Graphene and carbon nanotube nanocomposite (GCN) was synthesised and applied in gene transfection of pIRES plasmid conjugated with green fluorescent protein (GFP) in NIH-3T3 and NG97 cell lines. The tips of the multi-walled carbon nanotubes (MWCNTs) were exfoliated by oxygen plasma etching, which is also known to attach oxygen content groups on the MWCNT surfaces, changing their hydrophobicity. The nanocomposite was characterised by high resolution scanning electron microscopy; energy-dispersive X-ray, Fourier transform infrared and Raman spectroscopies, as well as zeta potential and particle size analyses using dynamic light scattering. BET adsorption isotherms showed the GCN to have an effective surface area of 38.5 m2/g. The GCN and pIRES plasmid conjugated with the GFP gene, forming π-stacking when dispersed in water by magnetic stirring, resulting in a helical wrap. The measured zeta potential confirmed that the plasmid was connected to the nanocomposite. The NIH-3T3 and NG97 cell lines could phagocytize this wrap. The gene transfection was characterised by fluorescent protein produced in the cells and pictured by fluorescent microscopy. Before application, we studied GCN cell viability in NIH-3T3 and NG97 line cells using both MTT and Neutral Red uptake assays. Our results suggest that GCN has moderate stability behaviour as colloid solution and has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity and good transfection efficiency. © 2014 Elsevier B.V.391288298Vardharajula, S., Ali, S.Z., Tiwari, P.M., Eroglu, E., Vig, K., Dennis, V.A., Functionalized carbon nanotubes: Biomedical applications (2012) Int. J. Nanomedicine, 7, pp. 5361-5374Zhou, X., Laroche, F., Lamers, G.E.M., Torraca, V., Voskamp, P., Lu, T., Ultra-small graphene oxide functionalized with polyethylenimine (PEI) for very efficient gene delivery in cell and zebrafish embryos (2012) Nano Res., 5, pp. 703-709Mastrobattista, E., Van Der Aa, M.A., Hennink, W.E., Crommelin, D.J., Artificial viruses: A nanotechnological approach to gene delivery (2006) Nat. Rev. Drug Discov., 5, pp. 115-121Lobo, A.O., Corat, M.A.F., Antunes, E.F., Ramos, S.C., Pacheco-Soares, C., Corat, E.J., Cytocompatibility studies of vertically-aligned multi-walled carbon nanotubes: Raw material and functionalized by oxygen plasma (2012) Mater. Sci. Eng. C-Mater., 32, pp. 648-652Putnam, D., Polymers for gene delivery across length scales (2006) Nat. Mater., 5, pp. 439-451Ryoo, S.R., Kim, Y.K., Kim, M.H., Min, D.H., Behaviors of NIH-3T3 fibroblasts on graphene and carbon nanotubes: Proliferation, focal adhesion, and gene transfection studies (2010) ACS Nano., 4, pp. 6587-6598Misra, S.K., Kondaiah, P., Bhattacharya, S., Rao, C.N., Graphene as a nanocarrier for tamoxifen induces apoptosis in transformed cancer cell lines of different origins (2012) Small, 8, pp. 131-143Harrison, B.S., Atala, A., Carbon nanotube applications for tissue engineering (2007) Biomaterials, 28, pp. 344-353Kaya, C., Singh, I., Boccaccini, A.R., Multi-walled carbon nanotube-reinforced hydroxyapatite layers on Ti6Al4V medical implants by electrophoretic deposition (EPD) (2008) Adv. Eng. Mater., 10, pp. 131-138He, S., Song, B., Li, D., Zhu, C., Qi, W., Wen, Y., A Graphene nanoprobe for rapid, sensitive, and multicolor fluorescent DNA analysis (2010) Adv. Funct. Mater., 20, pp. 453-459Jang, H., Kim, Y.K., Kwon, H.M., Yeo, W.S., Kim, D.E., Min, D.H., A graphene-based platform for the assay of duplex-DNA unwinding by helicase (2010) Angew. Chem. Intern. Ed., 49, pp. 5703-5707De La Zerda, A., Zavaleta, C., Keren, S., Vaithilingam, S., Bodapati, S., Liu, Z., Carbon nanotubes as photoacoustic molecular imaging agents in living mice (2008) Nat. Nanotechnol., 3, pp. 557-562Liu, Z., Robinson, J.T., Sun, X., Dai, H., PEGylated nanographene oxide for delivery of water-insoluble cancer drugs (2008) J. Am. Chem. Soc., 130, pp. 10876-10877Hu, W., Peng, C., Luo, W., Lv, M., Li, X., Li, D., Graphene-based antibacterial paper (2010) ACS Nano., 4, pp. 4317-4323Park, K.H., Chhowalla, M., Iqbal, Z., Sesti, F., Single-walled carbon nanotubes are a new class of ion channel blockers (2003) J. Biol. Chem., 278, pp. 50212-50216De Andrade, L.R., Sandin Brito, A., De Souza Melero, A.M.G., Zanin, H., Jose Ceragioli, H., Baranauskas, V., Silva Cunha, K., Pierre Irazusta, S., Absence of mutagenic and recombinagenic activity of multi-walled carbon nanotubes in the Drosophila wing-spot test and Allium cepa test (2014) Ecotoxicol. Environ. Saf., 99, pp. 92-97Bottini, M., Bruckner, S., Nika, K., Bottini, N., Bellucci, S., Magrini, A., Multi-walled carbon nanotubes induce T lymphocyte apoptosis (2006) Toxicol. Lett., 160, pp. 121-126Worle-Knirsch, J.M., Pulskamp, K., Krug, H.F., Oops they did it again! Carbon nanotubes hoax scientists in viability assays (2006) Nano Lett., 6, pp. 1261-1268Casey, A., Davoren, M., Herzog, E., Lyng, F.M., Byrne, H.J., Chambers, G., Probing the interaction of single walled carbon nanotubes within cell culture medium as a precursor to toxicity testing (2007) Carbon, 45, pp. 34-40Casey, A., Herzog, E., Davoren, M., Lyng, F.M., Byrne, H.J., Chambers, G., Spectroscopic analysis confirms the interactions between single walled carbon nanotubes and various dyes commonly used to assess cytotoxicity (2007) Carbon, 45, pp. 1425-1432Hurt, R.H., Monthioux, M., Kane, A., Toxicology of carbon nanomaterials: Status, trends, and perspectives on the special issue (2006) Carbon, 44, pp. 1028-1033Zanin, H., Peterlevitz, A.C., Ceragioli, H.J., Rodrigues, A.A., Belangero, W.D., Baranauskas, V., Magnetic and cytotoxic properties of hot-filament chemical vapour deposited diamond (2012) Mater. Sci. Eng. C-Mate. Biol. Appl., 32, pp. 2340-2343Casey, A., Herzog, E., Davoren, M., Lyng, F.M., Byrne, H.J., Chambers, G., Spectroscopic analysis con.rms the interactions between single-walled carbon nanotubes and various dyes commonly used to assess cytotoxicity (2007) Carbon, 45 (7), pp. 1425-1432Isobe, H., Tanaka, T., Maeda, R., Noiri, E., Solin, N., Yudasaka, M., Preparation, purification, characterization, and cytotoxicity assessment of water-soluble, transition-metal free carbon nanotubes aggregates (2006) Angew. Chem. Int. Ed. Engl., 45 (40), pp. 6676-6680Bhirde, A.A., Patel, S., Sousa, A.A., Patel, V., Molinolo, A.A., Ji, Y.M., Distribution and clearance of PEG-single-walled carbon nanotube cancer drug delivery vehicles in mice (2010) Nanomedicine - Uk., 5, pp. 1535-1546Foldvari, M., Bagonluri, M., Carbon nanotubes as functional excipients for nanomedicines: II. Drug delivery and biocompatibility issues (2008) Nanomed-Nanotechnol., 4, pp. 183-200Maynard, A.D., Baron, P.A., Foley, M., Shvedova, A.A., Kisin, E.R., Castranova, V., Exposure to carbon nanotube material: Aerosol release during the handling of unrefined single-walled carbon nanotube material (2004) J. Toxicol. Env. Heal A., 67, pp. 87-107Tejral, G., Panyala, N.R., Havel, J., Carbon nanotubes: Toxicological impact on human health and environment (2009) J. Appl. Biomed., 7, pp. 1-13Zhang, Y.B., Xu, Y., Li, Z.G., Chen, T., Lantz, S.M., Howard, P.C., Mechanistic toxicity evaluation of uncoated and PEGylated single-walled carbon nanotubes in neuronal PC12 cells (2011) ACS Nano., 5, pp. 7020-7033Pacurari, M., Yin, X.J., Zhao, J., Ding, M., Leonard, S.S., Schwegler-Berry, D., Raw single-wall carbon nanotubes induce oxidative stress and activate MAPKs, AP-1, NF-kappaB, and Akt in normal and malignant human mesothelial cells (2008) Environ. Health Perspect., 116, pp. 1211-1217Shvedova, A.A., Pietroiusti, A., Fadeel, B., Kagan, V.E., Mechanisms of carbon nanotube-induced toxicity: Focus on oxidative stress (2012) Toxicol. Appl. Pharmacol., 261, pp. 121-133Yuan, J., Gao, H., Sui, J., Duan, H., Chen, W.N., Ching, C.B., Cytotoxicity evaluation of oxidized single-walled carbon nanotubes and graphene oxide on human hepatoma HepG2 cells: An iTRAQ-coupled 2D LC-MS/MS proteome analysis (2012) Toxicol. Sci., 126, pp. 149-161Yuan, J., Gao, H., Ching, C.B., Comparative protein profile of human hepatoma HepG2 cells treated with graphene and single-walled carbon nanotubes: An iTRAQ-coupled 2D LC-MS/MS proteome analysis (2011) Toxicol. Lett., 207, pp. 213-221Murray, A.R., Kisin, E., Leonard, S.S., Young, S.H., Kommineni, C., Kagan, V.E., Oxidative stress and inflammatory response in dermal toxicity of single-walled carbon nanotubes (2009) Toxicology, 257, pp. 161-171Ding, L., Stilwell, J., Zhang, T., Elboudwarej, O., Jiang, H., Selegue, J.P., Molecular characterization of the cytotoxic mechanism of multiwall carbon nanotubes and nano-onions on human skin fibroblast (2005) Nano Lett., 5, pp. 2448-2464Yan, L., Zhao, F., Li, S., Hu, Z., Zhao, Y., Low-toxic and safe nanomaterials by surface-chemical design, carbon nanotubes, fullerenes, metallofullerenes, and graphenes (2011) Nanoscale, 3, pp. 362-382Yan, X.B., Gu, Y.H., Huang, D., Gan, L., Wu, L.X., Huang, L.H., Binding tendency with oligonucleotides and cell toxicity of cetyltrimethyl ammonium bromide-coated single-walled carbon nanotubes (2011) Trans. Nonferrous Met. Soc., 21, pp. 1085-1091Chan, W.C.W., Elucidating the Interactions of Nanomaterials with Biological Systems (2010) Nemb 2010: Proceedings of the Asme First Global Congress on Nanoengineering for Medicine and Biology - 2010, pp. 111-112Lam, C.W., James, J.T., McCluskey, R., Hunter, R.L., Pulmonary toxicity of single-wall carbon nanotubes in mice 7 and 90 days after intratracheal instillation (2004) Toxicol. Sci., 77, pp. 126-134Sato, Y., Yokoyama, A., Shibata, K., Akimoto, Y., Ogino, S., Nodasaka, Y., Influence of length on cytotoxicity of multi-walled carbon nanotubes against human acute monocytic leukemia cell line THP-I in vitro and subcutaneous tissue of rats in vivo (2005) Mol. Biosyst., 1, pp. 176-182Wei, H., Wei, J., Wu, Y., Liu, L., Fan, S., (2013) Jiang K, , High-Strength Composite Yarns Derived From Oxygen Plasma Modified Super-aligned Carbon Nanotube Arrays Nano Res 1-9Singh, R., Pantarotto, D., McCarthy, D., Chaloin, O., Hoebeke, J., Partidos, C.D., Binding and condensation of plasmid DNA onto functionalized carbon nanotubes: Toward the construction of nanotube-based gene delivery vectors (2005) J. Am. Chem. Soc., 127, pp. 4388-4396Liu, Y., Wu, D.C., Zhang, W.D., Jiang, X., He, C.B., Chung, T.S., Polyethylenimine-grafted multiwalled carbon nanotubes for secure noncovalent immobilization and efficient delivery of DNA (2005) Angew. Chem., 44, pp. 4782-4785Zangmeister, R.A., Maslar, J.E., Opdahl, A., Tarlov, M.J., Adsorption behavior of DNA-wrapped carbon nanotubes on self-assembled monolayer surfaces (2007) Langmuir, 23, pp. 6252-6256Tasis, D., Tagmatarchis, N., Bianco, A., Prato, M., Chemistry of carbon nanotubes (2006) Chem. Rev., 106, pp. 1105-1136Zheng, M., Jagota, A., Semke, E.D., Diner, B.A., McLean, R.S., Lustig, S.R., DNA-assisted dispersion and separation of carbon nanotubes (2003) Nat. Mater., 2, pp. 338-342Ju, S.Y., Doll, J., Sharma, I., Papadimitrakopoulos, F., Selection of carbon nanotubes with specific chiralities using helical assemblies of flavin mononucleotide (2008) Nat. Nanotechnol., 3, pp. 356-362Sanchez-Pomales, G., Santiago-Rodriguez, L., Cabrera, C.R., DNA-functionalized carbon nanotubes for biosensing applications (2009) J. Nanosci Nanotechnol., 9, pp. 2175-2188Ghosh, S., Dutta, S., Gomes, E., Carroll, D., D'Agostino, R., Olson, J., Increased heating efficiency and selective thermal ablation of malignant tissue with DNA-encased multiwalled carbon nanotubes (2009) ACS Nano., 3, pp. 2667-2673Cheung, W., Pontoriero, F., Taratula, O., Chen, A.M., He, H.X., DNA and carbon nanotubes as medicine (2010) Adv. Drug Deliv. Rev., 62, pp. 633-649Firme, C.P., Bandaru, P.R., Toxicity issues in the application of carbon nanotubes to biological systems (2010) Nanomed-Nanotechnol., 6, pp. 245-256Lundqvist, M., Stigler, J., Elia, G., Lynch, I., Cedervall, T., Dawson, K.A., Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts (2008) Proc. Natl. Acad. Sci. U. S. A., 105, pp. 14265-14270Cedervall, T., Lynch, I., Lindman, S., Berggard, T., Thulin, E., Nilsson, H., Understanding the nanoparticle-protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles (2007) Proc. Natl. Acad. Sci. U. S. A., 104, pp. 2050-2055Ge, C.C., Du, J.F., Zhao, L.N., Wang, L.M., Liu, Y., Li, D.H., Binding of blood proteins to carbon nanotubes reduces cytotoxicity (2011) Proc. Natl. Acad. Sci. U. S. A., 108, pp. 16968-16973Nel, A.E., Madler, L., Velegol, D., Xia, T., Hoek, E.M.V., Somasundaran, P., Understanding biophysicochemical interactions at the nano-bio interface (2009) Nat. Mater., 8, pp. 543-557Shim, M., Kam, N.W.S., Chen, R.J., Li, Y.M., Dai, H.J., Functionalization of carbon nanotubes for biocompatibility and biomolecular recognition (2002) Nano Lett., 2, pp. 285-288Zhang, L., Zhao, G.C., Wei, X.W., Yang, Z.S., A nitric oxide biosensor based on myoglobin adsorbed on multi-walled carbon nanotubes (2005) Electroanal., 17, pp. 630-634Feazell, R.P., Nakayama-Ratchford, N., Dai, H., Lippard, S.J., Soluble single-walled carbon nanotubes as longboat delivery systems for Platinum(IV) anticancer drug design (2007) J. Am. Chem. Soc., 129, pp. 8438-8439Lobo, A.O., Zanin, H., Siqueira, I.A.W.B., Leite, N.C.S., Marciano, F.R., Corat, E.J., (2013) Mater. Sc. Eng. C-Mater. Biol. Appl., 33, pp. 4305-4312Grinet, M.A.V.M., Zanin, H., Granato, A.E.C., Porcionatto, M., Marciano, F.R., Lobo, A.O., Fast preparation of free-standing nanohydroxyapatite-vertically aligned carbon nanotube scaffolds J. Mater. Chem. B., , http://dx.doi.org/10.1039/C3TB21525CFletcher, D.A., Mullins, D., Cell mechanics and the cytoskeleton (2010) Nature, 463, pp. 485-492Machado, C.M., Schenka, A., Vassallo, J., Tamashiro, W.M., Goncalves, E.M., Genari, S.C., Morphological characterization of a human glioma cell line (2005) Cancer Cell Int., 5, p. 13Izidoro, M.S.J., Varela, J.N., Alves, D.A., Pereira, R.F.C., Brocchi, M., Lancellotti, M., Effects of Salmonella enteritidis serovar typhimurium Infection in Adenocarcinomic human alveolar basal epithelial cells A549 in vitro: Bacteria induce apoptosis in adenocarcinomic cell (2012) J. Bacteriol. Parasitol., p. 3Mosmann, T., Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays (1983) J. Immunol. Methods, 65, pp. 55-63Borenfreund, E., Puerner, J.A., A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90) (1985) J. Tissue Cult. Methods, 9, pp. 7-9Machado, D., Shishido, S.M., Queiroz, K.C., Oliveira, D.N., Faria, A.L., Catharino, R.R., Irradiated riboflavin diminishes the aggressiveness of melanoma in vitro and in vivo (2013) PLoS ONE, 8, p. 54269Antunes, E.F., Lobo, A.O., Corat, E.J., Trava-Airoldi, V.J., Martin, A.A., Verissimo, C., Comparative study of first- and second-order Raman spectra of MWCNT at visible and infrared laser excitation (2006) Carbon, 44, pp. 2202-2211Zanin, H., May, P.W., Hamanaka, M., Corat, E.J., Field emission from hybrid diamond-like carbon and carbon nanotube composite structures ACS Appl. Mater. Iinterfaces, 5 (23), pp. 12238-12243Zanin, H., Teofilo, R.F., Peterlevitz, A.C., Oliveira, U., De Paiva, J.C., Ceragioli, H.J., Diamond cylindrical anodes for electrochemical treatment of persistent compounds in aqueous solution (2013) J. Appl. Electrochem., 43, pp. 323-330Zanin, H., Saito, E., Marciano, F.R., Ceragioli, H.J., Campos Granato, A.E., Porcionatto, M., Lobo, A.O., Fast preparation of nano-hydroxyapatite/superhydrophilic reduced graphene oxide composites for bioactive applications J (2013) Mater. Chem. B, 1, pp. 4947-4955Compton, O.C., Jain, B., Dikin, D.A., Abouimrane, A., Amine, K., Nguyen, S.T., Chemically active reduced graphene oxide with tunable c/o ratios (2011) ACS Nano., 5, pp. 4380-4391Zhanga, Y., Zhaoa, J., Sunb, B., Chena, X., Lia, Q., Qiua, L., Performance enhancement for quasi-solid-state dye-sensitized solar cells by using acid-oxidized carbon nanotube-based gel electrolytes (2012) Electrochim. Acta, 61, pp. 185-190Zanin, H., Saito, E., Ceragioli, H.J., Baranauskas, V., Corat, E.J., Reduced graphene oxide and vertically aligned carbon nanotubes superhydrophilic films for supercapacitors devices (2014) Mater. Res. Bull., 49, pp. 487-493Ramalingam, P., Pusuluri, S.T., Periasamy, S., Veerabahu, R., Kulandaivel, J., Role of deoxy group on the high concentration of graphene in surfactant/water media (2013) Rsc Adv., 3, pp. 2369-2378De Nicola, M., Gattia, D.M., Bellucci, S., De Bellis, G., Micciulla, F., Pastore, R., Effect of different carbon nanotubes on cell viability and proliferation (2007) J. Phys-Condens Mater., p. 19Mwenifumbo, S., Shaffer, M.S., Stevens, M.M., Exploring cellular behaviour with multi-walled carbon nanotube constructs (2007) J. Mater. Chem., 17, pp. 1894-1902Kalbacova, M., Kalbac, M., Dunsch, L., Hempel, U., Influence of single-walled carbon nanotube films on metabolic activity and adherence of human osteoblasts (2007) Carbon, 45, pp. 2266-2272Zhang, D.W., Yi, C.Q., Zhang, J.C., Chen, Y., Yao, X.S., Yang, M.S., The effects of carbon nanotubes on the proliferation and differentiation of primary osteoblasts (2007) Nanotechnology, p. 18Macdiarmid, J.A., Mugridge, N.B., Weiss, J.C., Phillips, L., Burn, A.L., Paulin, R.P., Haasdyk, J.E., Brahmbhatt, H., Bacterially derived 400 nm particles for encapsulation and cancer cell targeting of chemotherapeutics (2007) Cancer Cell, 11 (5), pp. 431-445Zhang, L., Lu, Z., Zhao, Q., Huang, J., Shen, H., Zhang, Z., Enhanced chemotherapy efficacy by sequential delivery of siRNA and Anticancer drugs using pei-grafted graphene oxide (2011) Small, 7, pp. 460-464Chang, Y., Yang, S.-T., Liu, J.-H., Dong, E., Wang, Y., Cao, A., Liu, Y., Wang, H., In vitro toxicity evaluation of graphene oxide on A549 cells (2011) Toxicol. Lett., 200, pp. 201-210Liao, K.-H., Lin, Y.-S., Macosko, C.W., Haynes, C.L., Cytotoxicity of graphene oxide and graphene in human erythrocytes and skin fibroblasts (2011) ACS Appl. Mater. Interfaces, 3, pp. 2607-2615