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Biointerface by Cell Growth on Graphene Oxide Doped Bacterial Cellulose/Poly(3,4-ethylenedioxythiophene) Nanofibers Chuntao Chen,† Ting Zhang,§ Qi Zhang,*,‡,§ Xiao Chen,† Chunlin Zhu,† Yunhua Xu,†,∥ Jiazhi Yang,† Jian Liu,§ and Dongping Sun*,† †

Institute of Chemicobiology and Functional Materials, School of Chemical Engineering, Nanjing University of Science and Technology, 200 Xiao Ling Wei Street, Nanjing, Jiangsu Province, China ‡ School of Radiation Medicine and Protection and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Medical College of Soochow University, 199 Ren Ai Road, Suzhou Industrial Park, Suzhou, Jiangsu Province, China § Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University, 199 Ren Ai Road, Suzhou Industrial Park, Suzhou, Jiangsu Province, China ∥ Lianyungang Normal College, Lianyungang, Jiangsu, China ABSTRACT: Highly biocompatible advanced materials with excellent electroactivity are increasingly meaningful to biointerfaces and the development of biomedicine. Herein, bacterial cellulose/poly(3,4-ethylene dioxythiophene)/graphene oxide (BC/PEDOT/GO) composite nanofibers were synthesized through the in situ interfacial polymerization of PEDOT with the doping of GO. The abundant free carboxyl and hydroxy groups offer the BC/PEDOT/GO film active functional groups for surface modification. We demonstrate the use of this composite nanofiber for the electrical stimulation of PC12 neural cells as this resultant nanofiber scaffold could closely mimic the structure of the native extracellular matrix (ECM) with a promoting cell orientation and differentiation after electrical stimulation of PC12 cells. It is expected that this biocompatible BC/PEDOT/GO material will find potential applications in biological and regenerative medicine. KEYWORDS: bacterial cellulose nanofibers, graphene oxide, poly(3,4-ethylenedioxythiophene), biointerface, electrical stimulus

1. INTRODUCTION

There is a significant impact on the cell/substrate interaction and biomacromolecules adsorption on topographic structure of the substrate surface.4 Moreover, there are three nature unique features at biological systems in this aspect from the biomimetic point of view:8 (1) to utilize the multilevel structural effect in substrate, (2) to use the hypersensitized weak interactions of biomolecule interactions, and (3) the special chiral recognition mechanism exist in biological systems. These effects bring much enlightenment for the design of novel biointerface materials with distinctive functions. All these derive three important research directions: structural biointerface materials,9 smart biointerface materials,10 and chiral biointerface materials.11 With the single-atom thickness and the micrometer scale lateral dimension, the new two-dimensional (2D) carbon nanomaterial graphene has drawn tremendous research attention recently.12,13 Thanks to its prominent advantages

The interface between cell and material is one of the most important considerations in designing a high-efficiency tissue engineering scaffold, as cellular microenvironment provides a wide range of signals that can guide and direct cell function.1 These include biophysical and biochemical cues that exist in natural extracellular matrices (ECM) and can be implemented into tissue engineering scaffolds and biomedical devices to improve the function.2−4 With the development of modern life science, an increasing number of artificial materials and devices have been used in biological applications and in human bodies.5 For many applications, there are two essential interactions directly making a difference at the interface of living organisms and materials.2 One is between cells and materials interface,6 and the other is between biomolecules and materials.7 Therefore, controlling the interfacial properties of materials and modulating the interfacial behaviors between biomolecules and cells are becoming more and more important in developing new biomaterials and biodevices. © 2016 American Chemical Society

Received: January 29, 2016 Accepted: April 7, 2016 Published: April 7, 2016 10183

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further purification. Deionized water was used throughout the experiments. 2.2. Method. 2.2.1. Fabrication of the BC/PEDOT Film. The BC dry film was prepared after vacuum drying of BC hydrogel (about 0.5 mm in thickness) at 60 °C for 2 h, and then immersed into a EDOTFeCl3 ethanol solution (EDOT, 0.015, 0.03, 0.05 g mL−1; FeCl3, 0.05 g mL−1). During the process of ethanol evaporation, the EDOT monomer on the surface of BC nanofibers started to form a PEDOT shell. 2.2.2. Fabrication of the BC/PEDOT/GO Film. We synthesized the GO sheets by a modified Hummer method using natural graphite. A 0.5 mg mL−1 GO water solution was prepared by sonicating GO in water for 2 h (amplitude 40%, 1 s pulse, 1 s stop), and then 30 μL of the as-prepared GO solution was dropped onto the BC/PEDOT film (1.2 cm samples in diameter) by spin−spray method. The samples were kept at 4 °C for further use after drying. 2.3. Characterization. Infrared spectra were obtained using a Nicolet iS10 FTIR instrument (Thermo Fisher Scientific, Waltham, MA) with a wavenumber ranges from 500 to 4000 cm−1 by accumulating 32 scans at a resolution of 4 cm−1. Raman spectra were recorded on a Renishaw Invia Raman Microprobe using a 532 nm argon ion laser from 250 to 2000 cm−1. XPS spectra were obtained using a RBD upgraded PHI-5000C ESCA system (PerkinElmer) with Mg K radiation (h = 1253.6 eV). Microscopic images were acquired by an optical microscope (Eclipse TE 200, Nikon, Melville, NY). Scanning electron microscopy (SEM) was performed using a Zeiss electron microscope (Supra 55, Carl Zeiss). The surface morphology and average diameter of BC/PEDOT nanofibers were determined by Multimode Nanoscope Scanning Probe Microscopy System (Bruker Dimension 3100 SPM, Billerica, MA) using tapping mode operated at the room temperature. Zeta potential analyses were performed with the Malvern Zetasizer Nano ZS90. Transmission electron microscope (TEM) images was performed operated at 200 kV on a JEM-2100 (Japan) and samples were deposited onto the gold grids covered with holey carbon support films. The water contact angle was measured using a contact angle goniometer (SL 200B, Solon Technology Co., Ltd., China) at room temperature with a water drop volume of 3 μL. 2.4. Electrochemical and Mechanical Measurements. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were performed by an electrochemical workstation (CHI660e, CH Instruments, Inc.). The BC, BC/PEDOT, and BC/PEDOT/GO nanofibers were used as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum electrode as counter electrodes. Electrolyte solution was 0.1 M phosphate buffer solution (PBS; pH = 7.4). The CVs were scanned in potentials from −0.6 to 0.8 V at a scan rate of 100 mV s−1, while EIS were measured over frequency range from 0.1 to 100,000 Hz. A Microcomputer Control Electronic Universal Testing Machine (RGWT-4000−20, China) was used to do the tensile stress−strain tests. Tensile test specimens were prepared by cutting the membranes to stripes (10 mm wide and 65 mm long) according to a standard procedure described by the protocol of ASTM D-882-97. 2.5. Cells Proliferation and Cytotoxicity Assays. The asprepared composites were evaluated for biocompatibility with PC12 neural cells. The samples were immersed in of 75% ethanol−water (v/ v) solution for 30 min, and ultraviolet radiated for 30 min before cell seeding. TCPs, BC, BC/PEDOT, and BC/PEDOT/GO were examined in groups (n = 9 in repetition) using microplates for cell culture. PC12 neural cells were cultured at 37 °C with 5% CO2 in medium (DMEM, 10% v/v FBS and 1% v/v antibiotic/antimycotic solution). They were transferred into a 24-well microplate with about 1.5 × 104 cells per well. The cell samples were washed with PBS for three times before use. Then the cells were fixed with a 4% formaldehyde solution and stained with a 4′,6-diamidino-2-phenylindole (DAPI) solution. Immunostaining the cytoskeleton of PC12 neural cells with FITC-labeled anti-Actin antibody was done as the well-documented methods.33,34 The standard methyl thiazolyl tetrazolium (MTT) assay35 was used to examined the viabilities of cells cultured on different substrates. The fluorescent images were taken by a fluorescence microscope (Olympus) configured with a

including outstanding electrical conductivity, excellent mechanical strength, large contact surface area and tailorable surface properties, graphene oxide (GO) has been explored for broad potential applications in energy,14 supercapacitors,15 and sensors.10 Therefore, GO is a superior candidate for building a smart cell-material interface.16−18 It was reported that mixing graphene, especially water-soluble GO, as counterions into polymer nanofibers forming new composite materials will improve their performance further.12,19 Bacterial cellulose (BC) is composed of three-dimensional (3D) natural nanofibers produced by many species of microorganisms. It exhibits remarkable properties including high purity and crystallinity in chemical composition, a high polymerization degree (2000− 8000), excellent biocompatibility and biodegradability.20,21 Among the functionalized nanofibers based on BC, there is a unique choice to integrate electroactivity with the inherent properties of BC materials including the flexibility, large lengthdiameter ratio, and high specific surface.22 It may assist in building biocompatible scaffolds by mimicking the native extracellular matrix (ECM) and interfacing the cells better for biomedical research or tissue engineering.23−26 There are already some studies on the bioelectrodes based on graphene/graphene oxide and poly(3,4-ethylenedioxythiophene) (PEDOT). And the main contribution of this paper is the introduction of BC nanofibers. We applied the BC nanofibers as a natural nanotemplate to construct graphene/ graphene oxide and PEDOT nanofibers. As reported,27 nanofibers can be a good native material in guiding growing cells, which may assist in building biocompatible scaffolds by mimicking the native extracellular matrix (ECM) and interfacing the cells better for biomedical research or tissue engineering.8,25 In our previous work, we have successfully fabricated a 3D electroactive and flexible BC/PEDOT nanofibers with high performance, high specific surface area, good mechanical properties, excellent electroactive stability, and low cell cytotoxicity.28,29 Herein, we report a GO-doped hybrid BC/PEDOT nanofibrous scaffolds for biointerfacing. As well as reinforcing a highly elastic and flexible structure, GO-coated substrates have been demonstrated to promote a variety of stem cell lines growth and differentiation.27,30,31 On the basis of these concerns, we demonstrate the use of GO as an effective doping material in combination with BC/PEDOT for the electrical stimulation of PC12 neural cells. Its abundant free carboxyl and hydroxyl groups offer the BC/PEDOT/GO film active functional groups for surface modification. With a promoting cell orientation and differentiation after electrical stimulation of PC12 cells, we expect that this BC/PEDOT/GO material will find important biological and regenerative medicine applications.

2. MATERIALS AND METHODS 2.1. Materials. 3,4-Ethylenedioxythiophene (EDOT) (99%) (molecular weight, 142.17 g mol−1) and iron(III) chloride (99%) were purchased from Sigma-Aldrich. BC hydrogel samples were obtained through a static fermentation process at 30 °C by Acetobacter xylinum NUST4.2 (keeping in our Lab).32 The BC samples were treated with 0.1 M of sodium hydroxide solution at 80 °C for 2 h, and then brought to a neutral pH value by the washing steps with distilled water several times in order to rinse off the bacteria. Cell culture reagents RPMI-1640 was supplied by Abcam, Inc. (Cambridge, MA). Fetal bovine serum (FBS) was obtained from GIBCO. MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was obtained from Sigma. Calcein-AM/PI was obtained from Dojindo Laboratories (Kumamoto, Japan). All chemicals were used as received without 10184

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ACS Applied Materials & Interfaces Scheme 1. Schematic Illustration of Fabricating BC/PEDOT/GO Film

a

The process of in situ polymerization of EDOT occurs on the surface of BC nanofibers assisted by FeCl3, producing BC/PEDOT composite nanofibers with high electroactivity. bThe in situ polymerization of EDOT occurs on BC nanofibers. cThe process of GO combining onto the surface of BC/PEDOT. dElectrostatic binding between PEDOT and GO. eThe zeta potential of GO, PEDOT and their composite of different ratio.

Nuance CCD camera (CRi, USA). The PC12 cell samples on the substrates were fixed with 3% glutaraldehyde for 2 h, dehydrated by incremental concentrations of ethanol solution (25, 50, 70, 80, 90, 95%, and absolute ethanol for 0.5 h each, v/v), and subsequently freeze-dried in a vacuum chamber for 24 h before SEM imaging. In addition, cytotoxicity of samples were evaluated by a LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen, Carlsbad, CA). The cells cultured on TCPS in the microplate were tested as the blank control. 2.6. Electrical Stimulation of PC12 Cells. To test the electrical performance of the as-prepared BC/PEDOT/GO nanofibers film, we seeded PC12 cells on the BC/PEDOT/GO scaffold, on which a homemade plastic culture ring (10 mm O.D., 10 mm height) was mounted.31,36 An electrical lead was prepared by immobilizing a copper wire with the silver paste onto the edge of the BC/PEDOT/ GO substrate. The input electrical stimulation was applied with an electrochemical workstation (CHI660e, CH Instruments, Inc.) A platinum foil connected with cathode of electrical stimulator and saturated calomel electrode were also immersed in PBS as counter electrode and reference electrode, respectively. A series of 1−100 ms monophasic anodic pulses were administrated on PC12 cells of noncontact stimulus and a stimulus of 0.5 V cm−1 was applied on the contact stimulus. The stimulation potential was limited to lower than 0.6 V to avoid any undesired effects of Faradaic electrolyte reactions. Two electrical leads were prepared by immobilizing two copper wires with the silver paste onto the edges of the BC/PEDOT/GO substrate and a potential of 100 mV cm−1 was adapt between the two electrical leads. PC12 cells were monitored by fluorescence microscopy. Quantitative analysis of fluorescence change in calcium imaging of the PC12 cells was performed using the ImageJ software. Statistical analysis of the data was completed using the Origin software.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of BC/PEDOT/GO Nanofibers. During the EDOT polymerization procedure (Scheme 1a,b), the color of BC/PEDOT gradually turned from white to black at room temperature in the ambient atmosphere. This color change indicated progression of EDOT polymerization on the BC nanofibers. The coating procedure could be quickly terminated when the composite nanofibers were removed from the monomer solution and washed with ethanol. Standing as counterion with PEDOT, GO was combined onto the surface of BC/PEDOT by electrostatic adsorption (Scheme 1c). As illustrated in Scheme 1, the monomer EDOT was in situ polymerized to form conducting polymer chains first on the BC nanofibers surface. Then, positively charged PEDOT chains were combined, by ionic bonds, with negative groups of GO (Scheme 1d). Thus, a PEDOT/GO composite film was formed and subsequently accumulated on the surface of BC nanofibers. The zeta potential of GO and PEDOT in ethanol are −51 and +17 mV, respectively. With an increasing percentage of GO added, the zeta potential of GO/PEDOT drew near GO as a result of the enhancing electrostatic interactions between PEDOT and GO. In this composite film, BC disordered as the structural material and formed threedimensional crossover networks, while PEDOT, serving as the stable charge transfer medium, was interspersed among the interspaces of graphene nets. GO doping enhanced the electrical property of conducting polymer film. Meanwhile, the conducting polymer encapsulation prevented GO from 10185

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Figure 1. (a) SEM images of the as-prepared BC/PEDOT films with increasing EDOT concentrations of 0.015, 0.03, and 0.05 g mL−1; (b) SEM and (c) AFM images of BC, BC/PEDOT, and BC/PEDOT/GO films.

Figure 2. (a) FT-IR and (b) Raman spectra of the BC, BC/PEDOT, and as-prepared BC/PEDOT/GO film. (c) The relationship between EDOT concentration and water contact angle of BC/PEDOT films. (d) Shapes of water drops and contact angle on the glass, pure BC, BC/PEDOT, and BC/PEDOT/GO substrates. 10186

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Figure 3. Mechanical properties and electrical properties of the BC, BC/PEDOT, and BC/PEDOT/GO. (a) Stress−strain curves for tensile tests of BC, BC/PEDOT, and BC/PEDOT/GO films. (b) Cyclic voltammogram curves of BC, BC/PEDOT, and BC/PEDOT/GO films. (c) Nyquist curves of BC/PEDOT and BC/PEDOT/GO films. (d) Electrochemical impedance spectroscopy impedance curves and phase curves of BC/ PEDOT and BC/PEDOT/GO films.

cm−1; the C−S peaks at 987 cm−1, 926 and 845 cm−1; and C− O−CH2−CH2−O-C vibration peak at 1150 cm−1 the C−S peaks at 987 cm−1, 926 and 845 cm−1.37 The absorption peaks at 1642 and 1433 cm−1 were assigned to the thiophene ring, which confirms the existence of PEDOT. The peaks of BC at 1648 and 1523 cm −1 disappeared after PEDOT was incorporated, which might be due to shelter from PEDOT and GO after polymerization. Raman analysis confirmed the formation of PEDOT shell on the surface of BC nanofibers and the GO dopant. As shown in Figure 2b, the as-prepared BC/ PEDOT nanofibers exhibit a strong vibrational Raman peak at 1435 cm−1, corresponding to the symmetric stretching mode of the aromatic C−C band. Three other Raman peaks at 1513, 1367, and 1262 cm−1 are contributed to the antisymmetric Cα=Cα, the stretching deformations of Cβ−Cβ, and the Cα−Cα inter-ring stretching vibrations, respectively.38,39 For the spectra of GO, the G band displays at 1597.2 cm−1, the D band at 1358.4 cm−1, while in the BC/PEDOT/GO film, the peaks of D and G of GO are obvious, thus confirming the doping of GO nanosheets. Water contact angle (WCA) analysis provided an important strategy to evaluate surface chemical properties of biomaterials. Figure 2c,d shows the shapes of water drops and the contact angles on glass, pure BC, BC/PEDOT, and BC/ PEDOT/GO substrates. The water contact angle of pure BC is 22.8 ± 0.7°, much lower than that of glass, suggesting good hydrophilic properties. The significant decrease in the contact

dispersing to the tissue during recording or stimulation process, which greatly abated the possibility of cytotoxicity induced by carbon nanomaterial diffusion while contacting with tissue directly. The morphology of bare BC, BC/PEDOT and BC/ PEDOT/GO films were imaged by SEM (Figure 1a,b). A wellorganized core−shell structure was presented in these two samples. BC/PEDOT composite nanofibers exhibited a significantly larger average diameter than that of bare BC samples. The image data provided good evidence of successful coating of PEDOT on the surface of BC nanofibers by in situ interfacial polymerization. With the increasing of EDOT concentration, the average diameter of BC/PEDOT diameter increased, while the nanotopography of BC/PEDOT nanofibers tended to damage. So, a suitable EDOT concentration of 0.030 g mL−1 was chosen at last. From the AFM images (Figure 1c) test, we can see that the quantified roughness (Ra) of BC, BC/PEDOT and BC/PEDOT/GO are 18.189, 29.028, and 37.379, respectively. The largest specific surface area of BC/ PEDOT/GO maybe a key factor in improving the performance of the bioelectrode. 3.2. Properties Characterization. FT-IR was used to analyze the chemical structures of the as-prepared BC/ PEDOT/GO nanofibers. Figure 2a shows the characteristic peaks of molecular vibrations such as CC and C−C of thiophene at stretching vibration peaks of 1523 and 1314 cm−1, respectively; the C−O−C peaks at 1202 cm−1, 1150 and 1057 10187

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Figure 4. (a) MTT assays of PC12 on TCPs, BC, BC/PEDOT and BC/PEDOT/GO films substrates after 24 h, normalized by the measurements in parallel on the substrate of TCPs. (b) The comparison of PC12 cell viability on TCPs, BC, BC/PEDOT and BC/PEDOT/GO in different culture time. Error bar: standard deviation. (c) Cell viability assays of PC12 cells on TCPS, BC, BC/PEDOT, and BC/PEOT/GO by a dual color staining with Calcein-AM and PI. Culture time: 2 days. Arrows point to the dead cells stained in red. The column graph depicts the percentage of live cells on TCPS, BC, BC/PEDOT, and BC/PEOT/GO.

strength and Young’s modulus increased, as a result of counterion GO combining onto the surface of BC/PEDOT by electrostatic adsorption, enhancing the BC chain effectively. We next characterized the electrical properties of the BC, BC/ PEDOT, and BC/PEDOT/GO using EIS and CV. The electrochemical impedance performance of bioelectrodes is a determining factor influencing the quality of electrophysiological signal recording and the effects of bioelectrical stimulation. Cyclic voltammogram (CV, Figure 3b) was recorded to evaluate the redox characteristics and the charge storage capacity of the electrodes. The CV curves approach to parallelograms without obvious redox peaks indicates that the BC/PEDOT/GO film acted as an electric double-layer capacitor during the charge transfer procedure of CV scanning, which was in accordance with the EIS (Figure 3c) results. As shown in Figure 3d, the impedance at 1 kHz decreases sharply from 40 000 Ω of BC/PEDOT to 15 000 Ω of BC/PEDOT/ GO. The phase plots show that the frequency angles are approximately 0° at high frequencies and 90° at low frequencies, which demonstrates that the BC/PEDOT/GO acts as the resistive material and capacitive material, respectively. In Figure 3c, the Nyquist plots showed that small rise at high frequencies and straight lines at low frequencies indicate the charge transfer procedure was controlled by electrochemical reaction and ion diffusion, respectively. In addition, the steeper gradient of BC/

angle of BC is due to its rich hydroxyl and groups. With the increase of EDOT concentration, the lower water contact angle BC/PEDOT was shown. However, the contact angle of BC/ PEDOT of 0.03 g mL−1 EDOT was 63.2 ± 1.8° and turned to 53.2 ± 1.2° after GO doping with improved hydrophilic properties. This change of BC/PEDOT was due to the presence of a large of oxygen-containing groups on the surface of the GO. The difference between the BC, BC/PEDOT, and BC/PEDOT/GO films was in agreement with the following studies where we have demonstrated the difference in biocompatibility. The results of wettability measurement suggested that, due to the hydrophilicity of GO, the surface wettability effectively improving biocompatibility. 3.3. Electrochemical and Mechanical Measurements. For a tissue engineering scaffold to be effective, it must withstand long-term hemodynamic stress and have an appropriate strength compliant enough to mismatch between the interface of the graft and the tissue cells.40 The mechanical properties of the BC, BC/PEDOT, and BC/PEDOTGO films were compared, as shown in Figure 3a. In contrast, Bare BC film show typical brittle behavior material. The Young’s modulus of BC, BC/PEDOT, and BC/PEDOT/GO were 1.632 ± 0.216, 1.253 ± 0.226, and 1.445 ± 0.112 GPa, respectively, drawing near to collagen protein (1.2 GPa).41 Although the BC/PEDOT film decreased in tensile strength and Young’s modulus. After doping with GO, the tensile 10188

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Figure 5. Fluorescence and SEM images of PC12 cells cultured on (a) TCPs, (b) BC, (c) BC/PEDOT and (d) BC/PEDOT/GO films after 24 h. Green fluorescence, Alexa Fluor 488 conjugated phalloidin for actin cytoskeleton; blue fluorescence, DAPI DNA stain for cell nuclei. The cell exhibits extensive neurite branching and forms contacts with other cells, demonstrating the biocompatibility of the BC/PEDOT/GO film.

PEDOT/GO film shows higher specific surface area, facilitating the electron transfer between the solution and electrode interface and closer similarity to the ideal capacitor, which is desired to perform electrical stimulation safely.42 3.4. Cytotoxicity of BC/PEDOT/GO Nanocomposite. The cytotoxicity of different film samples were evaluated using the MTT cell viability assay, the Live/Dead cell dual-staining assay, and immunocytochemistry (ICC). The PC12 was chosen as a model cell line because of its versatility in the research of neural electrophysiology, neuronal differentiation and neurosecretion.36 As shown in Figure 4a, PC12 cells maintained nearly 95% of cell viability on the substrates of BC/PEDOT and BC/PEDOT/GO composite nanofibers (EDOT concentration 0.015 g mL−1) but only 75% of bare BC. The blank control experiments were included using the standard tissue culture plates (TCPs) for normalization compared with. It suggested that both BC/PEOT and BC/PEOT/GO composite nanofibers prepared in an optimized condition exhibited very low cytotoxicity for PC12 cells. The effect of different culture times, including 24 and 48 h, were monitored for PC12 cells on different types of substrates by MTT assays (Figure 4b). The results demonstrated good biocompatibility of BC/PEDOT and BC/PEDOT/GO composite nanofibers. The cells cultured on BC/PEDOT/GO nanofibers exhibited slightly higher cell

viability than bare BC or BC/PEDOT and even TCP substrates, probably due to promoted adhesion of PC12 cells on the relatively rougher and more hydrophilic surface of composite nanofibers doped with GO. Furthermore, the dualcolor staining of Calcein-AM and PI also confirmed the low cytotoxicity of the BC/PEDOT/GO substrates on cells (Figure 4c). PC12 cells were imaged with a fluorescent microscope after they were cultured on TCPs, BC, BC/PEDOT, or BC/ PEDOT/GO films for 48 h. Cellular skeleton structure was immuno-stained with the primary antibody (Rabbit, antitubulin antibody) and the secondary antibody (Goat anti Rabbit IgG, FITC-labeled by Phalloidin-Oregon). Cell nuclei was stained by Hoechst (DAPI). The images displayed that PC12 cells grew very well on all of these four different substrates. The percentages of cell confluency were similar (approximately 50%) among these substrates, indicating that the proliferation rates of PC12 cells were comparable on either BC, BC/ PEDOT, or BC/PEDOT/GO nanofibers. Good cellular morphology was maintained on either type of these substrates. Interestingly, many pseudopodia were present for the cells cultured on the BC/PEDOT substrate (Figure 5, the SEM images), indicating good cell adhesion due to the increased roughness of composite nanofibers during interfacial polymer10189

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Figure 6. (a) Schematic diagram depicting the fabrication and application of BC/PEDOT/GO hybrid scaffolds. The in situ polymerization of EDOT occurs on the surface of BC nanofibers assisted by FeCl3, doped with graphene oxide (GO) and seeded with PC12 cells. PC12 cells cultured on the hybrid scaffolds show enhanced proliferation and differentiation after electrical stimulated of PC12 cells; (b) Neuron attachment and neurite outgrowth on BC/PEDOT/GO surfaces with contact stimulus and noncontact stimulus or bare material without stimulus at 24 h. Representative 20 × fluorescent images of b-III-tubulin immunofluorescent reactivity of neurons; (c) Average neurite length (n = 3, * = p < 0.05) of neurons growing on the three electrical stimulus patterns; (d) Neuron density (n = 3) growing on the three electrical stimulus patterns; (e) Cell alignment on three different substrates.

3.5. Cell Growth on BC/PEDOT/GO Nanocomposite. The cell attachment and average cell length of PC12 cells were quantified and compared among BC/PEDOT/GO films with no stimulus, contact stimulus and noncontact stimulus (Figure

ization. These results consistently supported each other, suggesting that BC/PEDOT/GO 3D composite nanofibers can provide biocompatible and well-controlled microenvironments for the growth and proliferation of PC12 neural cells. 10190

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(RFDP, Grant 20123219110015), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, China).

6a,b) after 24 h in culture. Cell length was measured by a linear distance from the tip of the neurite to the cell junction. Generally speaking, length of PC12 cells (Figure 6c) was significantly greater on BC/PEDOT/GO films with stimulus than no stimulus, while the density (Figure 6d) showed no difference. This result proved that the working electrode of BC/PEDOT/GO nanofibers film can provide an elevated current density at the electrode−cell interface, which facilitates electrophysiological investigation of neural cells as a power tool. We can also observe that after the contact-stimulus on BC/ PEDOT/GO substrate, the majority of cells tended to expose to medium perpendicular to the direction of the current at an angle between 50 and 90° (Figure 6e). This result identified, along with previous studies,43,44 that cells would reorient themselve and align perpendicular to the electrical current in order to minimize the voltage drop across the bodies. Consistent with our proposed theory,28,36 BC/PEDOT/GO nanofibers can allow for cells electrical stimulation at a highcurrent density. As a result of the influx of Ca2+ induced by the depolarizing current, which activated the calmodulinkinases to elicit neurites outgrowth and expedite neurites development (Figure 6a).30 So, overall, this BC/PEDOT/GO composite film can act as an attractive method to the challenge of designing outstanding electrode−cell interfaces, promising many important applications in biological and regenerative medicine.

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4. CONCLUSIONS We have successfully prepared conducting polymer PEDOTcoated BC nanofibers doped with GO. The in situ polymerized PEDOT on BC nanofibers film showed good conductivity, and the combination of electrostatic GO can significantly lower the impedance of the biomaterial. Moreover, the as-prepared BC/ PEDOT/GO films possess many free carboxyl and hydroxy groups, which offer the BC/PEDOT/GO films active functional groups for surface modification. Otherwise, a promoting cell orientation and development of the PC12 cells were achieved by using the GO-doped BC/PEDOT nanofibers for cellular electrical stimulation. We believe this biocompatible BC/PEDOT/GO material will find important applications in biological and regenerative medicine.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: 25 8443 1939. Tel: 25 8431 5079. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Advanced Catalysis and Green Manufacturing Collaborative Innovation Center (Changzhou University), Synergetic Research Center for Advanced MicroNano Materials and Technology of Jiangsu Province, Collaborative Innovation Center of Suzhou Nano Science and Technology, National Natural Science Foundation of China (Grants 51272106 and 21275106), the Major State Basic Research Development Program (973 program, Grant 2013CB932702), the Fundamental Research Funds for the Central Universities (Grant 30920130121001), Research Fund for the Doctoral Program of Higher Education of China 10191

DOI: 10.1021/acsami.6b01243 ACS Appl. Mater. Interfaces 2016, 8, 10183−10192

Research Article

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