Abstract
With the development of nanotechnology, myriad types of novel materials have been discovered at the nanoscale, among which the most interesting material is graphene. However, the toxicity data available on graphene are extremely limited. In this study, we explored toxic response of commercially available graphene nanoplatelets (GNPs) in vivo and in vitro. The GNPs used in this study had a high surface area and feature considerably few defects. In mice, GNPs (2.5 and 5 mg/kg) remained in the lung until 28 days after a single instillation, and the secretion of inflammatory cytokines reached the maximal level at Day 14 and then decreased over time. In vitro study using BEAS-2B cells, a human bronchial epithelial cell line, GNPs located within autophagosome-like vacuoles 24 h after exposure. The GNPs (2.5, 5, 10, and 20 μg/mL) also dose-dependently reduced cell viability, which was accompanied by an increase in the portion of cells in the subG1 and S phases. Moreover, the GNPs down-regulated the generation of reactive oxygen species, suppressed ATP production, caused mitochondria damage, and elevated the levels of autophagy-related proteins. Based on these results, we suggest that GNPs provoked a subchronic inflammatory response in mice and that GNPs induced autophagy accompanying apoptosis via mitochondria damage in vitro.
Similar content being viewed by others
References
Brownson DA, Banks CE (2010) Graphene electrochemistry: an overview of potential applications. Analyst 135(11):2768–2778
Bussy C, Ali-Boucetta H, Kostarelos K (2013) Safety considerations for graphene: lessons learnt from carbon nanotubes. Acc Chem Res 46(3):692–701
Chandra V, Park J, Chun Y, Lee JW, Hwang IC, Kim KS (2010) Water-dispersible magnetite-reduced graphene oxide composites for arsenic removal. ACS Nano 4(7):3979–3986
Chang Y, Yang ST, Liu JH, Dong E, Wang Y, Cao A, Liu Y, Wang H (2011) In vitro toxicity evaluation of graphene oxide on A549 cells. Toxicol Lett 200(3):201–210
Chang SH, Hong SH, Jiang HL, Minai-Tehrani A, Yu KN, Lee JH, Kim JE, Shin JY, Kang B, Park S, Han K, Chae C, Cho MH (2012) GOLGA2/GM130, cis-Golgi matrix protein, a novel target of anticancer gene therapy. Mol Ther 20(11):2052–2063
Choucair M, Thordarson P, Stride JA (2009) Gram-scale production of graphene based on solvothermal synthesis and sonication. Nat Nanotechnol 4(1):30–33
Christen V, Capelle M, Fent K (2013) Silver nanoparticles induce endoplasmatic reticulum stress response in zebrafish. Toxicol Appl Pharmacol 272(2):519–528
Chung KF (2001) Cytokines in chronic obstructive pulmonary disease. Eur Respir J Suppl 34:50s–59s
Di Gioacchino M, Petrarca C, Lazzarin F, Di Giampaolo L, Sabbioni E, Boscolo P, Mariani-Costantini R, Bernardini G (2011) Immunotoxicity of nanoparticles. Int J Immunopathol Pharmacol 24(1 Suppl):65S–71S
Ferrari AC, Meyer JC, Scardaci V, Casiraghi C, Lazzeri M, Mauri F, Piscanec S, Jiang D, Novoselov KS, Roth S, Geim AK (2006) Raman spectrum of graphene and graphene layers. Phys Rev Lett 97(18):187401
Ferri KF, Kroemer G (2001) Organelle-specific initiation of cell death pathways. Nat Cell Biol 3(11):E255–E263
Giulivi C, Poderoso JJ, Boveris A (1998) Production of nitric oxide by mitochondria. J Biol Chem 273(18):11038–11043
Gurunathan S, Han JW, Eppakayala V, Kim JH (2013) Green synthesis of graphene and its cytotoxic effects in human breast cancer. Int J Nanomed 8:1015–1027
Hou CC, Tsai TL, Su WP, Hsieh HP, Yeh CS, Shieh DB, Su WC (2013) Pronounced induction of endoplasmic reticulum stress and tumor suppression by surfactant-free poly(lactic-co-glycolic acid) nanoparticles via modulation of the PI3K signaling pathway. J Nanomed 8:2689–2707
Huang H, Xia Y, Tao X, Du J, Fang J, Gan Y, Zhang W (2012) Highly efficient electrolytic exfoliation of graphite into graphene sheets based on Li ions intercalation-expansion-microexplosion mechanism. J Mater Chem 22:10452–10456
Kim R, Emi M, Tanabe K (2006) Role of mitochondria as the gardens of cell death. Cancer Chemother Pharmacol 57(5):545–553
Kroll A, Pillukat MH, Hahn D, Schnekenburger J (2009) Current in vitro methods in nanoparticle risk assessment: limitations and challenges. Eur J Pharm Biopharm 72(2):370–377
Kroll A, Pillukat MH, Hahn D, Schnekenburger J (2012) Interference of engineered nanoparticles with in vitro toxicity assays. Arch Toxicol 86(7):1123–1136
Lammel T, Boisseaux P, Fernández-Cruz ML, Navas JM (2013) Internalization and cytotoxicity of graphene oxide and carboxyl graphene nanoplatelets in the human hepatocellular carcinoma cell line HepG2. Part Fibre Toxicol 10(1):27
Lanneau D, de Thonel A, Maurel S, Didelot C, Garrido C (2007) Apoptosis versus cell differentiation: role of heat shock proteins HSP90, HSP70 and HSP27. Prion 1(1):53–60
Lee HK, Jones RT, Myers RA, Marzella L (1992) Regulation of protein degradation in normal and transformed human bronchial epithelial cells in culture. Arch Biochem Biophys 296(1):271–278
Lee SH, Seo SD, Park KS, Shim HW, Kim DW (2012) Synthesis of graphene nanosheets by the electrolytic exfoliation of graphite and their direct assembly for lithium ion battery anodes. Mater Chem Phys 135(2–3):309–316
Li Y, Liu Y, Fu Y, Wei T, Le Guyader L, Gao G, Liu RS, Chang YZ, Chen C (2012) The triggering of apoptosis in macrophages by pristine graphene through the MAPK and TGF-beta signaling pathway. Biomaterials 33(2):402–411
Linkermann A, De Zen F, Weinberg J, Kunzendorf U, Krautwald S (2012) Programmed necrosis in acute kidney injury. Nephrol Dial Transplant 27(9):3412–3419
Manucha W, Vallés P (2008) Hsp70/nitric oxide relationship in apoptotic modulation during obstructive nephropathy. Cell Stress Chaperones 13(4):413–420
Marques MRC, Loebenberg R, Almukainzi M (2011) Simulated biological fluids with possible application in dissolution testing. Dissolut Technol 8:15–28
Park EJ, Cho WS, Jeong J, Yi J, Choi K, Park K (2009) Pro-inflammatory and potential allergic responses resulting from B cell activation in mice treated with multi-walled carbon nanotubes by intratracheal instillation. Toxicology 259(3):113–121
Park EJ, Shim HW, Lee GH, Kim JH, Kim DW (2013) Comparison of toxicity between the different-type TiO2 nanowires in vivo and in vitro. Arch Toxicol 87(7):1219–1230
Park EJ, Zahari NE, Lee EW, Song J, Lee JH, Cho MH, Kim JH (2014) SWCNTs induced autophagic cell death in human bronchial epithelial cells. Toxicol In Vitro 28(3):442–450
Peng H, Mo Z, Liao S, Liang H, Yang L, Luo F, Song H, Zhong Y, Zhang B (2013) High performance Fe- and N- doped carbon catalyst with graphene structure for oxygen reduction. Sci Rep 3:1765
Pham VH, Cuong TV, Hur SH, Oh E, Kim EJ, Shin EW, Chung JS (2011) Chemical functionalization of graphene sheets by solvothermal reduction of a graphene oxide suspension in N-methyl-2-pyrrolidone. J Mater Chem 21:3371–3377
Qu G, Liu S, Zhang S, Wang L, Wang X, Sun B, Yin N, Gao X, Xia T, Chen JJ, Jiang GB (2013) Graphene oxide induces toll-like receptor 4 (TLR4)-dependent necrosis in macrophages. ACS Nano 7(7):5732–5745
Richter C, Gogvadze V, Laffranchi R, Schlapbach R, Schweizer M, Suter M, Walter P, Yaffee M (1995) Oxidants in mitochondria: from physiology to diseases. Biochim Biophys Acta 1271(1):67–74
Roy A, Kolattukudy PE (2012) Monocyte chemotactic protein-induced protein (MCPIP) prototes inflammatory angiogenesis via sequential induction of oxidative stress, endoplasmic reticulum stress and autophagy. Cell Signal 24(11):2123–2131
Sampson AP (2000) The role of eosinophils and neutrophils in inflammation. Clin Exp Allergy 30(Suppl 1):22–27
Sasidharan A, Panchakarla LS, Chandran P, Menon D, Nair S, Rao CN, Koyakutty M (2011) Differential nano-bio interactions and toxicity effects of pristine versus functionalized graphene. Nanoscale 3(6):2461–2464
Sasidharan A, Panchakarla LS, Chandran P, Menon D, Nair S, Rao CN, Koyakutty M (2012) Hemocompatibility and macrophage response of pristine and functionalized graphene. Small 8(8):1251–1263
Schinwald A, Murphy FA, Jones A, MacNee W, Donaldson K (2012) Graphene-based nanoplatelets: a new risk to the respiratory system as a consequence of their unusual aerodynamic properties. ACS Nano 6(1):736–746
Stankovich S, Dikin DA, Piner RD, Kohlhaas KA, Kleinhammes A, Jia Y, Wu Y, Nguyen ST, Ruoff RS (2007) Synthesis of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide. Carbon 45(7):1558–1565
Stern ST, Adiseshaiah PP, Crist RM (2012) Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Part Fibre Toxicol 9:20
Van Hall G (2000) Lactate as a fuel for mitochondrial respiration. Acta Physiol Scand 168(4):643–656
Vial T, Descotes J (1995) Immune-mediated side-effects of cytokines in humans. Toxicology 105(1):31–57
Wang Y, Shi Z, Fang J, Xu H, Ma X, Yin J (2011) Direct exfoliation of graphene in methanesulfonic acid and facile synthesis of graphene/polybenzimidazole nanocomposites. J Mater Chem 21:505–512
Wang X, Podila R, Shannahan JH, Rao AM, Brown JM (2013) Intravenously delivered graphene nanosheets and multiwalled carbon nanotubes induce site-specific Th2 inflammatory responses via the IL-33/ST2 axis. Int J Nanomed 8:1733–1748
Wei L, Wu F, Shi D, Hu C, Li X, Yuan W, Wang J, Zhao J, Geng H, Wei H, Wang Y, Hu N, Zhang Y (2013) Spontaneous intercalation of long-chain alkyl ammonium into edge-selectively oxidized graphite to efficiently produce high-quality graphene. Sci Rep 3:2636
Wu W, Liu P, Li J (2012) Necroptosis: an emerging form of programmed cell death. Crit Rev Oncol Hematol 82(3):249–258
Yang K, Wan J, Zhang S, Zhang Y, Lee ST, Liu Z (2011) In vivo pharmacokinetics, long-term biodistribution, and toxicology of PEGlated graphene in mice. ACS Nano 5(1):516–522
Yang K, Li Y, Tan X, Peng R, Liu Z (2013) Behavior and toxicity of graphene and its functionalized derivatives in biological systems. Small 9(9–10):1492–1503
Yoshimura A, Muto G (2011) TGF-β function in immune suppression. Curr Top Microbiol Immunol 350:127–147
Yoshimura A, Wakabayashi Y, Mori T (2010) Cellular and molecular basis for the regulation of inflammation by TGF-beta. J Biochem 147(6):781–792
Yue Z, Levchenko I, Kumar S, Seo D, Wang X, Dou S, Ostrikow KK (2013) Large networks of vertical multi-layer graphenes with morphology-tunable magnetoresistance. Nanoscale 5(19):9283–9288
Zhang HB, Chen C, Wang JW, Yang Y, Lu ZH, Yan Q, Zheng WG (2011) A facile approach to the synthesis of graphene nanosheets under ultra-low exfoliation temperature. J Nanosci Nanotechnol 11(12):10868–10870
Zhang Y, Nayak TR, Hong H, Cai W (2012) Graphene: a versatile nanoplatform for biomedical applications. Nanoscale 4(13):3833–3842
Acknowledgments
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology (2011-35B-E00011, 2012R1A2A2A01045382).
Conflict of interest
The authors declare that they have no conflicts of interest.
Author information
Authors and Affiliations
Corresponding authors
Additional information
Eun-Jung Park and Gwang-Hee Lee have contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Park, EJ., Lee, GH., Han, B.S. et al. Toxic response of graphene nanoplatelets in vivo and in vitro. Arch Toxicol 89, 1557–1568 (2015). https://doi.org/10.1007/s00204-014-1303-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00204-014-1303-x