Biocompatibility effects of biologically synthesized graphene in primary mouse embryonic fibroblast cells
© Gurunathan et al.; licensee Springer. 2013
Received: 10 July 2013
Accepted: 2 September 2013
Published: 23 September 2013
Due to unique properties and unlimited possible applications, graphene has attracted abundant interest in the areas of nanobiotechnology. Recently, much work has focused on the synthesis and properties of graphene. Here we show that a successful reduction of graphene oxide (GO) using spinach leaf extract (SLE) as a simultaneous reducing and stabilizing agent. The as-prepared SLE-reduced graphene oxide (S-rGO) was characterized by ultraviolet–visible spectroscopy and Fourier transform infrared spectroscopy. Dynamic light scattering technique was used to determine the average size of GO and S-rGO. Scanning electron microscopy and atomic force microscopy images provide clear surface morphological evidence for the formation of graphene. The resulting S-rGO has a mostly single-layer structure, is stable, and has significant water solubility. In addition, the biocompatibility of graphene was investigated using cell viability, leakage of lactate dehydrogenase and alkaline phosphatase activity in primary mouse embryonic fibroblast (PMEFs) cells. The results suggest that the biologically synthesized graphene has significant biocompatibility with PMEF cells, even at a higher concentration of 100 μg/mL. This method uses a ‘green’, natural reductant and is free of additional stabilizing reagents; therefore, it is an environmentally friendly, simple, and cost-effective method for the fabrication of soluble graphene. This study could open up a promising view for substitution of hydrazine by a safe, biocompatible, and powerful reduction for the efficient deoxygenation of GO, especially in large-scale production and potential biomedical applications.
KeywordsAtomic force microscopy Biocompatibility Graphene oxide Graphene Scanning electron microscopy UV-visible spectroscopy
Recently, carbon-based nanomaterials such as carbon nanotubes, graphene oxide, and graphene have been explored extensively by researchers as well as the industry. Graphene is an emerging nanomaterial which has greater scientific and commercial advantages. Recently, single-layer and few-layer graphenes received great interest due to its exceptional characteristics including high surface area as well as strong electronic, mechanical, thermal, and chemical properties in various fields such as materials science, physics, chemistry, biotechnology, and nanomedicine [1–3]. Particularly, graphene has been attracted to the scientific community for numerous potential applications in biotechnology, including biosensing [4, 5], disease diagnostics , antibacterial [7–11] and antiviral materials , cancer-targeting  and photothermal therapy [14, 15], drug delivery [16–18], and tissue engineering [19, 20] due to these unique physical, chemical, and biocompatibility properties. During the past few years, several procedures have been established for the synthesis of graphene and its derivatives, including mechanical exfoliation, epitaxial growth, unzipping carbon nanotubes, exfoliation of GO, and liquid-phase exfoliation of graphite . Moreover, several other methods were implemented to prepare high-quality graphene such as chemical vapor deposition onto thin films of metal, epitaxial growth on electrically insulating surfaces like silicon carbide, and the scotch tape method . All of these methods can produce highly crystalline graphene but are not suitable for mass production [22, 23]. Several researchers have attempted to propose environmentally friendly and green approach including flash photo reduction  hydrothermal dehydration , solvothermal reduction , and catalytic  and photocatalytic reduction . The most promising method for the large-scale production of graphene is the chemical oxidation of graphite, conversion of the resulting graphite oxide to GO, and subsequent reduction of GO. The exfoliation of GO is one of the well-established methods for the mass production of graphene in the presence of some chemical reducing agents such as hydrazine and sodium borohydride [27, 28]. The usage of strong chemical reducing agents such as hydrazine is found to be corrosive, highly explosive, and highly toxic . In addition, hydrazine seems to be a hepatotoxic and carcinogenic agent in the kidney, and liver damage can result in blood abnormalities, irreversible deterioration of the nervous system, and even DNA damage . In this context, many studies used the green chemistry approach for the reduction of GO to overcome the toxicity problem using various biological molecules as reducing agents such as vitamin C , melatonin , sugars , polyphenols of green tea [34, 35], bovine serum albumin , and biomass of bacteria [37, 38]. The biologically derived graphene nanomaterials are biocompatible, stable, and soluble.
Biocompatibility is an essential factor for tissue engineering applications. Recent studies suggest that the biocompatibility of carbon-based nanomaterials depends strongly on mass, purity, ratio, and surface functional groups. A variety of biological applications depend on the functionalization of graphene. The ability of the functionalization of graphene and its derivatives brought the attention of nanomaterials in various applications including biosensors and tissue engineering. Several studies have reported the biocompatibility of graphene derivatives in proximity of mammalian cells. Biris et al.  demonstrated that osteoblast cells (MC3T3-E1) have a high ability to grow on graphene film. Agarwal et al.  reported that reduced graphene oxide (rGO) is more biocompatible than single-wall carbon nanotubes using different cell lines including neuroendocrine PC12 cells, oligodendroglia, or osteoblasts. Recently, Gurunathan and coworkers reported that microbially reduced graphene oxide shows significant biocompatibility with primary mouse embryonic fibroblast (PMEF) cells. Chen et al.  cultured PC12 cells with carbon nanotubes and rGO films with the same initial seeding density for 5 days, and the results suggest that the cells cultured with rGO enhanced proliferation, whereas nanotubes inhibited the proliferation of cells. Nayak et al.  reported that G-coated substrates accelerated osteogenic differentiation of human mesenchymal stem cells (hMSCs) compared to uncoated substrates. Lee et al.  reported that GO films enhanced adipogenesis of hMSCs due to their high affinity with insulin. Chen et al.  reported that G-coated substrates maintained induced pluripotent stem cells in the undifferentiated state.
Regarding synthesis of nanomaterials, various phytochemicals have been used from different natural sources like leaves, peels, roots, seeds, and other parts of plants as reducing agents for the synthesis of different metal nanoparticles like silver and gold [44–47]. Here we attempted to use spinach leaves because it is nontoxic and an edible plant which has high nutritional value and is extremely rich in antioxidants; therefore, the leaf extracts of spinach could be potential alternative reducing agents for the synthesis of soluble graphene. In the present report, we investigated a greener approach for the reduction of GO using spinach leaf extracts (SLE), and also we analyzed the biocompatibility effect of SLE-reduced graphene oxide (S-rGO) in PMEFs.
Graphite powder was purchased from Sigma-Aldrich (St. Louis, MO, USA). NaOH, KMnO4, anhydrous ethanol, 98% H2SO4, 36% HCl, and 30% hydrogen peroxide aqueous solution were purchased from Sigma-Aldrich (USA) and used directly without further purification. All aqueous solutions were prepared with deionized (DI) water. All other chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) unless stated otherwise.
Spinach leaves were obtained from the local market and stored at 4°C until needed. Twenty grams of spinach leaves was washed thoroughly with double distilled water and was then sliced with a sharp stainless steel knife into fine pieces, about 1 to 5 cm2. The finely cut spinach leaves were mixed in 100 mL of sterile distilled water and then boiled for 2 min. After boiling, it was filtered through Whatman filter paper no. 1. Further, the extracts were used for synthesis of graphene. The extracts were stored at 4°C until further use.
Synthesis of GO
Natural graphite powder was utilized as the raw material to prepare graphite oxide by suspension through a modified Hummers’ method  and according to Esfandiar et al. . The prepared graphite oxide powder was dispersed in DI water to obtain an aqueous graphite oxide suspension with a yellow-brownish color. The suspension was centrifuged at 3,000 rpm/min for 10 min to eliminate unexfoliated graphitic plates and then at 10,000 rpm/min for 10 min to remove tiny graphite particles. Finally, a GO suspension was achieved by exfoliation of the filtered graphite oxide suspension through its sonication. Reduction of graphene oxide was followed as described earlier  with slight modification.
Synthesis of reduced graphene oxide
Reduced graphene oxide was obtained from the reaction of a plant extract with graphene oxide. In the typical reduction experiment, 10 mL of spinach leaf extract was added to 40 mL of 0.5 mg/mL aqueous GO solution and then the mixture was kept in a tightly sealed glass bottle and stirred at 30°C for 24 h. Then, using a magneto-stirrer heater, reduced graphene oxide suspension was stirred at 400 rpm at a temperature of 30°C for 30 min. A homogeneous S-rGO suspension was obtained without aggregation. Then, the functionalized S-rGO was filtered and washed with DI water. Finally, a black S-rGO dispersion was obtained.
Ultraviolet–visible (UV–vis) spectra were obtained using a WPA (Biowave II, Biochrom Cambridge, UK). The aqueous suspension of GO and S-rGO was used as UV–vis samples, and deionized water was used as the reference. The particle size of dispersions was measured by Zetasizer Nano ZS90 (Malvern Instruments Limited, Malvern, UK). X-ray diffraction (XRD) analyses were carried out on an X-ray diffractometer (Bruker D8 DISCOVER, Bruker AXS GmBH, Karlsruhe, Germany). The high-resolution XRD patterns were measured at 3 kW with Cu target using a scintillation counter, and λ = 1. 5406 A at 40 kV and 40 mA was recorded in the range of 2θ = 5° − 80°. The changes in the surface chemical bonding and surface composition were characterized using a Fourier transform infrared spectroscopy (FTIR) instrument (PerkinElmer Spectroscopy GX, Branford, CT, USA). A JSM-6700F semi-in-lens FE-SEM operating at 10 kV was used to acquire SEM images. The solid samples were transferred to a carbon tape held by an SEM sample holder for analyses. The analyses of the samples were carried out at an average working distance of 6 mm. Raman spectra of graphene oxide and reduced graphene oxide were measured by WITec Alpha300 (Ulm, Germany) with a 532-nm laser. The calibration was initially made using an internal silicon reference at 500 cm−1 and gave a peak position resolution of less than 1 cm−1. The spectra were measured from 500 to 4,500 cm−1. All samples were deposited on glass slides in powder form without using any solvent. Surface images were measured using tapping-mode atomic force microscopy (SPA 400, SEIKO Instruments, Chiba, Japan) operating at room temperature. Height and phase images were recorded simultaneously using nanoprobe cantilevers (SI-DF20, SEIKO Instruments).
Exposing PMEFs to GO and S-rGO
PMEF cells were routinely cultured in Dulbecco’s modified Eagle’s low-glucose medium (DMEM/low, Gibco BRL Life Technologies, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum, plus 2 mM of l-glutamine, and 1% (v/v) penicillin-streptomycin (10 U mL−1 penicillin and 0.1 mg mL−1 streptomycin) and grown at 37°C in a 5% CO2 humidified environment. When the cells had reached 70% confluence, they were trypsinized (0.25% trypsin and 0.04% EDTA, Sigma-Aldrich) and passaged (1:3). Cells within three passages were used for experiments. GO or S-rGO suspensions were freshly prepared before the cells were exposed and diluted to appropriate concentrations from 20 to 100 μg mL−1 with the culture medium; they were then immediately applied to the cells. DMEM without GO and S-rGO supplements served as a negative control in each experiment.
Cell viability assay
WST-8 assay was followed as described earlier by Liao et al. . Typically, 1 × 104 cells were seeded in a 96-well plate and cultured in DMEM supplemented with 10% at 37°C under 5% CO2. After 24 h, the cells were washed with 100 μL of serum-free DMEM two times and incubated with 100 μL of different concentrations of GO or S-rGO suspensions in serum-free DMEM. After a 24-h exposure, the cells were washed twice with serum-free DMEM, and 15 μL of WST-8 solution was added to each well containing 100 μL of serum-free DMEM. After 1 h of incubation at 37°C under 5% CO2, 80 μL of the mixture was transferred to another 96-well plate because residual GO or S-rGO can affect the absorbance values at 450 nm. The absorbance of the mixture solutions was measured at 450 nm using a microplate reader. Cell-free control experiments were performed to see if GO and rGO react directly with WST-8 reagents. Typically, 100 μL of GO or S-rGO suspensions with different concentrations (20 to 100 μg/mL) was added to a 96-well plate and 10 μL of WST-8 reagent solution was added to each well; the mixture solution was incubated at 37°C under 5% CO2 for 1 h. After incubation, GO or S-rGO was centrifuged and 50 μL of the supernatant was transferred to another 96-well plate. The optical density was measured at 450 nm.
Cell membrane integrity of PMEF cells was evaluated by determining the activity of lactate dehydrogenase (LDH) leaking out of the cell according to manufacturer’s instructions (in vitro toxicology assay kit, TOX7, Sigma-Aldrich). The LDH assay is based on the release of the cytosolic enzyme, LDH, from cells with damaged cellular membranes. Thus, in cell culture, the course of GO- and S-rGO-induced cytotoxicity was followed quantitatively by measuring the activity of LDH in the supernatant. Briefly, cells were exposed to various concentrations of GO and S-rGO for 24 h, and then 100 μL per well of each cell-free supernatant was transferred in triplicates into wells in a 96-well plate, and 100 μL of LDH assay reaction mixture was added to each well. After 3 h of incubation under standard conditions, the optical density of the color generated was determined at a wavelength of 490 nm using a microplate reader.
Alkaline phosphatase activity
PMEF cells were cultured in a 48-well culture dish at a density of 5 × 103 cells per well for 4 days. Then medium was replaced with treatment solution, which was DMEM containing 5% serum plus GO or S-rGO. After 4 days, the alkaline-phosphatase activity was measured according to the method described by manufacturer’s instructions (DALP-250, QuantiChrom™ Alkaline Phosphatase Assay Kit, Gentaur, Belgium). The plates were incubated at 37°C for 30 min. The amount of released p-nitrophenol was measured at 405 nm in a 96-well microplate reader. Enzyme activity was evaluated as the amount of nitrophenol released through the enzymatic reaction, and absorbance was recorded using a microplate reader (Bio-RAD 680, Hercules, CA, USA) at 405 nm. For normalization, the total protein content was measured using a bicinchoninic acid protein assay kit. Thus, the alkaline phosphatase (ALP) activity was expressed and normalized by the total protein content (U/mg).
Results and discussion
Reduction of GO by SLE
Raman spectroscopy study
Biocompatibility of S-rGO
Impact of GO and S-rGO on membrane integrity
Impact of GO and S-rGO on ALP activity
We demonstrated a simple and green approach for the synthesis of water-soluble graphene using spinach leaf extracts. The transition of GO to graphene was confirmed by various analytical techniques such as UV–vis spectroscopy, DLS, FTIR, SEM, and AFM. Raman spectroscopy studies confirmed that the removal of oxygen-containing functional groups from the surface of GO led to the formation of graphene with defects. The obtained results suggest that this approach could provide an easy technique to produce graphene in bulk quantity for generating graphene-based materials. In addition, SLE can be used as an alternative reducing agent compared to the widely used and highly toxic reducing agent called hydrazine. Further, the cells treated with S-rGO show a significant compatibility with PMEF cells in various assays such as cell viability, LDH leakage, and ALP activity. The significance of our findings is due to the harmless and effective reagent, SLE, which could replace hydrazine in the large-scale preparation of graphene. The biocompatible properties of SLE-mediated graphene in PMEFs could be an efficient platform for various biomedical applications such as the delivery of anti-inflammatory and water-insoluble anticancer drugs, and also it can be used for efficient stem cell growth and differentiation purposes.
This paper was supported by the SMART-Research Professor Program of Konkuk University. Dr. Sangiliyandi Gurunathan was supported by Konkuk University SMART-Full time Professorship. This work was supported by Woo the Jang Choon project (PJ007849) and next generation of Biogreen 21 (PJ009625).
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