Article pubs.acs.org/Biomac
In Vitro Study of Electroactive Tetraaniline-Containing Thermosensitive Hydrogels for Cardiac Tissue Engineering Haitao Cui,†,‡ Yadong Liu,† Yilong Cheng,†,‡ Zhe Zhang,† Peibiao Zhang,† Xuesi Chen,*,† and Yen Wei*,§ †
Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China § Department of Chemistry, Tsinghua University, Beijing 100084, People’s Republic of China ABSTRACT: Injectable hydrogels made of degradable biomaterials can function as both physical support and cell scaffold in preventing infarct expansion and promoting cardiac repair in myocardial infarction therapy. Here, we report in situ hydrogels consisting of thermosensitive PolyNIPAM-based copolymers and electroactive tetraaniline (TA). Studies showed that the addition of 2-methylene-1,3-dioxepane (MDO) provided the PolyNIPAM-based gel with biodegradability, and the introduction of tetraaniline endowed these copolymers with desirable electrical properties and antioxidant activities. The encapsulated H9c2 cells (rat cardiac myoblast) remained highly viable in the gel matrices. In vivo gel formation and histological analyses were performed in rats by subcutaneous injection and excellent biocompatibility was observed. Furthermore, the proliferation and intracellular calcium transients of H9c2 cells were also studied with (and without) electrical stimuli. Both in vitro and in vivo results demonstrated that electroactive hydrogel may be used as a promising injectable biomaterial for cardiac tissue engineering.
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INTRODUCTION Myocardial infarction (MI) is among the major causes of morbidity and mortality in the developed countries and is now becoming a serious concern even in the developing countries.1,2 MI sets off a series of complicated processes, including cell death, scar formation, and ventricular dysfunction that alter the overall cellular, structural, and mechanical properties of the heart.2,3 Adult cardiac muscle is thought to lack the ability to regenerate after injury, and death of cardiomyocytes involves activation of an irreversible cascade of events leading to heart failure.2,4 Various drugs and surgical interventions for patients with heart failure have been developed. However, current drug therapies can increase life expectancies by only a few years and other conventional treatments are limited due to the inadequate supply of donor hearts or inability to restore cardiac function.1,5 Based on the understanding of the pathogenesis of MI and the need to improve traditional approaches for treatment, especially for myocardial ischemia-reperfusion injury, tissue engineering approaches with cellular therapy and biomaterials have been explored to reduce ventricular remodeling and to restore cardiac functions.3,6,7 With the recent development of new technologies, intelligent biomaterials combining drug delivery and biomimetic support for the cells allow the modulation of their immediate microenvironment to favor cell engraftment.8−12 Injectable hydrogels that possess sitespecificity, effective mass transport, tunable mechanical proper© 2014 American Chemical Society
ties, and biocompatible gelation process with minimal invasion have played a critical role in treating cardiac failure.5,13,14 Thermosensitive hydrogels are especially attractive as injectable biomaterials owing to their ability to undergo spontaneous gelation at body temperature, lack of any requirement for extra chemical treatment.13,15,16 Previous studies have demonstrated that mesenchymal stem cells (MSCs) or cardiosphere-derived cells could differentiate into cardiomyocytes when encapsulated in PNIPAM-based copolymer hydrogels that mimics the modulus of native heart tissue.4,17 Cardiac muscle is an electroactive tissue capable of transferring electrical signals and allowing the heart to beat.7,18 MI is often associated with abnormalities in electrical function due to a massive loss of functioning cardiomyocytes.7,19 Therefore, orderly coupling between electrical pacing signals and macroscopic contractions is crucial for the heart development and function.20 Electrical stimuli induce hyperpolarization and depolarization of the cell, such that the cells aligning with the electrical field lines are likely to generate action potentials, contract, and couple with other cells.7 Many studies have demonstrated that electrical stimuli can promote Received: August 26, 2013 Revised: February 19, 2014 Published: March 5, 2014 1115
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NIPAM, MAA, and mPEGMA were purified before use. All other chemicals were of analytical grade or higher. Synthesis of Degradable PolyNIPAM-Based Copolymer (PN). The copolymerization of NIPAM, MAA, mPEGMA, and MDO were performed in DMF using 2 mol % of the initiator AIBN under a nitrogen atmosphere. The system was cooled in liquid nitrogen and air was removed by three freeze−thaw cycles. The mixture was stirred at 60 °C for 72 h. The resulting copolymer was dissolved in methanol, precipitated in cold n-hexane, and then dried under vacuum for 48 h. Yield of purified product was 55%. Synthesis of Electroactive Grafted Polymer (PN-TA). Tetraaniline (TA) was synthesized according to a similar procedure reported in the literature.34 PolyNIPAM-based material, NHS, and EDC·HCl were dissolved in DMF. After the mixture was stirred at room temperature for 24 h, TA in DMF solution was added into the above mixture and the stirring was continued for 24 h at 50 °C. After the reaction, the solution was cooled to room temperature and poured into cool nhexane to precipitate the product. The crude product was then dissolved in methanol, filtered and precipitated in n-hexane. Such a dissolution−precipitation process was repeated three times to purify the product. The final precipitate was collected by filtration and dried under vacuum at room temperature for 24 h. The yield of the product was 70%. Characterization. 1H NMR spectra were recorded on a Bruker AV 400 MHz spectrometer. FT-IR spectra of samples 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 CHI660A 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) was carried out with a Waters GPC instrument and CHCl3 was used as an eluent. The molecular weights were calibrated with polystyrene standards. In Vitro Thermosensitive Hydrogel Formation and Characterization. The UV−vis spectra were used to analyze the change in transmittance associated with phase transition of the samples from liquid to gel. The samples were dissolved in distilled water to a concentration of 1 mg mL−1. Samples were subjected to a heating rate of 0.5 °C min−1 from 25 to 70 °C. The vial inverting approach was employed to determine whether the sol−gel transition occurred at body temperature. The vials with 0.5 mL 20 wt % copolymer in phosphate buffer saline (PBS) solutions (pH = 7.4) 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. Rheology experiments were performed on a MCR 302 Rheometer (Anton Paar). The copolymer solution was placed between cone plates of 25 mm diameter and a gap of 103 μm. The data were collected under a controlled strain γ of 1% and a frequency of 1 Hz. The heating rate was 0.5 °C min−1. G′ is an elastic component of the complex modulus and is a 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. The copolymer solution (20 wt %, 0.5 mL) was injected into a test tube and incubated in a shaking bath at 37 °C. After 10 min, 3 mL of PBS solution (pH = 7.4) was added to the formed gel and the tube was shaken at 80 stroke min−1. At a predetermined time, sample was taken out of the shaking bath. Following buffer removal, the remaining gel was freeze-dried until constant weight. The sample weight was measured and the weight containing equal volume of a PBS solution was used as a blank control. In Vitro Cell Encapsulation and Viability. Before encapsulation, the hydrogel solution (20 wt %) was sterilized under UV light for 30 min at 4 °C. H9c2 cell (rat cardiac myoblast) suspension with a density of 1 × 105 per well in 24-well plates was then added into hydrogel solution. After thorough mixing, the mixture was transferred to a 37 °C incubator for gelation. Then 500 μL of DMEM (Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% fetal calf serum (Gibco) and 100 U mL−1 penicillin−streptomycin (SIGMA))
cardiomyogenesis of embryonic stem cells and enhance cardiomyocyte phenotype.10,21−23 To re-establish the contractile function of an infarcted heart, electroactive materials such as carbon nanotubes, polypyrrole (PPy), and polyaniline (PANI) have been widely studied and applied in cardiac tissue engineering.24−27 Previous studies have investigated electroactive materials with or without electrical stimuli for evaluate their abilities to support the attachment and proliferation of cardiomyocytes and promote MSCs differentiation toward a cardioprogenitor phenotype.19,24,28−30 Despite the application of various approaches for incorporating electroactive segments within in vitro systems,31−35 a straightforward strategy with a fabricated material which has biocompatibility, biodegradability and sufficient cell loading still remains a challenge for tissue engineering application. The incorporation of electroactive segments into thermosensitive hydrogels for cardiac tissue engineering has rarely been reported. With this goal in mind, we designed and synthesized a novel electroactive, biodegradable, and thermosensitive copolymer P(NIPAM-mPEGMA-MDO-MATA) (PN-TA), and here we report our findings. At above human physiological temperature, the copolymer solution immediately formed a hydrogel. Tetraaniline as an electroactive material was introduced to the thermosensitive system, because it possessed a well-defined structure, good biocompatibility and excellent electroactivity similar to that of polyaniline.35 Moreover, its use significantly reduced some of the undesirable features of the conducting polymers such as poor solubility, poor processability and clearance in vivo.36 In this study, we sought to investigate the use of electroactive hydrogel as an injectable biomaterial for cardiac repair. Both in vitro and in vivo biocompatibility of hydrogels was evaluated. The proliferation and differentiation of cardiomyocytes in the gels were also investigated upon stimulation with pulsed electrical signals. Furthermore, considerable data have indicated that reactive oxygen species (ROS) produced either by the myocardium itself or by infiltrating inflammatory cells led to cellular damage through a number of pathways, including direct damage to the membranes and proteins or indirect damage through the activation of pro-apoptotic pathways in cardiovascular diseases.37 Antioxidant therapies in clinical trials have not shown optimal results on cardiovascular events, probably due to the fact that no specific antioxidants can selectively scavenge pathological (instead of physiological) ROS.38,39 Since polyaniline and their oligomers were reported to be antioxidants for biomedical applications,40,41 we postulated that synthesized antioxidant polymer could reduce free radical-mediated oxidative cardiac damage through removing O2•− in the process of ROS extracellular transmission for targeted sites, while not interfering with the beneficial function of intracellular ROS in physiological states.22,42
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EXPERIMENTAL SECTION
Materials. N-Isopropylacrylamide (NIPAM), methacrylic acid (MAA), methoxy(polyethylene glycol)methacrylate (mPEGMA; Mn = 454 g mol−1), 2,2-azobisisobutyronitrile (AIBN), N-phenyl-1,4phenylenediamine, ammonium persulfate (APS), 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC·HCl), and 2,2diphenyl-1-picrylhydrazyl (DPPH) were purchased from Aldrich. Pluronic F-127 (Sigma) and N-hydroxysuccinimide (NHS; Fluka) were used as received. 2-Methylene-1,3-dioxepane (MDO) was synthesized as previously described.43 Methanol, n-hexane, and N,Ndimethylformamide (DMF) were dried and distilled prior to use. 1116
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Scheme 1. Preparation of an Electroactive Thermosensitive Hydrogel
was added and changed every other day. After 1, 3, and 7 days of culture, a qualitative viability assay was performed by Live/Dead staining. At predesigned days postencapsulation, cell/hydrogel complex film was washed in PBS 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 “NIH Image J” software. 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 %, 0.2 mL) was subcutaneously injected into rats by a syringe with a 23-gauge needle. At the designated time intervals, the animals (n = 3) were sacrificed, and then the photographs of in situ gel formation were taken. The surrounding tissues of gel were surgically removed and histologically processed using hematoxylineosin (HE) stains for the examination of inflammatory responses of synthesized copolymers in rats. Antioxidant Activity Evaluation and Cell Viability Assay. The antioxidant assays (radical scavenging activity) of the material was measured using the stable DPPH free radical as a model. A total of 1.0 mg PN-TA was applied to 3.0 mL of 100 μM DPPH solution in methanol. The reaction mixture was vortexed for 30 s and its absorbance was measured at 516 nm. In order to study the time dependence of the antioxidant activity, the maximum concentration of each samples was applied to the 3 mL DPPH solution in cuvette and the spectra were recorded with in the time limit of t = 0 min to 2 h. The cell responses to radical scavenging were assessed by the MTT assay. The copolymer was dissolved in DMEM with the concentration of 1, 0.5, 0.25, 0.125, 0.0625, 0.0313, and 0.0156 mg mL−1, respectively. H9c2 cells were seeded in 96-well plates at a density of 7 × 103 cells per well. After 24 h incubation, various concentrations of copolymer solution and DPPH solution (100 μM) were added to the wells. After incubating for 24 h, MTT stock solution in PBS was added into each well and then incubated for 4 h. The medium was removed and DMSO was added to dissolve the formazan crystals. The optical
density (OD) was measured at 490 nm by a microplate reader (BioRad 680). Electrical Stimuli and Biological Assay. H9c2 cells were used to investigate the influence of electroactive hydrogel on the proliferation and differentiation stimulated by pulsed electrical signals. The solutions of F127, NP, NP-TA and NP-TA doped with HCl were sterilized by exposure to UV radiation for 1 h. Cells were seeded into various gels at a density of 1 × 105 cells per well in 24-well plates and cultured at 37 °C. The electrical stimuli were applied by a 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, 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). Calcium transient is an essential event in excitation-contraction (EC) coupling and the intracellular calcium cycling has been reported to be dependent upon cardiomyocyte maturation.29,44 We investigated the calcium transients in H9c2 cells grown on the various gels and TCPs with or without electrical stimuli. H9c2 cells were seeded onto the surface of various materials at a density of 2 × 104 cells per well in 24-well plates and cultured at 37 °C. After the predetermined period, the medium was removed and washed three times. Fluo-4/AM substrate (Fanbo biochemical) was added and cells were incubated for 30 min. Images of calcium signal at 3 days were obtained under an inverted fluorescent microscope (TE2000U, NIKON). The intracellular calcium concentration ([Ca2+ (i)]) and [Ca2+ (i)] changes of H9c2 cells were monitored and assessed quantitatively at 3 and 7 d. Fluorescence intensity was measured on multifunction microplate scanner using an excitation wavelength of 494 nm and an emission wavelength of 516 nm. 1117
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Table 1. Characterizations of PN and PN-TA Copolymers
a
sample
NIPAM (mol)
MAA (mol)
mPEGMA (mol)
MDO (mol)
PN1 PN2 PN3 PN-TA1 PN-TA2 PN-TA3
0.85 0.9 0.9 0.85 0.9 0.9
0.06 0.02 0.04 0.06 0.02 0.04
0.09 0.08 0.06 0.09 0.08 0.06
0.2 0.2 0.2 0.2 0.2 0.2
TA feeding (mol)
0.06 0.02 0.04
TA yieldinga (mol)
0.048 0.017 0.032
Mn (g mol−1)/PDIb 3.6 4.4 4.2 3.8 4.6 4.3
× × × × × ×
104/2.5 104/2.4 104/2.7 104/2.7 104/2.5 104/2.9
Determined by UV−vis spectra. bDetermined by GPC.
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.
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RESULTS AND DISCUSSION Synthesis of Electroactive Thermosensitive Material. The synthetic procedure of the electroactive thermosensitive copolymer (PN-TA) is shown in Scheme 1. Copolymerization of NIPAM, MAA, and mPEGMA with MDO contributed ester linkages to the polymer backbone chains through incorporation of caprolactone (CL) units and therefore led to the formation of a degradable polyNIPAM backbone. In order to obtain different low critical solution temperatures (LCSTs) and mechanical properties of copolymers, feed ratios of the monomers and TA were varied. The compositions and properties of PN and PN-TA copolymers 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. Overall, higher contents of NIPAM as mainly crosslinking points are required; combining hydrophilic mPEGMA with hydrophobic MAA-TA is necessary to adjust the assembly of copolymers and phase transition temperature. The 1H NMR and FT-IR spectra supported the proposed structure of the copolymer (Figure 1). The peak at 0.95 and 3.55 ppm were attributed to methyl protons of PNIPAM units and methylene protons of mPEGMA units, respectively. The peaks at 1.65− 1.25 ppm and 3.90 confirmed the presence of the CL units in polyNIPAM backbone. The protons on the TA blocks appeared at 7.80−7.20 ppm. We first introduced carboxyl group into copolymers, and the TA segments were then chemically linked to the copolymer by the coupling reaction. Since TA is a free radical inhibitor, using free radical polymerization to directly incorporate TA into polymers must adopt a rigorous and complex approach with the protection and deprotection of the −NH− groups in the polymer, which is not desirable for the synthesis of biomaterials here. Electrochemical Characterization of Copolymer Solution. It is well-known that aniline oligomers such as polyaniline have different oxidation states (that is, leucoemeraldine state (LM), emeraldine base state (EMB or EM), and pernigraniline (PN)) when they are treated by different voltages or oxidizing and reducing agents. The electrochemistry of PN-TA was investigated by UV−vis and CV. Figure 2a shows the UV−vis spectra of the PN-TA2 copolymer in aqueous solution at different oxidative states. The LM sample showed only one peak at 327 nm, which was associated with the π−π* transition of the benzene ring. Further oxidation caused a blue-shift of the peak at 327 nm and the appearance of a new peak at 570 nm was attributed to the excitonic transition
Figure 1. The 1H NMR and FT-IR spectra confirmed the structure of copolymer.
of EMB from benzene ring to quinoid ring. When EMB PN-TA copolymer was doped with 1 M HCl aqueous solution, the peaks at 425 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 copolymer. The electroactivity of PN-TA2 copolymer in the 1 M HCl electrolyte solution was also observed in CV, as shown in Figure 2b. After the removal of oxygen in the solution, a pair of reversible redox peaks was observed, which corresponded to the transitions between LM state and EMB state with the mean peak potential E1/2 of 0.35 V. From the UV−vis and CV results it could be concluded that the tetraaniline segments in the copolymer have kept their electroactivity in aqueous solution. Phase Transition and Gelation Property of Copolymer Solution. The temperature-induced phase transition behavior is crucial for in situ thermosensitive hydrogel. We used the UV−vis spectra to monitor the copolymer critical solution 1118
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Figure 2. (a) UV−vis spectra of PN-TA2 in LM, EM, and EMS states (doped with 1 M HCl aqueous solution). (b) Cyclic voltamogram of the PN-TA2 copolymer in 1.0 M HCl electrolyte solution.
Figure 3. Phase transition temperatures curves of (a) PN and (b) PNTA copolymer solutions.
temperature in water with varying proportions of hydrophilic and hydrophobic blocks (Figure 3). The results showed that the phase transition temperatures directly corresponded to the amount of hydrophobic blocks in the copolymers, as marked by the sudden increase in the particle size caused by hydrophobic aggregation. This downshift of LCST from 39 to 31 °C for PNTA copolymers was indeed expected as the proportion of hydrophobic fragment TA increases, whereas higher LCST was observed for PN copolymers. All samples underwent rapid phase transition at their LCST, as indicated by the sudden decrease in transmittance, but only the LCST of PN3 was closer to human physiological temperature for nongrafted copolymer. Direct gelation test at 37 °C was carried out for different samples. The gelation behavior was observed and was found to be dependent on temperature and concentration. At physiological temperature, gelation time was reached in
