Carbon Nanofiber Reinforced Nonmulberry Silk Protein Fibroin

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Carbon Nanofiber Reinforced Nonmulberry Silk Protein Fibroin Nanobiocomposite for Tissue Engineering Applications Deboki Naskar,† Promita Bhattacharjee,‡ Ananta K. Ghosh,*,† Mahitosh Mandal,§ and Subhas C. Kundu*,† †

Department of Biotechnology, ‡Materials Science Centre, and §School of Medical Science and Technology, Indian Institute of Technology Kharagpur, West Bengal 721302, India S Supporting Information *

ABSTRACT: Natural silk protein fibroin based biomaterial are exploited extensively in tissue engineering due to their aqueous preparation, slow biodegradability, mechanical stability, low immunogenicity, dielectric properties, tunable properties, sufficient and easy availability. Carbon nanofibers are reported for their conductivity, mechanical strength and as delivery vehicle of small molecules. Limited evidence about their cytocompatibility and their poor dispersibility are the key issues for them to be used as successful biomaterials. In this study, carbon nanofiber is functionalized and dispersed using the green aqueous-based method within the regenerated nonmulberry (tropical tasar: Antheraea mylitta) silk fibroin (AmF), which contains inherent − R-G-D- sequences. Carbon nanofiber (CNF) reinforced silk films are fabricated using solvent evaporation technique. Different biophysical characterizations and cytocompatibility of the composite matrices are assessed. The investigations show that the presence of the nanofiber greatly influence the property of the composite films in terms of excellent conductivity (up to 6.4 × 10−6 Mho cm, which is 3 orders of magnitude of pure AmF matrix), and superior tensile modulus (up to 1423 MPa, which is 12.5 times more elastic than AmF matrix). The composite matrices (composed of up to 1 mg of CNF per mL of 2% AmF) also support better fibroblast cell growth and proliferation. The fibroin-carbon nanofiber matrices can lead to a novel multifunctional biomaterial platform, which will support conductive as well as load bearing tissue (such as, muscle, bone, and nerve tissue) regenerations. KEYWORDS: carbon nanofiber, nonmulberry silk fibroin, nanocomposite, cytocompatibility, hemocompatibility, electrical conductivity, tensile modulus

1. INTRODUCTION Over last few decades, many companies have invested large amounts of money on biomaterials research for developing more suitable, biocompatible products. During this period, the rapid surge in biomaterials demands its engineering and advancement for its application in tissue bioengineering and healthcare sector. It is observed that pure materials alone are incapable of serving a particular problem.1 Researchers are attracted to combine more than one component to fabricate the multifunctional composite biomaterials as close as the required human tissue. The attractive unique features of each of the materials enhance their inefficiencies as an individual. In the last few decades, different forms of silk biomaterials such as suture, sponge, film, hydrogel, micro/nanosphere, nanoparticle, membrane, and tubes is investigated in search of perfect artificial tissue support.2−4 Silk fibroin from silkworm species is well-known high molecular weight protein polymer. The natural protein, particularly from nonmulberry origin renders superior mechanical strength, ease in fabrication into several multifunctional matrices (using aqueous-based processing), excellent cytocompatibility and tissue growth.5,6 Fibroin protein from Antheraea mylitta has its own cell adhesion promoting tripeptide (-R-G-D-) © XXXX American Chemical Society

motif, which ensures its potential scaffold based applications for cell-based tissue engineering and drug delivery.7,8 However, considering bone, nerve, and muscle repairing, pure fibroin based matrices are not enough mechanically strong or electrically conductive to support the mentioned tissue defects/damages. For this reason, several doping strategies including hydroxyapatite,9 gold nanoparticle,10 graphene oxide,11 CNT (Table S1), and CNF (Table S2) have been investigated to date to increase mechanical modulus, flexibility, and conductivity of the fibroinbased matrices for better cell growth, proliferation, and/or differentiation on it. Carbon nanofiber (CNF) is a nanoscale carbon fiber synthesized through catalytic chemical vapor deposition process with different temperature variations.12 A single carbon nanofiber is formed by stacking several curved nanocones made of graphene nanosheets in certain angle. CNF is already reported as they are Special Issue: Focus on India Received: April 21, 2016 Accepted: July 29, 2016

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ACS Applied Materials & Interfaces highly flexible, possess low mass density, and have large aspect ratio. They possess a unique combination of mechanical, thermal, and electrical properties.13 Carbon nanotube (CNT) is a much popular cousin of CNF, as widely used throughout the world.14 They are highly ordered rolled up single graphene sheet either in the form of single wall (SWCNT) or spiral multiwall (MWCNT). CNF is less expensive, less toxic, easily functionalized, organochemically modifiable, and better dispersible than CNT.15 Some attempts to reinforce CNT within mulberry silk protein fibroin are made and their cytocompatibility is also investigated (Table S1). Notable applications of CNF include composite materials, gene delivery vehicle, electrode, atomic force microscopic tip, synthetic membrane, biosensor, hydrogen and charge capacitor, and electron field emitters.16,17 CNF is synthesized in powder form and used as a filler material. This provides reinforcement within the polymer matrix. Some synthetic polymers such as poly(lactic−coglycolic acid),18 poly(acrylonitrile),19 and poly(carbonate)urethane20 along with natural polymers such as chitosan, cellulose acetate, and sodium alginate21 are already utilized as base materials for engineering a wide variety of artificial extra cellular matrices (Table S2). The reinforcement provides additional structural stability, electrical conductivity, and mechanical strength to the base matrix.22 Additionally, they can serve as a delivery medium for the sustained release of drugs and biological molecules.23 The biocompatibility of CNT and CNF based materials remains an all-time popular question.24 To address this issue, several scientific groups investigate the ultimate fate of these materials on the cells in vitro as well as in vivo system25−27. Moreover, the CNF is hydrophobic in nature. Proper functionalization is required to make them hydrophilic and compatible with base matrix for successful engineering of the composite material. The studies listed in Tables S1 and S2 are the complete list of the work based on mulberry silk fibroin, CNT, CNF, and polymer based materials. It may be observed that there is a gap of proper CNF functionalization and complexity in material fabrication along with lack of a complete biophysicalbiocompatibility study. To the best of our knowledge, the fabrication as well as cyto- and hemo-compatibility studies of the nonmulberry silk fibroin-CNF based nanocomposite biomaterials are not reported comprehensively until date. This information becomes major motivation of our current study. In this study, we report the fabricated multifunctional silk protein fibroin nanocomposite by combining the attractive features of both silk fibroin and carbon nanofiber. For the first time, CNF reinforced nonmulberry silk fibroin, a natural polymer based matrix is fabricated using easy functionalization and green processing steps. The significant effects of reinforcement of nanofiller material within the fabricated nanocomposites are investigated for their mechanical, electrical, biocompatible, and hemocompatible properties for their potential application in the next-generation biomedical field.

absorption. Five different concentrations 0.1 mg/mL, 0.5 mg/mL, 1.0 mg/mL, 5.0 and 10.0 mg/mL were prepared by dispersing the nanofiber powder in 1% t-octylphenoxypolyethoxyethanol (TritonX100) in ultrapure deionized water (Milli-Q 18.2Ω).30 The solutions were then sonicated using a probe type high energy sonicator (Q125 Sonicator, Qsonica Llc, USA) at 60% amplitude for 30 min with 40s ON and 20s OFF cycle. Fabrication of the Matrices. An equal volume of fibroin solution (2% w/v in SDS) was blended with CNF solutions (1% v/v TritonX100) separately. The solutions were then subjected to dialysis against 16 L of Milli-Q water for 8 h (2 L/h Milli-Q change) to remove the excess surfactants.28 The dialyzed solutions were then concentrated to 50% of total volume using 30% poly ethylene glycol (PEG) 6000 solution. The final solutions were cast on Teflon boats and dried overnight under laminar flow hood to prepare CNF reinforced fibroin thin nanocomposite films. Hereafter the samples are named as ‘AmF-0.1CNF’, ‘AmF-0.5CNF’, ‘AmF-1.0CNF’, ‘AmF-5.0CNF’ and ‘AmF-10.0CNF’, depending upon the previously mentioned five doping concentrations, where the mass ratios of AmF/CNF were 100/0.5, 100/2.5, 100/5, 100/25, and 100/50, respectively. Pure silk protein fibroin was taken as a control for every experiment and abbreviated as “AmF”. Only CNF-TritonX100 solution was also dialyzed as same as the blend solutions for using directly in some experiments. All the films were treated with 100% ethanol for 5 min followed by 70% ethanol for 15 min. Finally, the films were kept in 1X phosphate buffered saline (PBS buffer) and used for all the characterization experiments either in dry or in wet condition. 2.2. Characterizations of the Matrices. Transmission Electron Microscopy and Field-Emission Scanning Electron Microscopy. The distribution of CNF in the surfactant and in the silk fibroin blend was studied using an analytical transmission electron microscopy (TECNAI G2 20S−TWIN, FEI, USA). The samples were diluted 25 times of the original dispersion (1 mg/mL) in TritonX100 and a single drop was cast on a carbon-coated Cu grid (mesh size 300). Vacuum dried samples (24 h) were then visualized under 120 kV acceleration voltage in a TEM. Another drop was cast from the fibroin blend on a thin glass coverslip, dried, and sputter-coated with Au−Pd before visualizing and taking images under a field emission scanning electron microscope (Merlin, Zeiss, Germany). Size Distribution by Dynamic Light Scattering. The size distribution and dispersity of CNF in Triton X100 and in fibroin blend were monitored using a noninvasive backscatter dynamic light scattering instrument (Zetasizer nano ZS, Malvern). The scattering of the molecules was analyzed at room temperature using 630 nm laser and 90° detection angle. Atomic Force Microscopy for Revealing the Surface Structure. The surface nanoroughness of the nontreated as prepared and alcohol treated films (10 × 10 μm2 area) was measured using the atomic force microscope (Agilent 5100, Agilent Technologies, USA). The samples were mounted on mica plates. The nanoroughness was measured with nose cone cantilever using intermittent contact mode having ∼40 N/m constant force and ∼169.52 kHz resonant frequency. The RMS (root-mean-square) [Rq] roughness of the matrices was found by analyzing the images with PicoView Software (Agilent Technologies, USA). Contact Angle Measurement. In vitro film wettability was examined by PBS dynamic contact angle measurement setup of an optical tabletop contact angle Gonio/Tensiometer (ramè-hart Model 290, USA). The measurements were performed using sessile drop method along with different standard parameters such as room temperature, drop volume, and cleanliness of the sample. At least 3 measurements of advancing and receding angles were performed for each sample using ImageJ software (NIH, USA). Fourier Transform Infrared (FTIR) Spectroscopy. To analyze the blends and dispersion, we transcribed the IR spectra using an FTIR spectrophotometer (Nexus 870, Thermo Nicolet Corporation, WI). Pure CNF powder was mixed with potassium bromide (KBr) powder and the pellet was prepared in the form of a disc using a hydraulic press. Other test matrices were analyzed in their thin film form. The IR spectra were recorded within a range of 400−4000 cm−1 at a resolution of 4 cm−1 with

2. EXPERIMENTAL SECTION 2.1. Fabrication of the Composite. Isolation of Silk Protein Fibroin from Antheraea mylitta Silkworms. The silkworm species Antheraea mylitta was reared in our Biotechnology Departmental Farm, Indian Institute of Technology Kharagpur. The mature fifth instar larvae were collected from the garden for isolation of the silk protein fibroin. The protein was isolated and dissolved in 1% SDS buffer following the protocol described earlier.28,29 Functionalization of Carbon Nanofiber. Carbon nanofiber (Pyrograf-III, PR-24-XT-PS) was kindly gifted by Professor J. A. Szpunar. Carbon nanofiber (CNF) was functionalized using surfactant B

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ACS Applied Materials & Interfaces attenuated total reflectance probe with Germanium as an internal reflectance element. The analysis region was selected within 650−1800 cm−1. Thermogravimetric Analysis. The thermal property and degradation behavior of the nanocomposites based on different CNF/protein composition were analyzed using thermogravimetric analysis (PerkinElmer Pyris Diamond TG-DTA). The behavior of the matrices was monitored for a range of 45−700 °C with a temperature increase rate of 10 °C/min under gaseous N2 atmosphere. The initial weights of all the samples were around 9−10 mg. Three samples of each composition were considered for analysis of the data. Hall Effect to Measure the Conductivity of the Films. The electrical conductivity of the films was measured at dry condition by a four-probe method using Keithley 6514 electrometer. The experiment was operated at room temperature. The resistivity of the materials was analyzed using the following formula

Spectrum, Japan). Distilled water and 1× PBS was taken instead of samples as positive and negative control, respectively. Experiments were done in triplicates for each of the samples. 2.3. Cell Culture. Cell Culture and Maintenance. Mouse fibroblast cell line, L929 (NCCS, Pune, India) and Human dermal fibroblast cell line, HdFib (Himedia, India) were routinely maintained in DMEM media (ThermoFisher Scientific, Gibco, USA) supplemented with 10% heat-inactivated FBS (American origin, Gibco, USA), 2 mM glutamine, 100 μg/mL penicillin-streptomycin antibiotic mixture solution (Himedia, India). The cells were cultured in 75 cm2 flask and kept in a humidified incubator with 37 °C and 5% CO2 (Hera Cell 150, ThermoScientific, USA). Semiconfluent cells were detached using 1.5% trypsin-EDTA (Himedia, India) solution and seeded at an appropriate density on the matrices for different cell culture studies. Preparation of Matrices for Cell Culture. The thin films were cut into 8 mm × 8 mm small pieces. The pieces were taken in a 24 well sterile tissue culture plate (TCP) and sterilized by adding 70% ethanol to each well and left for 15 min. Subsequently, they were washed with excess amount of PBS solution to remove the traces of ethanol. Each washing step took 15 min and was performed inside laminar flow cabinet with UV radiation switched on. After third wash, matrices were conditioned with complete DMEM media and kept inside incubator until seeding. Each matrix was seeded with a density of 1000 cells for L929 and 800 cells for HdFib. Phase Contrast Microscopy. The initial attachment of the L929 cells on the solution (final dialyzed solutions) coated wells was monitored after day 1, 3, and 5 by phase contrast microscopy (Nikon Eclipse, Japan). The coating was prepared by putting the blended dialyzed solutions in the wells of a 96 well plate and dried inside the laminar hood. The rest of the washing steps were same as mentioned in section 2.3, part 2. MTT Assay. The cell viability on all the matrices were determined by MTT [3-(3,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay following manufacturer’s protocol (Sigma, USA), where the yellow dye is reduced to purple formazan crystal by cellular mitochondrial dehydrogenase activity. Briefly, each matrix was taken at different day points in a new 24-well plate and incubated with 0.5 mg/mL dye in PBS solution inside incubator under the dark condition for 4 h. After incubation, the dye was discarded carefully and the purple crystals were dissolved using DMSO. The purple supernatant solution was collected in a new 96 flat bottom microwell plate and absorbance was measured spectrophotometrically at 595 nm using a microplate reader (Thermo Scientific Multiskan Spectrum, Japan). The data of 6 values were acquired from each set of samples, plotted and analyzed using Microsoft excel. Effect of Functionalized CNF (fCNF) on the Cells by MTT Assay. Sonicated CNF solution of 10 mg/mL in TritonX100 was dialyzed extensively against Milli-Q water for 8 h as described earlier in this study. Serially diluted different concentrations of fCNF solution were prepared from the dialyzed solution using PBS such as 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.03, 0.05, 0.075, and 0.1 mg/mL. TCP only without fCNF was served as positive control. L929 cells were seeded at a density of 4000/well of a 96 well TCP. After 12 h of cell seeding, the different formulations of fCNF were added to the wells and the effect was monitored over a period of 72 h using MTT assay following same steps as described in previous section. The data were collected in triplicate at 12, 24, 48, and 72 h. Live−Dead Assay. Cell viability and proliferation on the matrices were examined by live−dead assay (Molecular Probes, Invitrogen, USA). After the fourth day of cell seeding of L929 cells and seventh day of cell seeding of HdFib cells, the matrices were incubated in a 10 mL solution mixture of 2 μM Calcein AM and 4 μM ethidium homodimer (EthD) in PBS. After incubating the samples in incubator at dark condition for 30 min, the samples were fixed using 4% paraformaldehyde and visualized using 488 nm (Calcein AM for imaging live cells) and 543 nm (EthD for imaging the dead cells) under confocal laser scanning microscope (Olympus, Fluoview 1000, Japan). Confocal Laser Scanning and Scanning Electron Microscopic Images for Cellular Morphology. Cellular distribution with actin morphology on the matrices was analyzed using confocal laser scanning

ρ (ohm − cm) = (wtR )/(d10) Where R = V/I; R = resistance, V = voltage, I = current; A = wt; w = width of the film, t = thickness of the film, and d = distance between two probes. The conductivity was then derived from the obtained resistivity by reciprocating the values. Three samples were analyzed of each category. As PBS itself is a saline solution, it is highly conductive in an electrical environment. Thus, the same experiment in wet condition is not performed. Mechanical Properties of the Matrices. The mechanical property in terms of a tensile test of the fabricated matrices was determined using a universal testing machine (UTM; Instron Electropuls; E1000). Each film strips were cut as per ASTM 638−5 standard with a size of 6 cm × 1 cm. The length between the two clamps was 2.8 cm. The thickness of the films was measured using digital slide calipers with sensitivity 0.02 mm. The films were then gripped in pneumatic grips and pulled apart with a crosshead speed of 1 mm/min using a 5 kN load cell. Tensile data were collected and evaluated in terms of tensile strength and Young’s modulus at room temperature (25 ± 5 °C) and 50 ± 5% relative humidity. Three replicates were performed for each sample. In Vitro Enzymatic Degradation Property. The film degradation property was investigated by incubating the matrices in PBS solution and in Proteinase K (Tritirachium album origin; Sigma-Aldrich, USA with specific activity: ≥30 units per mg protein) enzymatic solution. The enzyme solution (0.1 U/mL) was prepared in 1X PBS solution (pH 7.4). The study was conducted for 28 days at 37 °C. All the preweighed samples (∼120 mg) were dipped in the solutions and degraded weight was determined after drying the samples at desired incubation intervals. The solutions were changed every third day. The weight loss of each samples (with three replicates) were calculated in terms of percentage of initial weight using the following formula

%degraded weight = (Wi − Wf /Wi )100 Where Wi = initial sample weight and Wf = final degraded sample weight. The substrate degradation rate throughout the incubation period was determined in terms of mg matrix degraded per unit of enzyme per min (mg U−1 min−1). The reaction rate kinetics was determined by plotting a graph of dC (substrate degraded at a given time: C0 − Ct) versus dt (at particular time point), where C0 and Ct are the amount of substrate at initial and time t of the reaction, respectively. In Vitro Hemocompatibility (Red Blood Cell Compatibility). The anticoagulated blood was collected from the medication free healthy human volunteer donor at Dr. B. C. Roy Hospital, Indian Institute of Technology, Kharagpur. The donors’ approval was obtained in writing as per Institute ethical norms. The freshly collected blood was spun down at 700 rpm for 10 min. The plasma layer was removed and red blood cell (RBC) pellet at the bottom was washed with 1× PBS by centrifugation (at 700 rpm for 10 min) until the supernatant (PBS) becomes clear. Finally the cells were suspended in 1:4 ratio in PBS. The films were submerged completely in the diluted RBC and incubated for 2 h at 37 °C. The solutions were then centrifuged at 1200 rpm for 5 min. The supernatants were collected and the absorbance was measured at 540 nm in an Elisa plate reader (Thermo Scientific Multiskan C

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ACS Applied Materials & Interfaces microscope (Olympus, Fluoview 1000, Japan) and scanning electron microscope (Zeiss EVO 60 and Supra 40, Carl ZEISS SMT, Germany). After fourth day and seventh days of cell seeding (same as live-dead assay), the cells on the matrices were fixed using 4% paraformaldehyde at room temperature for 2 h. The cells were then permeabilized using 0.5% Triton X100 for 5 min. Permeabilized cells were then blocked using 2% BSA solution in PBS for overnight at 4 °C. Next day, the samples were incubated in Alexa fluor 488 (1:500 in PBS) for 1.5 h and subsequently in Hoechst 33258 (1:800 in PBS) for 4 min. The samples were then visualized using 350 nm (for Hoechst) and 488 nm (for Alexa fluor) lasers of a confocal microscope (CLSM). The same samples were dehydrated using graded ethanol series (30−100%) and vacuum drier. The critical point dried samples were sputter-coated with Au−Pd using a sputter coater (SC7620, Polaron, Quorum Technologies, UK) and visualized under scanning electron microscope (SEM). 2.4. Statistical Analysis. All the quantitative experiments were performed with at least 3 replications of each sample categories. The statistical significance of each experiment within the data set and among the groups was computed using one-way analysis of variance (ANOVA) and Tukey HSD posthoc test of R statistical environment, respectively. The level of significance was considered as “NS” for p > 0.05; ‘*’ for p ≤ 0.05; ‘**’ for p ≤ 0.01; ‘***’ for p ≤ 0.001; and ‘****’ for p ≤ 0.0001.

Then the local Triton X100 micelle wraps the individual CNF fiber along its free edges (Scheme 2) and resulting in homogeneous stable suspension. Bath type sonicator is not used here as requisite energy for exfoliation is not obtained and the CNF get precipitated out. Both the dispersity and the stability of the CNF increase when the solution is blended further with the SDS solubilized fibroin solution. The dispersed solution remains stable up to 24 h in Triton X100 and longer period in Triton X100 plus silk protein fibroin solution. Carbon nanofibers are graphene-based nanostructures. As purchased CNF powder does not disperse directly in distilled water or in dialyzed or undialyzed protein solution because of their hydrophobicity and high surface energy. Even after adding the CNF powder in protein solution followed by sonication, the protein gets coagulated and aggregated because of the strong shear force and molecular repulsion of the CNF. At this point, surface functionalization is required. CNF can be functionalized using several already established methods such as acid etching,31 plasma modification,32 sol−gel coating,33 electro-/electro-less plating, wet etching, photochemical functionalization, thermal treatment and addition of linker and polymer molecule.16 Most of these methods are quite complicated and not preferred as they are expensive. Also, harsh chemicals and/or harsh treatments do not support the final purpose of cytocompatibility. We thus follow the cheap, green and easy methods of ultrasonication (mechanical) and noncovalent surfactant absorption (chemical) methods to functionalize the CNF. Fibroin protein is dissolved in SDS buffer, which is an anionic surfactant. The dissolved protein solution also serves as a surfactant. Triton X100 is a nonionic surfactant. CNF interacts with Triton X100 molecules with noncovalent π−π interaction and forms a more stable dispersion up to 10 mg/mL for a longer time than SDS, Tween 20 and only water (data not shown). This finding is similar to another reported method by Rastogi et al, 2008.34 The stability increases more when the solution is blended with the SDS-solubilized

3. RESULTS AND DISCUSSION 3.1. Fabrication of the Composites. The rationale behind this study is to evaluate the potential of carbon nanofiber along with natural biopolymer in relation to the applications focused on tissue bioengineering. The composite fabrication process is illustrated in the Scheme 1. Five test compositions are prepared randomly (from 0.1 mg/mL to 10 mg/mL) by determining the maximum dispersibility of the functionalized carbon nano fiber (CNF) within Triton X100. Pure nonmulberry A. mylitta silk protein fibroin made films are prepared as a positive control without CNF. From the schematic diagram, it can be noted that CNF forms a homogeneous dispersion within the surfactant after sonication. The enough shear energy is created by the probe, which exfoliates individual CNF fiber from the CNF bundle.

Scheme 1. Schematic Representation of the Process of Fabrication of Carbon nanofiber (CNF) Reinforced Nonmulberry Silk Protein Fibroin (AmF) Nanocomposite Films

D

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Scheme 2. Schematic Illustration of Molecular Arrangements of Functionalized CNF and Fibroin Protein in Individual Solutions, Blended Solution, and in Composite Film

protein solution (2% w/v). The large protein molecules noncovalently wind up the nanofibers and make a homogeneous solution upon blending35 (Scheme 2). 3.2. Matrix Characterizations. Distribution and Dispersion of CNF in Surfactant and Protein Solution. The distribution of the CNF within the solutions was observed under TEM and FESEM. Figure 1a−c shows the TEM images of the CNF dispersion within the surfactant and surfactant plus protein. Figure 1d shows the FE-SEM image of a single fiber within the fibroin thin layer coating on glass coverslip. CNF needs proper functionalization as they possess high bundle forming tendency due to strong van der Waals force of attraction.35 For making a perfect dispersion, strong shear flow such as ultrasonication is required. The resulting solution after sonication shows a good dispersion of CNF in the surfactant as observed from the TEM image (Figure 1a). Individual exfoliated fiber can be seen in completely detangled form. The stability of the CNF in the form of individual fiber within the fibroin solution can also be observed in Figure 1c. The empty protein bed without CNF looks like thin layer having the carbon coating of Cu grid as a border Figure 1b. The images also reveal the size of the nanofibers as an average diameter of 100 nm with variable length, which is similar as mentioned in the product data sheet (PYROGRAF -III CARBON NANOFIBER). The size distribution of CNF within the solutions is monitored by measuring the fluctuations of scattered laser. This study shows that upon blending, monodispersions of protein (z-average diameter 25 nm) and CNF (z-average diameter 81 nm) become a polydispersed mixture of 22 and 97 nm, respectively (Figure 1e). The size of CNF increases because protein molecules entrap the

CNF inside its micelle. Protein micelles are also present in the mixture with the almost similar size of the monodispersed solution. The slight change in peak position may be due to the repulsion and/or interaction of Triton X100 with the protein micelles.36 Surface Roughness. The degree of surface irregularity exerts a great influence on the cellular adherence on it. AFM study provides additional information about phase state and topography of the film surface. The surface nanoroughness of the blended 2D film matrices appears to change qualitatively as well as quantitatively with root-mean-square values (Figure 1f−m) in comparison to the pure fibroin matrix with unaltered microroughness. After modification by means of both CNF doping (Figure 1g−i) and ethanol treatment, the surface becomes more uneven (Figure 1j−m) with protrusions of pits and valleys relative to the pure films (Figure 1f). The study concludes that both the treatments caused a change in the surface nanoroughness of the composite matrices, which is a good parameter for cells to adhere on the surface. The nanorough surface (with roughness 5%, then the sample can be considered as nonhemolytic, slightly

Figure 4. Analysis of the degradation behavior of the fabricated AmF, AmF-0.1CNF, AmF-0.5CNF, AmF-1.0CNF, AmF-5.0CNF, and AmF-10.0CNF nanocomposite matrices by (a) incubation in PBS pH 7.4 and (b) in Proteinase K solution (0.1 U/ml PBS pH 7.4) for 28 days. The degradation values are plotted in terms of percentage of degraded weights at different day points. (c) Plot of degradation rate of the matrices (mg U−1, min−1) versus time (t) and (d) plot of degraded substrate (mg) vs time (t) with zero-order rate constant (k) for the pure and composite matrices. I

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The initial mouse fibroblast cell adherence on the solution coated plates are monitored under phase contrast microscope. From the images (Figure S1), it is seen that the cells attach on all the composite coatings. The morphology of the cells is comparable to the control AmF one, except the AmF-10.0 CNF composite. In this coating, very few number of cells spread their actin filament whereas maximum cells are round shaped. They do not spread on the matrix properly and also not divided and not distributed throughout the matrix. This observation is further verified by MTT assay. From Figure 5a, b, the proliferation rates of the mouse fibroblast and human dermal fibroblast on the TCP, AmF, AmF-0.1CNF, AmF-0.5CNF, and AmF-1.0CNF are observed as comparable. The proliferation rates on AmF5.0CNF and AmF-10.0CNF are not very promising. Human dermal fibroblast is a slow growing cell line and comparatively larger in size than L929. So less number of cells for seeding and longer culture time are adopted. In the initial 4 days, there is no significant difference in their growth pattern on all the matrices. Later on, a significant difference is observed for last two compositions, where a decrease in growth pattern is observed. The same trend is also observed in the case of L929 cells. After the fifth day of culture, the cells gradually die on the matrices (AmF, AmF-0.1CNF, AmF-0.5CNF, and AmF-1.0CNF) due to overgrowth and lack of space, except TCP and last two compositions (AmF-5.0CNF and AmF-10.0CNF). A significantly slower growth rate is observed on AmF-5.0CNF and AmF10.0CNF. The effect of functionalized carbon nanofiber directly on the cells is analyzed to answer the question of cytotoxicity of the CNF. The composites may degrade and release CNF particles

7 days and subsequently the trend reaches the plateau with gradual decrease in the rate of degradation (Figure 4c). The surface catalyzed reaction model is suitable for the analysis of the degradation behavior of the nanocomposite films. The substrate degradation rate versus time for a constant amount of enzyme generally follows a linear pattern. The linearity of the reactions is confirmed by analyzing the data with zero-, first-, and second-order reaction kinetics. In our case, the reaction parameters are well supported by zero-order kinetics. The enzymatic degradation reaction of the pure and composite matrices is analyzed using this kinetics. From the plot of dC vs dt (Figure 4d) the rate constant is found to be same (k = 0.0022 mg/min) for AmF, AmF-0.1CNF, and AmF-0.5CNF. For subsequent reinforced samples, the rate constant becomes lower. The enzyme specifically can degrade only the fibroin protein but not the CNF as CNF does not contain the enzyme-specific cleavage site in it. The CNF effectively hinders the protein molecules making the composites more stable. 3.3. Cell Culture Experiments. Cell Viability. In spite of having excellent biophysical properties, still questions about biocompatibility or cytocompatibility of carbon nanofibers and tubes are not properly answered. Different research groups mention different positive51,52 or negative results53 while working with CNFs and/or CNTs as biomaterials. A green process of fabrication of CNF doped inherent cell adhesion promoting in-built RGD tripeptide containing fibroin nanocomposite is adopted to know their biological responses. Two types of fibroblast cells (i.e., mouse and human origin) are chosen for this purpose to study their behavior on these fabricated and yet unknown materials.

Figure 5. Analysis of the toxicity of the fabricated matrices through MTT assay using (a) human dermal fibroblast (HdFib) cell line and (b) mouse fibroblast (L929) cell line. Data plotted as n = 6, mean ± SE. (c) The cytotoxicity analysis of the functionalized CNF through MTT assay using L929 cell line. The direct effect for different concentration doses of CNF (ranging from 0 to 0.1 mg per mL of media) on the cells is monitored for up to 72 h. Data plotted as n = 3, mean ± SE. J

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Figure 6. Cellular viability analyses on TCP, AmF, AmF-0.1CNF, AmF-0.5CNF, and AmF-1.0CNF matrices in terms of Live/Dead assay using confocal laser scanning microscope (CLSM). The fluorescence micrographs of the matrices are captured (a) after 7 days of culture of human dermal fibroblast (HdFib) cells and (b) 4 days of culture of mouse fibroblast (L929) cells. The 488 nm laser and 543 nm laser are used to detect the green live cells (to detect Calcein AM) and red dead cells (to detect ethidium homodimer) respectively. The left and right panel of each group represents the fluorescent Live/Dead overlapped and DIC/live/dead merged images, respectively. Magnification = 20× and scale bar = 100 μm. K

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Figure 7. Visualization of the detailed cell structure and actin stress fiber distribution on the nanocomposites using (a) the confocal laser scanning microscope (CLSM) and (b) The scanning electron microscope (SEM) (both left column: human dermal fibroblast (HdFib) and right column: mouse fibroblast (L929) cells). The cells are stained with Alexa fluor 488 and Hoechst 33258 at day point 7 (for HdFib) and day point 4 (for L929) for CLSM imaging and gold sputtered for SEM imaging. The 488 and 350 nm lasers are used to detect the green fluoresced actin and blue fluoresced nucleus. CLSM magnification = 20× with scale bar = 100 μm; SEM magnification = 1000× with scale bar = 20 μm.

either as free or may be embeded within fibroin fragments when placed inside an in vivo system. Different doses of functionalized CNF concentrations are chosen randomly from 0.0001 to 0.1 mg per ml of culture media. From the absorption graph (Figure 5c), no drastic cell death is observed in all the CNF formulations.

As such, no detrimental effect of CNF is found. With the increase in CNF concentration, the cell proliferation rate decreases for some unknown reason. The cytotoxicity and bioactivity of the carbon materials depend on the method of synthesis, proper functionalization/solubilization, geometric shape/size, and purity L

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nonmulberry silk (Antheraea mylitta) protein fibroin matrix using easy functionalization and green aqueous-based method. The effect of the introduction of CNF reinforcement in the fabricated fibroin nanocomposites is demonstrated for the first time biophysically and biochemically for their potential as a biomaterial. The CNF reinforced nanocomposites are found to be superior to pure fibroin matrix in terms of hydrophilicity, tensile modulus, electrical conductivity, and thermal stability. From the conductivity study, the critical filler concentration is found to be 5 mg of CNF per ml of 2% w/v silk protein fibroin (with mass ratio 25:100). The nanocomposite films are cytocompatible if reinforcement is made up to 1 mg/mL of 2% w/v protein (with mass ratio 5:100). Aqueous base functionalized CNF does not possess vulnerable effect to the cells when administered directly in in vitro conditions. The identified characteristics of the CNF based fibroin nanomatrices may lead them to be engineered in different forms of supporting scaffolds for conductive and load bearing tissue (such as nerve, muscle, and bone tissue) regeneration as well as small biomolecule delivery vehicle. This study delivers a very explorative preliminary idea about the possible multifunctionality and candidature of the nanobiocomposites in biomedical and healthcare fields.

of the molecules.25 It is previously reported that moderately soluble aqueous functionalized CNT (fCNT) suspension preserve the functionality of primary immune cells by stimulating the secretion of proinflammatory cytokines by the macrophages while the highly soluble aqueous fCNT does not induce the immune cells.26 The in vivo study shows that the multiwall CNT (MWCNT) molecules are engulfed by the alveolar macrophages and located to the peritracheal lymph nodes and spleen when it is administered through inhalation by rat.25 In another in vivo study, the stereotactically injected MWCNT molecules into the mouse brain cortex are severely deformed and degraded by the microglia cells present in the local brain tissue.27 It is already mentioned that CNF is larger in size, more pure than CNT, easily functionalized and solubilized (PYROGRAF -III CARBON NANOFIBER). These may lead them as less toxic to the cells in comparison to CNT. It may be expected that the degraded CNF may follow the lymphatic clearance pathway without eliciting major immune response of the body as per the above-mentioned reported studies. The viability of the cells is also determined by the qualitative method. The aliveness and mortality of the cells grown on the matrices are observed by staining their esterase activity fluorescently. The more green color influenced by calcein indicates more esterase activity of the live cells. The dead cells appear as red for binding the EthD to the dead nuclei. From the live dead images (Figure 6a, b), a maximum number of live cells are visible on the TCP, AmF and first three composites (AmF-0.1CNF, AmF-0.5CNF, and AmF-1.0CNF) after fourth day of culture for L929 cells and seventh day of culture for HdFib cells. Interestingly, no dead cells are found on all these supports. Mass green color sheet like detached extracellular matrix is observed for L929 cells from the matrices after fifth day of culture (which can be seen after 4 th day on AmF-0.5CNF and AmF1.0CNF). This signifies that the matrices aid the survival of the cells. In AmF-5.0CNF and AmF-10.0CNF composites, less number of viable cells are observed. These two composites do not enhance significant cell growth and proliferation may be due to the less availability of the cryptic -R-G-D- tripeptide integrin binding sequence of fibroin and more availability of the CNF on the matrix surface. The matrices do not exert any toxicity as there is no mass death of cells; instead, a slow growth rate is observed. This result is similar to the effect of fCNF on the cells. Cellular Morphology. The morphology and organization of the cell structure are directly visualized and analyzed from fluorescence and scanning electron micrograph images (Figure 7a, b). For confocal based line scanning images, the fluorescent stains Alexa fluor 488 (green fluorescent stain for cellular F-actin) and Hoechst 33258 (blue fluorescent stain for nucleus) are used. The morphometric analysis shows a maximum number of broader and flatter cells with uniform cytoskeletal filaments along with regular cell−cell filopodic interacting appendages on TCP, AmF, AmF-0.1CNF, AmF-0.5CNF, and AmF-1.0CNF composites. The tendency of forming an extracellular matrix by the cells can also be observed on these matrices. The two remaining compositions (AmF-5.0CNF and AmF-10.0CNF) show a fewer number of cells with poor, abrupt, and radically distinct F-actin stress fiber distribution without any cell−cell interaction. This finding supports the data obtained in the Live/Dead assay (Section 3.3, part3).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b04777. Figure S1, phase contrast micrograph of L929 cell attachment on different fibroin/CNF solution coated tissue culture surfaces; Tables S1 and S2, summaries of some relevant work from throughout the world related to this work (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +91 3222 283762. Fax: +91 3222 278700. *E-mail: [email protected]. Tel.: +91 3222 283764. Fax: +91 3222 278700. Author Contributions

D.N. designed the study. D.N. and P.B. performed the experiments and data analysis and drafted the manuscript. A.K.G., M.M., and S.C.K supervised the research. All the authors reviewed and revised the manuscript. Funding

Department of Biotechnology (BT/PR10941/MED/32/333/ 2014) and Indian Council of Medical Research (5/13/12/2010/ NCD-III), Govt. of India. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by Department of Biotechnology and Indian Council of Medical Research, Government of India. We are thankful to Professor J. A. Szpunar (Department of Mechanical Engineering, University of Saskatchewan, Canada) for gifting us the carbon nanofiber and helpful scientific discussion with Deboki during her stay in his laboratory; Ms. Sunaina Sapru and Mr. Sibaram Behera of our laboratory for scientific comments and suggestions during the course of investigation.

4. CONCLUSION In this study, carbon nanofiber (CNF) is exploited as a reinforcement filler nanomaterial and successfully doped within M

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