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Dec 7, 2015 - Center for Biomedical Engineering and Regenerative Medicine, Frontier Institute of Science and Technology, ... Biomaterials have played a crucial role in tissue repair and ... (ACAT) is employed as an electroactive hard segment to offer ..... The PUUL copolymer was dissolved in THF, and the Pt disk was.
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Biocompatible, Biodegradable, and Electroactive Polyurethane-Urea Elastomers with Tunable Hydrophilicity for Skeletal Muscle Tissue Engineering Jing Chen,† Ruonan Dong,† Juan Ge,† Baolin Guo,*,† and Peter X. Ma*,†,‡,§,∥,⊥ †

Center for Biomedical Engineering and Regenerative Medicine, Frontier Institute of Science and Technology, Xi’an Jiaotong University, Xi’an, Shaanxi 710049, China ‡ Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States § Department of Biologic and Materials Sciences, University of Michigan, 1011 North University Ave., Room 2209, Ann Arbor, Michigan 48109, United States ∥ Macromolecular Science and Engineering Center, University of Michigan, Ann Arbor, Michigan 48109, United States ⊥ Department of Materials Science and Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States S Supporting Information *

ABSTRACT: It remains a challenge to develop electroactive and elastic biomaterials to mimic the elasticity of soft tissue and to regulate the cell behavior during tissue regeneration. We designed and synthesized a series of novel electroactive and biodegradable polyurethane-urea (PUU) copolymers with elastomeric property by combining the properties of polyurethanes and conducting polymers. The electroactive PUU copolymers were synthesized from amine capped aniline trimer (ACAT), dimethylol propionic acid (DMPA), polylactide, and hexamethylene diisocyanate. The electroactivity of the PUU copolymers were studied by UV−vis spectroscopy and cyclic voltammetry. Elasticity and Young’s modulus were tailored by the polylactide segment length and ACAT content. Hydrophilicity of the copolymer films was tuned by changing DMPA content and doping of the copolymer. Cytotoxicity of the PUU copolymers was evaluated by mouse C2C12 myoblast cells. The myogenic differentiation of C2C12 myoblasts on copolymer films was also studied by analyzing the morphology of myotubes and relative gene expression during myogenic differentiation. The chemical structure, thermal properties, surface morphology, and processability of the PUU copolymers were characterized by NMR, FT-IR, gel permeation chromatography (GPC), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray diffraction (XRD), scanning electron microscopy (SEM), atomic force microscopy (AFM), and solubility testing, respectively. Those biodegradable electroactive elastic PUU copolymers are promising materials for repair of soft tissues such as skeletal muscle, cardiac muscle, and nerve. KEYWORDS: soft tissue regeneration, elastomers, biomimetic materials, aniline oligomer, polyurethane, electroactivity articular cartilage,14 the cardiovascular system,15 and various soft tissues repair and regeneration.16−19 However, degradable PUs with good electroactivity for tissue repair have been paid rare attention. For their excellent soft tissue-like properties, electroactive PUs can be promising regeneration materials for soft tissues such as muscles and nerves which respond to electrical signals. Previous studies indicate that cell proliferation and differentiation can benefit from an electrical stimulus; therefore, conductive or electroactive polymers are employed to provide the surface delivery of an electrical signal for cell culture.20−22

1. INTRODUCTION Biomaterials have played a crucial role in tissue repair and regeneration.1,2 Biodegradable polymers provide temporary sustentation during the healing procedure of tissues and meanwhile enhance the ability of self-repair from injury.3,4 The compatibility evaluation of biomaterials also refers to elasticity when they are applied for elastic tissues such as muscle and tendon. Lots of studies therefore focus on development of elastomeric biomaterials which mimic the behavior of native body parts.5,6 Polyurethanes (PUs) are one of the widely studied synthetic elastic polymers in tissue engineering applications because of their diverse compositions, mechanical flexibility, and biocompatibility.5,7 Biodegradable PUs have been developed for artificial catheters,8 biosensors,9 shape-memory materials,10 drug delivery materials,11,12 bone,13 © 2015 American Chemical Society

Received: September 2, 2015 Accepted: December 4, 2015 Published: December 7, 2015 28273

DOI: 10.1021/acsami.5b10829 ACS Appl. Mater. Interfaces 2015, 7, 28273−28285

Research Article

ACS Applied Materials & Interfaces

Generally, p-phenylenediamine and 2 eqiv. of aniline were dissolved in a mixed solution of ethanol and 1 M HCl and cooled to −5 °C in a NaCl-crushed ice bath. Ammonium persulfate in 1 M HCl solution was added dropwise in 1 h, and the mixture was stirred vigorously for another 4 h to obtain a dark blue suspension. The particles were collected by filtration and washed with 1 M HCl followed by deionized water. The product was then treated with 1 M aqueous ammonia for 2 h and filtrated. The residual solid was washed with deionized water until the filtrate was neutral. The product was finally vacuum-dried at 40 °C for 24 h. Characterization. 1H NMR (400 MHz, DMSO-d6, δ): 5.45 (s, 4H, −NH2), 6.95 (s, 4H, Ar−H), 6.79 (d, 4H, Ar−H), 6.62 (d, 4H, Ar− H). IR (neat, cm−1) 3310 (m, νNH), 3206 (m, νNH), 1598 (s, νCC of quinoid ring), 1504 (s, νCC of benzenoid rings). 2.2.2. Synthesis of Hydroxyl-Capped PLLA. PLLA precursors were synthesized, and their feed ratios were listed in Table 1. L-Lactide,

Polyaniline (PANI) is a promising conductive polymer which has numerous applications including anticorrosion coatings, lightweight battery electrodes, sensors, and electromagnetic shielding because of its electrical conductivity and unique oxidation and reduction transition chemistry.23−25 PANI and PANI variants have recently been studied as novel intelligent polymers for cardiac and neuronal tissue engineering or sensors.26−28 However, PANI’s application in tissue engineering is restricted by several problems. Firstly, because of its nondegradable nature, PANI may cause choric inflammation as in vivo biomaterials. On the other hand, its poor processability and solubility still need to be improved by modifications26,29,30 or forming composites with other polymers.31−33 Aniline oligomers, such as aniline trimer,26,34,35 aniline tetramer,36−39 and aniline pentamer7,40−42 with similar electroactivity to polyaniline, have great biocompatibility and better solubility and processability than PANI. Therefore, they have been used to synthesize functional polymers and hydrogels. In addition, amine capped aniline trimer (ACAT) is a trimer with double terminated amines which can be easily incorporated into multiblock copolymers to endow the materials with electroactivity.26,43 The aim of the present work is to design and synthesize a series of novel biocompatible biodegradable and electroactive polyurethane-urea (PUU) copolymers with elasticity for soft tissue regeneration by combining the advantages of polyurethane and conducting polymers. We hypothesize that those electroactive degradable PUU copolymers with elastic properties represent a new class of biomaterials, and they are good candidates for muscle, cardiovascular, and nerve regeneration where electroactivity is desired. Poly(L-lactide) (PLLA) serves as soft segment in the copolymers because of its good biodegradability and biocompatibility. Dimethylol propionic acid (DMPA) is introduced to improve the surface hydrophilicity of copolymers, and amine capped aniline trimer (ACAT) is employed as an electroactive hard segment to offer electroactivity and physical cross-links to PUUs. All of the segments mentioned above are combined by a diisocyanate through a two-step polymerization. The chemical structure, electroactivity, thermal properties, hydrophilicity, degradability, and mechanical properties of the PUU copolymers are investigated. The potential application of PUU copolymers for skeletal muscle regeneration is studied by using C2C12 myoblast cells. Compared to PLLA and a medical grade polyurethane Tecofelx (PU), the proliferation and myogenic differentiation can be greatly promoted by these electroactive copolymers.

Table 1. Nomenclature and Feed Ratio of Precursor PLLA and Polyurethane-Urea (PUU) Copolymers feed ratio

precursor

n(EG):n(LLA) of PLLA

Mn of PLLA from 1 H NMR

PLLAH PLLAM PLLAL PLLAH

1:48.9 1:16.3 1:9.7 1:48.9

8000 3500 1500 8000

PUU copolymer PUUH PUUM PUUL PUUH0 PUUH1 PUUH2 PUUH3 PUUH4 PUUH5

n(PLLA):n(DMPA):n(HDI):n(ACAT) 1 1 1 1 1 1 1 1 1

1 1 1 0 2.0 2.3 2.7 3.1 3.4

3 3 3 2 4.0 4.3 4.7 5.1 5.4

1 1 1 1 1 1 1 1 1

ethylene glycol, and 0.1% Sn(Oct)2 were transferred into a flask in a glovebox (MBraun labstar). The flask was then immersed in an oil bath at 110 °C with magnetic stirring for 48 h. The product was dissolved in chloroform and precipitated into cold diethyl ether. This dissolution−precipitation procedure was repeated three times to purify the product. PLLA powders were filtered and vacuum-dried for 3 d at 40 °C. Characterization. 1H NMR (400 MHz, CDCl3, δ): 5.18 (t, 2H, poly −CH−), 4.38 (t, 4H, −CH2− of EG), 4.37 (t, 2H, end −CH−), 1.60 (d, 6H, poly −CH3), 1.51 (d, 6H, end −CH3). IR (neat, cm−1) 3690 (m, νOH), 2996 (w, νas CH3), 2959 (w, νOH), 1754 (s, νCO), 1455 (s, δs CH3), 1383 (w, δas CH3), 1370, 1359 (w, δs C−CH3), 1181, 1085 (m, νO−C−O), 871, 755 (s, γ C−CH3). 2.2.3. Synthesis of Degradable Electroactive Polyurethane-Urea Copolymers. A series of biodegradable electroactive polyurethane-urea (PUU) copolymers with different molecular weight of PLLA and varied ACAT content were synthesized through a two-step polymerization (Scheme 1). The feed molar ratios for the PUUH, PUUM, and PUUL (H, M, and L mean the high, medium, and low molecular weight of PLLA used) are listed in Table 1. In the first step, PLLA and DMPA were placed in a two-necked flask equipped with a condenser and dehydrated at 110 °C for 2 h. The powders were dissolved in a mixture of THF and NMP (vol:vol = 5:1) after cooling to 70 °C. HDI and 0.5% Sn(Oct)2 were then added under nitrogen protection. The prepolymerization was carried out at 70 °C for 4 h. In the second step, the temperature was maintained at 60 °C, and ACAT was dissolved in NMP and injected into the above mixture. After reaction for another 4 h, the product was precipitated into cold ether. After filtration, the obtained PUU copolymer was purified and vacuum-dried for 3 d. 2.3. Preparation of PUU Copolymer Thin Films. The obtained PUU copolymers were dissolved in dioxane to form 5 wt % solutions. The solutions were casted into a superflat glass Petri dish which was placed for 72 h under room temperature to form thin films. The films

2. EXPERIMENTAL SECTION 2.1. Materials. Aniline from J&K Scientific Ltd. was distilled twice under reduced pressure before use. L-Lactide (LLA) was recrystallized in dry toluene and subsequently dried under reduced pressure (10−2 mbar) at room temperature for at least 2 days before polymerization. Stannous octoate, Sn(Oct)2 (Aldrich), was dried over molecular sieves and stored under a N2 atmosphere before use. Ammonium persulfate, p-phenylenediamine, ethanol, camphorsulfonic acid (CSA), ethylene glycol (EG), dimethylol propionic acid (DMPA), hexamethylene diisocyanate (HDI), tetrahydrofuran (THF), N-methyl-2-pyrrolidone (NMP), dioxane, dichloromethane, chloroform, diethyl ether, proteinase K, sodium azide, Tris base, and hydrochloric acid (HCl) were purchased from Aldrich or J&K Scientific Ltd. Tecoflex (PU) was purchased from the Lubrizol Corporation and was used as received. 2.2. Synthesis. 2.2.1. Synthesis of Amine Capped Aniline Trimer (ACAT). ACAT was synthesized according to the previous report.44 28274

DOI: 10.1021/acsami.5b10829 ACS Appl. Mater. Interfaces 2015, 7, 28273−28285

Research Article

ACS Applied Materials & Interfaces Scheme 1. Synthetic Route of PLLA, ACAT, and PUU Copolymer

were vacuum-dried at room temperature for 48 h to remove residual solvent. The thickness of the films was around 200 μm determined by a thickness meter (Mitutoyo). 2.4. Cell Adhesion and Proliferation. A 96-well plate (Costar) was first coated with silicone (Dow Corning184) to avoid the disintegration of the plate. The silicone was first mixed with crosslinking agent (Dow Corning, wt/wt =10:1). After stirring, the mixture was put into a vacuum oven to remove air bubbles. Then, the mixture was carefully poured into 96-well plate wells and was placed in a 50 °C oven overnight to obtain silicone coated plates. After that, 100 μL of the dioxane solutions of PLLA, PU, PUUH, PUUM, and PUUL at a concentration of 5 mg/mL was added into each well. The solvent was removed in the air for 2 days. The plate was sterilized with ethylene oxide for 5 h and then washed three times with DPBS and twice with cell culture medium for 30 min each time at 37 °C while rotating. The mouse C2C12 cell line was originally obtained from the ATCC (American Type Culture Collection) and incubated at 37 °C in a humidified incubator containing 5% CO2. The complete growth medium was Dulbecco’s Modified Eagle Medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (GIBCO), 1.0 × 105 U/L penicillin (Hyclone), and 100 mg/L streptomycin (Hyclone). 100 μL of the cell suspension containing approximately 1500 C2C12 cells was added in each well of the 96-well plate coated with PLLA, PU, PUUH, PUUM, and PUUL polymer. Ten μL of the alamarBlue (Invitrogen) reagent was added into each well after the cells were incubated for 24 h and removal of the culture media. The cells were cultured for another 10 h at 37 °C in a humidified incubator containing 5% CO2 protected from direct light. 90 μL of the medium of each well was removed into a 96-well black plate (Costar). Fluorescence was read by a microplate reader (Molecular Devices) with excitation and emission wavelengths of 567 and 594 nm. The cells were incubated for 1, 3, and 5 days and

tested, respectively. Tests were repeated six times for each polymer. Cells seeded on PLLA served as the positive control group. 100 μL of the solution of PLLA, PU, PUUH, PUUM, and PUUL was casted onto a 18 mm × 18 mm cover slide, respectively, and the solvent was removed completely. The cover slides were placed in a 6well plate (Costar) and washed three times with DPBS and twice with cell culture medium for 30 min each time at 37 °C after being sterilized. The C2C12 cells were seeded on the cover slides at a density of approximately 1.0 × 105 cells/well and incubated for 48 h at 37 °C in a humidified incubator containing 5% CO2. The cover slides of the first group were washed three times with DPBS and fixed with 2.5% glutaraldehyde in DPBS at room temperature for 30 min and then washed with DPBS twice. The plates were incubated for 5 min at room temperature with 1.5 mL of 0.1% Triton X-100 diluted in DPBS in each well. The cells were stained by DPBS solution with fluorescein isothiocyanate (FITC) labeled phalloidin (Sigma) at a concentration of 5 μg/mL for 90 min at room temperature, protected from direct light. The cells were washed by DPBS for three times and redyed with DPBS solution with DAPI (Sigma) at a concentration of 0.1 μg/μL for 10 min. The cover slides of the second group were washed three times with DPBS and stained with a LIVE/DEAD Viability/Cytotoxicity Kit (Molecular Probes) by following the protocol of the manufacturer. Cell adhesion and morphology was observed under the inverted fluorescence microscope (IX53, Olympus). 2.5. Myogenic Differentiation. PUU copolymers were dissolved in dioxane to form 5% solutions. A certain amount of CSA (weight ratio ACAT/CSA = 1:1.3) was added, and the solutions were casted on glass slides to form doped PUU films. Films of PLLA, PU, and PUU copolymers were placed in a 24-well plate. C2C12 cells were seeded on the films, and the culture medium was replaced with differentiation medium (DMEM + 2% horse serum) after 24 h. The cells were cultured for 7 days with renewal of the differentiation 28275

DOI: 10.1021/acsami.5b10829 ACS Appl. Mater. Interfaces 2015, 7, 28273−28285

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ACS Applied Materials & Interfaces

a nitrogen atmosphere (nitrogen flow rate of 50 mL/min). The samples were first heated from 25 to 200 °C, and the thermal history of the samples was removed by a 2 min equilibrium at 200 °C. Measurement was taken during the cooling from 200 to −20 °C and the heating scan from −20 to 200 °C at a heating rate of 10 °C/min. Data of Tg and Tm were obtained from the second heating curve. X-ray diffraction patterns were recorded with a Rigaku Smartlab. 5% PUU dioxane solutions were casted on glass slides and vacuum-dried to form films. The tensile test was carried out by a MTS Criterion 43 equipped with a 50 N tension sensor. Copolymer films were prepared into stripes (40 × 6 × 0.2 mm3), and the crosshead speed was set as 15 mm/min. The tensile strength, breaking elongation, and modulus data were obtained by averaging six samples. Copolymer films were cut into 1 × 1 cm2 specimens for enzymatic degradation. Tris/HCl buffer (pH 8.6 at 37 °C) was prepared from a water solution of Tris base and hydrochloric acid. 0.02 wt % of sodium azide was dissolved in the buffer. Each sample of copolymers was weighed and immersed in a vial of 5 mL Tris/HCl buffer, and 1 mg of proteinase K was added. The vials were placed in a 37 °C shaker with rotating speed of 100 rpm. The buffer and proteinase K were replaced every 24 h to maintain the activity. Copolymer films were withdrawn at an interval of 5 d and washed twice with deionized water. Films were dried in an oven at 50 °C overnight and vacuum-dried for 2 d to remove moisture. Dry specimens were weighed, and weight loss was calculated by the following formulation:

medium every 2 days. Immunofluorescence staining was used to analyze the myogenic differentiation of C2C12 cells on the films. Generally, cells were rinsed with PBS and fixed with 2.5% glutaraldehyde, followed by washing with PBS three times. The samples were then treated with 0.3% Triton X-100 for 0.5 h and BSA (1% PBS solution) for 1 h. All of the films were incubated in 0.2% mouse anti-TUBULIN monoclonal antibody (Sigma) (1% BSA PBS solution) overnight at 4 °C. After being washed with PBS, Alexa Fluor 488 conjugated secondary antibody (Molecular Probes) was added, and the mixture was incubated for 2 h at 37 °C. After washing with PBS, cells were counterstained with 0.1% DAPI (Glycerol/PBS 1:1 solution) and observed under an inverted fluorescence microscope (IX53, Olympus). The number, length, diameter, and maturation index data of myotubes were obtained from at least three images of each sample using AxioVision software (Carl Zeiss). 2.6. Characterization. FT-IR spectra of PLLA precursors, ACAT, and the PUU copolymers were obtained with a Nicolet 6700 FT-IR spectrometer (Thermo Scientific Instrument) in the 4000−600 cm−1 range. The spectra were taken as the average of 32 scans at a resolution of 4 cm−1. 1 H NMR (400 MHz) spectra were recorded on a Bruker Ascend 400 MHz NMR instrument with CDCl3 as the solvent for all PLLA samples and internal standard (δ 7.26 ppm). DMSO-d6 was used as the solvent for ACAT and all PUU copolymer samples at room temperature and as internal standard (δ 2.50 ppm). Gel permeation chromatography (GPC) measurements were carried out at 40 °C with a Waters system. THF (for PLLAs) or DMF (for PUUs) was chosen as an eluent at a flow rate of 1 mL/min. The standard curve of molecular weight was calibrated by linear polystyrene standards (Shodex SM-105). The hydroxyl number of PLLA precursors was determined using an esterification method with acetic anhydride and titration of KOH solution.45 For the solubility test, PUU bulk copolymers were added in varied solvents and stirred for 12 h at room temperature. The solutions were observed after a 48 h standing. The absence of any solid residuals was considered as soluble.46,47 The surface morphologies of PUUs were investigated by an atomic force microscope (AFM, Cypher, Asylum Research) and a field emission scanning electron microscopy (SEM, SU-8010, Hitachi). The films were sputter-coated with gold before the SEM observation. Static water contact angle measurements (CAMs) were used to evaluate the surface hydrophilicity of the copolymer films with a contact angle and surface tension meter (Kino). A drop of Mili-Q water was placed on the surface of the sample, and the picture of the water drop was taken by a digital camera. The images were then analyzed with Kino software to obtain the contact angle. The contact angle of each sample was taken as the average of five measurements at different positions on the film. The UV−visible spectra of ACAT and the copolymers in a DMSO solution were obtained from a UV−vis spectrophotometer (PerkinElmer Lambda 35). Cyclic voltammograms (CV) were recorded on an electrochemical workstation (CH Instruments) with a scanning rate of 10 mV/s. A platinum disk acted as working electrode, and a platinum wire and an Ag/AgCl were used as counter and reference electrodes, respectively. The PUUL copolymer was dissolved in THF, and the Pt disk was dipped into the above solution. After the solvent was evaporated, the PUUL copolymer film was coated on the Pt disk and employed as working electrode. The electrical resistance R of copolymer films was measured with an HP 4284A bridge, and the conductivity σ was calculated as σ = l/RS, where l and S are the length and sectional area of the materials, respectively. Thermogravimetric analysis (TGA) was used to determine the thermal stability of the copolymers and the weight content of the ACAT segment. The measurement was carried out with a Mettler-Toledo TGA/DSC 1 thermogravimetric analyzer under a nitrogen atmosphere (nitrogen flow rate of 20 mL/min) and a heating rate of 10 °C/min. The scan range was from 30 to 800 °C. The glass-transition temperature (Tg) and melting temperature (Tm) of PLLA precursors and their copolymers were measured by differential scanning calorimetry (DSC) using a TA Q200 DSC under

weight loss(%) = (W0 − Wt )/W0 where W0 stands for the original weight of copolymer film and Wt is the dry weight of specimen after degradation. 2.7. Statistic Analyses. All the data are expressed as mean ± standard deviation. The Student t test is used to determine the statistical significance, which is considered to be significant when p < 0.05.

3. RESULTS AND DISCUSSION 3.1. Synthesis of Degradable Electroactive PUU Copolymers. This study aims to develop a series of biodegradable electroactive polyurethane-urea (PUU) copolymers with tunable elasticity and hydrophilicity for tissue engineering applications. The synthesis of the PUU copolymers is shown in Scheme 1. Three PLLA precursors (PLLAH, PLLAM, and PLLAL) with different molecular weights are synthesized and employed to study the effect of the soft segment’s length on the properties of the copolymers (PUUH, PUUM, and PUUL). ACAT is introduced into the copolymers to provide the electroactivity of the materials because of its native electrochemical behavior. DMPA with a pendent carboxyl group is employed to improve the hydrophilicity of the materials. All the (macro)monomers are connected together with HDI by polyaddition. Because of the high reaction activity of isocyanate with hydroxyl and amine groups, the PUU copolymers are obtained with a high yield. With an equal feed molar ratio of ACAT and DMPA, the weight fractions of them in the copolymers increase with the decrease of the PLLA molecular weight. Furthermore, six copolymers (PUUH0-5) based on PLLAH are synthesized to study the effect of DMPA content on the hydrophilicity of copolymer avoiding the influence from the length of the PLLA chains. The success of the synthesis and structure of the PUU copolymers are demonstrated by FT-IR and 1H NMR analyses. Figure 1 shows the FT-IR spectra of PLLAL, ACAT, and PUUL. The characteristic absorption at 2260 cm−1 (NC stretching) of the −NCO group is not observed, indicating that all the −NCO groups are incorporated into the copolymers. The absorption peaks of the amine group in ACAT (curve a) at 28276

DOI: 10.1021/acsami.5b10829 ACS Appl. Mater. Interfaces 2015, 7, 28273−28285

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ACS Applied Materials & Interfaces

4H, −CH2−), 1.09 (s, 3H, −CH3) for DMPA segments. The PUUL copolymers simultaneously show the peaks from PLLA segments (δ 5.20, and 1.47 ppm), ACAT segments (δ 6.84 to 6.90 ppm), and DMPA segments (δ 1.09 and 4.02 ppm). The peak at 7.17 ppm is assigned to −NH− in the urethane and urea groups from the addition polymerization of −NCO groups (HDI) and hydroxyl groups (PLLA) and amine groups (ACAT). This demonstrates that the polyurethane-urea copolymer is formed. Furthermore, there is no signal at 3.25 ppm from methene groups conjoined to the −NCO group, which also confirms the result from FT-IR in Figure 1, indicating that the −NCO groups are completely consumed in the reaction. The molecular weight of PLLA precursors is first calculated from the data of 1H NMR and confirmed with the OH value measurement. The Mn of PLLAs calculated from the OH value is very close to the Mn from 1H NMR (Table S1). Therefore, prepolymer chains are successfully extended by ACAT. The molecular weights of the PLLA precursors and PUU copolymers are tested by GPC, and the results are listed in Table 2. Compared with their corresponding PLLA precursors

Figure 1. FT-IR spectra of (a) ACAT, (b) PLLAL, and (c) PUUL.

3378, 3309, and 3204 cm−1 transfer into a single absorption peak at 3351 cm−1 in the curve c of the PUUL copolymer, demonstrating the formation of urea groups. Most of the absorptions in the PUUL copolymer’s spectrum (curve c) agree with PLLAL except the absorptions at 1602 and 1507 cm−1 which are attributed to the quinoid and benzenoid ring structure from the ACAT segment. The carbonyl peak (CO stretching) from PLLA at 1755 cm−1 in curve b shifts to 1750 cm−1 in curve c and becomes wider, indicating that hydrogen bonds are formed between the ester, amide, and urea groups in the copolymer. These hydrogen bonds between these groups serve as physical cross-linking points to provide elasticity of the PUU copolymers. Figure 2 shows a representative 1H NMR spectrum of PUUL: 1H NMR (400 MHz, DMSO-d6, δ): 7.45 (s, 2H, −NH−), 6.90 (d, 2H, Ar−H), 6.84 (s, 2H, Ar−H), 6.80 (s, 2H, Ar−H) for ACAT segments, 5.20 (t, 2H, poly −CH2−), 4.33 (t, 2H, −CH2− of EG), 1.47 (d, 6H, poly −CH3), 1.28 (d, 6H, end −CH3) for PLLA segments, 7.15 (d, 2H, −NH−), 2.93 (t, 4H, −CH2−), 1.39 (t, 8H, −CH2−) for HDI segments, 4.02 (s,

Table 2. Molecular Weight of the PLLA Precursors and Polyurethane-Urea Copolymers Mn of PUU copolymer

Mn of employed PLLA sample name

Mn from 1H NMR

Mn from GPC

PDI

Mn from GPC

PDI

PUUH PUUM PUUL

8000 3500 1500

14 100 4900 2900

1.4 1.1 1.1

127 400 93 800 61 200

4.4 4.5 4.1

as shown in Table 2, all the PUU copolymers have much higher molecular weights and wider polydispersity ranging from 4.1 to 4.5. All these data from FT-IR, NMR, and GPC indicate the successful synthesis of the PUU copolymers. 3.2. Solubility and Surface Morphology of the PUU Copolymers. PLLA is soluble in most nonpolar solvents such as chloroform and dichloromethane, while ACAT dissolves into

Figure 2. 1H NMR spectrum of PUUL in DMSO-d6. 28277

DOI: 10.1021/acsami.5b10829 ACS Appl. Mater. Interfaces 2015, 7, 28273−28285

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This means that these PUU copolymers with tunable hydrophilicity can be employed as tissue engineering materials in various culture environments. They can find a wide application in the biomedical field. 3.4. Electrochemical and Conductivity Properties of the PUU Copolymer. The UV−visible spectra of ACAT, ACAT doped with CSA, PUUH, PUUM, PUUL, and PUUM doped with CSA are shown in Figure 3A. All of the samples are dissolved in DMSO, and the concentration of copolymer samples are kept the same. In case of ACAT, two absorption peaks appear at 345 and 589 nm corresponding to the π−π* transition of benzenoid ring and the excitonic transition from benzenoid to quinoid ring, respectively. Three PUU copolymers (curves c, d, and f) have respective absorptions at the same characteristic wavelengths at 317 and 564 nm, which are also assigned to the absorption of benzenoid ring and quinoid ring. The blue shift phenomenon is generated from the formation of urea groups which lessen the electron density of the quinoid rings.51,52 The UV absorption of the PUUH, PUUM, and PUUL at 317 and 564 nm are in accordance with feed ratios of ACAT; i.e., the ACAT content in these copolymers decrease accordingly. These data indicate the successful synthesis of the PUU copolymers. There are two new peaks at 410 and 730 nm in the spectrum of ACAT after doping with CSA due to the formation of delocalized polaron. In the case of the CSA doped PUUM sample (curve e), the peak at 410 nm is also observed in the spectrum, and the new peak at 983 nm is due to the delocalization of polaron along the backbone of copolymer after doping.53 These data demonstrate that the electroactivity of ACAT is remaining in the PUU copolymers. The cyclic voltammograms of ACAT and PUUL are shown in Figure 3B,C. There are two pairs of redox peaks for both the ACAT and PUUL copolymer, exhibiting a similar trend with other trimeric aniline electrochemical studies.54,55 The two repairs of redox peaks from PUU copolymer are assigned to the transitions from the leucoemeraldine oxidation state to the emeraldine oxidation state and then from the emeraldine oxidation state to the “pernigraniline” state, respectively, as shown in Figure 3C.35 All the UV and CV results indicate the good electroactivity of the copolymers. Meanwhile, the conductivity of copolymers increases with a higher concentration of ACAT segments in the copolymers. After doping with CSA, the conductivity is raised to the range of about 10−6 S/cm for the PUU copolymers (Table S4). 3.5. Thermal Analysis of the PUU Copolymers. TGA curves of PLLAs with different molecular weights and three PUU copolymers are displayed in Figure S3. The slight mass loss before 180 °C is ascribed to the loss of entrapped solvent and moisture for both PLLAs and their copolymers. At higher temperature, the first weight loss from 180 to 320 °C is attributed to degradation of PLLA main chains in the curves of PLLA precursors (curves a, b, and c). Thermal stability of three PLLA samples increases with larger polymerization degree, and it agrees with the molecular weights of PLLA as listed in Table 2 due to the stronger interaction and entanglement between the macromolecules with a higher molecular weight. The decomposition of PUU copolymers (curves d, e, and f) undergoes a two-stage process. The first stage ranges from 220 to 320 °C, which is assigned to the thermal degradation of PLLA segments. All the copolymers have higher degradation starting temperatures than their corresponding PLLA precursors, indicating that PUU copolymers are more thermally

polar solvents such as DMF and NMP. Table S2 lists the solubility of PUU copolymers in several polar and nonpolar solvents. The PUU copolymers are soluble in nonpolar solvents such as CHCl3 and CH2Cl2 due to the good solubility of the PLLA segment. The copolymers can also easily dissolve into polar solvents with a high boiling temperature such as dioxane and DMF. The morphology of PUU films fabricated from dioxane solution is further observed. On the one hand, AFM pictures of copolymer films show very smooth surfaces and very low Ra values between 0.93 and 3.93 nm due to the good solubility (Figure S1). On the other hand, very uniform surfaces are observed on a larger range with SEM pictures (Figure S2). The PUU copolymers’ good solubility in many common solvents provides good processability of the copolymers. This overcomes the poor processing ability of polyaniline and oligoaniline which can only dissolve into strong polar solvent such as DMSO and NMP. 3.3. Surface Hydrophilicity of the PUU Copolymers. Proper surface hydrophilicity plays a key role for cell attachment and cell adhesion proliferation and function.48 It has been reported that cell adhesion was maximized on a surface with a moderate hydrophilicity (50−70°).49 To study the effect of DMPA segment concentration on the wettability of copolymers, a series of PUUH copolymers based on PLLAH precursor with different concentrations of DMPA are synthesized. The water contact angles of the PUUH copolymers with different DMPA content are shown in Table S3. Pure PLLA film has a contact angle higher than 90°,50 which is considered unfavorable for cell adhesion. Without DMPA, the contact angle of sample PUUH0 has a similar value to PLLA. The PUUH copolymers containing different DMPA contents (PUUH1-5) have water contact angles between 69° and 82°, which are significantly lower than PUUH0. The contact angles of the samples decrease with an increase in the DMPA content in the copolymers, indicating a more hydrophilic surface, due to the higher carboxyl group content in the copolymers. Moreover, the contact angles decrease to 57−83° after they are doped with CSA, because the formation of ammonium salt from ACAT segments significantly increases the surface wettability of the PUU copolymers. The wettability of the PUUH, PUUM, and PUUL is also tested, and the results are listed in Table 3. Before doping with Table 3. Water Contact Angle Values of Three PUU Copolymers and Weight Ratio of DMPA and ACAT in the Copolymer sample code

wt % of DMPA

wt % of ACAT

contact angle before doping (deg)

contact angle after doping with CSA (deg)

PUUH PUUM PUUL

1.49 3.02 5.54

3.19 6.49 11.91

84 ± 4 79 ± 2 78 ± 1

73 ± 2 68 ± 1 49 ± 2

CSA, the contact angles of the copolymers containing different DMPA and ACAT content are between 78° and 84°, which are hydrophobic. However, they decrease dramatically when the copolymers are doped with CSA. Especially, the contact angles of PUUL and PUUM decrease to 49° and 68°, respectively, due to the high content of ammonium salt from the doped ACAT segments. These values are more suitable for cell adhesion and cell function. Therefore, by controlling the DMPA and ACAT content and by doping the copolymers with CSA, we tune the contact angle of the copolymer in a wide range from 49° to 85°. 28278

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Figure 3. (A) UV−vis spectra of (a) ACAT, (b) doped ACAT, (c) PUUH, (d) PUUM, (e) doped PUUM, and (f) PUUL. (B and C) Cyclic voltammograms of (B) ACAT and (C) PUUL in DMSO and molecular structure of copolymers at various oxidation states.

stable than pure PLLAs. The decomposition temperatures of PUUH, PUUM, and PUUL are at 254, 241, and 226 °C, respectively, meaning that the thermal stability of copolymers increases with an increase in the molecular weight of PLLA in the copolymers. The second stage takes place from 320 to 510 °C corresponding to decomposition of ACAT segments. The content of the ACAT segment is roughly calculated from the second weight loss stage,2 and the ACAT weight content values as shown in Figure S3 are close to the theoretical ones as listed in Table 3. The successful synthesis of copolymers is therefore further confirmed. The glass-transition temperature (Tg) and melting point (Tm) of PLLA and PUU copolymer samples are determined by DSC and listed in Table 4. The Tg and Tm of PLLAs both increase with increasing molecular weight, because the longer PLLA chain has less mobility due to the stronger interactions and chain entanglement between the macromolecules. The three copolymers have higher Tg than the corresponding pristine PLLA. This is because the presence of ACAT hard segments and the hydrogen bonds between ester, urethane, and urea groups hindered the movement of the PLLA segments in

Table 4. Thermal Properties of PLLAs and PUU Copolymers

a

samples

Tg (°C)

Tm (°C)

ΔHm (J/g)

Xca (%)

PLLAH PLLAM PLLAL PUUH PUUM PUUL

53.2 44.1 25.5 53.3 46.7 34.3

162.4 129.8 120.6 154.9 129.4 113.0

39.2 34.5 10.4 17.6 13.7 4.83

42.4 37.1 11.2 18.9 14.8 5.2

The melting enthalpy of 100% crystalline poly(L-lactide) is 93 J/g.56

the copolymers. There is no chemical cross-linking in these linear PUUs, so the Tg of PLLAs was not significantly enhanced after copolymerization. The physical cross-linking degree of PUUs is mainly influenced by the concentration of urethane and urea groups. PUUH has the lowest physical cross-linking degree. That is why the difference of Tg values between PUUH and PLLAH is not as significant as that between PUUL and PLLAL. At the same time, the introduction of hard segments reduces the copolymer’s degree of crystallinity (Table 4). The 28279

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ACS Applied Materials & Interfaces Tm of PUU copolymers are therefore lower than that of the pure PLLA counterparts. The Tg and Tm of the PUUH, PUUM, and PUUL copolymers decrease accordingly with the decrease of the molecular weight of the PLLA block. This is probably caused by the weaker interaction and entanglement of the PLLA chain with a decrease in the Mn of PLLA. The lower molecular weight also leads to a decrease of crystal formation in both PLLAs and copolymers (Table 4). These results are further proved by analyzing crystallinity with X-ray diffraction (XRD) patterns (Figure S4). There are characteristic peaks of crystal PLLA chains at 2θ values of around 17° and 19° in the curves of PUU copolymer samples. With the highest molecular weight and lowest content of ACAT segments, copolymer PUUH has the highest intensity, while those peaks are lower in the PUUM curve and barely observed in the PUUL curve. This indicates that the formation of PLLA segment crystals can be greatly influenced by Mn of PLLA and the content of other amorphous segments, which can also influence the mechanical properties of these copolymers. 3.6. Mechanical Properties of the PUU Copolymers. To mimic the elasticity of soft tissue during regeneration, electroactive and elastic PUU copolymers are synthesized, and the results of their mechanical properties are shown in Table 5

ester, urethane, and urea groups which serve as physical crosslinking points to enhance the intra- and intermolecular interaction between the PUU macromolecules. Furthermore, low crystallinity, high level of aniline trimer interaction via π−π stacking, and self-doping reaction between carboxyl group from DMPA and ACAT also contribute to the high elongation of these PUU copolymers.58 The elasticity of the PUU copolymer overcomes the brittleness of PLLA having a breaking elongation of less than 5.1%.59 These PUU copolymers with a moderate modulus and great elasticity hold a great potential for soft tissue regeneration. 3.7. Enzymatic Degradation of PUU Copolymers. The degradation behavior of PUU copolymers is observed in vitro using proteinase K, an enzyme with great activity toward degradation of PLLA. The enzymatic degradation profile of PUU copolymers is shown in Figure 5. The rate of in vitro

Table 5. Tensile Test Result of PUUH and PUUM Copolymer samples

tensile strength (MPa)

breaking elongation (%)

Young’s modulus (MPa)

PUUH PUUM PUUL

12.0 ± 1 5.5 ± 1 0.8 ± 0.1

145.2 ± 28 174.5 ± 36 641.7 ± 66

236.5 ± 22 15.0 ± 2 1.9 ± 0.1

Figure 5. Degradation profile of copolymer films at 37 °C.

degradation is significantly influenced by the length of PLLA segment and the concentration of urethane/urea groups. Samples of PUUL degrade continuously with a rapid speed from the start and lose 100% of their weights in 25 days. PUUM shows a slow degradation speed at the first 15 days with a weight loss of less than 15%. The degradation of PUUM speeds up after the 15th day and loses more than 50% weight in 30 days. However, there is no obvious weight loss of PUUH samples during this degradation measurement. The remaining mass of PUUH is still over 90% after a month. These data exhibit a similar phenomenon with former results that shorter PLLA segment60 and higher content of the urethane/urea group61 can increase the rate of PLLA degradation. 3.8. Biocompatibility of PUU Copolymers. Easily obtained and widely employed myoblasts, mouse C2C12 cells,62 are chosen to evaluate the biocompatibility of PUUs and to study the effect of electroactive PUUs on myogenic differentiation. C2C12 myoblast cells are seeded on PLLA, PUUH, PUUM, and PUUL polymer coated cover slides, respectively. After being incubated for 48 h, the cells viability is evaluated by Live/Dead assay (Figure S5a−d). Live cells (green) are dominant, and dead cells (red) are barely found for all the samples, indicating that the PUUH, PUUM, and PUUL polymers are nontoxic. Cell morphology can be influenced by the culture substrate. C2C12 cells are seeded on PLLA, PUUH, PUUM, and PUUL polymer coated cover slides, respectively, and stained by FITC labeled phalloidin and DAPI after being incubated for 48 h (Figure S5e−h). All the C2C12 cells exhibit a normal spindle

Figure 4. Representative stress−strain curves of (a) PUUH, (b) PUUM, and (c) PUUL copolymer.

and Figure 4. PUU elastomers show tensile stress between 12.0 and 0.8 MP. With the longest PLLA precursor, which forms the large region of PLLA crystal during the casting, the sample of PUUH has a Young’s modulus of 236.5 MP, which is much higher than that of the polylactide-aniline pentamer multiblock copolymer of about 33 MP.57 Moreover, the breaking elongation rate of these PUU copolymers is between 145.2% and 641.7%, which is quite elastic. These values are higher than that of the polylactide-aniline pentamer copolymer which is 95%.57 The high breaking elongation rate of these PUU copolymers is attributed to the hydrogen bonds between the 28280

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enhances the proliferation of C2C12 cells after 5 days culture. These results indicate that the PUUH, PUUM, and PUUL copolymers are nontoxic and can promote the C2C12 myoblast cell proliferation. This should be ascribed to the good wettability and existence of ACAT segments which provided electroactivity. 3.9. Myogenic Differentiation on Electroactive PUU Films. Due to the excellent biocompatibility, electroactivity, and tunable hydrophilicity of PUU copolymers, myogenic differentiation of C2C12 cells is further studied in vitro. C2C12 cells are cultured on TCP, PLLA, PU (Tecoflex), and electroactive copolymer films in differentiation medium. After 7 days, immunofluorescence staining is used to observe the morphology of myotubes. As shown in Figure 7, myotube area ratio of PUU films is much higher than that on PLLA and PU films. C2C12 cells cultured on PUU films tend to form many more myotubes than cells cultured on PLLA and PU film. The data of multinucleate myotubes are analyzed as shown in Figure 8. The PUU copolymer films have a much higher number of myotubes per 105 μm2 than the PLLA film (Figure 8a), indicating the positive effect of the electroactive segment containing copolymers on the formation of multinucleate myotubes. Their myotube number significantly increases from PLLA’s 1.35/105 μm2 to 4.1, 5.0, and 5.5/105 μm2, respectively, which is statistically significant (p < 0.01) (Figure 8a). Among them, the PUUL sample has the highest myotube number which is higher than the TCP control. This is because PUUL has both a high concentration of ACAT (Table 1) and proper water contact angle (Table 3). This synergetic effect of electroactivity and wettability greatly influences the process of myogenic differentiation and consequently influences myotube morphology and maturation index. For the films of PUUs, the length of myotubes is ∼310 μm, which is much longer than that of PLLA (244 μm) (Figure 8b). Meanwhile, the myotubes cultured on PUU copolymer films have similar diameters with PLLA and TCP without a significant difference (Figure 8c). The maturation index (the percentage of myotubes with more than 5 nuclei) of the materials shows a similar tendency with myotube number data. The maturation index of myotubes formed on pure PLLA film is 53%, and it increases to higher than 80% (p < 0.01) on copolymer films. The maturation index

shape, indicating that these polymers are ideal substrates for C2C12 cells to adhere and grow. To be an ideal biomaterial for tissue engineering, the polymers should be suitable for cells proliferation and differentiation. Aside from PLLA, TCP and a medical grade PU, Tecoflex (PU), are used as controls in the cell proliferation and differentiation experiment. Due to its great properties, Tecoflex is employed as medical devices or a control group in several biomaterial studies.63,64 C2C12 cells are seeded onto the TCP, PLLA, PU (Tecoflex), PUUH, PUUM, and PUUL copolymer films. Cell viability is measured by using alamarBlue assay, and the results are shown in Figure 6. A continuous

Figure 6. Cell viability of C2C12 cells on TCP, PLLA, PU (Tecoflex), PUUH, PUUM, and PUUL copolymer substrates. TCP, PLLA, and PU (Tecoflex) groups served as the control groups. Note: (∗∗) for p < 0.01.

increase of cell viability is seen for all groups. Cell viability of PUU groups on day one is more than 80% of the cell viability of the PLLA group. Cell viability of the PUUM and PUUL group is higher than that of the PLLA group from day 3 to day 5. All of the PUU samples have significantly higher cell viability than the biomedical grade PU group at day 5. Among the copolymer groups, PUUL shows the best effect on C2C12 proliferation which is comparable with the TCP group (Figure 6). Compared to PLLA and PU groups, PUUL significantly

Figure 7. Tubulin (green) and nuclei (blue) staining of C2C12 cells after 7 days of culture on PLLA and PUU films: (a) TCP, (b) PU (Tecoflex), (c) PLLA, (d) PUUH, (e) PUUM, and (f) PUUL. Scale bar: 50 μm. 28281

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Figure 8. Statistic data of myotubes after 7 days of culture for myogenic differentiation of C2C12 cells: (a) myotube number per 105 μm2, (b) myotube length, (c) myotube diameter, and (d) myotube maturation index (% myotubes with ≥ 5 nuclei). Note: (∗) for p < 0.05; (∗∗) for p < 0.01.

of myotubes incubated on PUUL is even as high as 93.3% (Figure 8d), higher than the TCP group. All these data demonstrate that myogenic differentiation of C2C12 cells can be promoted greatly by electroactive PUU copolymers, which may serve as excellent muscle tissue engineering scaffolds considering their great mechanical properties. Studies have already proved that cell behavior can be manipulated by electrical stimuli on a subcellular level including protein distribution, gene expression, metal ion content, and action potential,65 especially for myoblasts.66 We hypothesize that these electroactive PUU copolymers enhanced differentiation of C2C12 cells probably involving ion flows. K+ channel (Kir2.1)-induced hyperpolarization triggers myoblast differentiation via the activation of the calcineurin pathway and enhances expression/activity of myogenin and MEF2.67 The gene expression of C2C12 myogenic differentiation is further determined by real-time quantitative PCR (RT-PCR). Myogenin (MyoG) and troponin T1 are gene markers expressed at early and late phase of myogenic differentiation,62 respectively. After 7 days of culture on different substrates, the gene expression levels are obtained and shown in Figure 9. In agreement with the results of myotube morphology, the expression levels of both MyoG and troponin T1 on the electroactive PUU copolymer groups are significantly higher than that of the PLLA group. This means that electroactive copolymer films can promote myogenic differentiation through the whole culture period. Due to its great wettability and electroactivity, sample PUUL exhibits the highest myogenic gene expression, which is in accordance with the myotube number results in Figure 8. All these data reveal the important role of electroactivity from the ACAT segment and proper hydrophilicity of substrates greatly enhancing the myogenic differentiation for C2C12 cells, indicating that these electro-

Figure 9. Myogenin (MyoG) and troponin T1 (TNNT) gene expression of C2C12 cells on different substrates at day 7. Note: (∗) for p < 0.01.

active PUU copolymers have great potential for skeletal muscle tissue engineering.

4. CONCLUSIONS A series of biodegradable and electroactive elastic polyurethaneurea (PUU) copolymers based on PLLA, ACAT, and DMPA were successfully synthesized for electrically sensitive soft tissue regeneration. The electroactivity of PUU copolymers was confirmed by both UV−visible spectroscopy and cyclic voltammetry measurements. PUU copolymers exhibited a strain between 145% and 641% and a modulus of 1.5 to 236.5 MP. The water contact angle of the PUU copolymers was tuned from 49° to 91° with different DMPA and ACAT concentrations. The copolymers dissolved into many organic solvents, indicating a much better processability than polyaniline. The copolymers were generally more thermally stable than 28282

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ACS Applied Materials & Interfaces pristine PLLAs. The PUU copolymer films were not cytotoxic and could support the adhesion and proliferation of C2C12 myoblast cells. The enhanced effect of these elastic electroactive copolymers for myogenic differentiation of C2C12 myoblasts was demonstrated by the morphology and quantitative analysis of myotubes and gene expression. All these results suggested that the electroactive elastic degradable PUU copolymers have great potential for soft tissue engineering where electroactivity is desired.



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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/acsami.5b10829. OH number and molecular weight of copolymers, solubility and conductivity of the PUU copolymers, water contact angle values of PUUH copolymers, AFM/ SEM images of copolymer surfaces, TGA curves, and XRD patterns of the copolymers. (PDF)



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Corresponding Authors

*Tel.:+86-29-83395363. Fax: +86-29-83395131. E-mail: [email protected]. *Tel.:+86-29-83395363. Fax: +86-29-83395131. E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The authors thank the National Natural Science Foundation of China (Grant No. 21304073) and “the Fundamental Research Funds for the Central Universities” (Grant No. xjj2013029) for financial support of this work.



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DOI: 10.1021/acsami.5b10829 ACS Appl. Mater. Interfaces 2015, 7, 28273−28285