Phosphorylcholine-Based Zwitterionic Biocompatible Thermogel

Nov 9, 2015 - This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (2012M3A9C6049835 and ...
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Phosphorylcholine-Based Zwitterionic Biocompatible Thermogel Du Young Ko, Madhumita Patel, Bo Kyoeng Jung, Jin Hye Park, and Byeongmoon Jeong* Department of Chemistry and Nanoscience, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul, 120-750, Korea S Supporting Information *

ABSTRACT: Zwitterionic polymers have been investigated as surfacecoating materials due to their low protein adsorption properties, which reduce immunogenicity, biofouling, and bacterial adsorption of coated materials. Most zwitterionic polymers, reported so far, are based on (meth)acrylate polymers which can induce toxicity by residual monomers or amines produced by degradation. Here, we report a new zwitterionic polymer consisting of phosphorylcholine (PC) and biocompatible poly(propylene glycol) (PPG) as a new thermogelling material. The PC-PPG-PC polymer aqueous solution undergoes unique multiple sol−gel transitions as the temperature increases. A heat-induced unimer-to-micelle transition, changes in ionic interactions, and dehydration of PPG are involved in the sol−gel transitions. Based on the broad gel window and low protein adsorption properties, the PC-PPG-PC thermogel is proved for sustained delivery of protein drugs and stem cells over 1 week.

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INTRODUCTION In search of a new biomaterial, introduction of cell membrane components can be an attractive strategy. Surface functional groups of cell membranes can probably impart cytocompatible and biocompatible properties to the material. Noting that the major components of cell membranes such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and so on are zwitterionic molecules that possess both negative charge(s) and positive charge(s), various zwitterionic (co)polymers using 2-methacryloyloxethyl phosphorylcholine, sulfobetaine methacrylate, and carboxybetaine methacrylate have intensively been investigated for various biomedical applications.1−3 Due to the low protein adsorption and low cell adhesion properties of zwitterionic polymers, they were applied for surface coating of implants, biosensors, biomedical devices, ships, nanoparticles, and polymersomes.4−13 Zwitterionic polymers prevented these materials from biofouling and biofilm formation. For example, when a catheter coated with polysufobetaine was exposed to human blood, protein adsorption was reduced to 2% of commercialized catheters.14 Consequently, the activation of platelets, lymphocytes, monocytes, and neutrophils was significantly reduced. When cations or cation-inducing moieties were incorporated in zwitterionic polymers, the polymers developed bacteriocidal and antibiofouling properties.15,16 The low protein adsorption properties of the zwitterions and the bacteriocidal properties of the cations synergistically contributed to the properties of the materials. When zwitterionic poly(carboxybetaine) was conjugated to α-chymotrypsin, its stability improved significantly, without scarificing bioactivity of the protein.17 Thus, compared to the conjugation of PEG or PEGylation, zwitterionic conjugation to proteins can be more advantageous. PEGylation has widely been used to prolong the © 2015 American Chemical Society

plasma half-life of protein drugs, whereas a significant decrease in the bioactivity is still a concern.18 Due to intrinsic ionic interactions of zwitterionic polymers, many zwitterionic polymers exhibit upper critical solution temperature (UCST) in water.19 This behavior indicates that zwitterionic polymers are not soluble in water at a low temperature due to the strong ionic interactions among the zwitterions, but their solubility increases at a high temperature when thermal energy overcomes the ionic interactions. By screening the ionic interactions by adding salts, the UCST of zwitterionic polymers decreased, indicating that solubility increases at a low temperature.19 On the other hand, the UCST increased by copolymerization with hydrophobic monomers or hydrophobic modifications of the zwitterionic polymers.20 Polymers exhibiting both UCST and lower critical solution temperature (LCST) were prepared by copolymerizing zwitterionic monomers with N-isopropylacrlamide or Nvinylcaprolactam.21−24 N-Isopropylacrylamide copolymers have been successfully used for cell sheet engineering in vitro or ex vivo; however, the teratogenicity of acrylamide monomers and the toxicity of amine molecules, which are produced by hydrolysis of the polymer, still limits the parenteral applications of these copolymers.25,26 To the best of our knowledge, most zwitterionic polymers reported so far are synthesized from (meth)acrylates, such as 2-methacryloyloxethyl phosphorylcholine, sulfobetaine methacrylate, and carboxybetaine methacrylate. We are reporting here a new zwitterionic polymer consisting of phosphorylcholine (PC) and biocompatible poly(propylene Received: August 29, 2015 Revised: October 26, 2015 Published: November 9, 2015 3853

DOI: 10.1021/acs.biomac.5b01169 Biomacromolecules 2015, 16, 3853−3862

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acetonitrile (25 mL) in a pressure reactor. The reactor was immersed in a dry ice-acetone bath. Then, trimethylamine (0.88 mL, 9.38 mmol) was added. After sealing the pressure reactor, the reaction was carried out at 65 °C for 72 h. The reaction mixtures were cooled down to room temperature, and the remaining trimethylamine and solvent were removed by evaporation. After bubbling the nitrogen for 24 h, the final products were purified by dialysis (cutoff molecular weight: 1000 Da). Thereafter, the purified products were lyophilized. The final yield is 84%. NMR and FTIR Spectroscopy. The structure of the PC-PPG-PC was confirmed by 1H and 31P NMR spectroscopy (500 MHz NMR spectrometer; Varian, U.S.A.) in CDCl3, and FTIR spectroscopy (FTIR spectrophotometer FTS-800; Varian, U.S.A.). In addition, changes in 1H NMR and FTIR spectra of the PC-PPG-PC (45.0 wt % in D2O) were investigated as a function of temperature in a range of 10−80 °C. The temperature was equilibrated for 5 min at each temperature. Gel Permeation Chromatography (GPC). The GPC system consisting of a pump (SP930D; Younglin, Korea) and a refractive index detector (RI750F; Younglin, Korea) was used to determine the molecular weight and molecular weight distribution of polymers. N,NDimethylformamide was used as an eluent, and OHpak SB-803QH column (Shodex, Japan) was used for analysis. Poly(ethylene glycol)s (Polysciences, Inc., U.S.A.) with molecular weights in a range of 200− 20000 Da were used as molecular weight standards. Dynamic Light Scattering (DLS). The apparent size of PC-PPGPC and their self-assemblies were studied by a dynamic light scattering instrument (Zetasizer Nano; Malvern Instruments Inc., U.S.A.). A YAG DPSS-200 laser (Lange, Germany) operating at 532 nm was used as a light source. The scattering intensity of the polymer aqueous solutions was measured as a function of concentration within a range of 0.0005−1.0 wt % at 25 °C. In addition, the apparent size of the polymer and polymer self-assemblies were measured as a function of temperature at 10, 25, and 40 °C at a fixed concentration of 0.10 wt %. The scattered intensity was measured at an angle of 173° to the incident beam. The results of dynamic light scattering were analyzed by the regularized CONTIN method. From the diffusion coefficient, the apparent hydrodynamic size of the polymer assemblies could be obtained by Stokes−Einstein equation. Critical Micelle Temperature. The hydrophobic dye (1,6diphenyl-1,3,5-hexatriene, DPH) was dissolved in PC-PPG-PC aqueous solution (0.10 wt %) at a dye concentration of 4.0 μM. UV−visible spectral changes of the dye were measured by the UV− visible spectrophotometer (S3100, Sinco, Korea) as a function of temperature in a temperature range of 10−80 °C. The temperature was equilibrated for 5 min at each temperature. Transmission Electron Microscopy (TEM). The PC-PPG-PC aqueous solution (10 μL; 0.10 wt %) was placed on the 200 mesh carbon-coated copper grid, and water was evaporated slowly at room temperature. The microscopic images were obtained by using a JEM2100F microscope (JEOL, Japan) at an accelerating voltage of 200 kV. Phase Diagram. Sol−gel transition temperatures of polymer aqueous solutions were determined by the test tube inverting method. The polymer aqueous solution (0.5 mL) was placed in a test tube, having an inner diameter of 11 mm. The transition temperature was measured by the flow (sol) and nonflow (gel) criterion with a temperature increment of 1 °C per step. Each data point is the average of three time measurements. Dynamic Mechanical Analysis. The modulus of a PC-PPG-PC aqueous solution was investigated by a dynamic rheometer (Rheometer RS 1; Thermo Haake, U.S.A.) as a function of temperature. The aqueous polymer solution (45.0 wt %) was placed between parallel plates, having a diameter of 25 mm. A gap of 0.5 mm was maintained between these parallel plates. Before performing dynamic mechanical analysis, the samples were placed inside of a chamber containing totally wet cotton to minimize the evaporation of water. The data were collected under conditions of controlled stress (4.0 dyn/cm2) at a frequency of 1.0 rad/s and a heating rate of 0.2 °C/ min.

glycol) (PPG). Zwitterionic PC is the functional group of phosphatidylcholine, which is a major component of biological cell membranes. PPG is already approved by the Food and Drug Administration (FDA) of the United States for intravenous, subcutaneous, and oral applications in humans.27 PPG aqueous solutions exhibit a lower critical solution temperature (LCST), where the LCSTs depend on the molecular weight of the PPG. The LCSTs of PPG aqueous solutions were reported to be 65, 33, and 20 °C for 425, 1000, and 2000 Da of PPGs, respectively.28 Due to the LCST behavior of PPG and the low protein adsorption properties of zwitterionic PC, PPG end-capped by PC, PC-PPG-PC, is expected to be a temperature-sensitive biocompatible material (Scheme 1). Conjugation of hydrophilic PC to PPG is also Scheme 1. Chemical Structure of PC-PPG-PCa

a

PPG is approved by the U.S. FDA for intravenous, subcutaneous, and oral applications in humans. PPG is a polymer exhibiting LCST in water. PC is a functional group of phosphatidylcholine, which is a major component of cell membranes, and prevents protein adsorption and biofouling. PC end-capped PPG (PC-PPG-PC) provides a biomimetic and biocompatible thermogel.

expected to increase LCST of the PPG. Therefore, PPG of 2700 Da, which exhibits an LCST of 20 °C, was selected to prepare the PC-PPG-PC with a sol-to-gel transition temperature of 20−30 °C in this study in considering biomedical applications. Thermogels are polymer aqueous solutions that undergo sol-to-gel transition as the temperature increases. The thermogelling behavior is attributed to a delicate balance between hydrophilicity and hydrophobicity of the polymer.29−31 Drugs or cells can be incorporated into the hydrogel by heating the polymer aqueous solution to warm conditions, typically, of 37 °C. Due to their simple procedure and mild conditions for gel formation, thermogels have been suggested as a promising scaffold for the delivery of biopharmaceuticals.29−39

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EXPERIMENTAL SECTION

Materials. Polypropylene glycol (M.W. = 2700 Da), 2-chloro-2oxo-1,3,2-dioxaphospholane, triethylamine, trimethylamine, anhydrous toluene, and anhydrous acetonitrile were purchased from SigmaAldrich, U.S.A. Recombinant human insulin was purchased from Sigma-Aldrich, U.S.A., and the insulin ELISA kit was purchased from RnD systems, U.S.A. Mayer’s hematoxylin solution, eosin, and the Masson’s trichrome stain kit were purchased from Sigma-Aldrich, U.S.A. Synthesis of PC-PPG-PC. PC-PPG-PC was synthesized by modifying the reaction between alcohol and 2-chloro-2-oxo-1,3,2dioxaphospholane.40 Poly(propylene glycol) (10.00 g, 3.70 mmol) and triethylamine (1.30 mL, 9.33 mmol) were dissolved in dried toluene (100 mL). 2-Chloro-2-oxo-1,3,2-dioxaphospholane (0.68 mL, 7.40 mmol) was added to the above solution, and the reaction mixtures were stirred at room temperature for 20 h. The precipitated trimethylammnium chloride was removed by filtration, and the solvent was eliminated. The products were dissolved in anhydrous 3854

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sample was fixed to microscope slides, and then it was stained by the Mayer’s hematoxylin and eosin (H&E) method and the Masson’s trichrome staining method. Animal Procedure. All the experimental procedures involving animals were performed in accordance with the NIH Guideline for the Care and Use of Laboratory Animals. These procedures were approved by the Committee of Ewha Womans University (Code 14−092). Statistical Analysis. The differences in the mean values were evaluated using the one-way analysis of variance (ANOVA) and Tukey tests. The differences were considered to be statistically significant when the p value was less than 0.05.

In Vitro Drug Release. Insulin was dissolved in the PC-PPG-PC aqueous solution (45.0 wt %). The vial containing 0.5 mL of each formulation was preincubated in 37 °C for 10 min. Then, phosphatebuffered saline (pH = 7.4, 3.0 mL) was added to the top of the gel at 37 °C. The vials were shaken at a stroke of 20 strokes/min in a thermostatic bath at 37 °C. The fresh media (3.0 mL) at 37 °C were carefully replaced at the specified time intervals. The recovered samples were analyzed by HPLC systems (Waters 1525B, Korea) at a wavelength of 214 nm. A Jupiter 5 μ C18 300A column (250 × 4.60 mm 5micron, Phenomenex, U.S.A.) and the acetonitrile/water 30/70 (v/v) cosolvent system were used as the eluting solvent. In Vivo Insulin Delivery. Male Sprague−Dawley rats with an age of 5 weeks were purchased from Central Lab (Animal Inc., Korea). After stabilizing them for 1 week, a solution of streptozotocin (65 mg/ kg-rat) in 0.01 M citrate buffer of pH = 4.5 was administered to rats by intraperitoneal injection to induce diabetes. The rats were diagnosed with diabetes when the blood glucose level was higher than 350 mg/ dL for five consecutive days. Then, insulin/PC-PPG-PC formulation (45.0 wt %, 0.5 mL) at an insulin dose of 13.8 mg/kg-rat was subcutaneously injected into diabetic rats. In the control experiments, saline and PC-PPG-PC aqueous solution were subcutaneously injected into diabetic rats. All the rats were subjected to the alternating light/ dark (12 h/12 h) conditions, while water was supplied all the time. After 2 h of a fasting period, the blood glucose level was monitored by using a glucometer (Accu-Chek, Roche, Switzerland). The blood glucose level of nondiabetic rats was also monitored as a positive control; n = 4 for each group. 3D Cell Culture and Delivery. TMSCs were isolated from palatine tonsils of a 11 years old female donor (IRB approval code: ETC 11−53−02) at the Ewha Womans University Mokdong Hospital (Seoul, Korea) following the ethical guidelines of the University.41 After obtaining the informed consent form from the donor, the tonsils were collected from the patient by tonsillectomy. For proliferating these stem cells, they were 2D cultured on the polystyrene culture plates over passage 5 by using a high glucose Dulbecco’s modified eagle media (DMEM, Hyclone, U.S.A.) supplemented with 10% (v/v) fetal bovine serum (FBS, Hyclone, U.S.A.), 1.0% (v/v) Hyclone penicillin/streptomycin solution, and 1.0% (v/v) Gibco antibioticantimitotic solution under 5% CO2 atmosphere. The harvested TMSCs (passage 6, 0.4 × 106 cells) were suspended in PC-PPG-PC aqueous solution (45.0 wt %; 0.20 mL), and then they were injected into 24-well culture plates at 37 °C. Under these conditions, the TMSCs got encapsulated in the gel by the sol-to-gel transition of the system. DMEM (1.0 mL; 37 °C) containing 10% FBS and 1% penicillin/streptomycin was added to the cell-encapsulated PC-PPGPC thermogel under 5% CO2 atmosphere at 37 °C, and the medium was replaced every 3 days. Cell Viability and Proliferation. Cell viability in the PC-PPG-PC thermogels was determined by the Live/Dead kit (Life Technologies, U.S.A.) after 0 (1 h), 1 day, 3 days, and 7 days of incubation. TMSCs encapsulated in the thermogels were incubated at 37 °C for 15 min in a solution of ethidium homodimer-1 (4.0 μM) and calcein AM (2.0 μM), which was prepared using phosphate buffered saline as the solvent. The labeled cells were viewed under the Olympus IX71 fluorescence microscope, and the images were captured using Olympus DP2-BSW. Live cells were stained into green by calcein AM, while dead cells were stained into red by ethidium homodimer-1. Cell proliferation was monitored by the cell counting kit-8 (CCK-8) (Dojindo Co. Ltd., Kumamoto, Japan; n = 3). CCK-8 solution (1.5 mL, 10% v/v in medium) was added to each well of the plate. After 3 h of incubation, the absorbance was measured at 450 nm with an ELISA reader (Model 550; Bio-Rad, U.S.A.), where the absorbance at 655 nm was used as a baseline. Tissue Compatibility. Rats were stabilized for 1 week. Then, the PC-PPG-PC aqueous solution (45.0 wt %, 0.5 mL/rat) was subcutaneously injected into these rats. After 5 days of incubation, the tissues around the implanted site were removed for assay. Thereafter, the tissues were fixed with 4% paraformaldehyde solution for 24 h. Finally, they were embedded in paraffin. Sections of the paraffin block 6 μm thick were obtained using a microtome. The

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RESULTS AND DISCUSSION PC-PPG-PC was synthesized by the reaction between dihydroxy end groups of PPG and 2-chloro-2-oxo-1,3,2dioxaphospholane, followed by treating the intermediate with trimethylamine (Figure 1a).40 1H NMR spectra of PC-PPG-PC

Figure 1. (a) Synthetic scheme of PC-PPG-PC. (b) 1H NMR spectra of PC-PPG-PC and PPG (in CDCl3).

(in CDCl3) exhibit a peak at 1.0−1.2 ppm, which is attributed to the methyl groups present in the internal propylene glycol groups. The 1H NMR spectra also exhibit a small peak at 1.2− 1.4 ppm, which is attributed to the methyl groups present in the terminal propylene glycol groups connected to phosphoryl groups. The peak at 4.4−4.5 ppm is attributed to the methylene groups of PC connected to oxygen of phosphoryl groups. The peaks at 3.2−3.7 ppm are attributed to the methyl groups of PC, methylene groups of PC connected to nitrogen, methine groups of PPG, and methylene groups of PPG (Figure 1b). By comparing the peak at 1.0−1.4 ppm (methyl peaks of PPG) and the peak at 4.4−4.5 ppm (methylene peaks of PC), both end groups of PPG were confirmed to be capped by PC groups. A singlet peak of 31P NMR spectra also indicates the exact structure of PC-PPG-PC (Figure S1a). FTIR spectra of neat polymer exhibited the phosphorylcholine groups at 1242 cm−1 (PO stretching; Figure S1b).42 GPC chromatogram of the PC-PPG-PC exhibited a unimodal distribution of the polymer 3855

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Figure 2. (a) Determination of critical micelle concentration of PC-PPG-PC at 25 °C. The scattering intensity of polymer aqueous solution was investigated in a concentration range of 0.0005−1.0 wt %. (b) UV−vis spectra of a hydrophobic dye (1,6-diphenyl-1,3,5-hexatriene) dissolved in PCPPG-PC aqueous solution (0.10 wt %) as a function of temperature. (c) Apparent size distribution of aggregates or self-assemblies determined by dynamic light scattering of PC-PPG-PC aqueous solution (0.10 wt %) at 10 °C (peak average = 3 nm), 25 °C (peak average =220 nm), 40 °C (peak average = 1280 nm), and 60 °C (peak average = 820 nm). (d) TEM image of PC-PPG-PC micelles developed from its aqueous solution (0.10 wt %) by air-drying at room temperature. The scale bar is 200 nm.

spectra indicated that the micelle structure was preserved within a temperature range of 25−60 °C. Below 25 °C, the polymers remained as a random coil structure owing to the hydrophilic nature of both PC and PPG. As the temperature increased, PPG became hydrophobic, while PC remained as a hydrophilic zwitterion, therefore, the PC-PPG-PC formed micelles. The critical micelle temperature of the PC-PPG-PC aqueous solution (0.10 wt %) was found to be about 25 °C. The temperature-sensitive unimer-to-micelle transition is a unique characteristic of current PC-PPG-PC and PEG-PPGPEG systems.43 Above the 60 °C, PPG underwent further dehydration, and thermal energy weakened the ionic interactions of PC. The temperature-sensitive micelle formation was also confirmed by dynamic light scattering. The apparent size of self-assemblies of PC-PPG-PC was 220 nm (peak average) at 25 °C, which reversibly reduced to 2−5 nm (unimer) at 10 °C in the dynamic light scattering study of the PC-PPG-PC aqueous solution (0.10 wt %; Figure 2c). At 40 °C, the bigger polymer aggregates with 1280 nm (peak average) were observed, whereas the apparent size decreased to 820 nm (peak average) at 60 °C. At above 60 °C, the correlation curve in the light scattering analysis by the regularized CONTIN method was not good to get the meaningful information on apparent size of polymer aggregates. TEM images developed from the polymer aqueous solution (0.10 wt %) also confirmed the spherical micelles of PC-PPG-

molecular weights with an average molecular weight of 1300 against the PEG standards and polydispersity index of 1.1 (Figure S1c). Dynamic light scattering, hydrophobic dye solubilization, and transmission electron microscopy (TEM) were used to investigate the self-assembling behavior of PC-PPG-PC polymers at a low concentration in their aqueous solution. Due to the amphiphilic nature of the PC-PPG-PC at 25 °C, the polymer formed micelles. As the concentration of the polymer increased, the scattering intensity began to increase, suggesting micelle formation of the PC-PPG-PC with PPG core and PC shell at 25 °C. The critical micelle concentration of PC-PPGPC was measured to be about 0.05−0.10 wt % at 25 °C (Figure 2a). Using PC-PPG-PC aqueous solution (0.10 wt %) containing hydrophobic dyes of 1,6-diphenly-1,3,5-hexatriene, the self-assembly of PC-PPG-PC in water was also measured as a function of temperature. The dye exhibits a maximum absorbance at 300 nm in a polar aqueous environment, whereas a characteristic triplet band in 300−400 nm is observed in a hydrophobic environment.43 The appearance of triplet bands in 300−400 nm suggests the formation of hydrophobic domains, that is, micelles. As the temperature of PC-PPG-PC aqueous solution increased above 25 °C, the triplet band of the dye at 337, 356, and 375 nm appeared (Figure 2b). Above 60 °C, the triplet band disappeared, and significant scattering was observed in the UV−vis spectra (Figure S2). The UV−vis 3856

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Figure 3. (a) Phase diagram of PC-PPG-PC aqueous solutions determined by the test tube inverting method. tp and tl indicate transparent and translucent, respectively. The polymer aqueous solution undergoes transparent free-flowing (low viscous) sol → translucent gel → translucent viscous sol > transparent gel transitions as the temperature increases. (b) Photos of the PC-PPG-PC aqueous solution (45.0 wt %) at 10 °C (transparent sol), 25 °C (translucent gel), 60 °C (translucent sol), and 70 °C (transparent gel). (c) 1H NMR spectra of PC-PPG-PC aqueous solution (45.0 wt % in D2O) as a function of temperature. (d) FTIR spectra of PC-PPG-PC aqueous solution (45.0 wt % in D2O) as a function of temperature.

temperatures above 66 °C, it turned into a transparent semisolid gel. At concentrations lower than 40.0 wt %, there were changes in viscosity of the PC-PPG-PC aqueous solution with an increase in temperature, however, it did not form a strong gel to stop the flow against the tilting stress of the test tube, so it was considered to be a sol. On the other hand, at concentrations greater than 60.0 wt %, the PC-PPG-PC aqueous system existed as a nonflowing gel in the temperature range of 0−80 °C. The phase behavior of the polymer aqueous solution was clearly demonstrated by photos at 10 °C (transparent sol), 25 °C (translucent gel), 60 °C (translucent sol), and 70 °C (transparent gel; Figure 3b). To investigate the molecular behavior involved in the multiple transitions of the PC-PPG-PC aqueous solution, 1H

PC with a size of 100−250 nm. The micelle size in TEM images was similar to that encountered by performing a dynamic light scattering study, even though the shape or size of these polymer assemblies could be damaged by the water evaporation procedure during the TEM experiment (Figure 2d). As the temperature increased, the PC-PPG-PC aqueous solution exhibited multiple sol-to-gel-to-sol-to-gel transitions in a specific concentration range of 40−60 wt % (Figure 3a). For example, the polymer aqueous solution at 45 wt % was in a transparent and low viscous sol state at temperatures below 22 °C. However, it turned into a translucent semisolid gel in a temperature range of 22−56 °C. Thereafter, it became a translucent viscous sol in a temperature range of 56−66 °C. At 3857

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mechanisms of lower gel and upper gel formation of the PLGA-PEG-PLGA aqueous solutions were described by a micelle packing model and a percolated micelle network model, respectively.49−51 Changes in polymer−water interactions and consequent changes in self-assembly of the polymers as a function of temperature play a key role in the multiple sol−gel transition of the PLGA-PEG-PLGA aqueous solutions. The unique multiple sol-to-gel-to-sol-to-gel transitions of current PC-PPG-PC are related to the dehydration of PPG and the changes in ionic interactions of PC moieties in water. Considering the above experimental results, the molecular behavior of the PC-PPG-PC aqueous solution as a function of temperature is schematically suggested (Figure 4). At low

NMR spectra and FTIR spectra of the PC-PPG-PC aqueous solution (45.0 wt % D2O) were investigated as a function of temperature. 1H NMR spectra of PC-PPG-PC aqueous solution (45.0 wt % D2O) indicated that both PPG peaks at 0.8−1.4 ppm (−CH3) and 3.2−3.8 ppm (−OCHCH2−) and a small PC peak (−N+(CH3)3) at 3.0−3.2 ppm collapsed as the temperature increased up to the first gel region (gel, tl), however, the peaks increased their intensity and the peak shape became sharpened as the temperature increased above 60 °C (Figure 3c). In addition, a new peak of PPG appeared at 3.2− 3.4 ppm, indicating changes in the magnetochemical environment, which could be attributed to the significant dehydration of the PPG. In the 1H NMR spectra, the collapsing or sharpening of a peak is related to the molecular motion of the corresponding moiety. The 1H NMR spectra of PC-PPG-PC suggested that the molecular motion of PPG decreased in the first gel region, however, it increased again as the temperature increased above 60 °C. The water peak at 4.7−5.0 ppm in the 1 H NMR spectra steadily shifted to 4.0−4.4 ppm as the temperature increased. FTIR spectra of the PC-PPG-PC aqueous solution (45.0 wt % in D2O) were monitored as a function of temperature (Figure 3d). The FTIR spectra of PPG aqueous solution (45.0 wt %, Figure S3) and literatures, the band at 1230 cm−1 could be assigned to the PO stretching vibration.44 The band at 1230 cm−1 shifted to 1249 cm−1 as the temperature increased from 10 to 80 °C. As the temperature increases, the intermolecular ionic interactions between the negatively charged phosphoryl group and the positively charged choline group are weakened, resulting in the strengthening of PO stretching bond. The change in force constant or band position in the FTIR spectra is well-known for carbonyl groups (CO) of polypeptides when intermolecular hydrogen bonding is involved. The carbonyl band of 1630 cm−1 in βsheet peptides that have well-organized hydrogen bond shifts to 1640 cm−1 in random coiled peptides.45 The phase behavior of PC-PPG-PC aqueous solution can be also compared with the aqueous solutions of poly(Nisopropylacrylamide-co-sulfobetaine methacrylate) and poly(N-vinyl caprolactam-co-sulfobetaine methacrylate), which exhibit both UCST and LCST.24,46 The ionic interactions among the zwitterions are weakened as the temperature increases, and thus solubility of the polymer increases as the temperature increases, leading to UCST behavior of the polymer aqueous solution. At the same time, the solubility of poly(N-isopropylacrylamide) moiety decreases as the temperature increases, leading to LCST at higher temperature. Multiple sol−gel (lower gel)-sol−gel (upper gel)-sol transitions were reported for an aqueous solution of poly(ethylene glycol)polycaprolactone-poly(ethylene glycol) (EG13CL23EG13).47 The melting of the crystalline polycaprolactone block and the dehydration of PEG were suggested as the multiple transition mechanism of the polymer aqueous solution. The PCL is in a crystalline state in the lower gel, whereas it is in an amorphous state in the upper gel. An aqueous solution of poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) (EG17PG60EG17) also exhibited multiple sol−gel (lower gel)sol−gel (upper gel) transitions as the temperature increased. The micellar packing and phase separation model were suggested for sol-to-lower gel transition and sol-upper gel transition, respectively.48 Some PLGA-PEG-PLGA aqueous solutions also exhibited multiple sol−gel transitions by varying the block length of the polymer, end-group modification, or by adding salts.49−51 In a series of systematic studies, the

Figure 4. Schematic presentation of the phase transition of the PCPPG-PC aqueous solution. Blue cones indicate zwitterionic PC. At 10 °C, the PC-PPG-PC aqueous solution is in a low viscous sol state. PPG (blue curves) is hydrophilic at 10 °C. However, as the temperature increases, it turns into hydrophobic, and the PC-PPGPC form micelles with a hydrophobic PPG core (orange spheres) and hydrophilic PC shell (blue cones). The micelles aggregate to form a translucent gel at 25 °C. Ionic interactions are strengthened in a hydrophobic environment of the gel state. At 60 °C, further dehydration of PPG (red spheres) and weakening of ionic interactions among PCs lead to the turbid sol. At 70 °C, the micelle structure gets disintegrated and a transparent gel is formed. At 70 °C, PPG is indicated by red curves.

temperature (10 °C), both PPG (sky blue curves) and PC (blue cones) are hydrophilic, and thus PC-PPG-PC has a random coiled conformation. The ionic interactions among the scattered end groups of PC-PPG-PC in water are not strong enough to form a gel. Therefore, the polymer aqueous solution is in a transparent sol state at 10 °C. As the temperature increases to 25 °C, PPG dehydrates and the PC-PPG-PC forms micelles with a hydrophobic PPG core (orange spheres) and a hydrophilic PC shell (blue cones). The sol-to-gel transition occurs through the aggregation of micelles. In the gel state (25 °C), micelles are in an aggregated state. Moreover, PC around the hydrophobic PPG can closely assemble through the ionic interactions. The macroscopic translucent gel is observed at this temperature. As the temperature increases further (60 °C), the PPG in the micelle core is dehydrated more as described in red spheres, and ionic interactions among PC (blue cones) are weakened by thermal energy. Under these circumstances, the translucent gel-to-translucent sol transition occurs. The electrostatic interactions are inversely proportional to a dielectric constant by Coulomb’s law. E = Q1Q2/4πεr. Q1 and Q2 are the charges. ε and r are the dielectric constant and the distance between the charges. The dielectric constant is large in a polar environment and small in a nonpolar, hydrophobic environment. For example, a dielectric constant of water, methanol, and cyclohexane are 78.5, 32.6, and 2.0, respectively.52 As the 3858

DOI: 10.1021/acs.biomac.5b01169 Biomacromolecules 2015, 16, 3853−3862

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Biomacromolecules dehydration of PPG occurs, the charges of PC are located in a rather hydrophobic environment, therefore, the ionic interactions are strengthened, similar to a salt bridge in a hydrophobic environment of proteins.53 On the other hand, the dipole−dipole interactions between permanent dipoles as in the PO group of PC are inversely proportional to the temperature by the equation, E = −μ12μ22/{24π2ε2κTr6} ∼ a/ T.52 μ and k are dipole moment and Boltzmann constant, respectively. Thermal energy also increases the molecular motion of the polymer and increases the average distance among the dipoles. Therefore, the ionic interactions and dipole interactions decrease as the temperature increases. When the thermal energy or kinetic energy overcomes the hydrophobic effect, gel-to-sol transition can occur as in the PC-PPG-PC aqueous solution. As the temperature increased further (70 °C), a transparent gel forms, where the ionic interactions among the PC (blue cones) are weakened more and PPG (red curves) is dehydrated more seriously. Moreover, the molecular motion of both PC and PPG increases. Therefore, a random aggregation of the polymer chains might occur, while free PC moieties retain water to form a macroscopically transparent gel. The thermogelling PC-PPG-PC system was investigated as sustained delivery systems for protein drug and stem cells. First, feasibility of the system as an injectable delivery carrier was tested by monitoring the changes in modulus of the polymer aqueous solution (45.0 wt %) as a function of temperature (Figure 5a). Increases in storage modulus (G′) and loss modulus (G″) at the first sol-to-gel transition temperature around 20 °C were evident. In particular, crossing the G′ over G″ at this temperature clearly indicated the sol-to-gel transition. G′ and G″ are the viscous component and the elastic component of a complex modulus, respectively. The gel is maintained its modulus over the body temperature range of 36−43 °C. Therefore, it can be used as biomedical injectable systems. The in vitro release system of insulin was prepared by injecting the insulin/PC-PPG-PC aqueous solution into a warm well at 37 °C. Insulin was released from the in situ formed gel over 7 days in a diffusion controlled manner (Figure 5b). The gel preserved its physical integrity over the period. Zwitterionic polymer exhibits low protein adsorption properties, as discussed in the Introduction, and thus facilitates the steady release of insulin. An in vivo insulin release system was prepared by subcutaneously injecting the PC-PPG-PC aqueous solution (45.0 wt %) containing insulin into diabetic rats. The insulin dose was fixed at 13.8 mg/kg-rat.54 Control experiments were carried out by subcutaneously injecting a saline solution (negative) or polymer aqueous solution (45.0 wt %) without insulin (gel only) into diabetic rats. Normal rats without diabetes were included in the positive control group and subject to the same control experiment. A single injection of the insulin/PC-PPG-PC formulation exhibited about 3 days of therapeutic efficacy in rats (Figure S4). PC-PPG-PC thermogel was also investigated as a delivery system of tonsil derived mesenchymal stem cells (TMSCs). DMEM containing 10% FBS and 1% penicillin/streptomycin was used as a cell culture medium. FBS contains growth factors such as insulin like growth factor-1 (IGF-1), transforming growth factor-β1 (GF-β1), and fibroblast growth factor-2 (FGF2) in a pg/mL−ng/mL level, and the growth factors help proliferation of the cells.55 To induce differentiation of stem cells to a specific lineage of the cells, specific growth factors are required in a sufficient amount. For example, BMP-2 (100 ng/ mL) and insulin (10 μg/mL) should externally be added to

Figure 5. Applications of the PC-PPG-PC thermogel as a drug delivery system. The delivery systems are prepared by injecting the polymer aqueous solution containing insulin into vessels at 37 °C. (a) Storage modulus (G′) and loss modulus (G″) of PC-PPG-PC aqueous solution (45.0 wt %) as a function of temperature. The sol-to-gel transition at about 20 °C indicates the feasibility as an injectable system. (b) In vitro insulin release profile; n = 3.

induce differentiation of MSCs into osteocytes and adipocytes, respectively.56−58 DMEM supplemented with FBS (10%) and antibiotics (1%) is a typical growth medium of stem cells where the proliferation of the cells occurs.59−61 When, the TMSCs were 2D-cultured for 7 days by using the above growth medium enriched with FBS (10%), significant increases in the mRNA level of sex determining region Y-box-2 (Sox2), and homeobox protein Nanog (Nanog) were observed (Figure S5). They are stem cell biomarkers essential to maintain pluripotency and self-renewing phenotype, confirming the stemness of the cells.62 On the other hand, there were no significant changes in the mRNA expression level of osteocalcin (OCN), collagen type II (COl II), and peroxisome proliferator-activated receptor gamma (PPARγ), which are typical differentiation biomarkers of TMSCs for osteogenesis, chondrogenesis, and adipogenesis, respectively (Figure S5). In our previous study, mRNA level of above differentiation biomarkers did not significantly increase by using the same growth media of DMEM enriched with FBS (10%) during 3D culture of TMSCs in a PEG-poly(L-alanine) thermogel over 7 days of incubation.58 This fact suggests that proliferation of the stem cells without a significant differentiation occurred by using the current cell culture medium of DMEM enriched with FBS (10%). The cell-suspended PC3859

DOI: 10.1021/acs.biomac.5b01169 Biomacromolecules 2015, 16, 3853−3862

Article

Biomacromolecules

Figure 6. Applications of the PC-PPG-PC thermogel as a stem cell delivery system. (a) In vitro stem cell (TMSC) delivery profile. The cell number in the gel is monitored by the live/dead images after 0, 1, 3, and 7 days of 3D culture; n = 3. (b) Stem cell (TMSC) delivery system. The total cell density is measured by the CCK-8 method after 0, 1, 3, and 7 days of 3D culture. * indicates p < 0.05 by one way ANOVA analysis, n = 3. (c) Cell images in the PC-PPG-PC gel after 0, 1, 3, and 7 days of 3D culture using the growth medium. The scale bar is 50 μm.

PPG-PC aqueous solution was injected into a well at 37 °C to form a cell-encapsulating hydrogel matrix. Since PC is a major functional group in biological cell membranes, PC-PPG-PC was expected to provide a cytocompatible environment for the stem cells. The TMSCs maintained their original spherical phenotype in the PC-PPG-PC gel formed in situ. Dead cells (red) were not observed in the gel for 7 days, however, cell density steadily decreased over 7 days from the PC-PPG-PC gel (Figure 6a−c). The total number of live cells in a cell culture well was measured by the CCK-8 method. The total number of live cells increased over 7 days, suggesting the cells were slowly delivered to outside of the gel and proliferated over 7 days (Figure 6b). These findings indicated that the PC-PPG-PC thermogel provided a cytocompatible sustained delivery vehicle for the stem cells. Biocompatibility of the PC-PPG-PC thermogel was investigated for the in situ formed gel prepared by subcutaneously injecting the polymer aqueous solution (45.0 wt %) into rats. The gel exhibited mild tissue responses around the implant. Haematoxylin and eosin (H&E) staining exhibited a little inflammatory cells (purple dots) around the implant that was incubated for 5 days in rats (Figure 7; left). Masson’s trichrome stain (MT), which stains the collagen layer in blue, cytoplasm in red, and nucleus in black also showed mild inflammation responses of the PC-PPG-PC thermogel (Figure 7, right). In addition, incorporated cells in the hydrogel were not damaged over 7 days. The gel contains 55% of empty space filled with water, which can act as a mass transport channel. A hydrogel prepared from poly(ethylene glycol)-poly(lactide-coglycolide)-poly(ethylene glycol) (PEG-PLGA-PEG) aqueous solution (33 wt %) initially contains 77% of water, and water content decreased due to the preferential mass loss of PEG-rich

Figure 7. H&E (left) and Masson’s trichrome (right) stained images 5 days after subcutaneously injecting PC-PPG-PC aqueous solution (45.0 wt %) into rats. The scale bar is 100 μm.

segments.63 The PEG-PLGA-PEG hydrogel was reported as a biocompatible thermogel in subcutaneous layer of rats.63 The current PC-PPG-PC hydrogel was tissue-compatible in the subcutaneous layer of rats and the incorporated TMSCs was released without cell death for 7 days, indicating the biocompatibility of the PC-PPG-PC system. However, further studies are also needed to convince the long-term biocompatibility of the system. At 37 °C, PPG moieties become hydrophobic and form a core of micelles, whereas the zwitterionic PC moieties get exposed to the tissue. Zwitterionic polymers tend to have low protein adsorption properties as discussed in the Introduction.10,11 Since the protein adsorption is the first step in the collagen capsule formation, the zwitterionic PC-PPG-PC can reduce the foreign body reaction sequences and thus enhance the biocompatibility of the thermogel.3 A longer gel duration of PC-PPG-PC is also 3860

DOI: 10.1021/acs.biomac.5b01169 Biomacromolecules 2015, 16, 3853−3862

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Biomacromolecules

(5) Kobayashi, M.; Terayama, Y.; Kikuchi, M.; Takahara, A. Soft Matter 2013, 9, 5138−5148. (6) Cheng, G.; Li, G.; Xue, H.; Chen, S.; Bryers, J. D.; Jiang, S. Biomaterials 2009, 30, 5234−5240. (7) Chang, Y.; Shih, Y. J.; Lai, C. J.; Kung, H. H.; Jiang, S. Adv. Funct. Mater. 2013, 23, 1100−1110. (8) Muro, E.; Pons, T.; Lequeux, N.; Fragola, A.; Sanson, N.; Lenkei, Z.; Dubertret, B. J. Am. Chem. Soc. 2010, 132, 4556−4557. (9) Zhan, N.; Palui, G.; Safi, M.; Ji, X.; Mattoussi, H. J. Am. Chem. Soc. 2013, 135, 13786−13795. (10) Seetho, K.; Zhang, S.; Pollack, K. A.; Zou, J.; Raymond, J. E.; Martinez, E.; Wooley, K. L. ACS Macro Lett. 2015, 4, 505−515. (11) Jiang, S.; Cao, Z. Adv. Mater. 2010, 22, 920−932. (12) Dang, Y.; Quan, M.; Xing, C. M.; Wang, Y. B.; Gong, Y. K. J. Mater. Chem. B 2015, 3, 2350−2361. (13) Colley, H. E.; Hearnden, V.; Olias, M. A.; Cecchin, D.; Canton, I.; Madsen, J.; MacNeil, S.; Warren, N.; Hu, K.; McKeating, J. A.; Armes, S. P.; Murdoch, C.; Thornhill, M. H.; Battaglia, G. Mol. Pharmaceutics 2014, 11, 1176−1188. (14) Loose, C.; Jensen, K.; Rigoutsos, I.; Stephanopoulos, G. Nature 2006, 443, 867−869. (15) Cao, Z.; Mi, L.; Mendiola, J.; Menye, J. R. E.; Zhang, L.; Xue, H.; Jiang, S. Angew. Chem., Int. Ed. 2012, 51, 2602−2605. (16) Cao, B.; Tang, Q.; Li, L.; Humble, J.; Wu, H.; Liu, L.; Cheng, G. Adv. Healthcare Mater. 2013, 2, 1096−1102. (17) Keefe, A. J.; Jiang, S. Nat. Chem. 2011, 4, 59−63. (18) Pasut, G.; Veroese, F. M. Prog. Polym. Sci. 2007, 32, 933−961. (19) Seuring, J.; Agarwal, S. Macromolecules 2012, 45, 3910−3918. (20) Woodfield, P. A.; Zhu, Y.; Pei, Y.; Roth, P. J. Macromolecules 2014, 47, 750−762. (21) Chang, Y.; Yandi, W.; Chen, W. Y.; Shih, Y. J.; Yang, C. C.; Chang, Y.; Ling, Q. D.; Higuchi, A. Biomacromolecules 2010, 11, 1101− 1110. (22) Zhai, S. Y.; Ma, Y.; Chen, Y.; Li, D.; Cao, J.; Liu, Y.; Cai, M.; Xie, X.; Chen, Y.; Luo, X. Polym. Chem. 2014, 5, 1285−1297. (23) Zhao, Y.; Bai, T.; Shao, Q.; Jiang, S.; Shen, A. Q. Polym. Chem. 2015, 6, 1066−1077. (24) Yang, B.; Wang, C.; Zhang, Y.; Ye, L.; Qian, Y.; Shu, Y.; Wang, J.; Li, J.; Yao, F. Polym. Chem. 2015, 6, 3431−3442. (25) Nishida, K.; Yamato, M.; Hayashida, Y.; Watanabe, K.; Yamamoto, K.; Adachi, E.; Nagai, S.; Kikuchi, A.; Maeda, N.; Watanabe, H.; Okano, T.; Tano, Y. N. Engl. J. Med. 2004, 351, 1187−1196. (26) Liang, X.; Kozlovskaya, V.; Chen, Y.; Zavgorodnya, O.; Kharlampieva, E. Chem. Mater. 2012, 24, 3707−3719. (27) http://www.accessdata.fda.gov/scripts/cder/iig/getiigWEB.cfm. (28) Cho, J. Y.; Lee, J.; Gutowska, A.; Tarasevich, B. J.; Sohn, Y. S.; Jeong, B. J. Drug Delivery Sci. Technol. 2006, 16, 71−76. (29) Yu, L.; Ding, J. Chem. Soc. Rev. 2008, 37, 1473−1481. (30) Yu, L.; Zhang, H.; Ding, J. Angew. Chem., Int. Ed. 2006, 45, 2232−2235. (31) Zhang, H.; Yu, L.; Ding, J. Macromolecules 2008, 41, 6493− 6499. (32) Karim, A. A.; Loh, X. J. Soft Matter 2015, 11, 5425−5434. (33) Zheng, J. Y.; Tan, M. J.; Thoniyot, P.; Loh, X. J. RSC Adv. 2015, 5, 62314−62318. (34) Ye, E.; Chee, P. L.; Prasad, A.; Fang, X.; Owh, C.; Yeo, V. J. J.; Loh, X. J. Mater. Today 2014, 17, 194−202. (35) Loh, X. J. Mater. Horiz. 2014, 1, 185−195. (36) Chang, G.; Ci, T.; Yu, L.; Ding, J. J. Controlled Release 2011, 156, 21−27. (37) Shen, W.; Luan, J.; Cao, L.; Sun, J.; Yu, L.; Ding, J. Biomacromolecules 2015, 16, 105−115. (38) Dou, Q. Q.; Liow, S. S.; Ye, E.; Lakshminarayanan, R.; Loh, X. J. Adv. Healthcare Mater. 2014, 3, 977−988. (39) Ko, D. Y.; Shinde, U. P.; Yeon, B.; Jeong, B. Prog. Polym. Sci. 2013, 38, 672−701. (40) Svenson, S.; Thompson, D. H. J. Org. Chem. 1998, 63, 7180− 7182.

compared with a short gel duration of PEG-PPG-PEG thermogels, which persist less than 1 day in the subcutaneous layer of rats.64 The ionic interactions among the zwitterions of PC contributed to the persistence as well as biocompatibility of the in situ formed thermogel.

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CONCLUSIONS A new zwitterionic thermogel of PC-PPG-PC was designed by using the biomimetic PC and biocompatible PPG. PC-PPG-PC aqueous solutions in a specific concentration range of 40−60 wt % underwent multiple sol-to-gel-to-sol-to-gel transitions as the temperature increased. The changes in nanoassemblies accompanying unimer−micelle transitions, changes in ionic interactions, and the dehydration of PPG were suggested as the mechanism of the multiple transitions as described in Figure 4. In addition, we investigated the feasibility of using PC-PPG-PC in protein and cell delivery systems. The steady release of insulin as well as stem cells from the in situ formed zwitterionic gel was observed. When the polymer aqueous solution was subcutaneously injected into rats, a hydrogel was formed in the injection site. The gel exhibited very mild tissue responses around the injection site. Based on the biocompatibility, cytocompatibility, and delivery efficacy, the zwitterionic PCPPG-PC thermogel can be a promising sustained delivery carrier for biopharmaceuticals and cells.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b01169. Experimental protocol on 2D culture of TMSCs, 31P NMR spectrum of PC-PPG-PC (in CDCl3), FT-IR spectrum of PC-PPG-PC and PPG in D2O, gel permeation chromatogram of PC-PPG-PC, FTIR spectra of PPG, and PC-PPG-PC aqueous solutions (45. 0 wt %) as a function of temperature in a range of 10−80 °C, in vivo insulin delivery system of PC-PPG-PC, and mRNA expression of TMSCs cultured in DMEM enriched with FBS (10%) (PDF).

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]. Fax: +82 2 3277 2384. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (2012M3A9C6049835 and 2014M3A9B6034223). D.Y.K. is thankful to the Solvay Scholarship.

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REFERENCES

(1) Ishihara, K.; Ueda, T.; Nakabayashi, N. Polym. J. 1990, 22, 355− 360. (2) Ng, V. W. L.; Chan, J. M. W.; Sardon, H.; Ono, R. J.; Garcia, J. M.; Yang, Y. Y.; Hedrick, J. L. Adv. Drug Delivery Rev. 2014, 78, 46−62. (3) Zhang, L.; Cao, Z.; Bai, T.; Carr, L.; Menye, J. R. E.; Irvin, C.; Ratner, B. D.; Jinag, S. Nat. Biotechnol. 2013, 31, 553−556. (4) Xu, Y.; Jang, K.; Konno, T.; Ishihara, K.; Mawatari, K.; Kitamori, T. Biomaterials 2010, 31, 8839−8846. 3861

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Article

Biomacromolecules (41) Kim, J. Y.; Kim, H. S.; Ryu, K. H.; Woo, S. Y. Cytokine 2012, 59, 211−214. (42) Dreissig, I.; Machill, S.; Salzer, R.; Krafft, C. Spectrochim. Acta, Part A 2009, 71, 2069−2075. (43) Loh, X. J.; Tan, Y. X.; Li, Z.; Teo, L. S.; Goh, S. H.; Li, J. Biomaterials 2008, 29, 2164−2172. (44) Tardif, K.; Cloutier, I.; Miao, Z.; Lemieux, C.; Denis, C. S.; Winnik, F. M.; Tanguay, J. F. Biomaterials 2011, 32, 5046−5055. (45) Choi, Y. Y.; Jang, J. H.; Park, M. H.; Choi, B. G.; Chi, B.; Jeong, B. J. Mater. Chem. 2010, 20, 3416−3421. (46) Schilli, C. M.; Zhang, M.; Rizzardo, E.; Thang, S. H.; Chong, Y. K.; Edwards, K.; Karlsson, G.; Müller, A. H. E. Macromolecules 2004, 37, 7861−7866. (47) Hwang, M. J.; Joo, M. K.; Choi, B. G.; Park, M. H.; Hamley, I. W.; Jeong, B. Macromol. Rapid Commun. 2010, 31, 2064−2069. (48) Park, M. J.; Char, K. Macromol. Rapid Commun. 2002, 23, 688− 692. (49) Chang, G.; Yu, L.; Yang, Z.; Ding, J. Polymer 2009, 50, 6111− 6120. (50) Li, T.; Ci, T.; Chen, L.; Yu, L.; Yang, Z.; Ding, J. Polym. Chem. 2014, 5, 979−991. (51) Chen, L.; Ci, T.; Yu, L.; Ding, J. Macromolecules 2015, 48, 3662−3671. (52) Atkins, P. W. Physical Chemistry, 2nd ed.; W. H. Freeman and Company: San Francisco, CA, U.S.A., 1982. (53) Pless, S. A.; Leung, A. W. Y.; Galpin, J. D.; Ahern, C. A. J. Biol. Chem. 2011, 286, 35129−35136. (54) Jeong, Y.; Joo, M. K.; Bahk, K. H.; Choi, Y. Y.; Kim, H. T.; Kim, W. K.; Lee, H. J.; Sohn, Y. S.; Jeong, B. J. Controlled Release 2009, 137, 25−30. (55) Brunner, D.; Frank, J.; Appl, H.; Schöffl, H.; Pfaller, W.; Gstraunthaler, G. Altex 2010, 27, 53−62. (56) Park, J. S.; Yang, H. N.; Jeon, S. Y.; Woo, D. G.; Na, K.; Park, K. H. Biomaterials 2010, 31, 6239−6248. (57) Hoshiba, T.; Kawazoe, N.; Chen, G. Biomaterials 2012, 33, 2025−2031. (58) Park, J.; Kim, I. Y.; Patel, M.; Moon, H. J.; Hwang, S. J.; Jeong, B. Adv. Funct. Mater. 2015, 25, 2573−2582. (59) Crowder, S. W.; Prasai, D.; Rath, R.; Balikov, D. A.; Bae, H.; Bolotin, K. I.; Sung, H. J. Nanoscale 2013, 5, 4171−4176. (60) Brandl, F. P.; Seitz, A. K.; Tessmar, J. K.; Blunk, T.; Göpferich, A. M. Biomaterials 2010, 31, 3957−3966. (61) Park, M. H.; Choi, B. K.; Jeong, B. Adv. Funct. Mater. 2012, 22, 5118−5125. (62) Rodda, D. J.; Chew, J. L.; Lim, L. H.; Loh, Y. H.; Wang, B.; Ng, H. H.; Robson, P. J. Biol. Chem. 2005, 280, 24731−24737. (63) Jeong, B.; Bae, Y. H.; Kim, S. W. J. Biomed. Mater. Res. 2000, 50, 171−177. (64) Sun, K. H.; Sohn, Y. S.; Jeong, B. Biomacromolecules 2006, 7, 2871−2877.

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