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Mar 24, 2015 - A synthetic approach toward a pH and sugar-responsive diblock copolymer via post-polymerization modification. Heba Gaballa , Shaojian L...
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Glucose-Responsive Polymer Vesicles Templated by α‑CD/PEG Inclusion Complex Hao Yang, Chuan Zhang, Chang Li, Yong Liu, Yingli An, Rujiang Ma,* and Linqi Shi* State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, Collaborative Innovation Center of Chemical Science and Engineering, Nankai University, Tianjin 300071, China ABSTRACT: Polymeric nanoparticles with glucose-responsiveness are of great interest in developing a self-regulated drug delivery system. In this work, glucose-responsive polymer vesicles were fabricated based on the complexation between a glucosamine (GA)-containing block copolymer PEG45-b-P(Asp-co-AspGA) and a phenylboronic acid (PBA)-containing block copolymer PEG114-b-P(Asp-co-AspPBA) with α-CD/PEG45 inclusion complex as the sacrificial template. The obtained polymer vesicles composed of cross-linked P(Asp-coAspGA)/P(Asp-co-AspPBA) layer as wall and PEG chains as both inner and outer coronas. The vesicular morphology was observed by transmission electron microscopy (TEM), and the glucose-responsiveness was investigated by monitoring the variations of hydrodynamic diameter (Dh) and light scattering intensity (LSI) in the polymer vesicle solution with glucose using dynamic light scattering (DLS). Vancomycin as a model drug was encapsulated in the polymer vesicles and sugar-triggered drug release was carried out. This kind of polymer vesicle may be a promising candidate for glucose-responsive drug delivery.

1. INTRODUCTION Glucose-responsive materials have attracted great attention in recent years due to their potential applications in purification and recognition of sugars and glycoproteins,1−4 glucose sensing,5−7 and self-regulated drug delivery.8−11 Among different kinds of glucose-responsive materials,12−14 phenylboronic acid (PBA)-containing polymers5,15 have been mostly studied because of their versatility for different designs and better stability than protein-based systems.16 Different forms of PBA-based glucose-responsive materials including bulk gels,17,18 nanogels,18−20 and micelles21−27 have been constructed in the past decades. Recently, PBA-based glucose-responsive vesicles have aroused scientific interest because of an important superiority of their large loading capacity and several studies have been reported.28 Direct self-assembly of block copolymers into polymersomes in aqueous solutions was generally used to prepare glucose-responsive polymer vesicles, where vesicular membrane consisted only of PBA-containing blocks. A pioneering work by van Hest et al.29 was the fabrication of a polymersome nanoreactor with controllable permeability induced by sugar-responsive block copolymers. Their key concept was to use a sugar-responsive block copolymer poly(ethylene glycol)-b-poly(styrene boronic acid) (PEG-bPSBA) as pore-generating component in the vesicular membrane of self-assembled poly(ethylene glycol)-b-polystyrene (PEG-b-PS) vesicles. This kind of polymersome was successfully used as a nanoreactor by encapsulating an enzyme, where PSBA domains became voids that permitted trans© 2015 American Chemical Society

membrane diffusion of small reagents. By using a boroxolecontaining styrenic monomer which was an analogue of PBA, Kim and co-workers30 synthesized a block copolymer poly(ethylene glycol)-b-poly(styreneboroxole) (PEG-b-PBOx) which could self-assemble into a variety of nanostructures including spherical and cylindrical micelles and polymer vesicles. This kind of polymersomes exhibited monosaccharide-responsive disassembly in a neutral pH medium. Encapsulated insulin could be released from the polymersomes only in the presence of sugars under physiologically relevant pH conditions. In another case, Kim et al.31 synthesized a block copolymer composed of PEG and a sugar-responsive block which contained a glucose receptor of styreneboroxole alternating with a nonresponsive solubilizing group of Nfunctionalized maleimide throughout the polymer chain. This kind of block copolymer could self-assemble into polymersomes in water and encapsulate water-soluble molecules, such as fluorescein isothiocyanate-labeled insulin, within their inner compartment. Template method was also used to fabricate PBA-based sugar-sensitive polymer vesicles. De Geest and co-workers32 reported PBA-based glucose-responsive polyelectrolyte capsules fabricated by layer-by-layer self-assembly of a PBA-containing polycation poly(3-acrylamidophenylboronic acid-co-dimethylaminoethyl acrylate) (P(AAPBA-co-DMAEA)) and a polyanion Received: February 4, 2015 Revised: March 23, 2015 Published: March 24, 2015 1372

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P(Asp-co-AspGA)/P(Asp-co-AspPBA) as shell, and PEG114 as corona. After removing α-CD under elevated temperature, polymer vesicles were obtained with cross-linked P(Asp-coAspGA)/P(Asp-co-AspPBA) layer as wall and PEG chains as both inner and outer coronas. Glucose-responsiveness of the polymer vesicles was investigated and sugar-triggered drug release was carried out. To our best knowledge, glucoseresponsive polymer vesicles based on the complexation between GA- and PBA-containing block copolymers and using a sacrificial template of α-CD/PEG inclusion complex have not been reported.

poly(sodium-p-styrenesulfonate) (PSS) on a sacrificial template of monodisperse polystyrene microparticles. The obtained polymer capsules displayed glucose-responsiveness because of the glucose-induced change in electrostatic interactions between the PBA-containing polycation and PSS. Levy et al.33 assembled polymer multilayers of PBA-modified poly(acrylic acid) (PAA) and polysaccharide mannan onto colloidal CaCO3 particles based on the interaction between PBA groups on PAA and diols on polysaccharide. Sugar-sensitive polymer microcapsules were obtained by dissolving CaCO3 template in 0.1 M EDTA solution. Du and co-workers34 reported synthesis of glucose and temperature dual-responsive hollow nanospheres via a one-pot two-step “self-removing” approach based on the consecutive radical (co)polymerization, where PNIPAM oligomers were synthesized and precipitated as a template for the P(NIPAM-co-AAPBA) hollow nanospheres. It is has been reported that poly(ethylene glycol) (PEG) could form insoluble inclusion complex, that is, pseudopolyrotaxane, in aqueous solutions when α-cyclodextrin (α-CD) was added.35−37 This kind of insoluble inclusion complex could also be used as template for the fabrication of polymer vesicles because of the easy removal of α-CD under elevated temperature. In our previous work,38 α-CD was used to assist self-assembly of block copolymers PEG-b-P4VP and PEG-bPAA into α-CD/PEG-P4VP/PAA−PEG core−shell-corona micelles in aqueous media. Polymer vesicles with electrostatically cross-linked P4VP/PAA as wall and PEG chains as both inner and outer coronas were obtained after removal of α-CD. Similarly in this paper, we reported a facile strategy to fabricate glucose-responsive polymer vesicles in aqueous media with insoluble α-CD/PEG inclusion complex as the template as illustrated in Scheme 1. A glucosamine (GA)-containing block

2. EXPERIMENTAL SECTION 2.1. Materials. α-Methoxy-ω-amino poly(ethylene glycol) (PEG45NH2, Mn = 2000 Da, Mw/Mn = 1.05) and α-methoxy-ω-amino poly(ethylene glycol) (PEG114-NH2, Mn = 5000 Da, Mw/Mn = 1.05) were purchased from Aladdin and used after dried under vacuum. βBenzyl-L-aspartate NCA (BLA-NCA) were synthesized by the Fuchs− Farthing method using bis(trichloromethyl) carbonate (triphosgene) according to ref 39. N-Hydroxysuccinimide (NHS; 97%) and αcyclodextrin (α-CD; 98%) were purchased from Acros Organics. 1Ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC·HCl, 98%), D-glucosamine, 3-aminophenylboronic acid (APBA), α-D(+)-glucose (97%), and vancomycin hydrochloride were purchased from Aldrich and used without further purification. N,NDimethylformamide (DMF) and dichloromethane (CH2Cl2) were dried with CaH2 and distilled by a general method before use. Other reagents were of analytical grade and used as received. 2.2. Synthesis of Block Copolymers. Synthesis procedures for two block copolymers of PEG45-b-P(Asp-co-AspGA) and PEG114-bP(Asp-co-AspPBA) used in this study are shown in Figure 1. Block copolymers of poly(ethylene glycol)-b-poly(β-benzyl-L-aspartate) (PEG-b-PBLA) with different PEG lengths were synthesized by ringopening polymerization (ROP) of BLA-NCA according to ref 40 using PEG45-NH2 or PEG114-NH2 as an initiator. First, BLA-NCA was completely dissolved in dry CH2Cl2 followed by addition of initiator which had been dissolved in CH2Cl2. Then, the reaction mixture was stirred for 3 days at 35 °C under a dry argon atmosphere and the crude products were precipitated in 10-fold excess of cold diethyl ether and isolated by filtration. After washed twice with diethyl ether, the products were dried in vacuum. Block copolymers of poly(ethylene glycol)-b-poly(aspartic acid) (PEG-b-PAsp) with different PEG lengths were obtained by deprotection of PEG-b-PBLA in 1 N NaOH solution. After stirred for 10 h at room temperature, the solution was neutralized with 1 N HCl and concentrated by vacuum evaporation. The concentrated solution was dialyzed against water in a dialysis bag with a proper molecular cutoff and then solid PEG-b-PAsp was obtained by lyophilization. As for PEG45-b-P(Asp-co-AspGA) and PEG114-b-P(Asp-co-AspPBA), they were synthesized by partial modification of PEG45-bPAsp and PEG114-b-PAsp with glucosamine and APBA, respectively.21 First, PEG-b-PAsp was dissolved in DMF at 4 °C together with glucosamine or APBA, and then NHS/EDC in water was added to the reaction mixture under magnetic stirring. The solution was kept at 4 °C for 4 h followed by being dialyzed against deionized water for 3 days in a dialysis bag with a proper molecular cutoff. Finally, PEG45-bP(Asp-co-AspGA) and PEG114-b-P(Asp-co-AspPBA) were obtained by lyophilizing the corresponding polymer solution. 2.3. Preparation of Polymer Vesicles. Core−shell (CS) micelles of α-CD/PEG45-b-P(Asp-co-AspGA) were prepared by α-CD assisted self-assembly of the block copolymer in aqueous solution. First, α-CD and PEG45-b-P(Asp-co-AspGA) were separately dissolved in PBS 7.4 both with a concentration of 2 g/L. Then, a given volume of α-CD solution was dropwise added into the polymer solution under vigorous stirring according to desired feed ratio and the mixed solution was immersed in ultrasonic bath for 10 min. After standing overnight at room temperature, CS micelles were formed and the polymer concentration was finally fixed at 0.5 g/L by diluting with PBS 7.4.

Scheme 1. Schematic Illustration for the Fabrication of Polymer Vesicles by Using a α-CD/PEG Inclusion ComplexTemplated Strategy

copolymer poly(ethylene glycol)-b-poly(aspartic acid-co-aspartglucosamine) (PEG45-b-P(Asp-co-AspGA)) was self-assembled into core−shell (CS) micelles in the presence of α-CD based on the formation of insoluble α-CD/PEG45 inclusion complex. By addition of a phenylboronic acid (PBA)-containing block copolymer poly(ethylene glycol)-b-poly(aspartic acid-co-aspartamidophenylboronic acid) (PEG114-b-P(Asp-co-AspPBA)) and because of the complexation between P(Asp-co-AspGA) and P(Asp-co-AspPBA) via formation of GA/PBA cycloborate, core−shell-corona (CSC) micelles were prepared with insoluble α-CD/PEG45 inclusion complex as core, cross-linked 1373

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Figure 1. Synthesis of PEG45-b-P(Asp-co-AspGA) (A) and PEG114-b-P(Asp-co-AspPBA) (B). For the preparation of core−shell-corona (CSC) micelles of α-CD/ PEG45-b-P(Asp-co-AspGA)/P(Asp-co-AspPBA)-b-PEG114, the CS-1.5 micelles were used as template. First, PEG114-b-P(Asp-co-AspPBA) were completely dissolved in a NaOH solution of pH 10 with a concentration of 2 g/L. Then, a given volume of the polymer solution was quickly added into CS-1.5 solution under magnetic stirring. Finally, the CSC micelle solution was fixed at a polymer concentration of 0.50 g/L by diluting with PBS 7.4. Polymer vesicles were obtained with the CSC micelles by removing the assistant molecule α-CD through dialysis of CSC micelle solutions against PBS 7.4 at 50 °C for 2 days. The final vesicle solution was fixed at a polymer concentration of 0.30 g/L and kept under room temperature. 2.4. Characterizations. 1H NMR spectra of block copolymers as well as CS micelles of α-CD/PEG45-b-PAsp were recorded on a Varian UNITY-plus 400 M NMR spectrometer at room temperature using CDCl3, D2O, or NaOD/D2O as solvent, respectively. The degree of polymerization (DP) of the BLA units was determined from 1H NMR spectra with PEG as the inner standard. GPC of PEG-b-PBLA was measured at room temperature with a Waters 1515 chromatograph equipped with a Waters 2414 refractive index detector. DMF was used as eluents with a flow rate of 1.0 mL/min and narrowly distributed polystyrene was used as standard. The polymer assembles, including CS micelles, CSC micelles, and polymer vesicles, were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements. DLS measurement was performed on a laser light scattering spectrometer (BI-200SM) equipped with a digital correlator (BI-9000AT) at 636 nm under 37 °C. All samples were first prepared by filtering 2 mL of solutions through a 0.45 μm Millipore filter into a clean scintillation vial. TEM measurement was performed by using a JEM-100CXII electron microscope at an acceleration voltage of 100 kV. High resolution transmission electron microscopy (HRTEM) measurement was performed by using a Tecnai G2 F20 electron microscope at an acceleration voltage of 200 kV. Samples for

TEM were obtained by depositing diluted micelle solution (0.05 g/L) onto a carbon-coated copper EM grid and dried at room temperature in a vacuum. 2.5. Glucose-Responsiveness of Polymer Vesicles. The glucose-responsiveness of polymer vesicles in PBS 7.4 was investigated by DLS measurements in terms of the variations of hydrodynamic diameter (Dh) and light scattering intensity (LSI). To the vesicle solution with an initial polymer concentration of 0.5 g/L, a given volume of PBS solution with higher concentration of glucose was added to obtain mixed solutions with a polymer concentration of 0.3 g/L but different glucose concentrations from 2 to 10 g/L. LSI of the mixed solutions as a function of time was monitored and final Dh of polymer vesicles was recorded. 2.6. Drug Release Study. Vancomycin, a glycopeptide antibiotic useful for the treatment of a number of bacterial infections, was used as a model drug for glucose-triggered release experiment. The preparation of drug-loaded polymer vesicles was similar to that of unloaded ones, where vancomycin solution was added after the formation of α-CD/PEG45-b-P(Asp-co-AspGA) CS micelles. Briefly, 16.3 mL of CS-1.5 micelle solution containing 19.5 mg α-CD and 13 mg PEG45-b-P(Asp-co-AspGA) was prepared and 2 mL PBS 7.4 solution containing 25 mg vancomycin was added. Then 3.3 mL solution containing 6.5 mg PEG114-b-P(Asp-co-AspPBA) was added and CSC micelles of α-CD/PEG45-b-P(Asp-co-AspGA)/P(Asp-coAspPBA)-b-PEG114 were obtained with vancomycin encapsulated. The feeding amounts of vancomycin and block polymers were 25 and 19.5 mg, respectively. Finally, both α-CD and unloaded vancomycin were removed by dialysis of the drug-loaded CSC micelle solutions against PBS 7.4 at 50 °C for 2 days to obtain vancomycin-loaded polymer vesicles. The obtained drug-loaded polymer vesicles had the same polymer composition and concentration as unloaded ones. Sugar-responsive drug release was carried out by incubating the drugloaded polymer vesicles in PBS 7.4 with different sugars at 37 °C. 1374

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Biomacromolecules Vancomycin-loaded polymer vesicle solution was injected in a dialysis bag with a molecular weight cutoff of 12−14 kDa, and then the dialysis bag was immersed in PBS 7.4 with different sugars. At certain time intervals, 1 mL of the external buffer solution was taken to measure the vancomycin concentration by on a UV−vis spectrometer (UV-2550, Shimadzu) at the wavelength of 281 nm and 1 mL of fresh sugarcontaining solution was added. The cumulative release of vancomycin from the polymer vesicles was calculated by monitoring the changes of the UV−vis absorption intensity in the external media.

3. RESULTS AND DISCUSSION 3.1. Synthesis of Block Polymers. Structures of block copolymers used in this study as well as their 1H NMR spectra are shown in Figures 2 and 3. The degrees of polymerization

Figure 3. 1H NMR spectra of PEG114-b-PBLA in CDCl3 (A), PEG114b-PAsp in D2O (B), and PEG114-b-P(Asp-co-AspPBA) in NaOD/D2O (C).

intensity (integral area) of the protons of glucosamine units (peak f, contributed by 6n protons, except the 1-H which shown peak in the range of 5.0−5.5 ppm) to that of methylene protons of Asp units (peak c, contributed by 2 × 90 protons of PAsp90) based on the 1H NMR spectrum shown in Figure 2C. The number (i.e., n) of glucosamine modified on PAsp could be obtained by simple mathematics and the degree of PAsp modified by glucosamine was calculated to be 40%. The degree of PAsp modified by APBA was calculated by comparing the peak intensity (integral area) of the protons of PBA phenyl rings (peak g, contributed by 4n protons) to that of methylene protons of Asp units (peak c, contributed by 2 × 100 protons of PAsp100) based on the 1H NMR spectrum shown in Figure 3C. The number (i.e., n) of APBA modified on PAsp could be obtained by simple mathematics and the degree of PAsp modified by APBA was calculated to be 80%. 3.2. Self-Assembly of α-CD/PEG45-b-P(Asp-co-AspGA). The synthesized PEG45-b-P(Asp-co-AspGA) was a double hydrophilic block copolymer which could be molecularly dissolved in aqueous solution. When α-CD was added into the polymer solution under the influence of ultrasonic at room temperature, significant Tyndall effect was observed, which indicated the formation of colloidal particles in the mixed solution. This phenomenon could be attributed to the wellreported formation of α-CD/PEG inclusion complex because of α-CD threading onto PEG chains of PEG45-b-P(Asp-coAspGA) in the mixed solution.35−37 The α-CD/PEG inclusion complex, also called pseudopolyrotaxane, was relatively hydrophobic and tended to associated together, while the P(Asp-coAspGA) block was always hydrophilic. As a result, selfassembled core−shell (CS) micelles with a hydrophobically associated α-CD/PEG core surrounded by a P(Asp-co-AspGA) shell were obtained. In this study, PEG45 with a molecular

Figure 2. 1H NMR spectra of PEG45-b-PBLA in CDCl3 (A), PEG45-bPAsp in D2O (B), and PEG45-b-P(Asp-co-AspGA) in D2O (C).

(DP) of the BLA units in PEG45-b-PBLA and PEG114-b-PBLA were determined by using 1HNMR spectra shown in Figures 2A and 3A, where PEG45 and PEG114 were used as the inner standards, respectively. By comparing the peak intensities (integral area) of methylene protons of BLA units (peak c, contributed by 2n protons) to those of PEG (peak a, contributed by 4 × 45 or 4 × 114 protons of PEG45 or PEG114 respectively), the DP (i.e., n) of PBLA blocks were calculated by simple mathematics to be 90 and 100, respectively. The polydispersity indexes of PEG45-b-PBLA and PEG114-b-PBLA were determined by GPC to be 1.15 and 1.21, respectively. After deprotection of PBLA block, signals at about 7.2 ppm disappeared in 1H NMR spectra of Figures 2B and 3B, which indicated the complete removal of benzyl group and block copolymers of PEG-b-PAsp were obtained. PEG45-bP(Asp-co-AspGA) and PEG114-b-P(Asp-co-AspPBA) were synthesized by partial modification of PAsp block with glucosamine and APBA respectively. The degree of PAsp modified by glucosamine was calculated by comparing the peak 1375

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Figure 4. (A) 1HNMR spectra of block copolymer PEG45-b-PAsp90 in the absence (I) and in the presence (II) of α-CD in D2O under room temperature; (B) 2D NOESY spectrum of α-CD/PEG45-b-PAsp90 complex micelles in D2O under room temperature.

peak b (2.7 ppm) in spectrum II in the presence of α-CD reduced significantly in comparison with that in spectrum I without α-CD. This change suggested that the mobility of a portion of PEG chains was restricted because of threading into α-CD and the association of the formed pseudopolyrotaxane.38,42 2D NMR study on the α-CD/PEG 45-b-PAsp90 complex micelles was also performed and the 2D NOESY spectrum was provided in Figure 4B. Signals pointed by arrows in the 2D spectrum demonstrated correlation peaks between protons inside α-CD’s cavities and those of EG units, indicating both the occurrence of α-CD threading onto PEG chains and the formation of pseudopolyrotaxane-b-PAsp. By varying the feed ratio of α-CD/polymer, CS micelles with different compositions were prepared as CS-1.0, CS-1.5, and CS-2.0, as listed in Table 1, where 1.0, 1.5, and 2.0 referred to the weight ratios of α-CD to PEG45-b-P(Asp-co-AspGA). For CS-1.5 micelles, dynamic light scattering (DLS) measurements

weight (Mn) = 2000 instead of that with Mn = 5000 was used to form pseudopolyrotaxane because the ability of PEG threading into α-CD was molecular weight dependent and PEG with Mn in the range of 1000 to 3000 had the best ability to form relatively stable inclusion complex with α-CD.41 In order to prove the formation of α-CD/PEG inclusion complex, 1HNMR spectra of unmodified block copolymer PEG45-b-PAsp90 with and without α-CD in the solution were recorded as shown in Figure 4. PEG45-b-P(Asp-co-AspGA) was not used here on account of the overlap between peaks of GA group and α-CD. From spectrum I of molecularly dispersed PEG45-b-PAsp90 in Figure 4, the intensity ratio of peak at 3.7 ppm (a) contributed by protons of PEG to that at about 2.7 ppm (b) contributed by the methylene of Asp units was corresponding to the composition of the block copolymer and could be used to calculate the DP of Asp with PEG as the inner standard. However, the intensity ratio of peak a (3.7 ppm) to 1376

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Biomacromolecules Table 1. α-CD/PEG45-b-P(Asp-co-AspGA) Micelles with Different Compositions, Where All of the Micelle Solutions Had a Final Polymer Concentration of 0.5 g/L code

α-CD/polymer (w/w)

α-CD/EG (mol/mol)

Dh (nm)

CS-1.0 CS-1.5 CS-2.0

1.0:1 1.5:1 2.0:1

0.8:2 1.2:2 1.6:2

43.7 42.3 46.7

(Figure 6B) for the CSC micelles showed a well-defined spherical morphology with diameters in the range 35 to 50 nm. It should be noted that free α-CD existed in the micelle solution of CS-1.5 because of the dynamic equilibrium between α-CD and pseudopolyrotaxane of α-CD/PEG45. Addition of PEG114-b-P(Asp-co-AspPBA) may not result in the formation of too many α-CD/PEG114 pseudopolyrotaxane or the dissociation of the already formed CS-1.5 micelles because of the lower tendency of the longer PEG114 threading into α-CD.38,41 3.4. Formation of Polymer Vesicles. The threading process of α-CD onto PEG chains was reversible and the dethreading of α-CD can be easily achieved by heating.37 This was very valuable for the α-CD/PEG inclusion complex acting as the template for the preparation of polymer vesicles in our system due to the possibility of α-CD to be easily removed after the formation of CSC micelles. This method had been utilized in our previous work to construct polymer vesicles with crosslinked P4VP/PAA as wall from α-CD/PEG-P4VP/PAA−PEG CSC micelles.38 For the preparation of polymer vesicles, the CSC micelles were dialyzed against PBS 7.4 at 50 °C for 2 days. At this temperature, α-CD in the CSC micelle core should gradually slip away from PEG chains and diffuse out, resulting in cavity in the CSC micelles and thus the formation of polymer vesicles with a cross-linked P(Asp-co-AspGA)/P(Asp-coAspPBA) layer as wall and PEG chains as inner and outer coronas (Scheme 1). Though the α-CD/PEG inclusion complex was hydrophobic and surrounded by the cross-linked membrane of P(Asp-co-AspGA)/P(Asp-co-AspPBA), dethreading of α-CD from PEG may not be completely restricted. The encapsulated α-CD/PEG inclusion complex should be relatively a soft sphere and the pseudopolyrotaxane may be slightly mobile especially under elevated temperature (i.e., 50 °C). The cross-linked membrane of P(Asp-co-AspGA)/P(Aspco-AspPBA) was formed through a “self-assembly” process without inward contraction, which may not result in a very congested micelle core that could restrain the dethreading of αCD from PEG. Additionally, the cross-linked membrane may not be completely compact but be porous for α-CD diffusing through. Overall, it was possible for α-CD dethreading from PEG and diffusing out of the micelle core through the vesicular membrane and thus polymer vesicles could be obtained. The cycloborates formed by GA and PBA were hydrophilic but stable under these conditions and thus maintained the structure integrity of the polymer vesicles. Besides, both the inner and

indicated a narrow and monodisperse size distribution (Figure 5A) and a number-average diameter (Dh) of 42.3 nm at 37 °C. Variation of the composition of α-CD/polymer did not much affect the Dh of CS micelles as shown in Table 1. Transmission electron microscopy (TEM) image (Figure 5B) showed a welldefined and nearly spherical morphology for the dried CS-1.5 micelles with diameters ranging from 25 to 30 nm. 3.3. Formation of Core−Shell-Corona Micelles. Phenylboronic acid (PBA) and its derivatives, with a pKa about 8.2, have been well-reported in the synthesis of glucose-responsive materials because of the transformation from hydrophobic to hydrophilic state upon combining glucose.18,43 PBA-modified polymers have also been reported in the formation of glucoseresponsive materials by complexation with polyols, including poly(vinyl alcohol) (PVA)44 and glycopolymers.45,46 In our previous studies,21,23 two kinds of block copolymers, which were partially modified with PBA and glucosamine (GA), respectively, were self-assembled into complex micelles with PBA- and GA-containing blocks cross-linked as core and PEG as shell. This was caused by the combination between PBA and GA via formation of cycloborates. In this work, aqueous solution of PEG114-b-P(Asp-co-AspPBA) was added into the micelle solution of CS-1.5 with a P(Asp-co-AspGA) shell, complexation between P(Asp-co-AspGA) and P(Asp-co-AspPBA) led to the formation of core−shell-corona (CSC) micelles with α-CD/PEG45 inclusion complex as core, crosslinked P(Asp-co-AspGA)/P(Asp-co-AspPBA) as shell, and PEG114 as corona. A weight ratio of 2:1 for PEG45-b-P(Aspco-AspGA)/PEG114-b-P(Asp-co-AspPBA) was used in the fabrication of CSC micelles and the molar ratio of GA/PBA was calculated to be 1:0.8. DLS study on the CSC micelles demonstrated a narrow and monodisperse size distribution (Figure 6A) at 37 °C and the Dh was determined to be 77.6 nm which was larger than that of CS-1.5 micelles because of the incorporation of PBA-containing block copolymer. TEM image

Figure 5. Size distribution of α-CD/PEG45-b-P(Asp-co-AspGA) core−shell micelles (CS-1.5) in PBS 7.4 at 37 °C (A) and TEM image of the CS-1.5 micelles (B). 1377

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Figure 6. Size distribution of the α-CD/PEG45-b-P(Asp-co-AspGA)/P(Asp-co-AspPBA)-b-PEG114 CSC micelles in PBS 7.4 (A) and TEM image of the CSC micelles (B).

Figure 7. Size distribution of the polymer vesicles in PBS 7.4 (A) and TEM image (with an inset of HRTEM image) of the polymer vesicles (B).

polymer vesicle solution in the absence of glucose changed little with time, which indicated the stability of the polymer vesicles. When glucose was added, the LSIs increased quickly in the first 20 min and then slowly leveled off in 100 min. Higher glucose concentration led to faster increase of LSI in the first 20 min and larger increment of LSI in 100 min. The polymer vesicles demonstrated narrow and monodisperse size distributions after incubation with different concentrations of glucose for 100 min as shown in Figure 8B. The calculated Dhs of polymer vesicles in the presence of glucose with concentrations of 2, 5, and 10 g/L were 80.8, 117.9, and 155.3 nm, respectively, which increased with glucose concentration and agreed with the changes of LSIs. Both the increases of LSI and Dh could be attributed to the swelling of the polymer vesicles in response to glucose. When glucose was added into the polymer vesicle solution, the dynamic equilibrium between GA and PBA were broken and the competitive combination of glucose with PBA could replace GA, which could result in partial disintegration of the crosslinked P(Asp-co-AspGA)/P(Asp-co-AspPBA). Thus, the polymer vesicles would swell as illustrated in Figure 8C because of the decreased cross-linking degree of the vesicular wall. Higher glucose concentration could lead to more cross-linking points being destroyed and thus larger increases in LSI and Dh of the polymer vesicles. It should be pointed out that the polymer vesicles were stable when they were incubated with 2, 5, and 10

outer PEG chains of the polymer vesicles were hydrophilic, the cross-linked layer would not collapse and the polymer vesicles could still disperse well and keep their vesicle morphology in aqueous media. DLS study on the polymer vesicles showed a narrow and monodisperse size distribution (Figure 7A) at 37 °C and the Dh was determined to be 60.3 nm which was smaller than that of CSC micelles. This could be attributed to the shrinkage of the CSC micelles caused by leave of α-CD from the core of the CSC micelles and dissociation of relatively rigid pseudopolyrotaxane. TEM image (Figure 7B,) confirmed the formation of polymer vesicles and demonstrated clear hollow morphology with diameters in the range of 40−60 nm. The HRTEM image (inset in Figure 7B) showed distinct vesicular structure for the polymersomes. 3.5. Glucose-Responsiveness of Polymer Vesicles. As discussed above, the polymer vesicles had a cross-linked wall of P(Asp-co-AspGA)/P(Asp-co-AspPBA) based on the complexation between GA and PBA via formation of cycloborates. This kind of cycloborates was dynamically stable in PBS 7.4 but could be destroyed in the presence of glucose because of the GA moieties being replaced by glucose, which may endow the polymer vesicles with glucose-responsiveness.21,23 DLS measurements in terms of the variations of light scattering intensity (LSI) and hydrodynamic diameter (Dh) upon addition of glucose were used to evaluate the glucose-responsiveness of the polymer vesicles. As showed in Figure 8A, the LSI of the 1378

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Biomacromolecules

Figure 8. Glucose-responsiveness of the polymer vesicles in terms of normalized light scattering intensity as a function of time in aqueous solutions of PBS 7.4 at 37 °C (A), size distributions of polymer vesicles after incubation in PBS 7.4 with different glucose concentrations for 100 min at 37 °C (B), proposed structure changes of the polymer vesicles in the presence of glucose (C), and TEM image of polymer vesicles after incubation with 5 g/L glucose for 100 min (D).

g/L glucose for a longer time, as indicated by the constant LSI and Dh. Complete disassembly of the polymer vesicles was only observed in the presence of glucose with a concentration 50 g/ L as indicated by quickly decreased LSI and almost undetectable Dh. This was consistent with our previous study that higher glucose concentration, such as 50 g/L, was necessary for the complete disintegration of the cross-linked P(Asp-co-AspGA)/P(Asp-co-AspPBA) layer.21 TEM image (Figure 8D) of the polymer vesicles incubated with glucose for 100 min showed a well-defined hollow spherical morphology with diameter in the range of 80−110 nm, which was larger than that without glucose and also confirmed the swelling of polymer vesicles in response to glucose. 3.6. Sugar-Triggered Drug Release from Polymer Vesicles. Polymer vesicles have the superiorities of larger loading capacity and easier encapsulation of hydrophilic molecules. In this study, vancomycin, a water-soluble glycopeptide antibiotic with a molecular weight of 1449 Da, was loaded during the self-assembly of α-CD/PEG45-P(Asp-coAspGA) followed by addition of PEG114-b-P(Asp-co-AspPBA) and then removal of α-CD and unloaded drug through dialysis against PBS 7.4. The drug loading efficiency (LE) and loading content (LC) of the polymer vesicles were determined to be 48.3 and 38.2%, respectively, both were relatively high. This might be attributed that, on the one hand, there was stronger interaction between vancomycin and the inclusion complex of α-CD/PEG45, and on the other hand, the vesicular membrane could effectively intercept vancomycin during dialysis because of its large molecular weight. This suggested that the polymer vesicles could be excellent nanocarriers for the encapsulation of relatively large drug molecules. Sugar-triggered drug release

from the polymer vesicles was carried out under different conditions and the release profiles are shown in Figure 9. The

Figure 9. Release profiles of vancomycin from polymer vesicles under different conditions at pH 7.4.

encapsulated vancomycin was barely released out during the incubation in PBS 7.4 in 14 h, which suggested that the drugloaded polymer vesicles were very stable without sugars and could effectively avoid drug leakage during storage. When glucose was added with a concentration of 5 g/L, the drug release rate was fast in the first 3 h, then slowed down and leveled off in 15 h with a cumulative drug release amount of 50.9%. The glucose-triggered drug release may be attributed to the swelling of the vesicular membrane in response to glucose which decreased the cross-linking density and thus increased 1379

DOI: 10.1021/acs.biomac.5b00155 Biomacromolecules 2015, 16, 1372−1381

Article

Biomacromolecules

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the permeability in the vesicular membrane. As a result, the diffusion of vancomycin across the vesicular membrane became easier. When fructose was used, comparing with glucose, a faster drug release rate was observed and a larger cumulative drug release amount was obtained as 92.7% in 15 h. These could be ascribed to the fact that fructose had a much higher affinity for PBA than glucose,47 which led to a larger swelling degree and thus a higher permeability of the vesicular membrane. It is known that vancomycin must be given intravenously for systemic therapy and is often injected twicedaily because of its short half-life (4−8 h). Fast administration or high drug concentration frequently arouses thrombophlebitis and red man syndrome. When encapsulated in glucoseresponsive polymer vesicles and administrated intravenously, vancomycin could be slowly released during blood circulation and in the presence of glucose with a prolonged half-life and an avoidance of the above serious side effects. Further study on the vancomycin-loaded glucose-responsive polymer vesicles in the application of antibacterial is under design and will be reported in the future.

4. CONCLUSIONS In this study, glucose-responsive polymer vesicles were fabricated based on the complexation between a GA-containing block copolymer PEG45-b-P(Asp-co-AspGA) and a PBAcontaining block copolymer PEG114-b-P(Asp-co-AspPBA) by using a α-CD/PEG inclusion complex-templated method. The obtained polymer vesicles composed of cross-linked P(Asp-coAspGA)/P(Asp-co-AspPBA) layer as vesicular membrane and PEG chains as both inner and outer coronas. The polymer vesicles displayed significant glucose-responsiveness which was indicated by the increases of LSI and Dh upon incubating with glucose. The diameter of the polymer vesicles increased from the initial 60.3 nm to 80.8, 117.9, and 155.3 nm, respectively, after incubation with glucose with concentrations of 2, 5, and 10 g/L for 100 min. TEM images demonstrated well-defined hollow spherical morphology for the polymer vesicles either in the absence of glucose or incubating with glucose for 100 min. Vancomycin as a water-soluble model drug was effectively encapsulated in the polymer vesicles with very high LE and LC and sugar-triggered drug release was successfully achieved. This kind of polymer vesicles may be excellent nanocarriers for glucose-responsive drug delivery and may find application in the future.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 21274001, 91127045, and 51390483), the National Basic Research Program of China (973 Program, No. 2011CB932503), and PCSIRT (IRT1257).

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DOI: 10.1021/acs.biomac.5b00155 Biomacromolecules 2015, 16, 1372−1381