Visible-Light Neural Stimulation on Graphitic-Carbon Nitride

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Visible Light Neural Stimulation on graphiticCarbon Nitride/Graphene Photocatalytic Fibers Zhongyang Zhang, Ruodan Xu, Zegao Wang, Mingdong Dong, Bianxiao Cui, and Menglin Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12733 • Publication Date (Web): 20 Sep 2017 Downloaded from http://pubs.acs.org on September 21, 2017

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ACS Applied Materials & Interfaces

Visible Light Neural Stimulation on graphitic-Carbon Nitride/Graphene Photocatalytic Fibers Zhongyang Zhang,a Ruodan Xu,b Zegao Wang,a Mingdong Dong,a,c Bianxiao Cuic and Menglin Chena,b,d* a

Interdisciplinary Nanoscience Center, Aarhus University, DK-8000 Aarhus C, Denmark

b

Department of Engineering, Aarhus University, DK-8000 Aarhus C, Denmark

c

Department of Chemistry, Stanford University, Stanford CA 94305, USA

d

School of Medicine, Stanford University, Stanford CA 94305, USA

*Corresponding author: [email protected]

ACS Paragon Plus Environment


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Abstract Light stimulation allows remote and spatiotemporally accurate operation that has been applied as effective, non-invasive means of therapeutic interventions. Here, visible light neural stimulation of graphitic carbon nitride (g-C3N4), an emerging photocatalyst with visible-light optoelectronic conversion, was for the first time investigated. Specifically, g-C3N4 was combined with graphene oxide (GO) in a 3D manner on the surfaces of electrospun polycaprolactone/gelatin (PG) fibers and functioned as a biocompatible interface for visible-light stimulating neuronal differentiation. The enhanced photocatalytic function of g-C3N4 was realized by spreading g-C3N4 on coated electrospun (PG) microfibers to improve both charge separation and surface area. Ascorbic acid (AA) was used in the cell culture medium, not only as a photo-excited hole scavenger, but also to mediate GO reduction to further improve the electrical conductivity. The successful coatings of gC3N4, GO and AA-mediated GO reduction were confirmed using scanning electron microscopy (SEM), photoluminescence (PL), Raman spectroscopy and X-ray photoelectron spectroscopy (XPS). Biocompatibility of g-C3N4 (0.01 to 0.9 mg/ml) to PC12 cells was confirmed by the Lactate Dehydrogenase (LDH) assay, live dead staining and colorimetric cell viability assay CCK-8. Under a bidaily, monochromatic light stimulation at a wavelength of 450 nm at 10mW/cm2, a 18.5-fold increase of neurite outgrowth of PC12 was found on g-C3N4 coated fibers; while AA reduced GOgC3N4 hybrid brought a further 2.6-fold increase, suggesting its great potential as a visible-light neural stimulator that could optically enhance neural growth in a spatiotemporal-specific manner. Keywords: graphitic carbon nitride; graphene; electrospun fibers; visible light photocatalytic; neural stimulator


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Introduction Neurons, as electro-active cells, respond to exogenous electrical signals, which could influence cell proliferation, migration and even functions at molecular level.1 One of current trends in neural engineering focuses on developing novel materials that can be functionally and structurally interfaced with nerve system. In order to maintain natural behavior, cells prefer the full context of three-dimensional (3D) extracellular matrix,2 where cell attachment, cell shape, localization and functions are highly different with those in 2D culture.2-4 Therefore, 3D architectures providing ECM mimicking microenvironment are desired for neural interfacing, as they enable similar regulations of cell behaviors in a 3D manner as those in vivo.5 Graphene based materials have been widely investigated as 2D neural interfacial material for various neural applications, such as neural microelectrodes,6-7 monitoring of neuron stem cells (NSCs) differentiation8 and detection of neuronspecific enolase,9-10 due to its outstanding electrical properties, and most importantly, good biocompatibility to neurons. Electrospun fibrous scaffolds possess a relatively close 3D fibrous structural mimic of the native ECM that is advantageous for neural tissue engineering.11-13 Intriguingly, photo stimulation allows remote and spatiotemporally accurate operation to trigger electrical stimulation, which alleviates cellular damage associated with lasting electric field stimulation.14-16 Recently, reduced graphene oxide (rGO)/TiO2 heterojunction film was reported to be a biocompatible flash photo stimulator for effectively inducing differentiation of human NSCs into neurons under UV light stimulation.14 However, as a photocatalyst, TiO2 functions under the irradiation of UV light with a wavelength below 387 nm, the related cellular/genomic toxicity is a concern. Graphitic carbon nitride (g-C3N4), on the other hand, has been demonstrated to be an efficient metal-free polymeric photocatalyst under visible light irradiation (mainly above 400 nm) with good stability in a wide range of pH.17-19 Although its quantum efficiency is always limited due to high recombination rate of photogenerated electron-hole pairs,20-21 constructing



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heterojunctions between g-C3N4 and another semiconductor to promote the separation of electronhole pairs has been proven as a feasible method. For instance, it has been reported that graphene and its derivatives could promote the photocatalytic performance of g-C3N4 by retarding the recombination of electron-hole pairs via effective charge transfer across heterojunction between them.22-23 Here, visible-light-responsive photocatalytic function of gC3N4/rGO hybrid on neural stimulation was for the first time evaluated (Scheme 1). The enhanced photocatalytic function of g-C3N4 was realized by spreading g-C3N4 on GO coated electrospun polycaprolactone/gelatin (PG) microfibers to improve both charge separation and surface area. Ascorbic acid (AA) was used in the cell culture medium, not only as a photo-excited hole scavenger, but also to mediate GO reduction to further improve the photocatalytic electric neural stimulation. The coatings of g-C3N4, GO and AAmediated GO reduction were characterized using scanning electron microscopy (SEM), Atomic force microscopy (AFM), photoluminescence (PL), Raman spectroscopy and X-ray photoelectron spectroscopy (XPS). PC12 cells derived from rat adrenal pheochromocytoma were used as the model neural cell line. Biocompatibility of g-C3N4 to PC12 was investigated by the Lactate Dehydrogenase (LDH) assay and colorimetric cell viability assay CCK-8. Neural differentiation of PC12 cells under monochromatic light stimulation at a wavelength of 450 nm was evaluated by immunofluorescence staining and confocal imaging.


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Scheme 1 Schematic illustration of visible light neural stimulation of PC12 cells on graphitic-carbon nitride/graphene photocatalytic fibers.



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Materials and methods Synthesis of g-C3N4 and GO sheets The g-C3N4 was prepared by direct polymerization of urea at high temperature as previously reported.24-25 10g urea placed in an alumina crucible was thermo-treated under 550 °C for 4 h, and the ramp rate was set at ~5 °C/min. The obtained product was yellow powder. GO nanosheets were prepared by oxidation and exfoliation of graphite powder according to modified Hummers’ method26: 3 g of 3500 mesh graphite powder was dispersed into a 9:1 mixture of concentrated H2SO4/H3PO4 (360:40 mL) under ice bath. Then 18 g KMnO4 was added into the mixture gradually under vigorous stirring. After stirred for 12 h, the mixture was poured onto ice (400 mL) with 30% H2O2. Afterwards, the product was filtered over a 0.45 µm pore size polytetrafluoroethylene (PTFE) membrane. The remaining solid materials was then washed with water, HCl and ethanol, and dried overnight in a vacuum at 60 °C. GO sheet colloidal dispersion was obtained by exfoliation of the asprepared graphite oxide in high purity water under sonication for 30 min.

Fabrication of PG microfibers PCL polymer (Mn 80,000, Sigma-Aldrich) and gelatin type A (Sigma-Aldrich) were dissolved separately in hexafluoro isopropanol (HFP, purity ≥99.0%, Sigma-Aldrich) at a concentration of 12% (w/v) with sufficient stirring at room temperature overnight. The two solutions were mixed in 50:50 volume ratios and stirred for another one hour before electrospinning. The homogenous polymer solution was put into a 5 mL plastic syringe mounted with a 20 gauge needle. The polymer solution dispensed from a pump (New Era Pump Systems, Model: Ne-300, NY, USA) at a constant rate of 1 ml/h. A high voltage of 15 kV (Gamma High Voltage Research, Fl, USA) was applied to the needle, and a grounded metal mandrel rotating at ~300 rpm was placed 15 cm away from the needle. Experiments were carried out at room temperature with a relative humidity of 30%. The


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prepared PG fibrous membranes were then freeze-dried for over 48 hours to remove solvent residuals and stored at -20 ℃ before further use.

Coating procedures Both graphene and g-C3N4 coating were carried out through simple filtration method. The prepared PG fibrous membranes were cut into discs and placed in a Buchner funnel for filtration process. Firstly, GO coated microfibers were prepared by filtering a GO sheet colloidal dispersion (0.1 mg/mL) through the PG membranes. Then, g-C3N4 coated and GO&g-C3N4 coated microfibers (i.e. PG- C3N4 and PG-GO- C3N4) were obtained by filtering a g-C3N4 suspension (0.1 mg/mL) through pure PG or GO pre-coated fibrous membranes.

Materials characterization Surface topography and height profile of the prepared g-C3N4 nanoparticles and GO nanosheets were examined using AFM in tapping mode by a Nanoscope V Multimode SPM system with an Escanner (Bruck, Digital Instruments, USA) at ambient conditions. Contact angle measurements (Drop Shape Analyzer-DSA100-KRÜSS GmbH) were carried out by locating a drop of water (triplicates) on the fibrous membranes at room temperature. Surface morphology of the coated microfibers was characterized by using a scanning electron microscope (SEM, Hitachi TM3030). Diameter distribution of the pure microfibers was obtained by analyzing at least two hundred fibers based on the SEM images. The absorbance of g-C3N4 nanoparticles at 405 nm was measured by a Victor X5 microplate reader. Photoluminescence spectroscopy (PL) of the samples was carried out using a fluorescence spectrometer (FS5 Spectrofluorometer-Edinburgh Instruments) at room temperature. The X-ray photoelectron spectroscopy (XPS) was performed on a Kratos Axis-Ultra DLD instrument with a monochromated Al Kα X-ray source at a pressure of 7.6 × 10-9 Torr and a



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pass energy of 160 eV. Raman spectroscopic measurements were carried out to characterize structural changes during coating GO and g-C3N4 by a Renishaw inVia Raman microscope.

PC12 cell culture PC12 cells were cultured on collagen IV (Sigma-Aldrich) coated flasks (Nunc-easyflask) as reported in our previous work.27 Both the pure PG and coated fibrous membranes were punched out using a Ø6 mm biopsy puncher (Acu Puch, Acuderm, USA), and were then placed in 96 well plates and sterilized under a 254 nm UV source for 1 h. Before seeding cells, the fibers and TCP wells were coated by collagen (type IV, Sigma-Aldrich) solution at a concentration of 10 µg/mL overnight, and washed once with PBS afterwards. PC12 cells were seeded with growth medium on the fibers and TCP wells at a concentration of 104 per well in 96-well plates. For the neuronal differentiation experiments, the medium was replaced after the first day of culture by a differentiation medium27 with 50 ng/mL of 2.5S NGF (Gibco) supplements.27 Both the growth and differentiation mediums were changed every second day. For visible light stimulation, the differentiation medium was further supplemented by 3 mM Ascorbic Acid (AA). A high-intensity blue LED inspection flashlight kit (OPX-450, Optimax) that could provide a monochromatic light with a wavelength of 450 nm was used as the visible light source. The distance between the samples and the lamp was fixed at 30 cm, where the flash intensity applied to the samples was about 10 mW/cm2. Flash photo pulse duration was controlled to be 1 s with a time interval of 1 s for a total exposure time of 30 min every 12 h.

Cytotoxicity assay After 24 h of culture, the activity levels of lactate dehydrogenase (LDH), which is a cytosolic enzyme normally used as an indicator of cellular toxicity, in the collected culture media was measured to evaluate cell damage. According to the manufacture’s protocol (Roche Diagnostics,


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Mannheim, Germany), the LDH activity was obtained by assessing the absorbance of formazan product at 490 nm via the plate reader (Victor X5). The absorbance of the culture medium without cells was used as the background. The LDH activity in the medium of cells cultured on TCP wells was used as low toxicity control, while that of cells cultured on TCP wells and treated with 1% Triton X-100 was marked as high toxicity control. The relative toxicity levels in percentages was calculated using the equation below:

=

. − × 100 ℎℎ −

Cell viability Cell viability was evaluated by a Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) after 1, 3 and 7 days of culture following the manufacurer’s protocol. Briefly, cells were incubated for 4 hours in CCK-8 diluted (1:10) culture medium before the absorbance at 450 nm was measured by plate reader (Victor X5). Six replicate wells were used in the measurement for all the samples. Live&Dead assay was also carried out to assess the cell viability after 24h of culture in growth and differentiation medium, following the manufacurer’s protocol (Thermo Fisher Scientific). Briefly, the live green and dead red component were diluted with culture medium and mixed together just before use, and then cells were immersed with the mixed solution and incubated for 15 min. Finally, the live and dead cells were visualized by an Invitrogen EVOS FL Auto Cell Imaging System. Besides, in order to evaluate the potential photo-toxicity of the monochromatic light on PC12 cells, Live&Dead assay was also carried out before and after exposing the cells under the light irradiation for 1h in differentiation medium.

Immunostaining and fluorescence imaging



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Cells cultured on all the samples and TCP wells were fixed with 4% formaldehyde in PBS at room temperature, and then the cells were rinsed with 0.2% (v/v) Triton X-100 in PBS as a permeabilization solution for 15 min. After blocking with 1% BSA in PBS for 1 h at room temperature, immunostaining with primary antibody, mouse anti-βIII-tubulin (Abcam, 1:1000), was performed overnight at 4 ℃, followed by Alexa Fluor 594-conjugated anti-mouse IgG, secondary antibody, (Abcam, 1:1000) staining for 1h at room temperature. Then cells were counterstained with Hoechst 33258 (Life technologies) to visualize the nuclei. The cells were visualized under a Zeiss LSM 700 laser confocal microscope (Carl Zeiss Micro-Imaging GmbH, Germany). Based on the fluorescence images, ImageJ were used to assess the neurite length on each group. Neurites were measured for at least 300 cells from each sample. Whereas, for counting neurites per cell, neurites with a length longer than the cell body were taken into account.

Statistics The results were expressed as means and error bars. Groups were compared by ANOVA or t-test using IBM SPSS statistics 23 software. Statistical significance was considered at p < 0.05.


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Results and discussion Synthesis and characterization of graphene&g-C3N4 coated microfibers AFM measurements were performed to characterize the structure of as-prepared g-C3N4 nanoparticles and GO nanosheets. As shown in Figure 1b, the thickness of the g-C3N4 nanoparticles is about 6.34 nm with diameters ranging around 63 nm (as seen in the inset), which indicates the nanoparticles composed of several layers of graphitic planes stacking along the c-axis.22 Singlelayer GO nanosheets were synthesized via modified hummers method as confirmed by the thickness of about 1.5 nm in Figure 1c, while the nanosheets sizes are in several hundred nanometer scale. Figure 1a show that homogeneous PG microfibers with diameters around 1.36 µm were fabricated by the electrospinning. The incorporation of gelatin introduced abundant oxygen containing groups on the fiber surface, contributing to highly improved hydrophilicity of the hybrid fibers compared to PCL fibers (Figure S1b). Attributed to the hydrophilic surface of PG microfibers, GO nanosheets and g-C3N4 were successfully coated on the fibers, evidenced by the GO wrinkles (Figure 1e) and g-C3N4 nanoparticles (Figure 1d) on the PG fiber surface and the changed colors (Figure S1c). Using a standard curve (Figure S1a) that correlates absorbance values at 405 nm of g-C3N4 nanoparticles with concentration of its aqueous solution, coating amount of the g-C3N4 nanoparticles on the fibrous membrane was determined as ~ 7 ug/mg PG.



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Figure 1 SEM images of (a) PG microfibers with diameter distribution in the inset. AFM images of (b) gC3N4 nanoparticles and (c) GO nanosheets. Corresponding histograms of g-C3N4 nanoparticles diameter, and GO nanosheets size distributions were shown in the insets. SEM images of (d) PG-C3N4 and (e) PG-GOC3N4 microfibers.

Graphitic carbon nitride (g-C3N4) has recently emerged as a promising visible-light-responsive photocatalyst. Nevertheless, its efficiency is always limited by its high recombination rate of photogenerated electron-hole pairs.20-21 It has been reported that graphene and its derivatives could promote the photoelectroncatalytic performance of g-C3N4 by retarding the recombination of electron-hole pairs via effective charge transfer across heterojunction between them.22-23 Herein, PL sepectra was employed to investigate the efficiency of charge carrier transfer, trapping and separation. As shown in Figure 2a, the emission peak at 450 nm is attributed to the recombination of free charge carriers in g-C3N4 generated under visible light excitation; while the lower intensity reveals the higher separation efficiency of photogenerated electron-hole pairs. As the emission


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intensity is much lower for the g-C3N4&GO composite samples than that in the pristine g-C3N4, the separation of electrons and holes in the composites are more efficient in GO-C3N4. Ascorbic Acid (AA) is often used as a photoexcited hole scavenger. Meanwhile, AA has been reported to be able to reduce GO.28 As shown here, AA treated C3N4&GO-AA further lowered the PL intensity compared to C3N4&GO. One reasonable explanation is that GO was partially reduced by AA, resulting in even more efficient charge transfer between g-C3N4 and reduced graphene oxide. In order to fully demonstrate the reduction of GO during incubation with AA, PG-GO-C3N4 microfibers were treated with 3 mM AA overnight (labeled as PG-GO-C3N4-AA), and characterized by Raman and XPS spectra in comparison with that of PG-GO-C3N4. Raman measurements (Figure 2b) were performed with excitation laser beam wavelength of 785 nm for PG and PG-C3N4 (lower panel). Compared with that of PG, several characteristic peaks of gC3N4 at 707, 767 and 1233 cm-1 were observed in the spectra of PG-C3N4, indicating the successful coating of g-C3N4 particles on the fibers. Under excitation laser beam wavelength of 514 nm, the typical D and G bands of GO were present, proving the presence of GO coating on the fiber surface (upper panel). Furthermore, the ratio of D/G significantly increased on PG-GO-C3N4-AA from 0.42 to 0.864, compared to that on PG-GO-C3N4, which also reveals that the reduction of GO did take place during the AA treatment.



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Figure 2 (a) PL spectra of pristine g-C3N4, C3N4&GO, C3N4&GO-AA. (b) Raman spectra of PG, PG-C3N4 (lower panel, under 785 nm excitation), PG-GO-C3N4 and PG-GO-C3N4-AA (upper panel, under 514 nm excitation).

To further examine the chemical composition and chemical status of the constituent elements in the microfibers, XPS measurements were conducted (Figure 3). XPS wide scan spectra depicted that the C, N, O elements were present on PG microfibers, in which the presence of N 1s is attributed to the incorporation of gelatin. After coated by g-C3N4 nanoparticles, the content of N 1s was highly increased from 1.52 % to 17.47 %, while GO coating resulted in the increased content of O 1s with accordingly reduced nitrogen composition (Table 1). After AA treatment, the oxygen composition was decreased. The high-resolution C 1s spectra in g-C3N4 shows characteristic peaks of C-C (284.6 eV) and N-C=N (288 eV);24 while C-C (284.6 eV), C-O (286.6 eV) and C=O (288.2 eV) in GO.28 The decrease of C-O/C-C ratio from 1.11 to 0.37 confirms the reduction of GO under AA treatment. C1s spectra of PG fibers are consisted of both characteristic PCL peaks (C-C at 284.6 eV, C-O at 286.6 eV and O-C=O at 288.6 eV) and characteristic C-N peak at 285.5 eV from gelatin.29-30 The GO and g-C3N4 coated fibers further introduced their corresponding peaks. Again the AA treated


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group, PG-GO-C3N4-AA, shows decreased C-O and O-C=O peaks from GO, confirmed the reduction of GO. In addition, the high-resolution N 1s spectra (Figure S2) of coated microfiber samples consist of four peaks with binding energies of 398.6, 399.9, 401.2 and 404.2 eV. The dominant peaks at 398.6 and 399.9 eV were assigned to the triazine units (C-N=C) and the bridging N atoms in N-(C)3, respectively.31-34 The peak at 401.2 eV corresponded to the terminal amino groups (C-N-H) induced by the incomplete condensation during thermal polymerization,31 and the peak at 404.2 eV was characteristic of π–excitation.32-34

Figure 3 XPS wide scan and C1s high-resolution spectra of C3N4, GO, GO-AA, PG, PG-C3N4, PG-GOC3N4 and PG-GO-C3N4-AA



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Table 1 Atomic composition of pure PG microfibers, and g-C3N4 nanoparticle, GO&g-C3N4 and rGO&gC3N4 coated microfibers from the wide scan spectra. Sample

C 1s (%)

O 1s (%)

N 1s (%)

C3N4

44.06

2.59

53.35

GO

68.77

30.42

-

GO-AA

81.97

18.03

-

PG

77.01

21.47

1.52

PG-C3N4

65.79

16.74

17.47

PG-GO-C3N4

66.81

25.22

7.96

PG-GO-C3N4-AA

66.01

20.13

13.86

Biocompatibility and effect of g-C3N4 nanoparticles on PC12 cell growth and differentiation Biocompatibility of g-C3N4 for PC12 cells was examined by both LDH assay and CCK-8 assay. To explore any potential dose-dependent toxicity of g-C3N4, PC12 cells were maintained in growth medium containing various concentrations of g-C3N4 ranging from 0.01 to 0.9 mg/mL. Cell viability was analyzed by CCK-8, a colorimetric assay that measures metabolic activity (Figure S3a). After 24 h of culture, there is no significant difference on the viability levels among g-C3N4 groups with different concentrations and TCP groups, indicating that g-C3N4 was biocompatible to PC12 cells within the experimental concentration range. On the other hand, LDH activity analysis was used to analyze cellular toxicity of g-C3N4 by quantification of any leaking of a cytoplasmic enzyme LDH caused by membrane damage (Figure S3b). LDH release on both g-C3N4 groups and TCP groups were well below 20% without any significant difference. Moreover, Live&Dead staining was also performed to further assess the biocompatibility of g-C3N4 for PC12 cells after 24


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h of culture (Figure S4). Obviously, the number of dead cells detected with Live&Dead assay was very low in all samples. Therefore, g-C3N4 is proved to be highly biocompatible for PC12 cells and no dose-dependent toxicity was observed in the experimental concentration range (from 0.01 to 0.9 mg/ml). The coating amount of 0.0056 mg on 0.8 mg PG fibers per well (100 µl medium) gave the g-C3N4 concentration, 0.056 mg/ml, which is within this range. The effect of AA on PC12 cell differentiation under g-C3N4 mediated (0.06 mg/ml in medium) visible light stimulation was investigated on TCP. A monochromatic light with a wavelength of 450 nm at intensity of 10 mW/cm2 was used as the visible light source. No photo-toxicity was found.(Figure S5) A concentration of 3mM AA was used, as it has been reported that the incorporation of AA in the high serum growth medium with a limited concentration below 3 mM could support PC12 cell growth without any cytotoxicity, while higher concentration of AA would induce cell apoptosis.35 The cell morphology and the differentiation process were assayed by immunofluorenscence staining, in which an early differentiation marker tubulin for neurons were stained with anti-beta tubulin III antibody (red) and nuclei were stained with dapi (blue). It should be noted that nuclei are difficult to distinguish in the images, since g-C3N4 nanoparticles exert overlapping fluorescence. As seen in Figure 4, without NGF, the incorporation of AA could enhance the neuronal differentiation of PC12 cells to some extent as evidenced by the slight outgrowth and higher expression of tubulin. However, the total number of cells was much less, suggesting certain cytotoxicity of AA in the low serum differentiation medium. To further confirm this, Live&Dead staining and LDH activity analysis of PC12 cells were carried out after 1 day of culture in low serum differentiation medium with/without AA or NGF (Figure S6). Both the number of dead cells and LDH activity were higher when AA was added into the medium alone, while NGF addition could significantly decrease the toxicity of AA in differentiation medium. On the contrary, under the chemical stimulation of NGF,



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the neuronal differentiation of PC12 cells cultured in both the groups with/without AA were highly promoted. Most importantly, neurons in the group with both AA and NGF treatments showed the best outgrowth and expression of tubulin without any significant decrease in the cell numbers. It is thus NGF is determined necessary to avoid the potential cytotoxicity of AA on PC12 differentiation.

Figure 4 (a) Fluorescence images of PC12 cells after 5 days of culture on g-C3N4 coated TCP under light stimulation in differentiation medium with/without NGF and AA. All scale bars are 200 µm. Immunostaining markers are anti-β III tubulin (red color) for neurons and DAPI (blue color) for nuclei, scale bar 200 µm. (b) Average neurite length and (c) neurite number/cell analysis of cells cultured on g-C3N4 coated TCP under light stimulation in differentiation medium with/without NGF and AA. Statistical are denoted as *(p < 0.05), **(p < 0.005), ***(p < 0.0005).

PC12 cells growth and differentiation on GO&g-C3N4 coated PG microfibers Firstly, biocompatibility of the microfibers with/without various coatings for PC12 cells was examined by LDH and CCK-8 assay (Figure 5). After 1 day of culture, similar viability levels were


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obtained among all samples without any significant difference. After continuous culture for 7 days, all groups showed highly increased proliferation indexes after day 3, while cells cultured on the PGC3N4 exhibited higher proliferation than that on the PG-GO-g-C3N4. As seen in Figure 5b, LDH activity analysis revealed that LDH release on all the sample with/without coatings were well below 20% without any significant difference. Thus the microfibers with/without coatings were biocompatible for PC12 cells and could enhance PC12 cell proliferation in the growth medium.

Figure 5 Biocompatibility analyses of microfibers with various coatings. (a) Cell viability assays and (b) LDH activity analysis of PC12 cells on pure PG microfibers, and g-C3N4 nanoparticle, GO&g-C3N4 and rGO&g-C3N4 coated microfibers. Values are normalized to cell viability of pure fiber group on day 1respectively. Statistical significances are denoted as *(p < 0.05), **(p < 0.005).

Differentiation of PC12 cells was further investigated by replacing the culture medium with the differentiation medium supplemented with 3 mM of AA. After 11 days differentiation, immunofluorescence staining was used to evaluate the differentiation process, where tubulin was stained with anti-beta tubulin III antibody (red) and nuclei were stained with dapi (blue). The obtained images were used to quantify the average neurite length and the average number of neurites per cell. Figure 6a shows representative images of cells on all groups after 11 days



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differentiation with/without light stimulation. Figure 6b,c are quantifications of neurite lengths and numbers. Though tubulin expression could be observed from the cells cultured on PG fibers, neurite outgrowth was unseen with no significant promotion under light stimulation. In contrast, for the PG-C3N4 group, slight neurite outgrowth could be observed even without light stimulation, while introducing light stimulation enhanced neurite protraction by ~ 18.5-fold. Furthermore, addition of GO further enhanced neurite outgrowth by ~ 2.6-fold in cells on the PG-GO-C3N4 under light stimulation. As shown in PL (Figure 2a), the separation of photo-excited electrons and holes in the GO&g-C3N4 composites are more efficient than that in the pristine g-C3N4, especially after AA treatment. As confirmed in XPS (Figure 3) and Raman (Figure 2b), AA not only serves hole scavenger but also further reduced GO during PC12 differentiation. Thus, the PG-GO-C3N4 at AA supplemented differentiation medium exerted stronger electrical stimulation to PC12 cells, which leads to improved neuronal differentiation.

Figure 6 (a) Representative immunofluorescence images of PC12 cells after 11 days of culture in AA supplemented differentiation medium on PG, PG-C3N4, PG-GO-C3N4 with or without visible light


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stimulation. All scale bars are 20 µm. Immunostaining markers are anti-β III tubulin (red color) for neurons and DAPI (blue color) for nuclei. (b) Average neurite length and (c) neurite number/cell analysis of cells cultured on various fibers with/without visible light stimulation. Statistical significances are denoted as *(p < 0.05), **(p < 0.005), ***(p < 0.0005).

Conclusions With the increasingly extensive application of nanotechnology in biomedical scenarios, interfacing biocompatible nanomaterials with biological entities at three dimensions has demanded novel designs of nano–bio interfaces. Light, attributed to its remote and spatiotemporally accurate operation, is an intriguingly important tool and energy source in biophysics, therapeutics and diagnosis applications. In this study, the visible-light responsible photocatalytic g-C3N4 was combined with GO in a 3D manner on the surfaces of electrospun PG fibers and functioned as a biocompatible neuronal interface for accelerated neuronal differentiation. Neuronal differentiation on g-C3N4 coated PG fibers exhibited highly improved neurite outgrowth under visible light stimulation, while the AA mediated reduced GO/g-C3N4 coated fiber further enhanced neuronal differentiation due to the improved separation efficiency of photo-excited electron-hole pairs. The g-C3N4 nanoparticles were demonstrated to be biocompatible to PC12 cells, where no toxicity was observed in a wide range of concentration (from 0.01 to 0.9 mg/ml). While the underlying ion channels associated cellular mechanisms are still under investigation, AA mediated reduced GO/gC3N4 hybrid is confirmed as a visible-light neural stimulator that potentially could optically enhance neural growth in a spatiotemporal-specific manner.



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Supporting Information Standard curve correlating absorbance values at 405 nm of g-C3N4 nanoparticles aqueous solution; contact angle (CA) measurements of PCL and PG fibers; digital photograph of PG, PG-C3N4, PGGO, PG-GO-C3N4, PG-GO-C3N4-AA microfibers; high resolution XPS spectra of N 1s for C3N4, PG-C3N4, PG-GO-C3N4 and PG-GO-C3N4-AA; cell viability assays, LDH activity analysis and Live&Dead staining of PC12 cells maintained in growth medium containing various concentrations of g-C3N4 nanoparticle ranging from 0.01 to 0.9 mg/mL; Live&Dead cell staining of PC12 cells before and after light stimulation; Live&Dead cell staining and LDH activity analysis of PC12 cells after 1 day of culture on g-C3N4 coated TCP in differentiation medium with/without NGF and AA. Acknowledgements We gratefully acknowledge the funding from Danish Council for Independent Research (DFF 7017-00185), EU H2020 RISE 2016—MNR4SCell 734174, Aarhus University Research foundation and Carlsberg Foundation for their financial support. References (1) Thompson, B. C.; Murray, E.; Wallace, G. G. Graphite Oxide to Graphene. Biomaterials to Bionics. Adv. Mater. 2015, 27 (46), 7563-7582. (2) Stevens, M. M.; George, J. H. Exploring and Engineering the Cell Surface Interface. Science 2005, 310 (5751), 1135-1138. (3) Cukierman, E.; Pankov, R.; Stevens, D. R.; Yamada, K. M. Taking Cell-Matrix Adhesions to the Third Dimension. Science 2001, 294 (5547), 1708-1712. (4) Gumbiner, B. M. Cell Adhesion: The Molecular Basis of Tissue Architecture and Morphogenesis. Cell 1996, 84 (3), 345-357. (5) Li, N.; Zhang, Q.; Gao, S.; Song, Q.; Huang, R.; Wang, L.; Liu, L.; Dai, J.; Tang, M.; Cheng, G. ThreeDimensional Graphene Foam as a Biocompatible and Conductive Scaffold for Neural Stem Cells. Sci. Rep. 2013, 3, 1604. (6) Deng, M.; Yang, X.; Silke, M.; Qiu, W.; Xu, M.; Borghs, G.; Chen, H. Electrochemical Deposition of Polypyrrole/Graphene Oxide Composite on Microelectrodes Towards Tuning the Electrochemical Properties of Neural Probes. Sensors Actuators B: Chem. 2011, 158 (1), 176-184. (7) Chen, C.-H.; Lin, C.-T.; Hsu, W.-L.; Chang, Y.-C.; Yeh, S.-R.; Li, L.-J.; Yao, D.-J. A Flexible HydrophilicModified Graphene Microprobe for Neural and Cardiac Recording. Nanomed. Nanotechnol. Biol. Med. 2013, 9 (5), 600-604.


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Table Of Contents (TOC)

Graphitic carbon nitride (g-C3N4) was combined with graphene oxide (GO) in a 3D manner on the surfaces of polycaprolactone/gelatin (PG) fibers and functioned as a biocompatible interface for visible-light stimulating neuronal differentiation.


Visible-Light Neural Stimulation on Graphitic-Carbon Nitride

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