Self-Healing Supramolecular Self-Assembled Hydrogels Based on

Publication Date (Web): September 28, 2015 ... The hydrogel formation relied on the host and guest linkage between β-CD and Chol. ... The developed h...
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Self-Healing Supramolecular Self-Assembled Hydrogels Based on Poly(L‑glutamic acid) Guifei Li,† Jie Wu,† Bo Wang,† Shifeng Yan,† Kunxi Zhang,† Jianxun Ding,‡ and Jingbo Yin*,† †

School of Materials Science and Engineering, Shanghai University, Shanghai, 200444, P. R. China Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, P. R. China

‡

S Supporting Information *

ABSTRACT: Self-healing polymeric hydrogels have the capability to recover their structures and functionalities upon injury, which are extremely attractive in emerging biomedical applications. This research reports a new kind of self-healing polypeptide hydrogels based on selfassembly between cholesterol (Chol)-modified triblock poly(L-glutamic acid)-block-poly(ethylene glycol)-block-poly(L-glutamic acid) ((PLGA-bPEG-b-PLGA)-g-Chol) and β-cyclodextrin (β-CD)-modified poly(Lglutamic acid) (PLGA-g-β-CD). The hydrogel formation relied on the host and guest linkage between β-CD and Chol. This study demonstrates the influences of polymer concentration and β-CD/Chol molar ratio on viscoelastic behavior of the hydrogels. The results showed that storage modulus was highest at polymer concentration of 15% w/v and β-CD/ Chol molar ratio of 1:1. The effect of the PLGA molecular weight in (PLGA-b-PEG-b-PLGA)-g-Chol on viscoelastic behavior, mechanical properties and in vitro degradation of the supramolecular hydrogels was also studied. The hydrogels showed outstanding selfhealing capability and good cytocompatibility. The multilayer structure was constructed using hydrogels with self-healing ability. The developed hydrogels provide a fascinating glimpse for the applications in tissue engineering.

1. INTRODUCTION Self-healing polymeric hydrogels are able to restore the initial properties after the interior or exterior cracks attributed to the dynamic/reversible linkages in the hydrogel networks,1,2 which is similar to some living organisms, such as human skin.3 Although the traditional hydrogels exhibit superior performances, their desirable properties and integrity of network structure are often obviously deteriorated or even lost when they suffer from micro- or macroscale injury, limiting their lifetime.2 Since self-healing hydrogels exhibit improved safety and extended lifetime,4 various kinds have been designed for diverse applications, such as biosensors,5 drug delivery systems,6−8 wound healing,9,10 and shape memory materials.11,12 Dynamic covalent and noncovalent interactions are employed to design self-healing hydrogels. The self-healing hydrogels developed by dynamic covalent chemistry mostly demand additional interventions (e.g., heat, pH, or light) and healing agents to trigger their reversible process.13−19 This difficulty of manipulation in vivo impedes their biomedical application.20 A noncovalently bonded system is an efficient approach to prepare self-healable hydrogels,21−26 which can autonomously repair themselves and restore their initial structures and functions without external stimuli.2 Host− guest interaction is an important noncovalently interaction combining multiple dynamic interactions, which is essential in © XXXX American Chemical Society

the self-healing hydrogel formation. Cyclodextrin (CD) possessing hydrophobic internal cavity can encapsulate guest molecules to form inclusion complex, which endow its applications in host−guest hydrogels.27 As guest molecules, adamantane,28 azobenzene,29 ferrocene,30 n-butyl, and t-butyl groups31 have been applied in the preparations of self-healing hydrogels. In consideration of biocompatibility and biodegradability of guest moieties, cholesterol (Chol) as an essential structural component of animal cell membranes32 can be a good candidate for guest molecules.33,34 The materials of self-healing hydrogels nowadays relying on host−guest chemistry focus on poly(acrylic acid) and polyacrylamide.30,35 Although the healing efficiency of these hydrogels has been almost improved to 100%, there is still challenges in tissue engineering applications because they have not been proven to be biodegradable.36 Therefore, the constructions of self-healing hydrogels using host−guest chemistry and biodegradable polymers are of practical interest and highly attraction. Poly(L-glutamic acid) (PLGA) is an ideal biomedical polypeptide material, which exhibits the advantages of nontoxicity, hydrophilicity, biodegradability, and avoiding antigenicity or immunogenicity.37 Recently, we have reported the PLGA/alginate injectable hydrogels for cartilage tissue Received: July 14, 2015

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a flask. After dissolution, DMAP (4.5 mmol, 0.5514 g) and EDC·HCl (9.0 mmol, 1.73 g) were added. The molar ratio of −COOH in PLGA to Chol was set at 5:1. After reaction for 72 h at 25 °C, the system was entirely dialyzed against dichloromethane and deionized water followed by lyophilization. Coupling of Chol to both PLGA13494-bPEG2000-b-PLGA13494 and PLGA30250-b-PEG2000-b-PLGA30250 was done accordingly. 2.3. Synthesis of PLGA-g-β-CD. PLGA modified with β-CDNH2·HCl moiety was synthesized using condensing agent of EDC· HCl. PLGA (15.5 mmol, 2.0 g) and β-CD-NH2·HCl (7.75 mmol, 8.8 g) were dissolved in DMSO (100 mL). To this solution, NHS (7.75 mmol, 0.782 g) and EDC·HCl (15.4 mmol, 2.61g) were added. The molar ratio of −COOH in PLGA to β-CD-NH2·HCl was set at 2:1. After stirring for 72 h at 35 °C, the system thoroughly completed dialysis process in deionized water for another 72 h. The residue was freeze-dried to obtain the final purified product. 2.4. Characterization of Polymers. The (PLGA-b-PEG-bPLGA)-g-Chol and PLGA-g-β-CD were confirmed by proton nuclear magnetic resonance (1H NMR) (AV 500 MHz, Bruker, Switzerland), Fourier-transform infrared (FTIR) spectroscopy (AVATAR 370, Nicolet, USA), differential scanning calorimeter (DSC) (Q-2000, TA, USA) and thermogravimetric analyzer (TGA) (Q-500, TA, USA). 1 H NMR spectra of (PLGA-b-PEG-b-PLGA)-g-Chol in deuterated trifluoroacetic acid (CF3COOD) and PLGA-g-β-CD in deuterated water (D2O) were carried out to confirm the chemical structures and analyze the graft ratio of Chol and β-CD onto PLGA. FTIR spectra were obtained with a KBr pellet method between 4000 and 500 cm−1 using an AVATAR 370 FTIR spectrophotometer from Nicolet. DSC was examined by means of TA Q-2000 apparatus with a 10 °C min−1 heating speed between −50 and 280 °C. TGA was conducted on a TA Q-500 thermogravimetric apparatus at a 10 °C min−1 heating rate in N2 atmosphere. 2.5. Preparation of Supramolecular Hydrogels. Hydrogels were obtained by mixing (PLGA-b-PEG-b-PLGA)-g-Chol and PLGAg-β-CD in water. The typical procedure was as follows: 77 mg of (PLGA1433-b-PEG2000-b-PLGA1433)-g-Chol and 73 mg of PLGA-g-βCD were dissolved in deionized water, respectively. The pH values of above two solutions were adjusted to 7.4 using sodium hydroxide (NaOH) solution (2 mol L−1), and then gently mixed to form the PLGA1433 hydrogels with different polymer concentrations (10 and 15% w/v). The β-CD/Chol molar ratio was kept at 1:1. Similarly, the PLGA13494 and PLGA30250 hydrogels were obtained after mixing solutions of PLGA-g-β-CD with (PLGA13494-b-PEG2000-b-PLGA13494)g-Chol and (PLGA30250-b-PEG2000-b-PLGA30250)-g-Chol, respectively. To investigate the effect of molar ratio (n[β‑CD]/n[Chol]) in the hydrogels on the viscoelastic behavior, the β-CD/Chol molar ratio (3:1, 2:1, 1:1, 1:2, and 1:3) was changed to prepare the PLGA30250 hydrogels at the same polymer concentration (15% w/v). 2.6. Characterization of Hydrogels. Rheological experiments were performed on an AR2000 rheometer from TA Instruments with 12 mm diameter parallel plates and the oscillatory mode at 25 °C. To test the viscoelastic behaviors of the supramolecular hydrogels (12 mm in diameter, and polymer concentration at 10 or 15% w/v), the frequency sweep tests were operated, which covered angular frequencies from 1 to 100 rad s−1 at controlled regular strain of γ = 0.1. Mechanical properties tests were conducted on a Q-800 dynamic thermomechanical analysis (DMA) from TA Instruments. A 12 mm diameter and 8 mm thickness cylindrical hydrogel sample (15% w/v) was laid on the lower plate, and then the upper plate moved down to compress the hydrogel with a 5% min−1 strain speed. X-ray diffraction patterns (XRD) were conducted on a D/ MAX2550 diffractometer from Rigaku using CuK α radiation at 30 mA and 40 kV. The region of the scanning was recorded from 5 to 50° with a 5° min−1 scanning rate. Circular dichroism tests were measured by a spectropolarimeter (J815, JASCO, Japan) using a thermo-controlled 0.2 mm quartz cell (bandwidth: 2.0 nm, response: 1 s, scanning speed: 100 nm min−1, “continues scanning” mode, five accumulations).

engineering.38 Chen’s group has reported the injectable enzymatically cross-linked hydrogels based on PLGA as biomimetic scaffolds for cartilage tissue engineering.39,40 Herein, we reported self-healing supramolecular hydrogels derived from cholesterol (Chol)-modified triblock poly(Lglutamic acid)-block-poly(ethylene glycol)-block-poly(L-glutamic acid) ((PLGA-b-PEG-b-PLGA)-g-Chol) and β-cyclodextrin (β-CD)-modified poly(L-glutamic acid) (PLGA-g-β-CD). The formation procedure and self-healing capability of the supramolecular hydrogels are exhibited in Scheme 1. The self-healing Scheme 1. Preparation and Self-Healing Process of Supramolecular Hydrogels

supramolecular hydrogels were further examined with noncytotoxicity. In addition, with the self-healing property of hydrogels, a kind of multilayer structure model was prepared, which indicated that the hydrogels were promising scaffolds for multiple tissue structure in tissue engineering applications.

2. EXPERIMENTAL SECTION 2.1. Materials. PLGA (Mη = 6.0 × 104 Da) was synthesized inhouse.41 The triblock copolymers of PLGA1433-b-PEG2000-b-PLGA1433, PLGA13494 -b-PEG2000 -b-PLGA 13494, and PLGA 30250 -b-PEG2000 -bPLGA30250 were prepared according to the literature.42 Mono-6amino-6-deoxy-β-cyclodextrin hydrochloride (β-CD-NH2·HCl) was purchased from Shandong Binzhou Zhiyuan Bio-Technology Co., Ltd. (Shandong, P. R. China). 1-Ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC·HCl) was used directly from GL Biochem Co., Ltd. (Shanghai, P. R. China). N-Hydroxysuccinimide (NHS), 4-dimethylaminopyridine (DMAP), Chol, dimethyl sulfoxide (DMSO), dichloromethane (DCM), erioglaucine disodium salt (EDS), and tartrazine (TAR) were used directly from Aladdin Industrial, Inc. (Shanghai, P. R. China). 1,1-Dioctadecyl-3,3,3,3tetramethylindocarbocyanine perchlorate (DiL) and 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) were purchased directly from Sigma-Aldrich Co. LLC. (Shanghai, P. R. China). 2.2. Synthesis of (PLGA-b-PEG-b-PLGA)-g-Chol. Chol was coupled to PLGA1433-b-PEG2000-b-PLGA1433 to obtain (PLGA1433-bPEG2000-b-PLGA1433)-g-Chol by a condensation reaction. A typically synthetic procedure of (PLGA1433-b-PEG2000-b-PLGA1433)-g-Chol was given: PLGA1433-b-PEG2000-b-PLGA1433 (1.0 mmol, 5.0 g), Chol (4.5 mmol, 1.742 g), DMSO (50 mL), and DCM (40 mL) were added into B

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Figure 1. Synthetic routes toward (a) (PLGA-b-PEG-b-PLGA)-g-Chol and (b) PLGA-g-β-CD. Scanning electron microscopy (SEM) images recorded the morphology of the freeze-dried hydrogels. After coating the gold to the cross sections of lyophilized hydrogels, the samples were detected with a microscope. 2.7. Characterization of Self-Healing Ability. (1) (PLGA30250b-PEG2000-b-PLGA30250)-g-Chol (60.0 mg) and PLGA-g-β-CD (90.0 mg) were dissolved in 0.5 mL deionized water, respectively. The pH values of above two solutions were adjusted to 7.4 with 2.0 M NaOH solution. EDS (2.0 mg) and TAR (2.0 mg) were dispersed in PLGA-gβ-CD solutions.43 Then (PLGA30250-b-PEG2000-b-PLGA30250)-g-Chol and PLGA-g-β-CD were mixed in a vial to prepare hydrogels. Two cylindrical hydrogels separately stained with EDS and TAR were cut in half. The two semicylindrical supramolecular hydrogels leaned on each other at the site of fresh cuts. With the purpose of observing the selfhealing process, a 126.7 μm crack was punched in the connection position of the hydrogels. The healing process of the hydrogels was recorded on an optical microscopy at diverse time intervals. (2) According to the above-mentioned methods, hydrogels with EDS and TAR were prepared using rod-like vials, respectively. Hydrogels were cut into similar pieces. Then, the separated pieces were joined together in turn and merged into a continuous column. The column-constructed hydrogels could be bent to a semicircle or circle. (3) Rheological experiments were performed on an AR2000 rheometer from TA Instruments with 12 mm diameter parallel plates at 25 °C. The viscoelasticity of hydrogels (12 mm in diameter, and

polymer concentration at 15% w/v) was operated in strain amplitude sweep (0.1−500%) at a fixed angular frequency of 1.0 rad s−1. The hydrogels disks (12 mm in diameter, and polymer concentration at 15% w/v) were measured with the alternate step strain test (strain = 1 and 200%, angular frequency = 1.0 rad s−1). The continuous step strains were switched with 200 s for every strain interval. 2.8. Degradation of Hydrogels. The degradation of PLGA1433, PLGA13494 and PLGA30250 hydrogels (15% w/v) were carried out in transparent glass vials. The net weight of empty glass vial was recorded as W1, and the total mass of hydrogel and glass vial was labeled as W2. The hydrogel was immersed in 0.1 M phosphate-buffered saline (PBS) (pH = 7.4). The PBS was removed exhaustively prior to the measurement of glass vial and hydrogel mass (W3) every 12 h. The weight loss ratio (R) was obtained according to the formula: R = (W3 − W2)/(W2 − W1) × 100%.44 The amount of residual hydrogels changed as a function of time, which was defined as the degradation of the hydrogel. All results were confirmed in triplicate. 2.9. Cytotoxicity Measurement. Adipose-derived stem cells (ASCs) were used to examine the relative cytotoxicity of the polymers. ASCs were seeded in 96-well microplates after being incubated overnight at a density of 105 cells per well in 200.0 μL medium involving 50 μg mL−1 streptomycin, 50 μg mL−1 penicillin and 10% (v/v) fetal bovine serum (FBS). Then, ASCs were incubated for 24 h (37 °C, 5% CO2) in the polymer solutions with different concentrations (0.25−4.0 g L−1) in 96-well microplates. Furthermore, the solution of methyl thiazolyl tetrazolium (MTT) was added into C

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Figure 2. Synthesis and characterization of (PLGA-b-PEG-b-PLGA)-g-Chol. (a) 1H NMR spectra, (b) FTIR spectra, (c) DSC analyses, and (d) TGA curves of (A) PLGA1433-b-PEG2000-b-PLGA1433, (B) Chol and (C) (PLGA1433-b-PEG2000-b-PLGA1433)-g-Chol19.67%. each well, and ASCs were cultured for another 1 h. The absorbance assay was conducted on a SpectraMax M2 microplate reader from Molecular Devices. Asample was the absorbance of the sample, while Acontrol was that of the control well. Cells incubated in medium without any polymer solutions were used as control. The measurements were performed in three times. The cell viability was obtained by the formula: Cell Viability = (Asample/Acontrol) × 100%. 2.10. Construction of Multilayer Structure. (PLGA30250-bPEG2000-b-PLGA30250)-g-Chol (15% w/v) and PLGA-g-β-CD (15% w/ v) were dissolved in deionized water, respectively. The pH values of solutions were adjusted to 7.4 with 2.0 M NaOH solution. 1 × 106 ASCs stained with DiL dyes (red fluorescence) and DiO dyes (green fluorescence) were dispersed in PLGA-g-β-CD solutions, respectively, followed by mixing with (PLGA30250-b-PEG2000-b-PLGA30250)-g-Chol (15% w/v) solution. Fluorescence microscopy microimages were photographed to observe the cell dispersion and perform the constructing process of multiple structures by self-healing hydrogels supported different stained cells.

DMSO, the reaction was performed in the mixture solvent of DMSO/DCM. PLGA-g-β-CD was synthesized by a facile one-step amidation reaction using EDC·HCl and NHS as activators. The synthetic routes for (PLGA-b-PEG-b-PLGA)-g-Chol and PLGA-g-β-CD were shown in Figure 1. 1 H NMR spectra of PLGA1433-b-PEG2000-b-PLGA1433, Chol, and (PLGA1433-b-PEG2000-b-PLGA1433)-g-Chol were shown in Figure 2a. It was clear that the graft ratio of Chol moiety onto PLGA side group was 19.67%. For the graft ratios of the (PLGA13494-b-PEG2000-b-PLGA13494)-g-Chol and (PLGA30250-bPEG2000-b-PLGA30250)-g-Chol were 20.03 and 19.53%, respectively, as shown in Table 1. The representative FTIR spectra of PLGA1433-b-PEG2000-bPLGA1433, Chol, and (PLGA1433-b-PEG2000-b-PLGA1433)-g-Chol were presented in Figure 2b. For PLGA1433-b-PEG2000-bPLGA1433, the stretching vibration of C−H bond in PEG2000 could be characterized by the peaks at 2930 and 2850 cm−1, the peak at 1729 cm−1 was ascribed to the stretching vibration of carbonyl group in PLGA1433, and the characteristic absorption peaks at 1630 and 1521 cm−1 were attributed to amide I and amide II groups, respectively.38 For the native Chol, the peaks of C−H bond at 2930 and 2850 cm−1 were attributed to the stretching vibration of C−H bond. The bending vibration of C−H bond in Chol could be characterized by the peak at 1470 cm−1, and the absorption band appeared at 1380 cm−1 was assigned to the bending vibration of C−H bond of−CH3 group. The characteristic absorption band of primary alcohol at 1050 cm−1 was attributed to the bending vibration of C−O bond.45 Modification of (PLGA1433-b-PEG2000-b-PLGA1433)-g-Chol was confirmed by the fact that the peaks at 2930 and 2850 cm−1 were larger than

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of (PLGA-b-PEGb-PLGA)-g-Chol and PLGA-g-β-CD. The side carboxyl group in PLGA-b-PEG-b-PLGA could be activated by EDC·HCl and DMAP, and then involved in the reaction with Chol. EDC·HCl was an important coupling agent, and DMAP was a significant catalyst in esterification reaction. Since Chol was insoluble in Table 1. Graft Ratio of Chol no.

graft ratio of Chol

1 2 3

(PLGA1433-b-PEG2000-b-PLGA1433)-g-Chol19.67% (PLGA13494-b-PEG2000-b-PLGA13494)-g-Chol20.03% (PLGA30250-b-PEG2000-b-PLGA30250)-g-Chol19.53% D

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Figure 3. Synthesis and characterization of PLGA-g-β-CD. (a) 1H NMR spectra, (b) FTIR spectra, (c) DSC analyses, and (d) TGA curves of (D) PLGA, (E) β-CD-NH2·HCl, and (F) PLGA-g-β-CD48.31%.

stretching vibration appeared at 2873 cm−1. A broad absorption peak of the C−O stretching vibration in pyranose rings appeared at around 1000−1200 cm−1.46 The formation of new peaks of PLGA-g-β-CD at 2873 and 1000−1200 cm−1 indicated that PLGA was successfully modified by β-CD. Typical DSC analyses of PLGA, β-CD-NH2·HCl, and PLGAg-β-CD were shown in Figure 3c. PLGA formed a fusion peak at 240 °C during heating, while β-CD-NH2·HCl formed a broad endothermic peak in the region of 90−210 °C. The peak at 90−210 °C was visible in the DSC scan of PLGA-g-β-CD, which proved that β-CD reacted with PLGA. The representative TGA analyses of PLGA, β-CD-NH2·HCl, and PLGA-g-β-CD were presented in Figure 3d. Among these polymers, PLGA had the worst thermal stability. However, the thermal stability of PLGA-g-β-CD had a further improvement, which was attributed to the introduction of β-CD. 3.2. Formation and Characterization of Supramolecular Hydrogels. Hydrogels could be prepared by the physical cross-linking of PLGA-g-β-CD with (PLGA-b-PEG-b-PLGA)-gChol. The mechanism of gelation was attributed to the supramolecular host−guest interaction between β-CD group of PLGA-g-β-CD and Chol group of (PLGA-b-PEG-b-PLGA)g-Chol. Viscoelastic materials, such as hydrogels, containing dynamically viscous and elastic mechanical properties exhibit the sensitive responses to the mechanical disturbance. The storage modulus (G′) corresponding to elastic part reflects the energy storage of the materials after perturbation. The loss modulus (G″) of viscous part reports the energy loss of active network through the relaxation or dissipated heat. Oscillatory rheology was used to measure rheological properties of the hydrogels. In the frequency sweep test, G′

those of PLGA1433-b-PEG2000-b-PLGA1433, which indicated Chol was successfully grafted onto PLGA1433-b-PEG2000-bPLGA1433. The representative DSC analyses of PLGA1433-b-PEG2000-bPLGA1433, Chol, and (PLGA1433-b-PEG2000-b-PLGA1433)-g-Chol were presented in Figure 2c. Neat Chol formed a fusion peak at 148 °C during heating. It was the obvious characteristic heat energy signal about (PLGA1433-b-PEG2000-b-PLGA1433)-g-Chol as compared to PLGA1433-b-PEG2000-b-PLGA1433 at 148 °C, which proved Chol was conjugated onto PLGA1433-b-PEG2000b-PLGA1433. Figure 2d showed typical TGA curves of PLGA1433-bPEG 2000-b-PLGA 1433 , Chol, and (PLGA1433 -b-PEG2000 -bPLGA 1433 )-g-Chol. PLGA 1433 -b-PEG 2000 -b-PLGA 1433 and (PLGA1433-b-PEG2000-b-PLGA1433)-g-Chol exhibited a similar two stage thermal behavior, corresponding to the decomposition of PLGA (200−350 °C) and PEG (350−450 °C), respectively. The mass loss rate of (PLGA1433-b-PEG2000-bPLGA1433)-g-Chol (92%) was higher than that of PLGA1433-bPEG2000-b-PLGA1433 (89%) after Chol grafted. The 1H NMR spectra of PLGA, β-CD-NH2·HCl, and PLGAg-β-CD were presented in Figure 3a. 1H NMR spectra showed that β-CD moieties were coupled to −COOH groups of PLGA with 48.31% graft ratio. The FTIR spectra of PLGA, β-CD-NH2·HCl, and PLGA-g-βCD were described in Figure 3b. Corresponding to the bending vibration of O−H in the carboxyl group (−COOH) and the stretching vibration of CO, the absorption bands appeared at 3400 and 1720 cm−1, respectively. The peaks at 1625 and 1510 cm−1 could be assigned to amide I and amide II bands, respectively. For β-CD-NH2·HCl, the characteristic spectrum of oligosaccharide structure was presented. The peak of C−H E

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Figure 4. Characterization of hydrogels. G′ and G″ of (a) PLGA1433, (b) PLGA13494, and (c) PLGA30250 hydrogels with different concentrations (n[β‑CD]/n[Chol] = 1:1). (d) G′ of PLGA30250 hydrogels (15% w/v) with different ratios of β-CD to Chol. (e) Compression tests for the hydrogels of PLGA1433, PLGA13494, and PLGA30250 at 25 °C. (f) SEM microimage of PLGA30250 hydrogels (15% w/v).

Figure 5. Mechanism of supramolecular hydrogels. (a) X-ray diffraction patterns and (b) circular dichroism analyses of (PLGA30250-b-PEG2000-bPLGA30250)-g-Chol (A), PLGA-g-β-CD (B), and PLGA30250 hydrogels (C).

F

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Figure 6. Photographs of the self-healing process: (a) two cylindrical hydrogels (left: TAR, right: EDS); (b) hydrogels were cut in half; (c) two semicircle hydrogels healed completely into a full disk-shaped hydrogel within 1 min without any additional stimulus; (d) the healed hydrogel was vertically picked up with forceps. The details of the self-healing process were recorded by optical microscopy microimages: (c1) 0 s; (c2) 5 s; (c3) 10 s; (c4) 20 s; (c5) 60 s; (c6) 5 min.

Figure 7. Flexibility of self-healing hydrogels: (a) two rod-like hydrogels stained with EDS and TAR, respectively; (b and e) color alternating hydrogel columns; (c) the self-healed hydrogel column held vertically by forcep; (d and f) the healed hydrogel rod bent to a semicircle or a circle. The evolution process of the colorant diffusion: Photo images of color alternating hydrogel column stored for 1 (f), 12 (g), and 36 h (h).

Figure 8. Rheological characterization of self-healing hydrogels: (a) G′ and G″ of hydrogels as a function of strain (0.1−500%), and (b) G′ and G″ of hydrogels in alternate step strain test at a fixed time interval of 200 s. G

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PEG2000-b-PLGA30250)-g-Chol was found to be amorphous. A certain degree of crystallinity of PLGA-g-β-CD was demonstrated by three broad crystalline peaks at 2θ = 12.5, 18.5, and 24° in the diffractogram. For PLGA30250 hydrogels, the diffractogram did not show the presence of the typical peaks at 2θ = 12.5°, whereas a broad diffraction peak in the 2θ range of 14−26° was detected. It showed that the host−guest supramolecular assembly between (PLGA30250-b-PEG2000-bPLGA30250)-g-Chol and PLGA-g-β-CD was formed during the gelation, and PLGA30250 hydrogels were amorphous in nature.34 The formation of hydrogels relying on the host−guest interaction between β-CD and Chol was proved by circular dichroism spectroscopy. The results were shown in Figure 5b. It was reported that random conformation possessed two characteristic bands corresponding to the negative band at 190 nm and positive band at 205−230 nm in circular dichroism spectra.47 (PLGA30250-b-PEG2000-b-PLGA30250)-g-Chol and PLGA-g-β-CD mainly exhibited random conformation. For PLGA30250 hydrogels, the negative band slightly increased at 230 nm, and another negative band red-shifted to 205 nm compared to the component polymers at 200 nm, which indicated β-sheet conformation increased in the PLGA block after inclusion complex formation.48 3.4. Self-Healing Ability. To demonstrate the self-healing behavior of PLGA30250 hydrogels, two cylindrical hydrogels (Figure 6a) with EDS and TAR, respectively, were cut into two pieces (Figure 6b) and then put together, followed by punching a 126.7 μm crack (Figure 6c). After 60 s, the two pieces of hydrogel disks healed and repaired their original shape (Figure 6d). To record the self-repairing process of the supramolecular hydrogels, the optical microscopy images were taken at various time intervals (Figure 6c1−c6). The initial gap width of the fresh crack was 126.7 μm (Figure 6c1), and then narrowed down to 42.3 μm after 5 s (Figure 6c2). The crack disappeared fast within 10 s (Figure 6c3), and the injury healed completely within 60 s (Figure 6c4−c5). The EDS and TAR used to stain hydrogels interpenetrated quickly with each other within 5 min (Figure 6c6). The above results showed that the hydrogels possessed good self-healable ability as well as appropriate permeation for bioactive agent delivery. The excellent flexibility of the self-healing hydrogels was shown in Figure 7. Hydrogel blocks stained with EDS and TAR were connected together alternately and in sequence, and a column was constructed (Figure 7a,b). The self-healed hydrogel column was held vertically by forceps and kept its integrity (Figure 7c). The resultant column was very flexible, and the interface between hydrogel blocks was steadily jointed and even not damaged after being bent to a semicircle (Figure 7d) or a circle (Figure 7f). The evolution process of the diffusion was exhibited in Figure 7e−h. After the healed hydrogels stood for 36 h, EDS and TAR deeply diffused to form an integrated one (Figure 7h). The efficiency of the diffusion was higher than nanofiber network-assisted selfhealing hydrogels.49 The quick and efficient permeability was a vital factor for hydrogels used in tissue engineering, which indicated that the developed self-healable hydrogels held potential biomedical applications. Rheology tests were performed to measure the self-healing ability of hydrogels.17,26 The strain amplitude sweep was employed to monitor the G′ and G″ of PLGA30250 hydrogels. As shown in Figure 8a, under the strain from 0.1% to 100%, G′ and G″ remained constant or G′ was larger than G″, suggesting that the hydrogel networks were stiff and the cross-linkage

Figure 9. Degradation of hydrogels. (a) Weight loss ratios of PLGA1433, PLGA13494, and PLGA30250 hydrogels in time. (b) Volume changes of a 15% w/v PLGA30250 hydrogel as a function of time.

and G″ of the supramolecular self-assembled hydrogels were measured with a range of frequencies from 1 to 100 rad s−1 at controlled regular strain of 10%. The results were shown in Figure 4a−d. For all the hydrogels of PLGA1433, PLGA13494, and PLGA30250, G′ increased rapidly with the upregulation of concentration as performed in Figure 4a−c. The G′ of PLGA1433 hydrogels at polymer concentration of 15% w/v were 2−6 folds higher than those at 10% w/v, which indicated that the hydrogels prepared at a higher concentration was tougher (Figure 4a). When the hydrogels were prepared under the same condition of polymeric concentration (10 or 15% w/ v), G′ increased with the increasing of PLGA molecular weight, which could be ascribed to the improvement of cross-linking degree between β-CD and Chol (Figure 4b,c). Figure 4d showed the G′ of PLGA30250 hydrogels (15% w/v) with different ratios of β-CD to Chol. When the β-CD/Chol molar ratio was 1:1, the highest G′ was obtained, ascribing to the highest cross-linking density at 1:1. Excellent compressive property is an important portion in the evaluation of a space-filling hydrogel. The mechanical resistances of the hydrogels were evaluated by compressive stress−strain tests, as shown in Figure 4e. An increase in PLGA molecular weight led to increasing moduli from 6 kPa for PLGA1433 hydrogels to 46 kPa for PLGA30250 hydrogels, which could be ascribed to the increased cross-linking degree with PLGA blocks of different lengths. Figure 4f showed the native morphology of PLGA30250 hydrogels at a concentration of 15% w/v. The hydrogels exhibited a porous and interconnected structure with pore diameter of about 40 μm. For the hydrogels, the microstructure is prerequisite as it tunes mass delivery and offers a comfortable matrix for cell growth in tissue engineering. 3.3. The Formation Mechanism of Supramolecular Hydrogels. The supramolecular host−guest interaction mechanism of gelation could be studied by XRD and circular dichroism. Figure 5a showed the XRD spectra of the component polymers and the hydrogels. (PLGA30250-bH

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Figure 10. Construction of multilayer structure: (a) two pieces of hydrogels with ASCs stained with DiL and DiO dyes separately; (b) the healed hydrogels kept their integrity; (a1) fluorescence microscopy microimage of two pieces of hydrogels; (b1) fluorescence microscopy microimage of selfhealed hydrogels. (c) Cytotoxicity evaluation of mixture solutions of (PLGA-b-PEG-b-PLGA)-g-Chol and PLGA-g-β-CD.

(pH 7.4) at 37 °C. The degradation of the PLGA1433, PLGA13494, and PLGA30250 hydrogels at a concentration of 15% w/v were shown in Figure 9a,b. As shown in Figure 9a, after an initial swelling, the hydrogels started to degrade, and the stabilities and degradation time of the PLGA1433, PLGA13494, and PLGA30250 hydrogels relied on the molecular weight of PLGA. Hydrogels made with PLGA with a higher molecular weight exhibited a lower initial swelling and a higher stability compared to those prepared with lower molecular weight PLGA. The degradation time of the developed hydrogels reached upward of two months, which was longer than the physically cross-linked hydrogels previously reported,33,50 suggesting that the hydrogels formed from (PLGA-b-PEG-b-PLGA)-g-Chol and PLGA-g-β-CD had a more stable structure. The time-dependent volumetric variation of a 15% w/v PLGA30250 hydrogel in PBS at 37 °C was shown in Figure 9b. The visible changes suggested that the supramolecular hydrogel slightly swelled in the first 36 days, which was similar to the

remained undamaged under relatively large deformations. With the G′ and G″ curves crossed, a gel−liquid transition point occurred at the strain of 150%, resulting in the beginning of hydrogel networks destruction.26 It was shown that G′ was smaller than G″, and G′ dramatically decreased to 15 Pa at the large strain of 500%, suggesting the cross-linking networks were completely collapsed. According to the results of strain amplitude sweep, the self-repairing capacity of PLGA30250 hydrogels was confirmed by the alternate step strain test (strain = 1 and 200%) at a fixed time of 200 s, as shown in Figure 8b. By switching the strain from a large strain of 200.0% to a small strain of 1% at a fixed angular frequency (1.0 rad s−1), the G′ and G″ recovered quickly to the initial data without any significant decrease in each repeatable cycle of the recovery. The above studies showed that PLGA30250 hydrogels recovered their original properties rapidly after being sheared. 3.5. Degradation of Hydrogels. The degradation is an important factor for hydrogel materials applied in the biomedical field. The degradation behaviors of the supramolecular hydrogels were studied in vitro in buffer solutions I

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data demonstrated in Figure 8a, and totally disappeared in the following 36 days. 3.6. Construction of Multilayer Structure and Cytotoxicity. It is an emerging trend to develop stratified scaffolds to support different types of cells in each layer to simultaneously regenerate multiple tissues in hybrid tissue engineering. As a result, the interfacial joint of discrete layers must be a critical design consideration. To date, two methods of suturing51 and gluing52 were used to join two materials. However, the suturing would result in the formation of voids, which could destroy the transplantation outcome. The gluing method introduced adhesive materials between discrete layer compartments, which might impede nutrient delivery or cell movement at the interface.53 We proposed that using selfhealing hydrogels to construct multilayer structure could potentially be applied in hybrid tissue regeneration. The construction of multilayer structure employing selfhealing hydrogels was shown in Figure 10. After putting two pieces of ASC-encapsulated hydrogels stained with DiL and DiO, respectively, together (Figure 10a), the reconstructed hydrogels were healed, and a complete joint achieved (Figure 10b). As shown in fluorescence microscopy microimages, two parts of cells integrated perfectly (Figure 10a1 and 9b1). It was also confirmed that the introduction of cells did not undermine the self-healing behavior of hydrogels. To investigate the cytotoxicity of the materials, the ASCs were subjected to the mixture solutions of (PLGA-b-PEG-bPLGA)-g-Chol and PLGA-g-β-CD with different concentrations for 24 h, meanwhile DMSO was employed as a positive control. Figure 10c showed the relative viabilities of ASCs, which were analyzed by MTT assay. The polymers displayed outstanding cytocompatibility even with the concentrations of the polymers up to 4.0 g L−1. To investigate whether the hydrogels could provide a suitable environment for cell growth, ASCs were three-dimensionally encapsulated in the hydrogels. After culture for 48 h, ASCs encapsulated in the hydrogels spread freely, and hydrogels provided a relatively stable matrix for cell culture (Supplementary Figure S1). Taken together, the degradable and biocompatible self-healing hydrogels could potentially be applied in tissue engineering, drug delivery, and other related biomedical fields.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b01287. To investigate whether the hydrogels could provide a suitable environment for cell growth, ASCs were threedimensionally encapsulated in the hydrogels, as detailed in the Supporting Information. After culture for 48 h, ASCs encapsulated in the hydrogels spread freely, and hydrogels provided a relatively stable matrix for cell culture (Supplementary Figure S1). Taken together, the degradable and biocompatible self-healing hydrogels will be potentially applied in tissue engineering, drug delivery, and other related biomedical field. (PDF)

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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 Financial support was provided by the National Natural Science Foundation of China (Nos. 51173101, 51373094, and 51473090), the Natural Science Foundation of Shanghai City (No. 14ZR1414600), the Science and Technology Commission of Shanghai Municipality (No. 15JC1490400), and Shanghai University youth teacher training program. The authors acknowledged Mr. Qiang Li for his help in1H NMR and XRD measurements from Instrumental Analysis Research Centre (Shanghai University).

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REFERENCES

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4. CONCLUSION In summary, a kind of degradable and biocompatible PLGAbased self-healing hydrogels was developed. The hydrogel networks were constructed by host−guest interaction, which was proved by XRD and circular dichroism. The rheological properties, mechanical properties, and degradation rate could be adjusted by changing the polymers concentrations, the PLGA block molecular weight in (PLGA-b-PEG-b-PLGA)-gChol, and β-CD/Chol molar ratio. The self-healing ability of the hydrogels was performed by the macroscopic self-healing tests and rheological measurements. In addition, the hydrogels exhibited excellent flexibility and quick colorant diffusion. The novelly developed hydrogels were particularly used to construct the complex structure due to their excellent self-healing property. We anticipated that the PLGA-based biocompatible self-healable hydrogels provided potentially various applications in tissue engineering. J

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