Synthesis of Biodegradable and Electroactive Tetraaniline Grafted

Aug 21, 2012 - Nirmalya Tripathy , Elumalai Perumal , Rafiq Ahmad , Jeong Eun Song , Gilson ... Shaikh Ziauddin Ahammad , S. Wazed Ali , T.R. Sreekris...
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Synthesis of Biodegradable and Electroactive Tetraaniline Grafted Poly(ester amide) Copolymers for Bone Tissue Engineering Haitao Cui,†,‡,⊥ Yadong Liu,†,⊥ Mingxiao Deng,∥ Xuan Pang,† Peibiao Zhang,† Xianhong Wang,† Xuesi Chen,*,† and Yen Wei*,§ †

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China ‡ Graduate University of Chinese Academy of Sciences, Beijing 100039, P. R. China § Department of Chemistry, Tsinghua University, Beijing 100084, China ∥ Department of Chemistry, Northeast Normal University, Changchun 130022, P. R. China S Supporting Information *

ABSTRACT: Biodegradable poly(ester amide)s have recently been used as biomaterials due to their desirable chemical and biological characteristics as well as their mechanical properties, which are amendable for material processing. In this study, electroactive tetraaniline (TA) grafted poly(ester amide)s were successfully synthesized and characterized. The poly(ester amide)s-graf t-tetraaniline copolymers (PEA-g-TA) exhibited good electroactivity, mechanical properties, and biodegradability. The biocompatibility of the PEA-g-TA copolymers in vitro was systematically studied, which demonstrated that they were nontoxic and led to favorable adhesion and proliferation of mouse preosteoblastic MC3T3-E1 cells. Moreover, the PEA-gTA copolymers stimulated by pulsed electrical signal could serve to promote the differentiation of MC3T3-E1 cells compared with TCPs. Hence, the biodegradable and electroactive PEA-g-TA copolymers possessed the properties in favor of the long-time potential application in vivo (electrical stimulation directly to the desired area) as bone repair scaffold materials in tissue engineering.

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geometry, and surface topography.9 However, some drawbacks related to the practical application of these polymers still exist such as poor solubility and processability. Moreover, prolonged residence time of the conducting polymer in vivo may cause local inflammation and lead to the need for surgical removal. To solve these problems, degradable electroactive polymers were designed and synthesized to meet biomedical applications, especially in vivo tissue engineering.10,11 These polymers showed good electroactivity, but also exhibited biodegradability in vitro and in vivo. The oligomers formed during the degradation process of the copolymers can be consumed by macrophages and subsequently undergo renal clearance, avoiding any long-term adverse effect in vivo.12 Recently, Guo et al. synthesized linear and hyperbranched degradable conducting copolymer composed of aniline pentamer and PCL.13 The electrical conductivity of the hyperbranched degradable conducting polymers is much higher than that of their linear counterpart with similar content of aniline pentamer. They also synthesized degradable and electroactive hydrogels of acrylated PLA-PEG-PLA (PEG = poly(ethylene

INTRODUCTION Synthetic polymers with multiple functionalities and properties are desirable for applications in the biomedical fields. The main demands on biomaterials for tissue engineering scaffolds are that they serve the bulk mechanical and structural requirements of the target tissue, enable molecular interactions with cells that promote tissue healing, and are biocompatible.1 Synthetic scaffolds that incorporate multiple physical, chemical and biological functionalities may mimic the natural cell environment, and participate in a dynamic, bidirectional exchange of information with cells. In the past decades, many types of synthetic biodegradable polymers, such as polylactide (PLA),2 poly(ε-caprolactone) (PCL),3 polycarbonate,4 polypeptide,5 as well as their copolymers have been used as tissue engineering scaffold materials due to their good biocompatibility, biodegradability, and mechanical properties. Previous studies have shown that cells such as fibroblasts, neurons, and osteoblasts responded to electrical fields created by electrets or between electrodes in vitro and in vivo.6−8 Hence, the general properties of conducting polymers such as polypyrrole (PPy) and polyaniline (PANi) desired for tissue engineering applications include conductivity, reversible reoxidation, biocompatibility, hydrophobicity (40−70° water contact angle promotes cell adhesion), three-dimensional © 2012 American Chemical Society

Received: June 12, 2012 Revised: August 17, 2012 Published: August 21, 2012 2881

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glycol))with aniline tetramer.14 These hydrogels possessed tunable conductivity and swelling behaviors. Our group has synthesized many electroactive polymers containing oligoaniline with good electroactivity and biodegradability.15−18 Poly(ester amide)s (PEAs), α-amino acid-containing degradable polymers, have attracted much attention in the past decades.19 Compared with polyester, they show less prominent acidity during degradation and the addition of amino acids as monomer allows the introduction of functional groups in the polymer, which facilitates further modification with bioactive molecules. They combine the favorable properties of both polyesters and polyamides, i.e., they possess not only good biodegradability but also good mechanical and processing properties, such as thermal stability, tensile strength, and modulus.20,21 The amide moiety enables interchain linkage via hydrogen bonding for producing filaments with improved fiber strength and durability. Because of their low immunogenity, good biocompatibility, and degradability, PEAs have been widely used in pharmaceutical and other medical applications, such as sutures, implants, and temporary matrices or scaffolds in tissue engineering.22,23 The early success of experiments with direct current and electromagnetic induction finally led to widespread clinical treatments of nonunion bone fractures. In previous study, Fitzsimmons et al. reported low-amplitude, low-frequency electric field-stimulated bone cell proliferation mediated by increasing IGF-II release.24 Zhuang et al. found that capacitively coupled electric field-induced proliferation and differentiation of preosteoblastic cells (MC3T3-E1) accompanied increased levels of transforming growth factor-beta 1 (TGF-β1) mRNA by a mechanism involving calcium/calmodulin pathway.25 However, localization of the electrical stimulation, which is critical to effective treatment, still remains a challenge. Realizing the needs to develop new materials as scaffolds in tissue engineering to directly affect cell activity and/or function, we employed an approach with the goal to attach or associate the desired cells to (or with) a surface comprising an electroactive material, and apply electrical stimulation directly to the desired area. In this work, we designed and synthesized biodegradable and electroactive poly(ester amide)s containing conjugated segments of amino-capped tetraaniline (PEA-g-TA), and the graft copolymers were investigated in depth on their chemical and physical properties, along with their electroactive and biocompatibility. In addition, the influence on differentiation of cells stimulated by pulsed electrical signal was also evaluated.

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emeraldine (EM) base form of TA was obtained upon addition of ammonium persulfate (0.02 mol) as the oxidant at 0 °C under stirring for 3 h. The mixture was filtered to collect the TA, and the cake was then washed with 1 M HCl and distilled water. The TA was dedoped in 1 M NH4OH, followed by filtration and washing until the filtrate was neutral. Finally, the TA was lyophilized. By the oxidative coupling reaction of aniline dimer, TA was obtained because its EM base form cannot be further oxidized to longer oligomers in the presence of an excess oxidizing agent. The yield of the EM TA was 85%. 1H NMR (400 MHz, DMSO-d6, ppm): 8.38 (s, 1H), 7.24 (t, 2H), 7.09 (s, 4H), 7.03−6.96 (m, 5H), 6.91−6.80 (m, 2H), 6.82−6.79 (m, 2H), 6.61− 6.60 (m, 2H), 5.53 (s, 2H). The molecular weight of EM TA was measured by mass spectrometry to be 366.6 (MH+/e). Synthesis of Di-p-toluenesulfonic Acid Salt of Bis-leucine Butane-1,4-diester (I). Di-p-toluenesulfonic acid salt of bis-leucine butane-1,4-diester was synthesized according to the published experimental procedure.27 In brief, a mixture of 1,4-butanediol, L-leu, and p-toluenesulfonic acid (molar ratio 1/2.1/2.1) was dissolved in toluene, then the solution was refluxed until the evaporation of calculated amount of water. The mixture was then cooled to room temperature, and acetone was added to the reaction mixture for precipitating the product. The precipitated product was filtered and washed until the cake became white. After drying under vacuum for 24 h, the solid was recrystallized three times from distilled water followed by lyophilization. Yield of purified product I was 63%. 1H NMR (400 MHz, DMSO-d6, ppm): 8.27 (s, 6H), 7.48−7.04 (dd, 8H), 4.14(t, 4H), 3.95(t, 2H), 2.47(m, 4H), 2.25(s, 6H), 1.73−1.53 (m, 6H), 0.85 (d, 12H). Synthesis of Di-p-nitrophenyl Sebacate Diester (II). Di-pnitrophenyl sebacate diester was also synthesized according to the published experimental procedure.27 In brief, p-nitrophenol was soaked in dry acetone overnight and filtered. Triethylamine was added to the filtrate, and then sebacoyl chloride in acetone solution (m/v 1 g/7 mL) was added dropwise to the above mixture in an ice bath under vigorous stirring for 4 h. The supernatant was slowly poured into distilled water to precipitate the product. The precipitate was subsequently filtered and recrystallized three times from ethyl acetate. The product was dried under vacuum for 48 h. Yield of purified product II was 60%. 1H NMR (400 MHz, DMSO-d6, ppm): 8.58 (d, 4H), 7.44 (d, 4H), 2.68(t, 4H), 1.68−1.65 (m, 4H), 1.34− 1.32 (m, 8H). Synthesis of γ-Benzyl-L-glutamate N-Carboxyanhydride (BLG NCA) (III). BLG and triphosgene (weight ratio 1/0.6) were dissolved in anhydrous THF under nitrogen conditions. The mixture was stirred at 50 °C until the solution became clear and then was purged with nitrogen for 30 min. The solution was cooled to room temperature and poured into cool petroleum ether to precipitate the product. The mixture was kept in the refrigerator overnight. The precipitated product was filtered off and recrystallized three times from the mixture of ethyl acetate and n-hexane. The product was dried under vacuum for 24 h. Yield of purified product III was 77%. 1H NMR (400 MHz, CDCl3, ppm): 7.48−7.46 (m, 5H), 6.48(s, 1H), 5.17(s, 2H), 4.42(m, 1H), 2.58(t, 2H), 2.04−2.32 (tt, 2H). The Synthesis of PEA(Bz). Briefly, triethylamine (0.22 mol) was added dropwise into the mixture of monomers in predetermined molar ratio in dry DMA (monomer I 0.1 mol, monomer II 0.1 mol, and monomer III 0.025 mol). The solution was then heated to 80 °C under stirring for 16 h. The viscous reaction solution obtained was cooled to room temperature, diluted by ethanol, and poured into cool distilled water to precipitate the product. The precipitated PEA(Bz) was subsequently filtered, thoroughly washed with distilled water, and dried at 30 °C under vacuum. The precipitate was washed with warm ethyl acetate, filtered, then dissolved in chloroform, filtered, and cast as a thin film. CHCl3 was allowed to evaporate until dry film was obtained. The film was washed with fresh ethyl acetate and kept overnight. The product was dried under vacuum for 48 h. Yield of purified product PEA(Bz) was 57%. Deprotection of PEA(Bz). In a typical procedure, PEA(Bz) was dissolved in dichloroacetic acid (m/v 1 g/30 mL), followed by addition of 33% (wt.) HBr in acetic acid solution. The mixture was

EXPERIMENTAL SECTION

Materials. L-Leucine (L-leu) and γ-benzyl-L-glutamate (BLG) were purchased from Gill Biochem (Shanghai) Ltd., China. N-Phenyl-1,4phenylenediamine, ammonium persulfate, camphorsulfonic acid (CSA), and 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC·HCl) were purchased from Aldrich. N-Hydroxysuccinimide (NHS) (Fluka), sebacoyl chloride (Alfa Asia), HBr solution of 33% (wt.) in acetic acid (Acros) were used as received. Triethylamine, 1,4-butanediol, chloroform (CHCl3), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), dimethylformamide (DMF), ethyl acetate, nhexane and dimethyl acetamide (DMA) were dried and distilled prior to use. All other chemicals were of analytical grade and were used as received. Synthesis of Amine-Capped TA. TA was synthesized according to a similar procedure reported in the literature.26 First, N-pheny-1,4phenylenediamine (0.02 mol) was dissolved in a mixture solution of hydrochloric acid (HCl), acetone, and water (vol. ratio 1/4/4). The 2882

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Scheme 1. Synthesis Route of PEA-g-TA

gently stirred at ambient temperature overnight, and then precipitated with acetone. The resultant PEA-COOH was purified by washing with acetone repeatedly to remove the residue HBr and then dried under vacuum. Yield of purified product PEA(H) was 54%. Synthesis of PEA(H)-graf t-Tetraaniline (PEA-g-TA). The average molecular weight of PEA(H) was approximate to 100 kDa. In theory, PEA(H) had 50 carboxyl groups of each polymer chain. As shown in Scheme 1 and Table 1, PEA-g-TA copolymers were prepared

refractometer detector. DMF was used as an eluent containing 0.01 M LiBr at a flow rate of 1 mL min−1. The molecular weights were calibrated with polystyrene standards. The thermal stability was estimated by thermal gravimetric analysis (TGA) (TA Instrument Q500) under a nitrogen atmosphere. The measurements were performed incrementally (10 °C min−1) from 50 °C up to 600 °C. Differential scanning calorimetry (DSC) measurements were carried out on a TA Instrument Q100 under nitrogen atmosphere. Measurements during the first heating from −20 to 180 °C and then the first cooling from 180 to −20 °C as well as the second heating from −20 to 180 at 10 °C min−1 were performed. The contents of TA in the products were determined according to our previous method.16 The standard van der Pauw four-probe method was used to measure the electrical conductivity. The square samples were placed on the four-probe apparatus. Providing a voltage, a corresponding electrical current could be obtained. The electrical conductivity of samples was calculated by the following formula: σ (S cm−1) = (2.44 × 10/S) × (I/ E), where σ is the conductivity, S is the sample side area, I is the current passed through outer probes, and E is the voltage drop across inner probe. For the tensile strength test, the specimens were prepared as 40 mm × 5 mm × 0.2 mm. Normal tensile tests were conducted on an Instron 1121 machine at a crosshead speed of 1 mm min−1. The tensile strength and modulus data were both obtained by averaging over three specimens. Preparation of Graft Copolymer Thin Films. The copolymer solutions (5.0 wt % in CHCl3) were cast onto a super flat polytetrafluoroethylene plate and placed for 5 h under room temperature to form thin films. The films obtained were dried under vacuum at room temperature for 48 h to remove CHCl3. In Vitro Degradation of Polymers. For degradation studies, each specimen (10 mm × 10 mm × 0.2 mm) of the PEA-g-TA copolymer made from films was placed in tube filled with 10 mL of 0.1 M TrisHCl buffer solution (pH = 7.4) containing 0.2 mg mL−1 of proteinase K. The tubes were placed in the thermostatic shaker at 37 °C. The buffer solution containing proteinase K was changed every 2 days. After different time, the specimens were taken out and washed with distilled water for three times, then lyophilized before being subjected to weight loss analysis. Three samples were prepared in each time interval. The weight loss is calculated as the average value of measurements from three samples. In Vitro Cytotoxicity Assay. Cytotoxicity of the TA, PEA, and PEA-g-TA copolymers was assessed by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. First, TA, PEA, and PEA-g-TA powders were respectively put into culture medium (Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal calf serum (Gibco) and 100 U·mL−1 penicillin−streptomycin (Sigma)) for 48 h at 37 °C to get their extract liquid with the concentration of 10, 5, 2.5, 1.25, 0.625, 0.313, and 0.156 mg mL−1, respectively. Mouse preosteoblastic MC3T3-E1 cells were seeded in 96-well plates at a density of 1.2 × 104 cells per well and incubated in culture medium under standard conditions (a

Table 1. Properties of the PEA-g-TA Copolymers

sample PEA-gTA#1 PEA-gTA#2 PEA-gTA#3 a

[TA]/[COOH]

feeding weight ratio (%)

yielding weight ratio (%)a

1:5

3.5

1.9

1:2

8.4

5.4

1:1

15.5

10

feeding mole ratio

Mn(g/mol) /PDIb 1.023 × 105 (1.80) 1.056 × 105 (1.97) 1.126 × 105 (2.20)

electrical conductivity (S cm−1) 7.11 × 10−7 8.01 × 10−6 2.45 × 10−6

Determined by UV−vis spectra. bDetermined by GPC (DMF).

in different feeding ratios. Briefly, PEA(H), NHS, and EDC·HCl (molar ratio 1/10/10) were dissolved in DMSO. The mixture was stirred at room temperature for 24 h under nitrogen atmosphere. After 24 h, TA in DMSO solution was added dropwise into the above mixture, and the stirring was continued for 24 h at 50 °C under nitrogen. After the reaction, the solution was cooled to room temperature and poured into cool diethyl ether to precipitate the product. The crude product was dissolved in DMSO and precipitated in ethanol. Such a dissolution−precipitation process was repeated three times to purify the product. The precipitate was collected by vacuum filtration and dried under vacuum for 24 h. The yields of the products were 71%, 65% and 75%. Characterization. 1H nuclear magnetic resonance (1H NMR) spectra and 13C NMR spectra were recorded on a Bruker AV 400 MHz spectrometer. FT-IR spectra were recorded on a Bio-Rad Win-IR instrument in the range of 4000−500 cm−1. The UV−vis spectra were recorded on a UV-2401PC spectrophotometer. Cyclic voltammetry (CV) was conducted on a CHI20A electrochemistry system (CHI, U.S.A.) using Ag/AgCl and Pt as the reference and counter electrodes, respectively. The indium tin oxide (ITO) electrode was used as the working electrode, and the scan rate was 50 mV s−1. Environmental scanning electron microscopy (ESEM) was performed on an XL 30 ESEM PEG scanning electron microscope (Micrion FEI PHILIPS). Matrix-assisted laser desorption/ionization time-of-flight (MALDITOF) mass spectrum was obtained on an AXIMA-CFR laser desorption ionization time-of-flight spectrometer (COMPACT). Gel permeation chromatography (GPC) measurements were carried out at 50 °C with a Waters 505 GPC instrument equipped with three Waters Styragel columns (HT3, HT4, and HT5) and a differential 2883

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humidified incubator, 37 °C, 5% CO2). Then various concentrations of extract liquid were added to the wells. After incubating for 24 h, 20 μL of MTT stock solution in phosphate-buffered saline (PBS) was added into each well with a final concentration of 0.5 mg mL−1 MTT. The plate was then incubated for 4 h. The medium was removed, and 200 μL of DMSO was added to dissolve the formazan crystals. The optical density (OD) was measured at 490 nm by a Microplate Spectrophotometer. The relative cell viability (%) was calculated according to the following equation: Cell Viability (%) = ([Abs]sample/ [Abs]control) × 100%, where [Abs]sample is the absorbance of the cells treated with material solution and [Abs]control is the absorbance of the cells treated with culture medium. Cell Adhesion and Proliferation Assays. For PEA, EM PEA-gTA, and EM PEA-g-TA doped with CSA, every polymer was respectively dissolved in chloroform to form a 1.0 wt % solution. The solution was coated onto a diameter 15 mm cover slide (treated with 2% dimethyl dichlorosilane (Fluka)/chloroform solution in order to improve the contact angle and make the copolymer attach the cover slide more easily, then dried at 180 °C for 4 h before use), and the solvent was then removed by air drying for 12 h following by additional drying under vacuum for 48 h. The coated films were sterilized under UV radiation for 1 h. MC3T3-E1 cells were used to investigate the cell adhesion and proliferation on the materials. The cells were seeded onto various coated films and tissue-culture-treated polystyrene (TCPs) (the empty 24-well plates) at a density of 5 × 104 cells per well. The plates were incubated for 1 h, 6 h, 12 h, 24 h, 1 day, 3 days, and 5 days, respectively. The proliferation activity was determined quantitatively by means of the MTT assay at predesigned time points. Cell morphology on the surface of the materials was evaluated by a microscope (TE2000-U, Nikon). MC3T3-E1 cells were washed three times with PBS, fixed with 2.5% glutaraldehyde at room temperature for 8 min, dyed with a DMSO/H2O solution containing 2% fluoresceinisothiocyanate (FITC) for 8 min, and then washed with PBS several times. Cell attachment and proliferation were observed qualitatively under a reverse microscope (TE2000U, Nikon). The fluorescence pictures were taken by Digital Camera DXM1200F (Nikon) and analyzed with “NIH Image J” software (>20 per sample). Electrical Stimulation and Cell Differentiation Assays. MC3T3-E1 cells were used to investigate the influence of electroactive substrate on cell differentiation (to mature osteoblasts) stimulated by pulsed electrical signal. TCPs and cover slides coated with PEA, EM PEA-g-TA, EM PEA-g-TA doped with CSA were sterilized by exposure to UV radiation for 1 h. MC3T3-E1 cells were seeded on TCPs and various coated films at a density of 5 × 104 cells per well for 48 h prior to the induction of MC3T3-E1 cells differentiation by replacing the growth medium with differentiating medium (culture medium described above supplemented with 50 mg mL−1 L-ascorbic acid and 10 mM β-glycerophosphate). The electrical stimulation was carried on the Signal Generator (Suing TFG6030 DDS), and the signals were displayed and checked on the wave inspector (Rigol DS1022C digital oscilloscope). The square wave, frequency of 50 Hz, 50% duty cycle, and electrical potential of 0.2 V was adopted in the experiment. The electrical potential was added directly on the surface of TCPs and various coated films, respectively, through two microwire platinum electrodes (0.5 mm in diameter). The samples were respectively stimulated for 2 h every day. The intracellular free calcium concentration ([Ca2+ (i)]) of the differentiating MC3T3-E1 cells was assessed both qualitatively and quantitatively at 7 and 14 day intervals by fluo-4/AM substrate using calcium assay kits (Invitrogen). After careful removal of the medium from each well, MC3T3-E1 cells were washed three times with PBS. Fluo-4 substrate was added and cells were incubated for 30 min. Fluorescence intensity was measured on Multifunction microplate scanner (Tecan infinite M200) using an excitation wavelength of 480 nm and an emission wavelength of 524 nm. The presence of alkaline phosphatase (ALP) was evaluated quantitatively at 7 and 14 days after addition of the differentiation medium. ALP activity was determined by measuring the amount of pnitrophenol produced using p-nitrophenol phosphate substrate using ALP assay kits (Sigma-Aldrich, St. Louis, MO). The medium of each

well was carefully removed. Then MC3T3-E1 cells were washed three times with PBS and lysed in RIPA buffer, freezing at −80 °C for 30 min and thawing at 37 °C. ALP substrate was added in the dark at room temperature for 30 min. The absorbance at 405 nm was read by Multifunction microplate scanner. The differences of OD of the wells were used to reflect the level of ALP activity correspondingly. Statistical Analysis. The data presented are the mean (standard deviation ± SD). Independent and replicated experiments were used to analyze the statistical variability of the data, with p < 0.05 being statistically significant.

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RESULTS AND DISCUSSION Synthesis and Characterization of the PEA-g-TA Copolymers. The procedure for the synthesis of PEA-g-TA is shown in Scheme S1 (Supporting Information) and Scheme 1. Three types of monomers were synthesized and easily purified for preparing PEA with pendant −COOH groups. PEA was prepared by solution polycondensation, followed by acidolysis deprotection and then coupled with TA. The characteristics and properties of the PEA-g-TA samples, prepared under different feed weight percentages of TA, are summarized in Table 1. The content of TA in the products increased with the increase of feed weight fraction. Due to the low efficiency of the condensation coupling reaction and steric hindrance, the grafting degree is correspondingly lower than the theoretical value. The 1H NMR spectra confirmed the structure of PEA(Bz) and PEA(H) as shown in Figure S1. The disappearance of signals at 5.07 and 7.34 ppm illustrated the complete removal of the protecting benzyl groups. Then the condensation polymerization of PEA and TA with the predesigned feed molecular ratio was carried out in concentrated DMSO solution with EDC and NHS as the condensation reagents. In 1H NMR spectrum of PEA-g-TA#2 (Figure 1), proton signals at 8.36−

Figure 1. 1H NMR (in DMSO-d6) of PEA-g-TA#2.

7.92, 7.24−6.87 ppm (multiplet) corresponding to aromatic protons further confirmed the introduction of EM TA to PEA. The 13C NMR spectrum also confirmed the structure of PEA-gTA#2 as shown in Figure S2. The FT-IR spectra of the copolymer PEA and PEA-g-TA#2 are shown in Figure 2. In the spectrum of PEA, the absorption bands at 3310 and 3080 cm−1 were assigned to the N−H stretching of amide (II), while those at 2950(2830), 1720, 1640, and 1538 cm−1 for PEA were assigned to C−H stretching of the alkyl group, CO stretching of the ester, CO stretching of amide (I), and N−H bending of amide (II), respectively. In the spectrum of PEA-g-TA#2, the typical absorption peaks of TA observed at 2884

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EM state. The LM sample showed only one peak at 327 nm, which was associated with the π−π* transition of the aromatic benzene ring. Further oxidation caused blue-shift of the peak at 327 nm and the appearance of a new peak at 576 nm, which was attributable to the excitonic transition from benzene ring to quinoid ring. In general, when the EM TA was oxidized from an EM state to a PN state, the band at 576 nm would show a blueshift. However, this blue-shift was not observed for the PEA-gTA#2 copolymer, which implied that the graft copolymers could not reach its PN state. The reason was that the amide bond hindered further oxidation.17 The cyclic voltammogram spectrum is one of the best ways to illustrate the transitions among oxidation states. The chloroform solution of the PEA-gTA copolymer was cast on an ITO substrate and dried to a film. As shown in Figure 3B, the sample had one pair of reversible redox peaks, and the mean peak potential was 0.47 V (E1/2 = (Epa + Epc)/2). Our CV results were similar to the previous report,28 where only one oxidation peak was observed for TA segments. The results of UV−vis and CV demonstrated that our copolymers maintained a good level of electroactivity. Thermal Properties of the PEA-g-TA Copolymers. Figure S3 shows the glass-transition temperature (Tg) of PEAg-TA powders. For the PEA-g-TA copolymers, the Tg increased from 19 to 28 °C, which could be attributed to the introduction of hard TA segments in the PEA copolymer chain. The thermal stabilities of TA, PEA, and PEA-g-TA were studied under identical drying and heating conditions. The TGA curves of the samples obtained by heating the samples from room temperature to 600 °C are shown in Figure 4. The copolymer had a clear two-step decomposition path. The first weight loss step occurred in the temperature from 150 to 350 °C, which could be attributed to the decomposition of PEA main chains. The subsequent loss step in the temperature range of 350−470 °C should be due to the decomposition of tetreaniline segment, while the obvious weight loss began from 230 °C and the weight loss before 230 °C should be ascribed to the volatilization or decomposition of moisture and low-molecular-weight parts in the samples. The result demonstrated that PEA-g-TA copolymers had good thermal stability compared to PEA. Moreover, the copolymers with high ratio of TA possessed better thermal stability, because the TA segments contained a stable aromatic ring, which led to the PEA-g-TA

Figure 2. FT-IR spectra of (a) PEA and (b) PEA-g-TA#2.

1510 cm−1 (s, −N−B−N−) and 1601 cm−1 (s, −NQN−) were attributed to the benzenoid unit and the quinoid unit of the TA segments, respectively. These data showed the successful synthesis of the PEA-g-TA copolymers. As shown in Table 1, the conductivities of the PEA-g-TA samples were lower than that of TA (≈10−2 S/cm). The reduction of conductivity of the copolymers is, understandably, due to the low content of TA in the copolymers. It is difficult for an electron to transport from one polymer chain to another, that is, so-called “interchain transport” of electrons. However, these conductivity values are adequate to transfer bioelectrical signals in vivo, since the microcurrent intensity in the human body is quite low.15 Electrochemical Characterization of the PEA-g-TA Copolymers. It is well-known that aniline oligomers such as PANi have different oxidation states (that is, leucoemeraldine state (LM), emeraldine state (EM), and pernigraniline state (PN)), when they are treated by different voltages or oxidating and reducing agents. The electroactivity of PEA-g-TA was investigated by UV−vis and CV spectra. The UV−vis absorption spectra of the PEA-g-TA#2 copolymer oxidized by ammonium persulfate in DMF are shown in Figure 3A. The UV−vis spectra of the sample exhibited a stepwise oxidation process of TA segments in the sample from the LM state to the

Figure 3. (A) UV−vis spectra of PEA-g-TA#2 in DMF oxidized by ammonium persulfate. (B) Cyclic voltammogram of the PEA-g-TA#2 film obtained in 1.0 M HCl aqueous solution. 2885

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(about 35%) and PEA-g-TA#3 (about 25%) were slower because the content of the TA segments increased in graft polymers. For the PEA-g-TA polymers, the degradation rate decreased with the increase of the graft TA content. Evidently, introducing TA to the PEA polymer increased the hydrophobicity and steric hindrance, which decreased its degradation rate. The biodegradability of the PEA-g-TA copolymer provided a favorable prerequisite condition for the in vivo applications. Cytotoxicity of the PEA-g-TA Copolymers. To determine the cytotoxicity of the PEA-g-TA copolymers in comparison with PEA and TA, the MTT assay was performed, and the results are shown in Figure 6. MC3T3-E1 cells were

Figure 4. TGA curves of (a) PEA, (b) PEA-g-TA#1, (c) PEA-g-TA#2, (d) PEA-g-TA#3, and (e) EM TA.

copolymers being resistant to decomposition under higher temperature. Mechanical Properties of the PEA-g-TA Copolymer Thin Films. The mechanical properties of the PEA-g-TA copolymer films were investigated. The PEA-g-TA#2 copolymer was highly distensible with breaking elongation rate of 105 ± 10%, and the tensile modulus of copolymer was 20 ± 2.5 MPa but lower than pure PEA (120 ± 5%, 30 ± 1.2 MPa), because the intermolecular interaction of the PEA main chains was comparatively weakened by tetreaniline segment.15 The PEA-g-TA copolymer could be processed and applied in vivo with sufficient mechanical strength. In Vitro Biodegradation of the PEA-g-TA Copolymer Thin Films. In vitro degradation curves of the PEA-g-TA copolymer films are shown in Figure 5. Environmental

Figure 6. Cytotoxicity of (a) PEA, (b) PEA-g-TA#1, (c) PEA-g-TA#2, (d) PEA-g-TA#3 and (e) TA in vitro. Viability of mouse MC3T3-E1 cells is expressed as a function of polymer concentration.

incubated in culture medium with increasing elution solution concentrations of TA, PEA, and PEA-g-TA ranging from 0.156 to 10 mg mL−1. As expected, the cell viability of the PEA-g-TA copolymer was more than 90% in each concentration, which was similar to that of PEA. The cell viability of pure TA presented lower than 80% at a high concentration compared to that of PEA and PEA-g-TA, which indicated the successful reduction of cytotoxicity through grafting to PEA. Biocompatibility of the PEA-g-TA Copolymers. The biocompatibility was measured to demonstrate whether the materials could promote cell adhesion and proliferation in vitro. PEA-g-TA copolymers, PEA, and TCPs were subjected to the biocompatibility evaluation with MC3T3-E1 cells. In Figure 7A, MC3T3-E1 cells were seeded and cultured for 1, 6, 12, and 24 h on different substrates to observe the cell adhesion and proliferation. After culturing for 6 h, the cell average areas treated with different materials were almost the same. With the increase of culture time, the cell average areas showed differentiation distinctly. Especially for 24 h, the cell average area of PEA was the smallest, and that of PEA-g-TA copolymers and TCPs increased in order, and then PEA-g-TA doped with CSA was the highest, even exceeding that treated with TCPs. In Figure S4, the different microstructures of polymer films were observed by SEM; however, irrespective of whether polymers were doped with CSA, the differences of material surfaces were not noticeable. The cell average area was on one hand related to the surface characteristics (stiffness, roughness) of materials, and on the other hand, the important electroactivity effect (charge density and hydrophobicity) of the PEA-g-TA copolymers accelerated the adhesion and growth of cells. The

Figure 5. Degradation of PEA and PEA-g-TA films. Degradation studies of PEA-g-TA films (10 mm ×10 mm ×0.2 mm) were performed in Tris-HCl buffer containing proteinase K (pH = 7.4) at 37 °C.

conditions in vivo were simulated by Tris-HCl buffer solution containing proteinase K at 37 °C. The transparent degradation solution surrounding in the PEA-g-TA copolymer films appeared light blue after degradation for 24 h, and the color of the degradation solution changed to dark blue after degradation for 6 d. Compared with PEA (lost about 45%), the degradations of PEA-g-TA#1 (about 42%), PEA-g-TA#2 2886

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7B, when MC3T3-E1 cells were cultured for 1, 3, and 5 days on different substrates. MC3T3-E1 cells displayed two distinct growth phases during in vitro osteogenic differentiation. After MC3T3-E1 cells became attached to an osteoconductive surface, they entered a rapid proliferative growth phase in order to establish critical cell−cell interactions essential for the subsequent postconfluent differentiation growth phase.30 With the increase of culture time, the cell proliferation rate of TCPs was highest, and PEA-g-TA copolymers showed good proliferation ability similar to TCPs. The PEA-g-TA polymers doped with CSA were able to display their electroactivity and induced the chemical and energy exchange between cells and its surroundings, which benefit the cell growth. As previously known, the charges from TA segments could improve cell proliferation, but toxicity was a main concern; thus, to be useful as biomaterial, the percentage of TA segments would need to be at an optimal level to preserve the strength and electroactivity while avoiding undesirable side effects. Influence on Differentiation of Cells Stimulated by pulsed Electrical Signal. For PEA-g-TA copolymers, our ultimate goal was realizing their potential applications as scaffold materials in tissue engineering. It has been previously demonstrated that electroactive polymers can promote neuronal differentiation stimulated by electrical signal.15,16 As reported in the literature, MC3T3-E1 cells are sensitive to electrical field or eletromagnetic field, and it is easy to express their phenotype on the TCPs. We expected that the electroactive PEA-g-TA copolymers could be more effective in promoting the differentiation of the MC3T3-E1 cells stimulated by electrical signal. Three critical processes are required for bone formation: the presence of osteogenic stem and/or progenitor cells, osteoinductive factors to stimulate the differentiation of these cells along an osteoblastic pathway, and an osteoconductive surface to support cell growth and the deposition of a new bone matrix.30 After a 48 h culture stabilization period, the cells were cultured for a subsequent 2 week period in osteogenic differentiation media. Osteogenic differentiation of MC3T3E1 cells was assessed by the intracellular free calcium concentration ([Ca2+ (i)]) and ALP enzyme activity. Intracellular Ca2+ oscillation has crucial roles in regulation of various cellular processes including proliferation, differentiation, and apoptosis.29 Calcium concentration is also a functional variable used for measuring the osteogenic effect of the

Figure 7. The morphology (A) and proliferation (B) of MC3T3-E1 cells cultured on the surface of the PEA-g-TA polymers, PEA, and TCPs with various time points. (a) PEA, (b) PEA-g-TA#2, (c) PEA-gTA#2 doped with CSA, (d) PEA-g-TA#3 doped with CSA and (e) TCPs.

MC3T3-E1 cells readily demonstrated their elongated, spindlelike morphology on the electroactive material surfaces compared with PEA and TCP substrates. These cells assumed a cuboidal morphology with marked increase in cytoplasm area and numerous surface projections. The proliferation activity was quantitatively determined via MTT assay to measure the metabolic activity of the total population of cells growing on the surfaces as shown in Figure

Figure 8. [Ca2+ (i)] of MC3T3-E1 cells (A) and [Ca2+ (i)] of the unit cell (B) on the different substrates stimulated by the electrical signals for 7 and 14 days. (a) PEA, (b) PEA-g-TA#2, (c) PEA-g-TA#2 doped with CSA, (d) PEA-g-TA#3 doped with CSA, and (e) TCPs. 2887

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Figure 9. ALP enzyme activity of MC3T3-E1 cells (A) and ALP enzyme activity of the unit cell (B) on the different substrates stimulated by the electrical signals for 7 and 14 days. (a) PEA, (b) PEA-g-TA#2, (c) PEA-g-TA#2 doped with CSA, (d) PEA-g-TA#3 doped with CSA, and (e) TCPs.

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CONCLUSIONS In this work, biodegradable and electroactive PEA materials based on dual amino acids were synthesized and demonstrated excellent osteoinductivity in electrical field, i.e., the ability to act as a scaffold to support the robust adhesion, proliferation, and differentiation of preosteoblastic MC3T3-E1 cells. On the basis of the results of UV−vis spectra and CV, these materials possessed electroactivity and reversible redox property. The materials also showed good solubility, thermal stability, and mechanical properties, which gave them excellent properties for processing. The cell culture results showed that electroactive PEA-g-TA copolymers doped with CSA could be more effective to promote the differentiation of MC3T3-E1 cells stimulated by pulsed electrical signal compared with pure PEA and TCPs. Future work will focus on designing the appropriate electroactive three-dimensional scaffold models and evaluating the in vivo tissue compatibility and therapy effect through animal tests.

electrical signals on proliferation, differentiation, and mineralization in vitro. As shown in Figure 8A, cells were seeded for 7 d, irrespective of whether cells were exposed to electrical stimulation, the differences of [Ca2+ (i)] of MC3T3-E1 cells on different material surfaces were not noticeable. However, compared with cells on the TCPs surface, a higher [Ca2+ (i)] of unit cell (quantity via the MTT assay to measure the total population of cells) was observed for those exposed to electrical field when cells were seeded on the surface of the PEA-g-TA copolymers doped with CSA (Figure 8B). Moreover, higher [Ca2+ (i)] and [Ca2+ (i)] values of the unit cell were observed when cells exposed to electrical field were seeded on the surface of the PEA-g-TA copolymers doped with CSA for 14 d. Calmodulin is a ubiquitous intracellular calcium-binding protein that interacts with a wide range of enzymes. An increase in the concentration of cytosolic Ca2+ leads to an activation of calmodulin, which is responsible for many calcium-mediated processes including proliferation and differentiation of cells. Thus, modulation of calcium transport thorough voltage grated channels, as a result of the electrical stimulation, could be mediating certain cellular differentiation pathways.25 Increased ALP enzyme activity is an early marker of osteogenic differentiation.30 As shown in Figure 9, 1 week after the induction of osteogenic differentiation, ALP activity of cells on the surface of PEA-g-TA copolymers doped with CSA was slightly lower than that of cells on the TCPs, while the ALP activity of the unit cell was higher than that of cells on the TCPs. A distinct increase in ALP enzyme activity was observed for MC3T3-E1 cells exposed to electrical stimulation both on TCPs and on the surface of electroactive copolymers for 14 days. Especially ALP activity of the unit cell showed higher expression on PEA-g-TA copolymers doped with CSA for 14 days. The intracellular calcium concentration assay and ALP enzyme activity were highest in the PEA-g-TA#2 copolymer treated group, suggesting that an appropriate percentage of TA segments in the PEA-g-TA copolymer were essential. The effect of electrical stimulation on electroactive material surface may be due to the acceleration of interaction between materials and cells by changing surface properties of the electroactive materials. Our results described above indicate that the electroactive PEA-g-TA copolymer surface could indeed promote the differentiation of MC3T3-E1 cells stimulated by pulsed electrical signal as expected.

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ASSOCIATED CONTENT

S Supporting Information *

The synthesis Route of PEAs with pendant −COOH (Scheme S1), 1H NMR of PEA(Bz) and PEA(H) (Figure S1), 13C NMR of PEA-g-TA and TA (Figure S2), DSC heating curves of PEAg-TA and PEA (Figure S3), and SEM images of polymer films (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail address: [email protected] (X.C.); weiyen@tsinghua. edu.cn (Y.W.). Author Contributions ⊥

These two authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from National Natural Science Foundation of China (Project 21004061, 50973109, 21074018, 51021003). 2888

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