated solutions. The concentration of the different polymers solutions was fixed at 1 or 2 g L−1. Nanoassemblies were prepared by mixing modified dextrans and CD polymers solutions (0.6 mL of each solution) at room temperature under magnetic stirring at 150 rpm. Nanoassemblies Irradiation. DMPAP was used as a photoinitiator in the nanoassemblies irradiation experiments. A solution of DMPAP (80 μL, 3.2 × 10−2 mol L−1) in ethanol was deposited at the bottom of a flask and left to evaporate; then, 12.5 mL of cylodextrin polymer solution at 2 g L−1 was added to the flask and shaken for 2 days. The final concentration of DMPAP was 2 × 10−4 mol L−1, leading to an absorbance lower than 0.1 at 312 nm. The CD polymer solutions at 2 g L−1 had a CD cavities concentration around 1.1 × 10−3 mol L−1, which means that less than one cavity over five could be complexed with DMPAP. The nanoassemblies were prepared as described above but using the CD polymer solution containing DMPAP. Then, the nanoassemblies were irradiated at 312 nm using a Spectrolinker irradiation chamber. The irradiation time has been optimized and fixed at 15 min. Nanoassemblies Characterization. The mean hydrodynamic diameter and the polydispersity index (PdI) of the nanoassemblies were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS (model ZEN3500) from Malvern Instrument equipped with a He−Ne laser (λ = 633 nm, scattering angle 173°). Each sample was measured at least in duplicate (each measurement being the average of 8 runs of 10 s) at 25 °C. The standard deviation is ∼2%. The reported mean value (or Z-average size) and the PdI values are obtained by cumulant analysis (fit of the logarithm of the correlation function by a third-order polynomial). Each reported value is at least the average of three different experiments. Examples of intensity distribution are presented as Supporting Information in Figures S1, S2, and S3.

have been grafted through an esterification reaction with 1adamantanecarbonyl chloride.13,14 A degree of substitution around 5 mol % for Dext-Ada was targeted to ensure nanoassemblies formation. Then, acidic functions have been introduced by reaction with succinic anhydride to graft the PEPO chains through a peptidic coupling reaction in the last step. A degree of substitution around 11−13 mol % for DextAda-PEPO was achieved after PEPO grafting. The substitution level of the modified dextrans can be found as Supporting Information in Table S2 for the different groups. The amount of remaining COOH groups in Dext-Ada-PEPO was too low to be detectable by NMR analysis. Dext-C12 was obtained by modification of DT40 through an esterification reaction with lauroyl chloride.13 A substitution level of 6.9 mol % was obtained. To get a photo-cross-linkable dextran, Dext-C12 was further modified by reaction with GMA via a trans-esterification process as it has been reported for DT40.15,16 The grafted methacryloyl groups were characterized by 1H NMR (Figure 3A), and the substitution degree of DextC12-MA was ∼6.8 mol %.

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Figure 3. 1H NMR of (A) Dext-C12-MA in deuterated DMSO and (B) Poly(βCD-Ep)*-AE in D2O.

RESULTS AND DISCUSSION Polymers Synthesis and Characterization. The three different dextrans bearing adamantyl groups were synthesized as described in Figure 2. In a first step, the adamantyl groups

Introduction of terminal double bonds onto the CD polymers was performed using AGE in a DMSO/H2O mixture 530

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Figure 4. Size variation (A) as a function of time for the nanoassemblies formed at 1 g L−1 between Poly(βCD-Ep) and Dext-C12 (Δ) or Dext-Ada (◆) and (B) as a function of HOPrβCD concentration for the nanoassemblies formed at 1 g L−1 between Poly(βCD-Ep) and Dext-Ada without (gray) or with (black) Dext-Ada-PEPO at 0.12 g L−1 (diameter measured 3 h after HOPrβCD addition).

Figure 5. (A) Size variation as a function of time for the nanoassemblies formed at 1 g L−1 between Dext-Ada and Poly(βCD-Ep), without Dext-AdaPEPO (◆), and with Dext-Ada-PEPO at 0.04 (□), 0.08 (Δ), 0.12 (■), 0.16 (∗), and 0.20 g L−1 (○), respectively. (B) Size variation as a function of time for the nanoassemblies (Dext-Ada/Poly(βCD-Ep)/Dext-Ada-PEPO (0.12 g L−1)) formed in one step (gray) or in two steps (black).

after the mixing. The PdI values are ∼0.05. Additional modification of monosubstituted dextrans does not favor the formation of nanoassemblies. For instance, we previously showed that the introduction of carboxylic functions, like in Dext-Ada-COOH, prevents the formation of nanoassemblies.10 In pure water, the negative charges brought by the ionized carboxylic functions lead to soluble complexes that can be described as swollen aggregates. In this study, we noticed that the PEPO chains grafted onto Dext-Ada also prevent the formation of nanoassemblies when mixing Dext-Ada-PEPO with Poly(βCD-Ep). Two main reasons could explain this behavior. Few residual acidic functions can be present onto the Dext-Ada-PEPO chains, leading to a negatively charged polymer in pure water. As already mentioned, the presence of ionic groups hinders nanoassemblies formation.10 The PEPO side chains could induce a steric hindrance preventing adamantyl groups of Dext-Ada-PEPO from an efficient meeting with CD cavities of Poly(βCD-Ep). Conversely, mixing DextC12-MA and Poly(βCD-Ep) leads to nanoassemblies. Anyhow, the efficiency of formation is much lower with Dext-C12-MA than with Dext-C12 at 1 g L−1. (This is evidenced by the decrease in the signal intensity in DLS experiments by a factor at least of 6 between the nanoassemblies formed with Dext-C12 and those formed with Dext-C12-MA.)

in the presence of tetrabutylammonium hydroxide. This procedure was adapted from that described in the case of dextran modification by 1,2-epoxy-alkanes.17 Modification by AGE was first studied with low-molecular-weight Poly(βCDEp)*. Evidence of grafted AE groups was obtained by characterization of the terminal double bonds by 1H NMR spectrum (Figure 3B). Broadening of the signals due to the high molecular weight of Poly(βCD-Ep)-AE led to an imprecise determination of the substitution degree that can be estimated around 1 AE group per CD for Poly(βCD-Ep)*-AE, which means a molar yield around 50% for the reaction. Nanoassemblies Formation and Characterization. In the case of Dext-C12 and Poly(βCD-Ep) in pure water, we previously showed from phase diagram experiments that the two polymers are subject to associative phase separation.13,18,19 At low concentrations, typically between 0.5 and 2 g L−1, mixtures of both polymers show a peculiar behavior. No macroscopic phase separation is observed, but the mixtures present a Tyndall effect: metastable nanoassemblies are formed with a mean diameter around 200 nm.7,8,10,20 Among the dextrans described in this study, Dext-C12 and Dext-Ada lead to efficient formation of nanoassemblies when mixed with Poly(βCD-Ep). At a total concentration of 1 g L−1, the diameters of the nanoassemblies measured in this work are 190 and 165 nm with Dext-C12 and Dext-Ada, respectively, 1 h 531

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Figure 6. Schematic structures of the nanoassemblies modified (A) by a shell of Dext-Ada-PEPO or (B) by photo-cross-linking.

above (two steps) and those obtained by mixing directly DextAda, Dext-Ada-PEPO, and Poly(βCD-Ep) (one step). Under the same concentration conditions, the nanoassemblies formed in two steps present a much higher stability than those formed in one step, as shown Figure 5B. In other words, the shelf stability of the one-step nanoassemblies containing Dext-AdaPEPO is even worst than the one of bare nanoassemblies (Figure 4A). Additionally, one can roughly estimate the corona thickness in the two steps process. An average diameter of the nanoassemblies after their formation was estimated around 160 nm, and 1 h after addition of Dext-Ada-PEPO (at concentrations higher than 0.05 g L−1) the average diameter was ∼175 nm, leading to a corona thickness of 7.5 nm. This value is in good agreement with the Dext-Ada-PEPO hydrodynamic radius in solution. A value of ∼6 nm can be estimated by DLS, although presence of aggregates always disturbs the measurements. This gives a good indication that the Dext-Ada-PEPO conformation in the corona is coiled, similarly to the one adopted in solution, whereas some Ada moieties anchor the chains on the surface by making inclusion complexes with available CD cavities. The Dext-Ada-PEPO corona thus increases the shelf stability, and this effect is attributed to the repulsion brought by the hydrophilic PEPO grafts. It has been reported that more efficient steric stabilizations were provided by brush layers.21,22 The question is thus whether the surface density of the PEPO grafts onto the nanoassemblies is high enough to display a brush conformation. An estimate of the number of PEPO grafts per nanoassembly can be made assuming a concentration of adsorbed Dext-Ada-PEPO around 0.05 g L−1 (experimental conditions of Figure 5a) and a density of the nanoassemblies around 200 g L−1 (concentration of the polymer rich phase determined in previous phase diagram studies).13 This leads to ca. 3000 PEPO side chains per nanoassemblies. For nanoassemblies with a 160 nm diameter, this gives a distance d ≈ 5 nm between two PEPO groups. Because d is more than 2 times larger than the radius of gyration of the PEPO groups (∼2 nm), this rough estimate shows that PEPO side chains are not expected to form a dense brush in the outer layer of nanoassemblies but are more likely to adopt a mushroom conformation. A schematic structure is given in Figure 6A. The influence of salt addition has also been studied because the corona is expected to improve the stability in ionic environment. The nanoassemblies formed in two steps (even at the highest Dext-Ada-PEPO concentration, 0.20 g L−1) were

The size of the nanoassemblies strongly depends on the polymer concentration. Increasing the concentration increases the diameter and decreases the stability. Phase separation in the mixtures occurs in a few hours at concentration higher than 5 g L−1. Therefore, the nanoassemblies are more stable over time when the starting diameter is lower. Figure 4A reports the diameter variation versus time in the case of nanoassemblies formed between Dext-C12, or Dext-Ada, and Poly(βCD-Ep) at 1 g L−1. At this concentration, the diameter is usually around 170−190 nm after 1 h; then, it progressively increases, being multiplied by a factor of 3 after 1 week. The PdI index also increases, but the values are still lower than 0.2. The same behavior is also observed when the nanoassemblies are prepared with the modified CD polymer, Poly(βCD-Ep)-AE. As previously reported,10 these nanoassemblies are destabilized by salt addition, even at low concentration (0.03 mol L−1 of NaCl). We also studied the effect of a competitor addition, and hydroxypropyl-β-cyclodextrin (HOPrβCD) was chosen for that purpose because of its high water solubility and good affinity for adamantane derivatives. Figure 4B reports the diameter of nanoassemblies (formed between Dext-Ada and Poly(βCD-Ep) at 1 g L−1) 3 h after the addition of different HOPrβCD concentrations. One can easily notice that the nanoassemblies are destabilized at concentration higher than 10−3 mol L−1. Increasing the HOPrβCD concentration increases the number of adamantyl groups that will form complexes with HOPrβCD, decreasing the number of links between the polymers and therefore the shelf stability.11 Nanoassemblies Stabilization by PEPO Corona. As previously mentioned, mixing directly Dext-Ada-PEPO and Poly(βCD-Ep) does not form nanoassemblies. To test the ability of PEPO chains to stabilize the nanoassemblies, we used a two-step procedure. Various aliquots of a concentrated solution of Dext-Ada-PEPO were added to nanoassemblies solutions of Dext-Ada and Poly(βCD-Ep) previously prepared at a total concentration of 1 g L−1. The effect of the Dext-AdaPEPO addition is pointed out in Figure 5A. At concentrations up to 0.05 g L−1, the size of the nanoassemblies increases in the same way as without Dext-Ada-PEPO. Significant stabilization of nanoassemblies is observed over 4 days when Dext-AdaPEPO is added at concentrations larger than 0.05 g L−1. We suggest that the addition of Dext-Ada-PEPO leads to the formation of a corona around the nanoassemblies with PEPO chains facing the solution, therefore hindering the growth of nanoassemblies. This is supported by comparison of the shelf stabilities exhibited by the nanoassemblies formed as described 532

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Figure 7. Size variation as a function of time for the nanoassemblies formed at 2 g L−1 (A) between Poly(βCD-Ep) and Dext-C12/Dext-C12-MA and (B) between Poly(βCD-Ep)-AE and Dext-C12 without irradiation (◆) and after 15 min irradiation (□).

lower than 0.1 at 312 nm. To optimize the irradiation time, we irradiated five test tubes containing nanoassemblies formed between a 1/1 mixture of Dext-C12/Dext-C12-MA with Poly(βCD-Ep) at a total concentration of 2 g L−1 during 0, 5, 10, 15, and 20 min. The size stability of the nanoassemblies was followed over 1 week. The results were the same for the samples irradiated over 10 min; therefore, the irradiation time was fixed at 15 min in the following experiments. In a first series of experiments, the nanoassemblies have been formed from a 1/1 mixture of Dext-C12/Dext-C12-MA with Poly(βCD-Ep), the total concentration being 2 g L−1. As shown in Figure 7A, the irradiation led to a considerable stabilization of the nanoassemblies. The size remained unchanged after more than 7 days. It should also be noticed that after the irradiation the diameter slightly decreases from 160−170 nm to 130−140 nm, and the irradiation led to more compact nanoassemblies. In a second series of experiments, the nanoassemblies were formed between Dext-C12 and Poly(βCD-Ep)-AE; the total concentration is 2 g L−1. Again, a very good stability of nanoassemblies is observed (Figure 7B); their diameter remaining stable over more than 7 days. The irradiation has been performed just after the formation of nanoassemblies, and this allowed us to have and keep much smaller diameter (240 nm) than the one already observed after 2 h (370 nm) for the nonirradiated nanoassemblies. Irradiation thus drastically increases the shelf stability of nanoassemblies in a more efficient way than the one imparted to the Dext-Ada-PEPO shell. Moreover, cross-linking either the host or the guest polymer did not influence the stability. A network of host or guest polymers inside the nanoassemblies seems to prevent bridging attractions between them. The sensibility to salt or competitor addition has also been studied on the first set of irradiated nanoassemblies (made of Dext-C12/Dext-C12-MA and Poly(βCD-Ep)). Unfortunately, cross-linked nanoassemblies did not show better stability than un-cross-linked nanoassemblies in the presence of salt, even at low concentration (0.03 mol L−1 of NaCl).10 Even though the structure of the cross-linked nanoassemblies is frozen, the stability of the colloidal suspension is still ensured by electrostatic repulsions between the nanoassemblies that have a negative charge surface. Salt addition will again screen the electrostatic repulsions, leading to a precipitation. Cross-linked nanoassemblies were not destabilized by adding HOPrβCD as a competitor, even for concentrations as high as 7 × 10−3 mol L−1, where the nonirradiated nanoassemblies were destabilized.

unfortunately not stable in the presence of salt, even at low concentration (0.03 mol L−1 of NaCl). The nanoassemblies coalesce very rapidly; diameters over 1 μm are obtained. (See Figure S3 in the Supporting Information.) This behavior can be attributed to the too low surface density of the PEPO groups that are not enough efficient to protect the nanoassemblies from bridging attractions induced by the adamantyl groups of the Dext-Ada-PEPO chains. Indeed, the adamantyl groups can establish bridges between different nanoassemblies by forming inclusion complexes. Moreover, electrostatic repulsions being screened at low salt concentration, this allows the interplay between steric repulsions of PEPO chains and bridging attractions. The weak stabilization provided by the outer PEPO layer under salt conditions can also be attributed to a moderate hydrophilic character of PEPO chains that contain relatively high propylene oxide content in comparison with ethylene oxide. In this regard, we can imagine that the stabilization of nanoassemblies could be improved by using longer polyether side chains with higher ethylene oxide content. Figure 4B reports the effect of HOPrβCD addition up to concentrations of 7 × 10−3 mol L−1. After 3 h, the nanoassemblies without Dext-Ada-PEPO are already destabilized for concentration higher than 10−3 mol L−1. The nanoassemblies covered with Dext-Ada-PEPO (concentration of 0.12 g L−1) are almost not destabilized in the same range of HOPrβCD concentration. The shelf stability is preserved over several hours. A time range of ca. 12 h is needed to observe nanoassemblies destabilization at concentration higher than 10−3 mol L−1. The Dext-Ada-PEPO layer can partially slow the destabilization process of the nanoassemblies against a competitor. Nanoassemblies Stabilization by UV Reticulation. Cross-linking of methacrylated dextrans (Dext-MA) has been reported via radical polymerization using potassium peroxodisulfate as initiator. By this way, microparticules23 and nanocapsules for drug release24 were prepared. Cross-linking of Dext-MA under UV irradiation was also used to prepare hydrogels, either by irradiation at 365 nm using DMPAP as photoinitiator25,26 or by direct irradiation at 312 nm in presence of methacrylated polyaspartamide.16 In this work, we used DMPAP as photoinitiator, and the irradiation was performed at 312 nm to cross-link the nanoassemblies formed with one of the photocross-linkable polymers, Dext-C12-MA or Poly(βCD-Ep)-AE. DMPAP was added to the CD polymer solutions, and its concentration was fixed and chosen so that the absorbance of solutions remained 533

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Biomacromolecules

Article

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This shows that the chains of dextrans and CD polymers are irreversibly interconnected, as shown in the scheme in Figure 6B; the alkyl-modified dextrans do not have the possibility to escape from the nanoassemblies.

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CONCLUSIONS Two strategies aiming at stabilizing CD nanoassemblies in complex media have been developed. The first strategy, based on supramolecular chemistry, was to coat the nanoassemblies with another polymer (Dext-Ada-PEPO). This polymer bears both anchoring groups (Ada) and hydrophilic PEPO chains, which increase the steric repulsion between the nanoassemblies. The adlayer induced a large improvement of the shelf stability and largely increased the kinetic resistance against a competitor for host−guest interactions compared with uncoated nanoassemblies. However, it did not protect the nanoassemblies from salt-induced aggregation. The steric PEPO layer was not dense enough (and perhaps not hydrophilic enough) to prevent bridging attractions when electrostatic repulsions were screened. The second strategy was to post-reticulate the nanoassemblies upon UV irradiation. The internal structures of irradiated nanoassemblies were of semi-interpenetrated network type as either the host or the guest polymers were modified with photo-cross-linkable groups. The results are remarkable shelf stability, larger than with the first strategy, together with a large resistance to dissociation in the presence of a competitor. Cross-linked nanoassemblies are nevertheless destabilized in the presence of low amounts of salt. The first strategy shows that supramolecular chemistry can improve stabilization of nanoassemblies in complex media. Improvement of shelf stability and resistance to competitors can be reached in a time range that is suitable for biomedical applications. The strategy of post-reticulation has the advantage to freeze the nanoassembly integrity over a large range of competitor concentration.

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ASSOCIATED CONTENT

S Supporting Information *

Characteristics of Poly(βCD-Ep), substitution level of the different dextrans, and examples of intensity distribution. This material is free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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dx.doi.org/10.1021/bm201608n | Biomacromolecules 2012, 13, 528−534