PLA-PEG-PLA and Its Electroactive Tetraaniline Copolymer as Multi

Apr 24, 2013 - Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. ...
5 downloads 7 Views 4MB Size
Download PDF
Article pubs.acs.org/Biomac

PLA-PEG-PLA and Its Electroactive Tetraaniline Copolymer as Multiinteractive Injectable Hydrogels for Tissue Engineering Haitao Cui,†,‡ Jun Shao,†,‡ Yu Wang,† Peibiao Zhang,† Xuesi Chen,*,† and Yen Wei*,§ †

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

ABSTRACT: Injectable hydrogels have served as biomimic scaffolds that provide a three-dimensional (3D) structure for tissue engineering or carriers for cell encapsulation in the biomedical field. In this study, the injectable electroactive hydrogels (IEHs) were prepared by introducing electrical properties into the injectable materials. Carboxyl-capped tetraaniline (CTA) as functional group was coupled with enantiomeric polylactide−poly(ethylene glycol)−polylactide (PLA-PEG-PLA), and the electroactive hydrogels were obtained by mixing the enantiomeric copolymers of CTAPLLA-PEG-PLLA-CTA and CTA-PDLA-PEG-PDLA-CTA aqueous solutions. ultraviolet−visible spectroscopy (UV−vis) and cyclic voltammetry (CV) of the complex solution showed good electroactive properties. The gelation mechanism and intermolecular multi-interactions such as stereocomplextion, hydrogen bonding, and π−π stacking were studied by Fourier transform infrared spectroscopy (FT-IR), UV−vis, and wide-angle X-ray diffraction (WAXD). Gelation properties of the complexes were also studied by rheometer. The encapsulated cells remained highly viable in the gel matrices, suggesting that the hydrogels have excellent cytocompatibility. After subcutaneous injection, the gels were formed in situ in the subcutaneous layer, and hematoxylin−eosin (H&E) staining suggested acceptable biocompatibility of our materials in vivo. Moreover, these injectable materials, when treated with pulsed electrical stimuli, were shown to be functionally active and to accelerate the proliferation of encapsulated fibroblasts, cardiomyocytes, and osteoblasts. Hence, the IEHs possessing these excellent properties would be potentially used as in vivo materials for tissue engineering scaffold.

■

INTRODUCTION Currently, there is a high demand in the clinic for novel biomaterials that not only have specific target functions, but can also be externally controlled or tailored by surrounding stimulation. Many studies have demonstrated that electrical stimuli can adjust a range of cellular activities, such as cell adhesion, migration, proliferation and differentiation.1−4 Electrically conductive materials such as polypyrrole (PPy), polyaniline (PANI), carbon nanotubes, and conductive nanoparticles have been widely studied and applied in tissue engineering, due to the key advantage of permitting external control over the intensity and position of electrical stimuli.5−8 However, successful incorporation of electrically conductive materials into the biomaterials remains limited, due to their poor mechanical properties, nondegradability, and difficulty to process into complex three-dimensional (3D) structures.9−11 Similar to electrically conductive polymers, oligomers such as tetraaniline and aniline pentamer characteristically have a conjugated backbone with a certain degree of w-orbital overlap. Through a reversible process called “doping−dedoping”, the neutral chain can become positively charged with polarons and bipolarons as the charge carriers for electrical conduction.12,13 © XXXX American Chemical Society

When an electrical stimulation is applied, the changes of surface charges and polymer surface properties including wettability and conformation can influence the behavior of cells.9,14 Moreover, the use of oligomers solves drawbacks of the conducting polymers in biomedical application, such as poor solubility, poor processability, and unable clearance in vivo.11,15 Our group has synthesized many electroactive polymers containing oligoaniline with good electroactivity and biodegradability for nerve and bone tissue engineering.16−20 Albertsson’s group proposed a new concept of degradable electrically conducting hydrogels (DECHs) and have synthesized degradable and electroactive hydrogels of acrylated polylactide−poly(ethylene glycol)−polylactide (PLA-PEGPLA) with aniline tetramer. These hydrogels possess tunable conductivity and swelling properties.21 They have also obtained degradable and conductive polysaccharide hydrogels that possess the good film-forming properties and biocompatibility.22 Received: February 22, 2013 Revised: April 21, 2013

A

dx.doi.org/10.1021/bm4002766 | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Table 1. Characterizations of Partial PLA-PEG-PLA and CTA-PLA-PEG-PLA-CTA Copolymers sample

chain structure

PEG

PEG 4L6 4D5 4L8 4D8 4L11 4D10 4L′6 4D′5 4L′8 4D′8 4L′11 4D′10

EG91 LLA6-EG91-LLA6 DLA5-EG91-DLA5 LLA8-EG91-LLA8 DLA8-EG91-DLA8 LLA11-EG91-LLA11 DLA10-EG91-DLA10 CTA-LLA6-EG91-LLA6-CTA CTA-DLA5-EG91-DLA5-CTA CTA-LLA8-EG91-LLA8-CTA CTA-DLA8-EG91-DLA8-CTA CTA-LLA11-EG91-LLA11-CTA CTA-DLA10-EG91-DLA10-CTA

4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000

DPa PLA

EG/LAa

12 10 16 16 22 20 12 10 16 16 22 20

7.9 8.8 5.5 5.8 4.2 4.5 7.8 8.8 5.5 5.8 4.2 4.5

Mna (g mol−1)

Mn (g mol−1)/ PDIb

water-solubility

4800 4700 5100 5100 5600 5400 5700 5600 6000 6000 6500 6300

9100/1.1 10500/1.2 10300/1.1 11100/1.2 11000/1.1 12100/1.1 11800/1.2 11600/1.2 11600/1.4 12600/1.3 12400/1.3 13300/1.4 13400/1.3

Y Y Y Y Y Y Y Y Y Y Y Y Y

a Calculated from the integration of 1HNMR signals. DPPEG = MnPEG/44 DPPLA = DPPEG/(EG/LA). Mn = DPPEG × 44 + DPPLA × 72. bDetermined by GPC.

to advance this class of biomaterials for applications as injectable scaffolds. As our understanding of the biological process of tissue development and healing expands, the information must be incorporated into the scaffolds such that the specific signals are delivered in the appropriate spatial and temporal manner.24 In addition, more consideration needs to be made regarding the influence and requirements of external mechanical and electrical signals on development of engineered tissues.9,24,38,39 On the basis of previous studies, our objective was thus to develop injectable electroactive hydrogels (IEHs) by introducing electrical properties into the materials. We hypothesize that IEHs combining the advantages of degradable electrically conducting polymers with the unique properties of injectable hydrogels will open a novel area of biomaterials for tissue engineering. In this work, we synthesized tetraaniline-functional PLAPEG-PLA copolymers, and their hydrogels were obtained by mixing both CTA-PLLA-PEG-PLLA-CTA and CTA-PDLAPEG-PDLA-CTA copolymers in aqueous solution. The intermolecular multi-interactions and gelation properties of the complexes were studied in detail. The resulting electroactive hydrogels exhibited good cytocompatibility in vitro and acceptable biocompatibility in vivo. Moreover, these electroactive hydrogels could accelerate the proliferation of fibroblasts, cardiomyocytes, and preosteoblasts with pulsed electrical stimuli, which may serve as useful candidates for tissue engineering application.

One of the recent trends in biomaterials for tissue engineering is to develop biomimic functional scaffolds, which integrate the simple fabricated strategies with biological responsive signals. Therefore, the techniques incorporating signals such as topological, biological, chemical, and electrical cues into easy-molding scaffolds to stimulate repair or regeneration in vivo will become increasingly important in this research field.23−25 Injectable hydrogels with biodegradability have the ability to mimic many physical properties of tissues and in situ formability, which allows an effective and homogeneous encapsulation of drugs/cells and convenient in vivo surgical operation in a minimally invasive way at the target site.26,27 They can readily take the shape of ideal soft materials for biomedical applications, such as drug delivery, cell encapsulation, and tissue engineering.28 In situ hydrogels can be divided into two main categories: chemical gelling systems that are cross-linked by covalent bonds, and physical gelling systems that are formed via physical association between polymeric chains or nanoparticles.29 Physical interactions such as electrostatic interaction, hydrophobic interaction, hydrogen bonding, π−π stacking, and sterecomplextion can be exploited for the design of self-assembly of polymeric networks, which can form hydrogels in time or in response to a certain stimulus (e.g., temperature, pH value and ionic concentration). Stereocomplextion allows forming a physical hydrogel, but the sol− gel transition and physical properties are similar to chemical hydrogels.30 De Jong et al. prepared a self-assembled hydrogel from enantiomeric PLA oligomers grafted to dextran, and the hydrogel was formed from the stereocomplexation of poly(Llactide) (PLLA) and poly(D-lactide) (PDLA) blocks.31,32 Feijen et al. reported the formation of a hydrogel by stereocomplexation of PLLA-PEG/PDLA-PEG star-shaped copolymers and the photopolymerization of methacrylated stereohydrogels to improve mechanical strength.33,34 Fujiwara et al. introduced a stereocomplex mechanism to prepare thermosensitive hydrogels, but tetrahydrofuran was used to dissolve the copolymers.35 Other studies also have been reported on stereohydrogels for drug release and tissue engineering.30,36,37 However, to date, studies on the functional properties, such as cellular and histological responses to the hydrogels, were seldomly reported, despite the importance of such information

■

EXPERIMENTAL SECTION

Materials. N-Phenyl-1,4-phenylenediamine, 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC·HCl), 4-dimethylaminopyridine (DMAP), stannous octoate (Sn(Oct)2, 95%), polyethylene glycol (PEG) with molar masses of 4000 and 10,000 g mol−1, ammonium persulfate (APS) and succinic anhydride were purchased from Aldrich. L-Lactide (L-LA) and D-Lactide (D-LA) were obtained from Changchun SinoBiomaterials Co., Ltd. and recrystallized from ethyl acetate three times before polymerization. N,N-Dimethylformamide (DMF), toluene, and ethyl acetate were dried and distilled prior to use. Dichloromethane (CH2Cl2), chloroform (CHCl3), ethyl ether, ethanol, hydrochloric acid (HCl), and ammonium hydroxide (NH3·H2O) were used as received. All chemicals were of analytical grade or higher. Synthesis of CTA-PLA-PEG-PLA-CTA Copolymers. As shown in Scheme S1 (Supporting Information), the carboxyl tetraaniline− B

dx.doi.org/10.1021/bm4002766 | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

poly(L-lactide)−poly(ethylene glycol)−poly(L-lactide)−carboxyl tetraaniline (CTA-PLLA-PEG-PLLA-CTA) and the carboxyl tetraaniline−poly(D-lactide)−poly(ethylene glycol)−poly(D-lactide)−carboxyl tetraaniline (CTA-PDLA-PEG-PDLA-CTA) were prepared by a twostep synthetic procedure. Briefly, PEG was azeotropically dried with toluene for 8 h at first. Then predetermined amounts of L-LA (or D-LA), PEG, and Sn(Oct)2 (1 wt %) were added into an dried reactor and stirred in toluene at 120 °C for 24 h. The feed amounts of PEG and L-LA (or D-LA) were varied to provide tunable chain lengths and block ratios of block copolymers (Table 1 and Table S1). After the reaction was complete, the solution was precipitated in a mixture of cold ethyl ether/ethanol, and then dissolved in CHCl3 and precipitated twice in cold ethyl ether. The product was dried under vacuum for 48 h. Yield of purified product was 86%. Tetraaniline was synthesized according to a similar procedure reported in the literature.19 The carboxyl-capped tetraaniline (CTA) was synthesized from the carboxylation reaction of tetraaniline and succinic anhydride in CH2Cl2, and the crude product was washed with distilled water, followed by washing in a Soxhlet extractor with CH2Cl2 till the filtrate became colorless. The product was dried under vacuum for 48 h. Yield of purified product was 78%. 1H NMR (400 MHz, DMSO-d6, ppm): 12.11 (s, 1H), 9.71 (s, 1H), 7.77(s, 1H), 7.70−7.60 (d, 2H), 7.44−7.33 (d, 2H), 7.30−7.21 (s, 1H), 7.20−7.08 (m, 2H), 7.08−6.78 (m, 8H), 6.67 (m, 1H), 2.73 (m, 2H), 2.35−2.27 (m, 2H). The molecular weight of CTA was measured by mass spectrometry to be 465.2 (MH+/e). One millimole of PLLA-PEG-PLLA (L) (or PDLA-PEG-PDLA (D)), 3 mmol of CTA, 10 mmol of EDC, 3 mmol of DMAP and 20 mL of DMF were added into a dried reactor under nitrogen. The reactor was stirred for 48 h at room temperature. After the reaction, the mixture was precipitated in ethanol and was dissolved in CHCl3, filtered and precipitated in a mixture of cold ethyl ether/ethanol. Such a dissolution−precipitation process was repeated three times to purify the product. The product was dried under vacuum for 48 h. Yield of purified product was 83%. Characterization. 1H NMR spectra were recorded on a Bruker AV 400 MHz spectrometer. Fourier transform infrared (FT-IR) spectra of samples were recorded on a Bio-Rad Win-IR instrument in the range of 4000−500 cm−1. Matrix-assisted laser desorption/ionization timeof-flight (MALDI-TOF) mass spectra were performed on an AXIMACFR laser desorption ionization time-of-flight spectrometer (COMPACT). Environmental scanning electron microscopy (ESEM) was performed on an XL 30 scanning electron microscope (Micrion FEI PHILIPS). The ultraviolet−visible (UV−vis) spectra of samples and their solutions were recorded on a UV-2401PC spectrophotometer. Cyclic voltammetry (CV) of samples was conducted on a CHI 660 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 100 mV s−1. Gel permeation chromatography (GPC) measurements were carried out with a Waters GPC instrument and DMF was used as an eluent. The molecular weights were calibrated with polystyrene standards. The wide-angle X-ray diffraction (WAXD) measurement was carried out on a Bruker D8 Advance X-ray diffractometer, using Cu Kα radiation; the scattering angle ranged from 2θ =10° to 30° at a scan speed of 3° min−1 at room temperature. The conductivity of the samples at room temperature was measured using a broadband dielectric spectrometer between two-terminal Cu electrodes within the frequency range of 1 Hz ∼1 MHz (Novocontrol). In vitro Hydrogel Formation and Dynamic Mechanical Analysis. The vial inverting approach was employed to determine whether the sol−gel transition occurred with body temperature. Critical gel concentrations (CGCs) were determined as described in the literature.34 Briefly, CTA-PLLA-PEG-PLLA-CTA (L′) and CTAPDLA-PEG-PDLA-CTA (D′) copolymer solutions were prepared with concentration increments of 5% (w/v), by dissolving the polymers overnight. Subsequently, equal amounts of polymer solutions were mixed in the vial, and then the vials were immersed in a 37 °C

water bath and allowed to reach equilibrium. The sample was defined as a “gel” in the case of no visual flow within 30 s by inverting the vial. SEM was used to observe the morphology of lyophilized hydrogels in vitro. Rheology experiments were performed on a MCR 301 Rheometer (Anton Paar). The mixture solution of L (L′) and D (D′) copolymer was placed between parallel plates of 25 mm diameter and a gap of 0.5 mm. To prevent the evaporation of water, the outer edge of the sample was sealed by a thin layer of silicon oil. The data were collected under a controlled strain γ of 1% and a frequency of 1 Hz. G′ is an elastic component of the complex modulus for measure of the gel-like behavior of a system, whereas G″ is a viscous component of the complex modulus and is a measure of the sol-like behavior of the system. In Vitro Cell Encapsulation and Viability. Before encapsulation, L′ and D′ solutions were sterilized under UV light for 30 min. Mouse fibroblast L929 cells suspension with a density of 5 × 104 per well in 24-well plates was added into 200 μL of L′ and D′ copolymer mixture solution. After thorough mixing, the mixture was transferred to a 37 °C incubator for gelation. Then 500 μL of culture medium (Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal calf serum (Gibco) and 100 U mL−1 penicillin−streptomycin (SIGMA)) was added and changed every other day. After 1, 3, and 7 days of culture, a qualitative viability assay was performed using a LiveDead assay kit. At predesigned days postencapsulation, the cell/ hydrogel complexes were washed with phosphate-buffered saline (PBS) three times and treated with calcein AM (2 μM) and propidium iodide (4 μM) for 30 min. Cells were observed under an inverted fluorescent microscope (TE2000U, Nikon) and analyzed using the “NIH ImageJ” software. Electrical Stimuli and Proliferation Assays. Mouse fibroblast L929 cells, rat cardiomyocytes H9c2 cells, and mouse osteoblasts MC3T3-E1 cells were used to investigate the influence of electroactive hydrogel on the proliferation of various cells stimulated by pulsed electrical signals. The solutions of L/D, EM L′/D′, EM L′/D′ doped with HCl were sterilized by exposure to UV radiation for 1 h. Cells were seeded into various gels at a density of 2 × 104 cells per well in 24-well plates. The electrical stimuli were carried on the signal generator (Rigol DG1022 Function/Arbitrary Waveform Generator), and the signals were displayed and checked on the wave inspector (Rigol DS1022C Digital Oscilloscope). The square wave, frequency of 100 Hz, 50% duty cycle, and electrical potential of 0.5 V were adopted in the experiment. The electrical potential was added directly in gels through two microwire platinum electrodes (0.5 mm in diameter). The samples were respectively stimulated for 0.5 h every day. After the predetermined period, the incubation medium was removed, and the dishes were washed with PBS three times. In addition, WST-8 solution (10% v/v in medium) (Cell Counting Kit-8) was added to each well. After 4 h of incubation, the absorbance value at 450 nm was measured on multifunction microplate scanner (Tecan Infinite M200). Animal Procedure and In Vivo Evaluation. The animal experiments were carried out according to the NIH Guide for the Care and Use of Laboratory Animals, provided by Jilin University, Changchun, China. Sprague−Dawley (SD) rats were anesthetized, and then an aqueous solution of the mixture (20 wt %, 0.3 mL) was subcutaneously injected into rats by a syringe with a 23-gauge needle. At designated time intervals, the animals were sacrificed, and the injection site was carefully cut open. Then the photographs of in situ gel formation were taken. The surrounding tissues of gel were surgically removed and histologically processed using hematoxylin− eosin (H&E) stains for the examination of inflammatory responses of synthesized copolymers in rats. Statistical Analysis. The data presented as the mean ± standard deviation. One-way analysis of variance (ANOVA) was used to analyze the statistical variability of the data, with p < 0.05 being statistically significant.

■

RESULTS AND DISCUSSION Synthesis and Characterization of CTA-PLA-PEG-PLACTA Copolymers. The synthetic procedure of CTA-PLAC

dx.doi.org/10.1021/bm4002766 | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Figure 1. (a) UV−vis spectra of the 4L′11/4D′10 complex oxidized by APS. (b) UV−vis spectra of the 4L′11/4D′10 complex in the LM state, EMB state, and EMS state (doped with 1 M HCl aqueous solution). (c) Cyclic voltamogram of the 4L′11/4D′10 complex in 1 M HCl aqueous solution.

Scheme 1. Schematic Illustration of Intermolecular Multi-interactions between Copolymer Chains

resulting DP values and CTA/triblock ratio did not exactly coincide with the feed ratios. This was possibly due to the incomplete polymerization of PLA and limited efficiency of the condensation coupling reaction, but unreacted LA and CTA were eliminated during the purification procedure. The conductivity of the sample films was also evaluated, and the results are shown in Figure S2. Electrochemical Characterization of Copolymer Solution. The electrochemistry of L′ and D′ copolymers were investigated by UV−vis spectra and CV. 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. Figure 1a shows the UV−vis spectra of the 4L′11/4D′10 complex oxidized by APS in DMF. The UV−vis spectra of the complex exhibited a stepwise oxidation process of the CTA blocks in the complex from the LM to the emeraldine base (EMB) state. The LM complex showed only one peak at 310 nm, which is

PEG-PLA-CTA (L′ and D′) copolymers was shown in Scheme S1. First, triblock copolymers consisting of a PEG central block and two PLLA (or PDLA) lateral blocks were synthesized by the ring-opening polymerization of L-lactide (or D-lactide) in the presence of dihydroxyl PEG. Subsequently, CTA was reacted with terminal hydroxyl groups of triblock copolymers by EDC as the condensating agent and DMAP as the catalyst. As shown in Figure S1, the composition of the copolymers was determined using 1H NMR spectrum as reported in the literature.36 Table 1 and Table S1 present the chain structure, DPPLA, EG/LA ratio, Mn (PDI), and water-solubility data for the various copolymers. It should be noted that DPPLA represents the total length of the two PLA blocks. Based on the comparison of the molecular weight of L (or D) with the L′ (or D′) copolymers, these L′ (or D′) samples all had molecular weights greater than L (or D) copolymers, and the calculated CTA/triblock ratios were higher than 1.5. The combined data of the 1H NMR, GPC and FT-IR spectra (Figure 3) confirmed the successful synthesis of the copolymers. However, the D

dx.doi.org/10.1021/bm4002766 | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

associated with the π−π* transition of the benzene ring. Further oxidation caused the appearance of a new peak at 569 nm, which was attributed to the excitonic transition from benzene ring to quinoid ring. Figure 1b shows the characteristic absorbance spectra of the complex doped with 1 M HCl aqueous solution. During the doping process, the color of the solution changed from blue to green. The peak at 430 nm represented the polaron band, and the localized polaron peak at ∼800 nm confirmed the generation of emeraldine salts (EMS) and the ability of conducting electrons of complex. The electroactivity of the 4L′11/4D′10 complex in the 1 M HCl aqueous solution was also observed in CV, as shown in Figure 1c. After the removal of oxygen in the solution, one pair of reversible redox peaks was clearly observed, which corresponded to the transition between the LM state and the EM state with the mean peak potential E1/2 of 0.41 V. From the UV−vis and CV results, it could be concluded that the CTA blocks in the complex have maintained their good electroactivity in aqueous solution. Intermolecular Multi-interactions of Copolymer Chain. Scheme 1 gives a schematic illustration of intermolecular multi-interactions between copolymer chains. It is known that enantiomeric PLA exists in two opposite configurations of PLLA and PDLA and a special crystalline structure termed a stereocomplex can be obtained from coprecipitation or solution casting of PLLA and PDLA in solutions or through cooling from melts of their mixtures.36 It is considered that the CH3···CO interactions among the βform 31-helices between the PLLA and PDLA chains is the driving force for forming the racemic nucleation of the PLLA/ PDLA stereocomplex, which is attributed to a C−H···O hydrogen bond.40,41 And its crystallization behavior was obviously different from the pure PLLA, in which the dipole−dipole intermolecular interaction of the CH3 group among the α-form 103-helices.42,43 To confirm the gel formation by stereocomplex crystallization of the PLA blocks, WAXD experiments were performed on solid samples and lyophilized hydrogels. Figure 2a shows the

contrast, 4L6/4D5 exhibited no stereocomplex diffraction peaks because the shorter PLA blocks length could not form stereocomplex or homopolymer crystal. The 4L′8/4D8′ and 4L′17/4D16′ complexes also exhibited stereocomplex diffraction peaks ∼12.1° and 20.9°, indicating that the rigid CTA blocks did not eliminate the stereocomplexation of PLLA and PDLA blocks. WXRD analysis was also performed on lyophilized hydrogels of 4L′8/4D′8 aqueous solutions at different concentrations. As shown in Figure 2b, with increasing of solutions concentration, the crystallization of stereocomplex became stronger, which was attributed to the enhanced interaction of the copolymers with increasing viscosity. FT-IR spectroscopy was employed to verify hydrogen bonding between copolymers because of its sensitivity to hydrogen bond formation. As shown in Scheme 1, the Hbonding involves CO groups of PLLA (or PDLA), often regarded as hydrogen bond acceptor, together with CH3 groups of PDLA (or PLLA) and NH groups of undoped CTA, usually regarded as hydrogen bond donor.41,45 Figure 3 shows scale-

Figure 3. Scale-expanded infrared spectra recorded at room temperature for lyophilized samples of 4L10 (a), 4L10/4D10 (b), 4L′10 (c), 4L′10/4D′10 (d), and HCl doped 4L′10/4D′10 (e).

expanded infrared spectra recorded at room temperature for lyophilized samples of 4L11 (a), 4L11/4D10 (b), 4L′11 (c), 4L′11/4D′10 (d) and HCl-doped 4L′11/4D′10 (e). The broad absorption band at 3200−3700 cm−1 represented the different types of strong hydrogen bonding interactions, including intramolecular and intermolecular −O/H−N, −O/H−O or− N/H−N. Obviously, the CO asymmetric stretching mode of ester in PLA blocks shifted from 1758 to 1754 cm−1 (Figure 3a and 3b), and its intensity also increased with the formation of the PLA stereocomplex. The low-frequency shift of the CO stretching band is an important criterion for hydrogen bond formation of the CO group in the complex as reported in the literature.41 In Figure 3c,d, two new absorption bands centered at 1646 and 1566 cm−1 were assigned to the CO stretching of amide (I), and N−H bending of amide (II). Meanwhile, the bands at 1603 and 1508 cm−1 corresponded to the stretching vibration of the quinoid (NQN) and benzenoid (N−B− N) units of the CTA blocks in the chain, respectively. Direct evidence for enhanced hydrogen bonding in the copolymers containing CTA blocks was obtained by the comparison between undoped complex and HCl doped complex.45 In Figure 3c and 3d, the new weak peak at 1709 cm−1 should be characteristic of hydrogen-bonded CO groups (CO/H− N), since the hydrogen bond weakened the force constant of CO double bond. In Figure 3e, however, the peak of hydrogen-bonded CO groups disappeared, consistent with the presence of the protonated imine nitrogens (+N−H) of

Figure 2. (a) The WAXD profiles of various complexes as well as PEG and copolymers. (b) WAXD patterns of lyophilized hydrogels of 4L′8/ 4D′8 aqueous solutions at different concentration.

WAXD profile of PEG, various copolymers, and the complexes. The PEG exhibited two main diffraction peaks at ∼19.2° and ∼23.4°, and the two characteristic peaks were detected in all the copolymers and complexes, although the peak intensity of PEG blocks weakened with increasing of the length of PLA blocks. In 4L17 and 4L′17 copolymers, the PLA blocks showed a diffraction peak at ∼16.9°. Two new diffraction peaks appeared at ∼12.1° and 20.9° in the 4L8/4D8 complex, which were assigned to the PLA stereocomplex crystal,44 and their intensities increased as the length of PLA blocks increased. By E

dx.doi.org/10.1021/bm4002766 | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

doped CTA, which are devoid of hydrogen bonding capabilities due to their strong electrostatic repulsion interactions. Furthermore, π-conjugated molecules can form well-defined morphologies and molecular arrangements through π−π stacking interactions in a nonsolvent or in a mixture.46−49 Besides hydrogen bonding, the CTA blocks of the copolymers can also enhance aggregation of the chains through π−π stacking in aqueous solution.50,51 As shown in Figure 4, the

Figure 4. UV−vis spectra of the 4L′11 copolymer in DMF (a) and in aqueous solution (b).

π−π* and πB-πQ adsorption bands of 4L′11 in the good solvent DMF were located at 305 and 572 nm. However, in aqueous solution, the adsorptions decreased due to the formation of aggregates, the π−π* band showed a pronounced red-shifted from 305 to 310 nm, and πB−πQ-bands were redshifted from 572 to 580 nm, respectively, which indicated the strong π−π stacking interaction of tetraaniline blocks in the aggregates. This is because, in aqueous solution, solvophobic association could be formed between the aromatic rings of oligoaniline in self-assemble progress, which promotes extended π−π stacking in the amphiphilic copolymers. The ability of the block copolymer to form strong hydrogen bonding and π−π stacking along the polymer backbone suggested that it might be possible to utilize these relatively strong secondary forces to assemble to form the mutiinteractive hydrogel. Hydrogel Formation and Gelation Property of Copolymer Solution. When the copolymers with similar chain lengths of enantiomeric PLA were mixed with each other in an aqueous solution, interactions between PLLA and PDLA blocks can lead to stereocomplexation and the formation of hydrogel. In addition, the hydrophobic aggregation, hydrogen bonding, and π−π stacking also can affect the assembly of the copolymer and the properties of hydrogel. The tube inverting method was performed on the samples to study the influence of the CTA blocks and the PLA blocks length on hydrogel formation at body temperature. Figure 5a shows images of 4L′8 aqueous solutions (20 wt %) and the in situ hydrogel formation by mixing the 4L′8 and 4D′8 aqueous solutions (20 wt %) at 37 °C. Hydrogels were obtained from L′/D′ aqueous solutions by varying gelation conditions such as time and concentration. Figure 5b shows that the CGC of the stereocomplexation of L′/D′ was somewhat lower compared to that of L/D, which was attributed to the increasing interaction of hydrogen bond and π−π stacking of the CTA blocks. In the cases of 4L′17/4D′16 and 4L17/4D16, the copolymers were precipitated in water, because the chain lengths of hydrophobic PLA block were too long to be dissolved in aqueous solution. No hydrogel was formed with increasing the concentration and time of the 4L′6/4D′5 and 4L6/4D5 solutions at 37 °C.

Figure 5. (a) Images of 4L′8 (4L8) aqueous solution (20 wt %) and the in situ hydrogel formation by mixing the 4L′8 and 4D′8 (4L8 and 4D8) aqueous solutions (20 wt %) at 37 °C. (b) CGC of the L′/D′ and L/D hydrogels at 37 °C. (c) Images of 4L′6 aqueous solution (20 wt %) and 4L11 aqueous solutions (20 wt %) at room temperature.

However, the 4L′6 or 4D′5 solution (20 wt %) at room temperature showed gel-like behavior, compared with the similar chain length 4L11 solution that remained a free-flowing solution, as shown in Figure 5c. However, it did not show large enough storage modulus to be defined as gel when it was measured on the rheometer. As we know, the hydrogel formation and their respective properties might be affected by several factors, such as stereocomplextion of PLA blocks, hydrogen bonding and π−π stacking of the CTA blocks and the increasing hydrophobicity of the CTA blocks. The phenomenon could be explained by the presence of the CTA blocks which increased the solution viscosity. We found that the DP of PLA blocks in copolymers should be at least 8 to obtain a hydrogel in our experiment. In addition, the gelation time decreased with increasing PLA chain length, which was due to decreases in the capacity of the PLA blocks for stereocomplex formation. Therefore, the stereocomplexation of the PLA blocks plays more important role than CTA blocks in the muti-interactive gelation process. Hydrogel formation was also confirmed by rheological measurements. Figure 6a shows the evolution of both storage (G′) and loss (G″) modulus of the 4L′8/4D′8 and 4L′11/ 4D′10 (20 wt %) hydrogels as a function of time at 37 °C. After L/D mixing, the solutions were quickly applied to the rheometer. The crossover point of 4L′8/4D′8 and 4L′11/ 4D′10 were observed at 2.4 h and 3.5 min, respectively. The storage modulus increased in time due to the ongoing stereocomplexation. The storage modulus−temperature viscoelastic properties (G′−T) (20 wt %), and storage modulus− concentration properties (G′−C) of the 4L′11/4D′10 sample were also investigated in Figure S3. When the temperature increased from 15 to 60 °C at 0.5 °C min−1, both G′ and G″ decreased, G′ decreasing faster than G″. However, G′ remained F

dx.doi.org/10.1021/bm4002766 | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Figure 6. The dynamic mechanical analysis of hydrogels at 37 °C. (a) The evolution of both storage (G′) and loss (G″) modulus of the 4L′11/4D′10 and 4L′8/4D′8 (20 wt %) hydrogels as a function of time. (b) The storage modulus values of 10, 15 and 20 wt % samples at 5 h.

higher than G″. The modulus decrease could be explained by the weakened interchain interactions of both hydrogen bonding and π−π stacking, leading to the decreased cross-link density. The change of storage modulus of 4L′11/4D′10 was obtained for 10, 15, and 20 wt % samples as a function of time at 37 °C, respectively. With increasing of the samples concentration, the higher G′ values were obtained, which was attributed to the increased cross-link density. The storage modulus values of different samples in 10, 15 and 20 wt % at 5 h are shown in Figure 6b. The G′ value of the all L′/D′ hydrogels were lower than those of L/D hydrogels, upon increasing the polymer concentration from 10 to 20 wt %, suggesting that the CTA blocks weaken the stereocomplexation of PLLA and PDLA blocks despite the fact that they increased the interaction of the hydrogen bonding, π−π stacking, and hydrophobicity. To study the influence of CTA blocks on the overall morphology of the hydrogels, SEM measurements were performed on lyophilized 4L8/4D8 and 4L′8/4D′8 (20 wt %) hydrogels. Figure S4 shows that 4L′8/4D′8 hydrogels had pore sizes of ca. 18 μm, while 4L8/4D8 hydrogels had pore sizes of ca. 10 μm, indicating that CTA blocks had a significant influence on the pore size of the lyophilized hydrogels. Moreover, the images of 4L8/4D8 and 4L′8/4D′8 hydrogels showed different interconnected network structures. The intermolecular interactions of the copolymers and the rigidity of the CTA blocks resulted in formation of a more regular structure. The thickness of the 4L′8/4D′8 hydrogel walls increased compared with 4L8/4D8 hydrogel, leading to the assumption that the rigid and hydrophobic CTA blocks in the aqueous solution should be spontaneously encapsulated into the interior by the flexible PLA-PEG-PLA chain. In Vitro Cytocompatibility and Cell Proliferation Assay by Electrical Stimulation. In the research on the electroactive hydrogels, the ultimate goal is to utilize the signaling molecule (electroactive tetraaniline) of functional injectable materials and to realize their application as 3D carriers in tissue engineering and accelerating repair or regeneration with electrical stimuli. Because of the appropriate flowability and storage modulus values, 4L′11/4D′10 (20 wt %) hydrogels were evaluated in vitro and in vivo. In the biocompatibility study of the hydrogels, mouse fibroblast L929 cells were incorporated in the 4L′11/4D′10 hydrogels, and the cell-encapsulated hydrogel matrices were incubated in DMEM up to a week. Cell viability was determined by Live−Dead staining. The viable cells (stained green) and dead cells (stained red) were observed as shown in Figure 7a. Staining of cultures 1, 3, and 7 days after encapsulation indicated that the cell viability was preserved and not significantly influenced in the electroactive hydrogel

Figure 7. (a) Live (green)/Dead (red) staining images of L929 cells entrapped in gels for different times. (b) The cell proliferation activity of 4L11/4D10, 4L′11/4D′10 and 4L′11/4D′10 doped with HCl hydrogels with (without) electrical stimuli: b1: L929 cells, b2: MC3T3-E1 cells, and b3: H9c2 cells.

scaffolds. Initial death of cells was observed on the first day, and it was significantly reduced after 3 d. In addition, the proliferation of cells was obvious up to 7 days. These images demonstrated the good cytocompatibility of the hydrogel scaffolds. It has been demonstrated that the electroactive polymers can promote the proliferation of various cells such as fibroblasts, neurons, cardiomyocytes, and osteoblasts upon stimulation by electrical signals, as mentioned previously. The proliferation activity was quantitatively determined to measure the total population of cells growing into the hydrogels of 4L11/4D10, 4L′11/4D′10, and 4L′11/4D′10 doped with HCl with (or without) electrical stimuli as shown in Figure 7b, when various cells were cultured for 3, 5, and 7 days. During the cell proliferation process, on day 3, the proliferation rates of various cells in all hydrogels showed no obvious differences without electrical stimuli. A general increase in the cell proliferation rate was observed after treatment with electrical stimuli, when compared to untreated controls. The proliferation rates of H9c2 cells and MC3T3-E1 cells in all hydrogels after treatment with electrical stimuli were higher than that of L929 cells, indicating that the two types of cells in electroactive hydrogels were more sensitive to electrical signal. Moreover, cell proliferation rate at 5 d had more significant difference compared with that at 3 d when the cells were seeded in all electroactive hydrogels, indicating that the charges from CTA blocks could improve cell proliferation. Cell proliferation rate of the hydrogel 4L′11/4D′10 doped with HCl with electrical stimuli in various cells was highest as expected, especially H9c2 cells compared with that of other treated and untreated groups up to 20% (P < 0.05) at 7 d stimulation period. We postulated that our hydrogels are able to display their electroactivity and induce the chemical and energy exchange between cells and its surroundings with electrical stimuli, which benefit cell growth. These results indicate that these electroactive hydrogels indeed G

dx.doi.org/10.1021/bm4002766 | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

accelerated the proliferation of fibroblasts, osteoblasts and cardiomyocytes with electrical stimuli, which may be desirable and useful for excitable tissue engineering. In Vivo Gel Formation and Biocompatibility. In vivo gel formation and biocompatibility were evaluated in SD rats. The polymer solutions of 4L′11/4D′10 (20 wt %, 0.3 mL) were injected into the subcutaneous layer of rats. At 1 day and 3 weeks postinjection, the hydrogels were observed in situ in the subcutaneous layer (Figure 8a). After 3 weeks, the gels almost

blocks and was dependent on time, temperature, and the length of PLA blocks. Moreover, the introduction of CTA decreased critical gelation concentration, although the storage modulus of L′/D′ decreased compared with that of L/D. Due to the interactions of copolymers and the rigidity of CTA blocks, the hydrogels formed more regular structure. The biocompatibilities of the IEHs in vitro and in vivo were studied, which demonstrated that they were noncytotoxic and might be suitable for in vivo applications. Moreover, these hydrogels could accelerate the proliferation of fibroblasts, cardiomyocytes, and preosteoblasts with pulsed electrical stimulation. Future work will focus on evaluating the in vivo therapeutic effect through additional animal tests.

■

ASSOCIATED CONTENT

S Supporting Information *

The synthetic procedure of copolymer (Scheme S1), 1H NMR spectra of copolymer (Figure S1), characterizations of the residual copolymers (Table S1), the conductivity of the samples (Figure S2), the relation between storage modulus of sample and temperature as well as concentration (Figure S3), SEM of lyophilized hydrogels (Figure S4), and in vivo degradation of hydrogels at different time (Figure S5). This material is available free of charge via the Internet at http://pubs.acs.org.

■

Figure 8. (a) In vivo hydrogels formation in the subcutaneous tissue at different intervals. (b) Images of H&E stained surrounding tissues at indicated days for examination of the inflammation reaction.

AUTHOR INFORMATION

Corresponding Author

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

disappeared at the injection sites. In vivo degradation of hydrogels was quantitatively evaluated as shown in Figure S5. Moreover, the responses after subcutaneous injection were examined by H&E staining of the surrounding tissues including epidermis, the connective tissue, and muscle layers at different intervals, and some images are shown in Figure 8b. Considerable neutrophils were observed, indicating acute inflammatory reaction in the initial days after injection. With the size of the gel decreasing, a noticeable reduction in the number of inflammatory cells was observed, and only a few inflammatory cells were seen after 2 weeks, suggesting that the acute inflammatory reaction was gradually replaced by a mild chronic inflammation. Notably, after 4 weeks postinjection, the gels completely disappeared, and the histology of the tissue sample surrounding the injection site was almost restored to the normal tissue. Although acute inflammatory reaction was found surrounding the injection site, it was reduced significantly and eventually eliminated accompanying the degradation of the gels. This data is encouraging as acute injection site inflammation is a manageable side effect and easily treatable. Hence, we concluded that the electroactive hydrogels exhibited acceptable biocompatibility in vivo, which may be suitable for in vivo applications.

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (Project 51233004, 50973109, 51203152, and 51021003) and the Ministry of Science and Technology of China (International Cooperation and Communication Program 2011DFR51090).

■

REFERENCES

(1) Hwang, S. J.; Song, Y. M.; Cho, T. H.; Kim, R. Y.; Lee, T. H.; Kim, S. J.; Seo, Y. K.; Kim, I. S. Tissue Eng., Part A 2012, 18, 432−445. (2) Kaji, H.; Ishibashi, T.; Nagamine, K.; Kanzaki, M.; Nishizawa, M. Biomaterials 2010, 31, 6981−6986. (3) Park, J. S.; Yang, H. N.; Woo, D. G.; Jeon, S. Y.; Do, H. J.; Huh, S. H.; Kim, N. H.; Kim, J. H.; Park, K. H. Biomaterials 2012, 33, 7300− 7308. (4) Langelaan, M. L.; Boonen, K. J.; Rosaria-Chak, K. Y.; van der Schaft, D. W.; Post, M. J.; Baaijens, F. P. J. Tissue Eng. Regener. Med. 2011, 5, 529−539. (5) Runge, M. B.; Dadsetan, M.; Baltrusaitis, J.; Ruesink, T.; Lu, L.; Windebank, A. J.; Yaszemski, M. J. Biomacromolecules 2010, 11, 2845− 2853. (6) Shao, S.; Zhou, S.; Li, L.; Li, J.; Luo, C.; Wang, J.; Li, X.; Weng, J. Biomaterials 2011, 32, 2821−2833. (7) Li, M. Y.; Guo, Y.; Wei, Y.; MacDiarmid, A. G.; Lelkes, P. I. Biomaterials 2006, 27, 2705−2715. (8) Gomez, N.; Lee, J. Y.; Nickels, J. D.; Schmidt, C. E. Adv. Funct. Mater. 2007, 17, 1645−1653. (9) Guimard, N. K.; Gomez, N.; Schmidt, C. E. Prog. Polym. Sci. 2007, 32, 876−921. (10) Guimard, N. K.; Sessler, J. L.; Schmidt, C. E. Macromolecules 2009, 42, 502−511.

■

CONCLUSIONS CTA-PLA-PEG-PLA-CTA copolymers were prepared by condensation between the CTA and the end hydroxyl groups of PLA-PEG-PLA triblock copolymers. Both UV−vis spectra and CV results showed that the complexes were electroactive. Electroactive hydrogels were formed by mixing aqueous solutions of L′ and D′ block copolymers. The gelation was induced by stereocomplexation between PLLA and PDLA H

dx.doi.org/10.1021/bm4002766 | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

(11) Rivers, T. J.; Hudson, T. W.; Schmidt, C. E. Adv. Funct. Mater. 2002, 12, 33−37. (12) Zhang, W. J.; Feng, J.; MacDiarmid, A. G.; Epstein, A. J. Synth. Met. 1997, 84, 119−120. (13) Rebourt, E.; Joule, J. A.; Monkman, A. P. Synth. Met. 1997, 84, 65−66. (14) Huang, L. H.; Hu, J.; Lang, L.; Wang, X.; Zhang, P. B.; Jing, X. B.; Wang, X. H.; Chen, X. S.; Lelkes, P. I.; MacDiarmid, A. G.; Wei, Y. Biomaterials 2007, 28, 1741−1751. (15) Guo, Y.; Li, M. Y.; Mylonakis, A.; Han, J. J.; MacDiarmid, A. G.; Chen, X. S.; Lelkes, P. I.; Wei, Y. Biomacromolecules 2007, 8, 3025− 3034. (16) Huang, L. H.; Zhuang, X. L.; Hu, J.; Lang, L.; Zhang, P. B.; Wang, Y.; Chen, X. S.; Wei, Y.; Jing, X. B. Biomacromolecules 2008, 9, 850−858. (17) Hu, J.; Huang, L. H.; Zhuang, X. L.; Zhang, P. B.; Lang, L.; Chen, X. S.; Wei, Y.; Jing, X. B. Biomacromolecules 2008, 9, 2637− 2644. (18) Liu, Y. D.; Hu, J.; Zhuang, X. L.; Zhang, P. B.; Wei, Y.; Wang, X. H.; Chen, X. S. Macromol. Biosci. 2012, 12, 241−250. (19) Cui, H. T.; Liu, Y. D.; Deng, M. X.; Pang, X.; Zhang, P. B.; Wang, X. H.; Chen, X. S.; Wei, Y. Biomacromolecules 2012, 13, 2881− 2889. (20) Lang, L.; Zhuang, X. L.; Liu, Y. D.; Chen, X. S.; Wei, Y. Acta Polym. Sin. 2010, 956−960. (21) Guo, B. L.; Finne-Wistrand, A.; Albertsson, A. C. Chem. Mater. 2011, 23, 1254−1262. (22) Guo, B. L.; Finne-Wistrand, A.; Albertsson, A. C. Biomacromolecules 2011, 12, 2601−2609. (23) Mano, J. F. Adv. Eng. Mater. 2008, 10, 515−527. (24) Drury, J. L.; Mooney, D. J. Biomaterials 2003, 24, 4337−4351. (25) Tsang, V. L.; Bhatia, S. N. Adv. Drug Delivery Rev. 2004, 56, 1635−1647. (26) Van Tomme, S. R.; Storm, G.; Hennink, W. E. Int. J. Pharm. 2008, 355, 1−18. (27) Nguyen, M. K.; Lee, D. S. Macromol. Biosci. 2010, 10, 563−579. (28) Li, Y.; Rodrigues, J.; Tomas, H. Chem. Soc. Rev. 2012, 41, 2193− 2221. (29) Yu, L.; Ding, J. D. Chem. Soc. Rev. 2008, 37, 1473−1481. (30) Jun, Y. J.; Park, K. M.; Joung, Y. K.; Park, K. D.; Lee, S. J. Macromol. Res. 2008, 16, 704−710. (31) De Jong, S. J.; De Smedt, S. C.; Wahls, M. W. C.; Demeester, J.; Kettenes-van den Bosch, J. J.; Hennink, W. E. Macromolecules 2000, 33, 3680−3686. (32) Bos, G. W.; Hennink, W. E.; Brouwer, L. A.; Den Otter, W.; Veldhuis, T. F. J.; Van Nostrum, C. F.; Van Luyn, M. J. A. Biomaterials 2005, 26, 3901−3909. (33) Hiemstra, C.; Zhong, Z. Y.; Li, L. B.; Dijkstra, P. J.; Feijen, J. Biomacromolecules 2006, 7, 2790−2795. (34) Hiemstra, C.; Zhou, W.; Zhong, Z. Y.; Wouters, M.; Feijen, J. J. Am. Chem. Soc. 2007, 129, 9918−9926. (35) Abebe, D. G.; Fujiwara, T. Biomacromolecules 2012, 13, 1828− 1836. (36) Li, S. M.; Vert, M. Macromolecules 2003, 36, 8008−8014. (37) Zhang, Y.; Wu, X. H.; Han, Y. R.; Mo, F.; Duan, Y. R.; Li, S. M. Int. J. Pharm. 2010, 386, 15−22. (38) Brandl, F.; Sommer, F.; Goepferich, A. Biomaterials 2007, 28, 134−146. (39) Guiseppi-Elie, A. Biomaterials 2010, 31, 2701−2716. (40) Brizzolara, D.; Cantow, H. J.; Diederichs, K.; Keller, E.; Domb, A. J. Macromolecules 1996, 29, 191−197. (41) Zhang, J. M.; Sato, H.; Tsuji, H.; Noda, I.; Ozaki, Y. Macromolecules 2005, 38, 1822−1828. (42) Zhang, J. M.; Tsuji, H.; Noda, I.; Ozaki, Y. J. Phys. Chem. B 2004, 108, 11514−11520. (43) Sun, J. R.; Zhuang, X. L.; Chen, X. S.; Jing, X. B. Chem. J. Chin. Univ. (Chin. Ed.) 2005, 26, 956−959. (44) Shao, J.; Sun, J. R.; Bian, X. C.; Cui, Y.; Li, G.; Chen, X. S. J. Phys. Chem. B 2012, 116, 9983−9991.

(45) Stockton, W. B.; Rubner, M. F. Macromolecules 1997, 30, 2717− 2725. (46) Udeh, C. U.; Fey, N.; Faul, C. F. J. J. Mater.Chem. 2011, 21, 18137−18153. (47) Wang, Y.; Tran, H. D.; Liao, L.; Duan, X.; Kaner, R. B. J. Am. Chem. Soc. 2010, 132, 10365−10373. (48) Huang, L. H.; Hu, J.; Lang, L.; Chen, X. S.; Wei, Y.; Jing, X. B. Macromol. Rapid Commun. 2007, 28, 1559−1566. (49) Huang, L. H.; Hu, J.; Lang, L.; Zhuang, X. L.; Chen, X. S.; Wei, Y.; Jing, X. B. Macromol. Rapid Commun. 2008, 29, 1242−1247. (50) Xiong, W.; Wang, H. F.; Han, Y. C. Macromol. Rapid Commun. 2010, 31, 1886−1891. (51) Li, Y.; He, W.; Feng, J.; Jing, X. L. Colloid Polym. Sci. 2012, 290, 817−828.

I

dx.doi.org/10.1021/bm4002766 | Biomacromolecules XXXX, XXX, XXX−XXX