Phenylboronic Acid-Based Complex Micelles with Enhanced Glucose

Sep 7, 2012 - ABSTRACT: Polymeric nanoparticles with glucose-respon- siveness under physiological conditions are of great interests in developing drug...
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Phenylboronic Acid-Based Complex Micelles with Enhanced GlucoseResponsiveness at Physiological pH by Complexation with Glycopolymer Rujiang Ma, Hao Yang, Zhong Li, Gan Liu, Xiaocheng Sun, Xiaojun Liu, Yingli An, and Linqi Shi* Key Laboratory of Functional Polymer Materials, Ministry of Education, and Institute of Polymer Chemistry, Nankai University, Tianjin 300071, China S Supporting Information *

ABSTRACT: Polymeric nanoparticles with glucose-responsiveness under physiological conditions are of great interests in developing drug delivery system for the treatment of diabetes. Herein, glucose-responsive complex micelles were prepared by self-assembly of a phenylboronic acid-contained block copolymer PEG-b-P(AA-co-APBA) and a glycopolymer P(AA-co-AGA) based on the covalent complexation between phenylboronic acid and glycosyl. The formation of the complex micelles with a P(AA-co-APBA)/P(AA-co-AGA) core and a PEG shell was confirmed by HNMR analysis. The glucoseresponsiveness of the complex micelles was investigated by monitoring the light scattering intensity and the fluorescence (ARS) of the micelle solutions. The complex micelles displayed an enhanced glucose-responsiveness compared to the simple PEG-bP(AA-co-APBA) micelles and the sensitivity of the complex micelles to glucose increased with the decrease of the amount of P(AA-co-AGA) in the compositions. The cytotoxicity of the polymers and the complex micelles was also evaluated by MTT assay. This kind of complex micelles may be an excellent candidate for insulin delivery and may find application in the treatment of diabetes.

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PBA, that is, about pH 9,14,15 or in the presence of glucose with a higher concentration, for example, 20 g/L,16 which hindered their application in glucose-responsive delivery of insulin under the physiological condition (pH 7.4, 1−3 g/L glucose). Great efforts have been devoted to decrease the apparent pKa of PBAbased glucose-responsive materials and enhance their sensitivity to glucose. Kataoka et al6,17 synthesized glucose-responsive polymer and gels that could be operated at the physiological pH by using a new PBA derivative DDOPBA possessing an appreciably low pKa (∼7.8). Lately, amino groups were introduced either into the polymer or in the vicinity of the phenylboronic acid moiety to decrease the apparent pKa value of PBA because of the possible coordination between nitrogen and boron.5,18,19 But these techniques generally involved in complicated synthetic chemistry, which was unfavorable for the preparation and application of these glucose-responsive materials. Glucose-responsive materials were also prepared by complexation between PBA-contained polymer and polyol polymers, such as polyvinyl alcohol (PVA) and glycopolymers. The resultant complex can be dissociated in the presence of glucose which is able to form a stronger complex. Springsteen et al20 reported that the apparent pKa of phenylboronic acid/polyol

INTRODUCTION In the past decades, glucose-responsive materials have attracted great interest because of their potential applications in the construction of self-regulated insulin delivery systems for the treatment of diabetes. Among three kinds of typical glucoseresponsive materials,1−3 phenylboronic acid (PBA) and its derivatives are the most studied because of their versatility for different designs and better stability than protein-based systems (glucose oxidase and concanavalin A).4 Phenylboronic acid and its derivatives are a kind of weak acids with a reported pKa of 8.2−8.6.5,6 In aqueous solution, PBA exists in an equilibrium between a triangular form and a tetrahedral form.7 The triangular form is neutral and hydrophobic but the tetrahedral form is negatively charged and hydrophilic. The tetrahedral form of PBA can complex with cis-diol compounds, for example, glucose and form stable but hydrophilic phenylborates,8−10 which shifts the equilibrium in the direction of increasing hydrophilic form but decreasing the hydrophobic form. As a result, the PBA-based materials are endowed with glucose-responsiveness. A series of studies on the PBA-based glucose-responsive materials have been reported for the construction of selfregulated insulin delivery systems. Typical examples are gels7 and microgels composed of PBA and poly(N-isopropylacrylamide) (PNIPAM),11−13 which can swell and release insulin in response to glucose. However, these materials displayed effective glucose-responsiveness only slightly above the pKa of © 2012 American Chemical Society

Received: August 12, 2012 Revised: September 6, 2012 Published: September 7, 2012 3409

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Scheme 1. Schematic Illustration for the Formation and Glucose Responsive Disintegration of the PEG-b-P(AA-co-APBA)/ P(AA-co-AGA) Complex Micelles

7.4. However, higher glucose concentration such as 5 g/L was needed to trigger the release of insulin. Herein, PBA-contained block copolymer PEG-b-P(AA-coAPBA) was used to complex with a PAA-based glycopolymer poly(acrylic acid-co-acrylglucosamine) (P(AA-co-AGA)), forming complex micelles with a P(AA-co-APBA)/P(AA-co-AGA) core and a PEG shell as illustrated in Scheme 1. The complex micelles combined a variety of advantages such as stability against aggregation due to PEG shell, fast response to glucose due to nanoscale size and hydrophilic micelle core, and more importantly better glucose sensitivity ascribed to the decrease of apparent pKa of PBA as a result of complexation with glycopolymer.

complexes dropped 2−4 pKa units upon complexation, which could result in significant glucose-responsiveness under physiological pH. This drop in pKa is because the polyol complex itself is a stronger acid than the phenylboronic acid derivative.21 Zhang et al22−24 reported self-assembled hybrid nanoparticles based on the complexation between poly(3acrylamidophenylboronic acid) (PAPBA) and glycopolymers of poly(2-lactobionamidoethyl methacrylate) (PLAMA) or poly(maleimide glucosamine) (PMAGA). The insulin-loaded nanoparticles by intranasal administration led to a significant decrease in the physiological glucose levels. So far, most of the glucose-responsive materials reported in the literature are in the form of gels or microgels. Bulk gels are not ideal candidates for in vivo insulin delivery because of their slow response to glucose and the difficulty for administration. Glucose-responsive microgels of several nanometers in diameter can overcome the shortcomings of bulk gels but encounter with the plight of quick clearance in blood circulation because of the lack of protection as PEG on selfassembled micelles. Recently, glucose-responsive micelles selfassembled by PBA-contained amphiphilic block polymers have been reported as potential and excellent carriers for insulin delivery due to their faster response to glucose at neutral pH, longer circulation stability as a result of the protection by PEG, and easier drug loading upon self-assembly. Yang et al25,26 synthesized an amphiphilic block copolymer PEG-b-PPBDEMA with phenylborate ester as a leaving group in response to glucose in the hydrophobic block. This block copolymer could self-assemble into core−shell micelles, which could be used as a glucose-sensitive drug carrier and successfully realized glucoseresponsive release of insulin at neutral pH. In our previous work,27,28 PBA-contained block copolymer poly(ethylene glycol)-block-poly(acrylic acid-co-acrylamidophenylboronic acid) PEG-b-P(AA-co-APBA) was also synthesized by modification of PEG-b-PAA with 3-aminophenylboronic acid (APBA), which self-assembled into core−shell micelles but dissociated in response to glucose at pH 7.4. Solid 11B MAS NMR analysis indicated that coordination between carboxyl and PBA induced the transform of PBA from the trigonal planar to the tetrahedral form, resulting in the decrease of apparent pKa and glucose-responsiveness of the micelles at pH

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EXPERIMENTAL SECTION

Materials. Poly(ethylene glycol) monomethyl ether (CH3OPEG114−OH) (Mw = 5000 and polydispersity index (PDI) = 1.05) was purchased from Fluka. 2-Bromo-2-methylpropionyl bromide (BMPB, 98%) was purchased from Acros Organics and used without further purification. t-Butyl acrylate (tBA) (Aldrich, 98%) was dried with CaH2 and then distilled under vacuum. CuCl was purchased from Aldrich and purified according to ref 29. 3-Aminophenylboronic acid (APBA), D-glucosamine, Alizarin Red S (ARS), α-D(+)-glucose (97%), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl, 98%) were purchased from Aldrich and used without further purification. Tris-(2-dimethylaminoethyl) amine (Me6TREN) was prepared from tris(2-aminoethyl)amine (TREN, Aldrich) according to ref 30. Other reagents were of analytical grade and used as received. Synthesis of PEG-b-P(AA-co-APBA) and P(AA-co-AGA). Phenylboronic acid-contained block copolymer PEG114-b-P(AA47-coAPBA73)120 was synthesized by partial modification of the poly(acrylic acid) (PAA) block with 3-aminophenylboronic acid (APBA) in the presence of EDC as has been reported in out previous work.27 The composition was determined by HNMR analysis with the PEG as the inner standards. The glycopolymer P(AA14-co-AGA35) was synthesized by partial modification of homopolymer poly(acrylic acid) (PAA) with glucosamine in the presence of EDC. The composition was determined by a combination of GPC and HNMR analysis. PAA used in this study was obtained by hydrolysis of poly(t-butyl acrylate) (PtBA), which was synthesized by ATRP, in the presence of trifluoroacetic acid (TFA).31 Details for synthesis and characterization of the polymers used in this study can be seen Supporting Information. 3410

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Figure 1. Normalized light scattering intensity of the PEG-b-P(AA-co-APBA) micelle solutions at different pH values (A) in the absence of glucose and (B) in the presence of 5 g/L glucose as a function of time. Preparation of Micelles. First, the block copolymer was molecularly dissolved in a NaOH solution of pH 10 with a concentration of 2.5 g/L. For the preparation of PEG-b-P(AA-coAPBA) simple micelles, acidic water of pH 2 was then slowly added into the polymer solution under vigorous stirring until the appearance of opalescence, which indicated the formation of micelles. The pH value of the micelle solution was measured to be 8.2. Finally, the micelle solution was diluted to a concentration of 0.5 g/L by buffer solutions (with or without glucose) with targeted pH values for study. For the preparation of PEG-b-P(AA-co-APBA)/P(AA-co-AGA) complex micelles, P(AA-co-AGA) was first dissolved in PBS of pH 7.4 with a concentration of 1.5 g/L. Then, a given volume of PEG-bP(AA-co-APBA) solution at pH 10 was quickly added into the P(AAco-AGA) solution with designed ratio under vigorous stirring. The mixed solution became opalescent instantly due to the formation of complex micelles. Finally, the micelle solution was diluted to a concentration of 0.5 g/L by PBS of pH 7.4. MTT Assay for Cell Viability. 293T cells were seeded in a 96-well plate at an initial density of 104 cells/well in 100 μL RPMI1640 complete media containing 10% FBS at 37 °C in humidified 5% CO2 atmosphere. After incubation of 24 h, the culture medium of each well was replaced with 100 μL fresh medium containing various concentrations of polymers and micelles. Twenty-four hours later, the culture media were replaced with 25 μL of MTT solution (1 g/L final concentration) and the cells were further incubated for another 4 h. Then, the solution was replaced with 150 μL of DMSO and the plates were slightly shaken for 10 min. The optical absorbance was measured at 570 nm using a microplate reader (Labsystem, Multiskan, Ascent, Finland). Cells without materials were used as the control. Characterizations. The 1H NMR spectra of polymers and micelles were recorded on a Varian UNITY plus-400 M NMR spectrometer at room temperature with tetramethylsilane (TMS) as the internal standard and D2O as the solvent. The complex micelles were characterized by a combination of dynamic light scattering (DLS) and static light scattering (SLS) measurements, which were performed on a laser light scattering spectrometer (BI-200SM) equipped with a digital correlator (BI-9000AT) at 636 nm under room temperature. All samples were obtained by filtering through a 0.45 μm Millipore filter into a clean scintillation vial. Fluorescence emission spectra were recorded on a Hitachi F-4600 fluorescence spectrophotometer at the excitation wavelength of 468 nm under room temperature.

P(AA-co-APBA) with a molar ratio of 61% APBA units on the P(AA-co-APBA) chain was used to prepare complex micelles by complexation with a glycopolymer P(AA-co-AGA). Similar solution properties of the simple PEG-b-P(AA-co-APBA) micelles were also observed based on the time-dependent light scattering intensity as shown in Figure 1. When the pH value of the micelle solution was changed by addition of buffer solutions with different pH values, the simple PEG-b-P(AA-coAPBA) micelles were stable at and below pH 8.2 but were instantly disintegrated at pH 10.3. This could be attributed to the inherent pH-responsiveness of phenylboronic acid. PBA had a pKa value of 8.2−8.6 as mentioned above. In the solutions of pH value at and/or below pH 8.2, most of the PBA groups on the PEG-b-P(AA-co-APBA) chains were in the protonated and hydrophobic form, which tended to hydrophobically associate together and resulted in micelles due to the steric stabilization by PEG chains. While in more basic solution of pH 10.3, PBA groups were generally in the deprotonated and hydrophilic form, which led to the molecular dispersion of the block copolymer. When glucose with a concentration of 5 g/L was added, the micelles behaved differently at various pH values as indicated in Figure 1B. The pH-dependent glucose-responsiveness of the PEG-bP(AA-co-APBA) micelles could be explained as follows. It was reported that the glucose-responsiveness of PBA-based polymer was due to the reversible combination of PBA and glucose as illustrated in Supporting Information Scheme S3.8,9 The neutral and hydrophobic form of PBA could not stably combined with glucose while the negatively charged PBA could form stable and hydrophilic phenylborate with glucose, which shifted the equilibrium in the direction of increasing hydrophilic form but decreasing the hydrophobic form. At pH 6.8, almost all of the PBA groups were in the neutral and hydrophobic form, glucose had little influence on the PEG-b-P(AA-co-APBA) micelles as indicated in Figure 1B. At elevated pH value, a fraction of PBA groups was in the negatively charged form. The added glucose could combine with the negatively charged PBA and induce the increase of hydrophilic PBA. As a result, a fraction of micelles was disintegrated. The higher pH values, the more notable glucose-responsiveness of the micelles. The micelles were quickly and completely disintegrated in 25 min at pH 8.2 but only a very weak glucose-responsiveness was observed in 60 min at pH 7.4. Formation of the Complex Micelles. Enlightened by the work reported by Springsteen et al20 that complexation with polyol polymer could significantly enhance the glucoseresponsiveness of PBA-contained materials, hydrophilic glyco-

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RESULTS AND DISCUSSION pH- and Glucose-Responsiveness of the PEG-b-P(AAco-APBA) Micelles. As reported in our previous work,27 block copolymer PEG-b-P(AA-co-APBA), which had a molar ratio of 63% APBA units on the P(AA-co-APBA) chain, was soluble in basic solution but could self-assemble into core−shell micelles at and below pH 8.2. The micelles could be slowly destroyed in the presence of glucose with the lowest concentration of 5 g/L at pH 7.4. In this study, a similar block copolymer of PEG-b3411

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the size of the complex micelles would increase with the increasing P(AA-co-AGA) content. The complex micelles were stable with various feed ratios and would not fall apart even with large excess of glycopolymer. In this study, the glycopolymer always acted as the cross-linking agent for PBA-contained chains, which connected with the P(AA-co-APBA) chains forming the micelle core through the stronger PBA−diol interaction. When a large excess of glycopolymer existed, only a part of them would inevitably connect with the P(AA-co-APBA) chains and result in crosslinked network as the micelle core. The excessive glycopolymer would be free in the solution. It was believed that the micelle structure would be stable against the free glycopolymer because of the difficulty of the glycopolymer chains diffusing in or out the micelle core, which was the result of the stronger PBA−diol interaction and the entanglement between polymer chains. In fact, in the preparation of the complex micelles, Tyndall effect appeared at the very initial stage when a small volume of PEGb-P(AA-co-APBA) solution was added into the P(AA-co-AGA) solution, which indicated the formation of stable complex micelles with a very large excess of the glycopolymer. The formation of the complex micelles was also confirmed by HNMR analysis as shown in Figure 3. Spectra A and D were

polymer P(AA-co-AGA) was used to interact with PEG-bP(AA-co-APBA) for the preparation of glucose-responsive complex micelles in this study. A series of complex micelles with different compositions was prepared as listed in Table 1. Table 1. PEG-b-P(AA-co-APBA)/P(AA-co-AGA) Complex Micelles with Different Compositions (w/w), Where All of the Micelle Solutions Had a Final Concentration of 0.5 g/L code

composition (w/w)

PBA/AGA (mol/mol)

Dh (nm)

Rg/Rh

CM-0 CM-0.75 CM-1.0 CM-1.5 CM-2.5 CM-4.5

1:0 1:0.75 1:1 1:1.5 1:2.5 1:4.5

1:0 1:0.87 1:1.16 1:1.74 1:2.90 1:5.22

215 152 170 192 218 236

0.94 0.84 0.79 0.67 0.76 1.02

Under appropriate conditions, P(AA-co-AGA) acted as a multifunctional cross-linker of PEG-b-P(AA-co-APBA) because of the specific complexation between glycosyl and PBA. The cross-linked microgels were spontaneously segregated by PEG chains, forming complex micelles with a P(AA-co-APBA)/ P(AA-co-AGA) core and a PEG shell, which was very similar with PIC micelles reported by Kataoka et al.32 Figure 2A shows

Figure 2. Size distributions of the complex micelles with different compositions, where all of the micelle solutions had a final concentration of 0.5 g/L.

Figure 3. HNMR spectra of (A) molecularly dissolved PEG-b-P(AAco-APBA) at pH 10.0, (B) PEG-b-P(AA-co-APBA) micelles (CM-0) at pH 7.4, (C) complex micelles of PEG-b-P(AA-co-APBA)/P(AA-coAGA) with weight ratio of 1:1.5 (CM-1.5) at pH 7.4, and (D) molecularly dissolved P(AA-co-AGA) at pH 7.4 in D2O solutions.

the hydrodynamic diameter distributions f(Dh) of the complex micelles with different compositions at the scattering angle of 90°. The simple PEG-b-P(AA-co-APBA) micelles (CM-0) have an average hydrodynamic diameter (Dh) of 215 nm. When complex micelles were formed with a feed ratio of 1:0.75 (w/ w), the Dh was decreased to 152 nm. The increase of P(AA-coAGA) in the complex micelles led to a continuous increase of Dh as indicated in Table 1 and Figure 2A. The reason for the increase in diameter of the complex micelles with the increasing of P(AA-co-AGA) content could be explained from two aspects. On the one hand, more P(AA-co-AGA) would be incorporated in the micelle core at a higher P(AA-co-AGA) content, which resulted in complex micelles with higher weight and thus larger size. On the other hand, the incorporated glycopolymer would cause the micelle core to be not only hydrophilic but also swollen, which was confirmed by the increase of Rg/Rh values with the increasing of P(AA-co-AGA) content.33,34 As a result,

recorded for the molecularly dispersed PEG-b-P(AA-co-APBA) at pH 10.0 and P(AA-co-AGA) at pH 7.4 in D2O solutions. Sharp peaks were observed for phenyl ring of PBA (c, 7.3 ppm) and glycosyl (e, 3.0 − 4.0, and 5.2 ppm) respectively. When the PEG-b-P(AA-co-APBA) solution was adjusted to pH 7.4, the peaks contributed by PBA were extended and reduced in terms of intensity (Figure 3B) compared to those in Figure 3A, which indicated the decreased mobility of P(AA-co-APBA) chains because of the hydrophilic (pH 10.0) to hydrophobic (pH 7.4) transition.35−37 When PEG-b-P(AA-co-APBA) was added into the P(AA-co-AGA) solution to prepare complex micelles, the intensities of the peaks contributed by PBA were further reduced (Figure 3C, c), indicating a further loss of chain mobility due to the complexation with P(AA-co-AGA). At the same time, the peaks contributed by glycosyl were also 3412

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Figure 4. Glucose-responsiveness of the complex micelles with different compositions (A) CM-0, (B) CM-0.75, (C) CM-1.5, and (D) CM-4.5 in aqueous solutions of PBS 7.4 in terms of normalized light scattering intensity as a function of time.

ditions. In our systems, the enhancement of the glucoseresponsiveness may be both attributed to the coordination between carboxyl and PBA and the complexation of PBA with glycopolymer. When the feed ratio of P(AA-co-AGA) was further increased, the resultant complex micelles of CM-1.5 and CM-4.5 displayed a decreased glucose-responsiveness as shown in Figure 4C and 4D. The CM-4.5 complex micelles were entirely intact in the presence of 2 g/L glucose and cannot be completely disintegrated in the presence of 50 g/L glucose as indicated in Figure 4D. This may be attributed to the increased degree of complexation between the block copolymer and the glycopolymer. It can be seen from Figure 4A and 4B that the light scattering intensities of the micelle solutions of CM-0 and CM-1.5 decreased slowly in 60 min in response to 5 g/L glucose at pH 7.4, however, their hydrodynamic diameters almost kept constant as indicated in Figure 5. This may explained as follows. In the case of CM-0, the micelle core was hydrophobically associated, which might be relatively compact and not allow glucose to freely enter into and swell the micelles. Glucose would interact with the periphery of the core, which would be peeled off because of the leaving of water-soluble block copolymer with glucose attached. Once the block copolymer started to leave, the structure of micelles might become unstable and would be quickly destroyed. In the cases of complex micelles, the core was a cross-linked network, which would allow glucose to freely enter into. There might be an induction period during which the glycopolymer was replaced by glucose until the cross-linked network suddenly fall apart. Notable increase in diameter was also not detected for both CM-0 and CM-0.75 micelles based on the results of supplementary experiment with 20 g/L glucose. This suggested that both the simple micelles CM-0 and complex micelles CM0.75 were destroyed in a one-by-one manner, which was similar to that of PCL-cored micelles digested by enzymes, instead of

extended and reduced in terms of intensity (Figure 3C, e) compared to those in Figure 3D, which indicated the decreased mobility of P(AA-co-AGA) chains due to the complexation with P(AA-co-APBA). It should be noted that the peak contributed by the PEG block (a) changed little under different situations, which indicated that the PEG block was well-soluble before and after the complexation. These facts demonstrated the formation of the complex micelles with a structure of P(AA-co-APBA)/ P(AA-co-AGA) core surrounded by the PEG shell. Glucose-Responsiveness of the Complex Micelles. PBA-based polymers have been well studied as the promising material for self-regulated insulin delivery. However, effective response of PBA-based polymers to glucose was difficult under physiological conditions (pH 7.4, 1−3 g/L glucose) because of its higher pKa, which restricted their application in treatment of diabetes. We have reported that the PEG-b-P(AA-co-APBA) micelles displayed glucose-responsiveness at pH 7.4 because of the complexation between carboxyl and PBA, but higher glucose concentration was needed. It can be clearly shown in Figure 4A that the CM-0 micelles were almost intact in 60 min in the presence of 2 g/L glucose and the light scattering intensity decreased only about 20% with 5 g/L glucose. Notable glucose-responsiveness was observed only above the glucose concentration of 10 g/L, which was 5 times of hyperglycemia. The CM-0 micelles were completely disintegrated in 30 min in the presence of 50 g/L glucose. When complex micelles were formed with a weight ratio of 1:0.75 (CM-0.75), the light scattering intensity decreased about 47% in 60 min with 2 g/L glucose as indicated in Figure 4B. The CM-0.75 micelles were completely disintegrated in 10 min. These facts suggest an enhanced glucose-responsiveness of the PBA-based micelles due to complexation with glycopolymer. It was reported by Springsteen et al20 that complexation with polyol compounds could reduce the apparent pKa of PBA-based materials and thus increase its glucose-sensitivity under the physiological con3413

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CM-0, CM-2.5, and CM-4.5 responded to glucose weakly as shown in Figure 6B. As the glucose concentration continued to increase, all of the complex micelles displayed better glucoseresponsiveness than the simple micelles CM-0 at the initial stage as shown in Figure 6C and 6D. But in the latter half of the time range, the complex micelles CM-4.5 displayed poorer glucose-responsiveness than CM-0, indicating that too high content of glycopolymer in the complex micelles was unfavorable for the sensitivity to glucose. It should be pointed out that when the content of glycopolymer was lower than the case of CM-0.75, stable complex micelles were not obtained because of the decreased complexation between PBA and glycopolymer. It was shown in Figure 6A−C that, at a glucose concentration of less than or equal to 20 g/L, the light scattering intensities gradually leveled off in 60 min. This suggested that the micelles would not disappear eventually. When glucose with a concentration of 50 g/L was added, the micelles except CM4.5 were quickly destroyed as indicated by very low light scattering intensity in Figure 6D and complete transparence for the solutions. It has been well reported that cycloborates of phenylboronic acid and diols were negatively charged and wellsoluble in water. In this study, the block copolymer with glucose attached was indeed water-soluble. It was shown in Figure 4A, for example, that the light scattering intensity of the CM-0 solution in the presence of 50 g/L glucose decreased to about 2% of the initial value in 60 min, which indicated the complete disintegration of the micelles and good solubility of the glucose-attached block copolymer. Dynamic light scattering results showed a monodisperse size distribution around 10 nm as indicted in Supporting Information Figure S4. The average diameter was determined to be 9.5 nm, which suggested the block copolymer was molecularly dispersed.

Figure 5. Time-dependence of hydrodynamic diameters of the micelles of CM-0 and CM-0.75 in the presence of 5 g/L glucose in aqueous solutions of PBS 7.4.

being wholly swollen before disintegration.38,39 The decrease of light scattering intensity of the micelle solution can attributed to the disintegration of micelles in response to glucose. This enables us to easily observe the glucose-responsiveness of the complex micelles by monitoring the light scattering intensities of the micelle solutions. Figure 6 shows the glucose-responsiveness of the complex micelles in the presence of glucose with different concentrations. Only the complex micelles of CM-0.75 displayed notable glucose-responsiveness, while other complex micelles almost did not response to glucose with a concentration of 2 g/ L as indicated in Figure 6A. When the glucose concentration was increased to 5 g/L, the complex micelles of CM-1.0 and CM-1.5 displayed a moderate glucose-responsiveness while

Figure 6. Glucose-responsiveness of the complex micelles in the presence of glucose with different concentrations, (A) 2, (B) 5, (C) 20, and (D) 50 g/L in aqueous solutions of PBS 7.4 in terms of normalized light scattering intensity as a function of time. 3414

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Figure 7. Fluorescence emission spectra of ARS (λext = 468 nm) in micelle solutions of (A) CM-0, and (B) CM-0.75 as function of time in the presence of 2 g/L glucose at PBS 7.4. The Insets are time-dependent fluorescence intensity of ARS at 565 nm in the presence of glucose with concentrations 2, 5, and 10 g/L.

the 5.5% of the remnant PBA was calculated to be 2.35 × 10−5 M, which was still larger than that of ARS (1.0 × 10−5 M), which also ensured the complete combination of ARS by PBA. In fact, combination between PBA and AGA can not be ideal because of the steric hindrance of the polymer backbone, more PBA groups would be available for combination with ARS. As a result, the highest fluorescence intensity was also obtained with the CM-0.75 in the absence of glucose. When glucose with a concentration of 2 g/L was added, only about 18% of ARS was replaced in the case of CM-0. While about 29% of ARS was replaced for CM-0.75, which was due to more diols in this case. The enhancement of the glucose-responsiveness of the complex micelles may be attributed to the three following reasons. First, the presence of carboxyl in the vicinity of PBA can induce the transform of PBA from the uncharged trigonal planar to the negatively charged tetrahedral form by coordination.28 The tetrahedral form of PBA is easier to form stable and hydrophilic phenylborate with cis-diols. Second, the complexation between PBA-contained polymer and glycopolymer results in not only the formation of complex micelles but also the drop of the apparent pKa of PBA because of formation of cyclic phenylborate.20 The glycosyl in the resultant PBA/ AGA complexes can be easily replaced by glucose under physiological conditions, which leads to the disintegration and glucose-responsiveness of the complex micelles. Third, the core of the complex micelles is composed of hydrophilic PBA/AGA complexes, in which the diffusion of glucose is easier than in hydrophobically associated PBA core. Overall, the glucoseresponsiveness of the phenylboronic acid-contained materials is enhanced by complexation with glycopolymer. Biocompatibility of drug delivery system is always very important for practical application. It was reported by AlShabanah et al40 that most of the boronated moieties and their derivatives possessed cytotoxic activity in murine and human tumors and different cell lines. The cytotoxicity of phenylboronic acid derivatives were also reported in other studies.22,24,41 It is necessary to verify the cytotoxicity of materials used in this study. Figure 8 shows the cytotoxicity of the two polymers and the complex micelles CM-1.5 on the 293T cells measured by MTT assay. The cell viability of PEGb-P(AA-co-APBA) was generally lower than 100% and decreased with the increase of concentration, indicating that the block copolymer was cytotoxic especially at higher concentration. The glycopolymer P(AA-co-AGA) was noncytotoxic with the cell viability basically higher than 100% under different polymer concentrations. As far as the complex micelles of CM-1.5 concerned, the cell viability was higher than

The glucose-responsiveness of the complex micelles was also characterized by monitoring the fluorescence of Alizarin Red S (ARS) in the micelle solution as shown in Figure 7. It is known that ARS is nonfluorescent in aqueous solution but becomes fluorescent when combines with PBA.20 The competitive combination of glucose with PBA can lead to fluorescence quenching of ARS. In the absence of glucose, both CM-0 and CM-0.75 solutions had the same fluorescent intensity of ARS (see Supporting Information for explanation). When glucose was added, the decrease of the fluorescence intensities of ARS in CM-0.75 solution was faster and more than that of CM-0 in the presence of glucose with different concentrations. This can be explained as follows. When glucose with a concentration of 5 g/L was added into the micelle solution, they would competitively combine with PBA and replace ARS, leading to the decrease of fluorescence intensity. In the case of CM-0, the core was relatively compact due to the hydrophobic association and diffusion of glucose into the micelle core would be slower. While in the case of CM-0.75, the micelle core was believed to have a loose structure due to the hydrophilicity of P(AA-coAGA) and the interpenetration between P(AA-co-APBA) and P(AA-co-AGA) chains. Glucose could easily diffuse into the micelle core and result in a faster replacement of ARS. Except ARS, the total diol moieties in the CM-0.75 solution (including glucose and AGA groups) were more than those in the CM-0 solution (only glucose). As a result, more ARS would be replaced by diols in the case of CM-0.75 and it was easy to understand that the fluorescence intensity of ARS in the CM0.75 solution was lower than that in the case of CM-0 at a certain time. The fluorescence intensities of CM-0.75 decreased to 71%, 62%, and 53% in the presence of glucose of 2, 5, and 10 g/L respectively in 60 min, which were lower than those of CM-0 with 82%, 76%, and 59%. These also confirmed the fact that the complex micelles of CM-0.75 displayed a better glucose-responsiveness than CM-0 under the physiological conditions (PBS 7.4). In the CM-0 solution, most of the PBA groups were in the neutral form and hydrophobically associated together forming the relatively compact micelle core, while only about 5.5% (1.79 × 10−4 M, which was calculated by the Henderson− Hasselbalch equation with the pKa of PBA as 8.6) of PBA groups were in the negatively charged form, which were more than the amount of ARS. The excess of negatively charged PBA groups could combine all of the ARS due to strong interaction between them and result in the highest fluorescence intensity in the absence of glucose. In the case of CM-0.75 solution, assuming that all of the AGA groups were combined with PBA, 3415

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APBA) with glucose attached, and explanation for the same fluorescent intensity of ARS in Figure 7. This information is available 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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ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (No. 20904025, 21274001, 91127045, and 50830103) and the National Basic Research Program of China (973 Program, No. 2011CB932503) for financial support.

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Figure 8. Cell viability of 293T cells as a function of material concentration by the MTT assay after incubation of 24 h at 37 °C.

100% but decreased with the increase of the micelle concentration, indicating the complex micelles was slightly cytotoxic at higher concentration. It also can be seen from Figure 8 that the cell viability of the complex micelles ranged between those of PEG-b-P(AA-co-APBA) and P(AA-co-AGA), suggesting that the introduction of glycopolymer improved the biocompatibility of the resultant complex micelles. As the complex micelles investigated in this study were not well biocompatible, PBA-contained block copolymer PEG-bP(Asp-co-AspAPBA) and glycopolymer P(Asp-co-AspAGA) based on poly(aspartic acid) (PAsp) will be synthesized. These two polymers were expected to form complex micelles not only with enhanced glucose-responsiveness but also with better biocompatibility. Self-regulated insulin delivery based on these complex micelles will be studied in the future.

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CONCLUSIONS Glucose-responsive complex micelles were prepared by selfassembly of a phenylboronic acid-based block copolymer PEGb-P(AA-co-APBA) and a glycopolymer P(AA-co-AGA) due to the covalent bonding between phenylboronic acid and glycosyl. The complex micelles were stable under physiological condition (PBS 7.4, 150 mmol) but would dissociate in the presence of glucose due to the replacement of glycosyl groups by free glucose. The complex micelles were more sensitive to glucose than the simple PEG-b-P(AA-co-APBA) micelles (CM-0) and the sensitivity of the complex micelles to glucose could be easily manipulated by the varying the composition of the micelle core. The complex micelles of CM-0.75 PEG-b-P(AA-co-APBA)/ P(AA-co-AGA) = 1/0.75, w/w) had the highest glucosesensitivity, which could dissemble under physiological condition with a glucose concentration of 2 g/L. It was an important enhancement of glucose-sensitivity for the phenylboronic acid-based glucose-responsive system. This kind of complex micelles may be a promising carrier for self-regulated insulin delivery and find application in the treatment of diabetes.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Detailed synthesis and characterization of polymers, schematic illustration of interaction between glucose and phenylboronic acid at different pH values, size distribution of PEG-b-P(AA-co3416

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