Graphene-based nanomaterials received attention from scientists due to their unique properties: they are highly conductive, mechanically resistant and elastic. These materials can be used in different sectors of society from electronic energy storage in industry to biomedical applications. This study evaluates the influence of graphene nanoplatelets in vitro and in vivo. The toxicological influence of graphene nanoplatelets (GPs) was analyzed by cytotoxic methods, the change of cell proliferation was assessed in real-time, and the effect of GPs on a living organism was evaluated in an animal model using histopathological examination. We analyzed two types of GP administration: intratracheal and peroral. We found dose- and time-dependent cytotoxic effects of GPs in vitro; the concentration above 50 μg/mL increased the cytotoxicity significantly. The real-time analysis confirmed these data; the cells exposed to a high concentration of GPs for a longer time period resulted in a decrease in cell index which indicated lower cell viability. Histopathological examination revealed thickened alveolar septa and accumulation of GPs in the endocardium after intratracheal exposure. Peroral administration did not reveal any morphological changes. This study showed the dose- and time-dependent cytotoxic potential of graphene nanoplatelets in in vitro and in vivo models.

Department of Histology and Embryology Faculty of Medicine in Hradec Kralove Charles University 50003 Hradec Kralove Czech Republic

Institute of Clinical Immunology and Allergology University Hospital Hradec Kralove and Faculty of Medicine in Hradec Kralove Charles University 50005 Hradec Kralove Czech Republic

Institute of Preventive Medicine Faculty of Medicine in Hradec Kralove Charles University 50003 Hradec Kralove Czech Republic

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Levendorf M.P., Kim C.-J., Brown L., Huang P., Havener R.W., Muller D., Park J. Graphene and boron nitride lateral heterostructures for atomically thin circuitry. Nature. 2012;488:627–632. doi: 10.1038/nature11408. PubMed DOI

Raccichini R., Varzi A., Passerini S., Scrosati B. The role of graphene for electrochemical energy storage. Nat. Mater. 2015;14:271–279. doi: 10.1038/nmat4170. PubMed DOI

Thapa R.K., Ku S.K., Choi H., Yong C.S., Byeon J.H., Kim J.O. Vibrating droplet generation to assemble zwitterion-coated gold-graphene oxide stealth nanovesicles for effective pancreatic cancer chemophototherapy. Nanoscale. 2018;10:1742–1749. doi: 10.1039/C7NR07603G. PubMed DOI

Mao H.Y., Laurent S., Chen W., Akhavan O., Imani M., Ashkarran A.A., Mahmoudi M. Graphene: Promises, facts, opportunities, and challenges in nanomedicine. Chem. Rev. 2013;113:3407–3424. doi: 10.1021/cr300335p. PubMed DOI

Bullock C.J., Bussy C. Biocompatibility considerations in the design of graphene biomedical materials. Adv. Mater. Interfaces. 2019;6:1900229. doi: 10.1002/admi.201900229. DOI

Fadeel B., Bussy C., Merino S., Vázquez E., Flahaut E., Mouchet E., Evariste L., Gauthie L., Koivisto A.J., Vogel U., et al. Safety assessment of graphene-based materials: Focus on human health and the enviroment. ACS Nano. 2018;12:10582–10620. doi: 10.1021/acsnano.8b04758. PubMed DOI

Novoselov K.S., Geim A.K., Morozov S.V., Jiang D., Zhang Y., Dubonos S.V., Grigorieva I.V., Firsov A.A. Electric field effect in atomically thin carbon films. Science. 2004;306:666–669. doi: 10.1126/science.1102896. PubMed DOI

Schinwald A., Murphy F.A., Jones A., Macnee W., Donaldson K. Graphene-based nanoplatelets: A new risk to the respiratory system as a consequence of their unusual aerodynamic properties. ACS Nano. 2012;6:736–746. doi: 10.1021/nn204229f. PubMed DOI

Efremova L.V., Vasilchenko A.S., Rakov E.G., Deryabin D.G. Toxicity of graphene shells, graphene oxide, and graphene oxide paper evaluated with Escherichia coli biotests. Biomed. Res. Int. 2015;2015:869361. PubMed PMC

Mombeshora E.T., Stark A. Understanding oxidative reaction of carbon nanoplatelets toward tailored physicochemical properties. Mater. Chem. Phys. 2022;277:125535. doi: 10.1016/j.matchemphys.2021.125535. DOI

Tang N., Li Y., Chen F., Han Z. In situ fabrication of a direct Z-scheme photocatalyst by immobilizing Cds quantum dots in the channels of graphene-hybridized and supported mesoporous titanium nanocrystals for high photocatalytic performance under visible light. RSC Adv. 2018;8:42233–42245. doi: 10.1039/C8RA08008A. PubMed DOI PMC

Chen H., Chen Z., Yang H., Wen L., Yi Z., Zhou Z., Dai D., Zhang J., Wu X., Wu P. Multi-mode surface plasmon resonance absorber based on dart-type single-layer graphene. RSC Adv. 2022;12:7821–7829. doi: 10.1039/D2RA00611A. PubMed DOI PMC

Xiao L., Youji L., Feitai C., Peng X., Ming L. Facile synthesis of mesoporous titanium dioxide doped by Ag-coated graphene with enhanced visible-light photocatalytic performance for methylene blue degradation. RSC Adv. 2017;7:25314–25324. doi: 10.1039/C7RA02198D. DOI

Long F., Zhang Z., Wang J., Yan L., Zhou B. Cobalt-nickel bimetallic nanoparticles decorated graphene sensitized imprinted electrochemical sensor for determination of octylphenol. Electrochim. Acta. 2015;168:337–345. doi: 10.1016/j.electacta.2015.04.054. DOI

Li J., Jiang J., Zhao D., Xu Z., Liu M., Liu X., Tong H., Qian D. Novel hierarchical sea urchin-like Prussian blue palladium core-shell heterostructures supported on nitrogen-doped reduced graphene oxide: Facile synthesis and excellent guanine sensing performance. Electrochim. Acta. 2020;330:135196. doi: 10.1016/j.electacta.2019.135196. DOI

Goenka S., Sant V., Sant S. Graphene-based nanomaterials for drug delivery and tissue engineering. J. Control. Release. 2014;173:75–88. doi: 10.1016/j.jconrel.2013.10.017. PubMed DOI

Martín C., Kostarelos K., Prato M., Bianco A. Biocompatibility and biodegradability of 2D materials: Graphene and beyond. Chem. Commun. 2019;55:5540–5546. doi: 10.1039/C9CC01205B. PubMed DOI

Nurunnabi M.D., McCarthy J.R. Biomedical Applications of Graphene and 2D Nanomaterials. 1st ed. Elsevier; Amsterdam, The Netherlands: 2019.

Kaur T., Thirugnanam A., Pramanik K. Effect of carboxylated graphene nanoplatelets on mechanical and in-vitro biological properties of polyvinyl alcohol nanocomposite scaffolds for bone tissue engineering. Mater. Today Commun. 2017;12:34–42. doi: 10.1016/j.mtcomm.2017.06.004. DOI

Chng E.L.K., Chua C.K., Pumera M. Graphene oxide nanoribbons exhibit significantly greater toxicity than graphene oxide nanoplatelets. Nanoscale. 2014;6:10792–10797. doi: 10.1039/C4NR03608E. PubMed DOI

Akhavan O., Ghaderi E., Akhavan A. Size-dependent genotoxicity of graphene nanoplatelets in human stem cells. Biomaterials. 2012;33:8017–8025. doi: 10.1016/j.biomaterials.2012.07.040. PubMed DOI

Allen M.J., Tung V.C., Kaner R.B. Honeycomb carbon: A review of graphene. Chem. Rev. 2010;110:132–145. doi: 10.1021/cr900070d. PubMed DOI

Kurapati R., Mukherjee S.P., Martín C., Bepete G., Vázquez E., Pénicaud A., Fadeel B., Bianco A. Degradation of single-layer and few-layer graphene by neutrophil myeloperoxidase. Angew. Chem. Int. Ed. Engl. 2018;57:11722–11727. doi: 10.1002/anie.201806906. PubMed DOI

Svadlakova T., Hubatka F., Turanek-Knotigova P., Kulich P., Masek J., Kotoucek J., Macak J., Motola M., Kalbac M., Kolackova M., et al. Proinflammatory effect of carbon-based nanomaterials: In vitro study on stimulation of inflammasome NLRP3 via destabilisation of lysosomes. Nanomaterials. 2020;10:418. doi: 10.3390/nano10030418. PubMed DOI PMC

Svadlakova T., Kolackova M., Vankova R., Karakale R., Malkova A., Kulich P., Hubatka F., Turanek-Knotigova P., Kratochvilova I., Raska M., et al. Carbon-based nanomaterials increase reactivity of primary monocytes towards various bacteria and modulate their differentiation into macrophages. Nanomaterials. 2021;11:2510. doi: 10.3390/nano11102510. PubMed DOI PMC

Amrollahi-Sharifabadi M., Koohi M.K., Zayerzadeh E., Hablolvarid M.H., Hassan J., Seifalian A.M. In vivo toxicological evaluation of graphene oxide nanoplatelets for clinical application. Int. J. Nanomed. 2018;13:4757–4769. doi: 10.2147/IJN.S168731. PubMed DOI PMC

Wang K., Ruan J., Song H., Zhang J., Wo Y., Guo S., Cui D. Biocompatibility of graphene oxide. Nanoscale Res. Lett. 2011;6:8. doi: 10.1007/s11671-010-9751-6. PubMed DOI PMC

Ma Y., Shen H., Tu X., Zhang Z. Assessing in vivo toxicity of graphene materials: Current methods and future outlook. Nanomedicine. 2014;9:1565–1580. doi: 10.2217/nnm.14.68. PubMed DOI

Yan J., Chen L., Huang C.C., Lung S.C.C., Yang L., Wang W.C., Lin P.H., Suo G., Lin C.H. Consecutive evaluation of graphene oxide and reduced graphene oxide nanoplatelets immunotoxicity on monocytes. Colloids Surf. 2017;153:300–309. doi: 10.1016/j.colsurfb.2017.02.036. PubMed DOI

Seabra A.B., Paula A.J., Lima R.D., Alves O.L., Durán N. Nanotoxicity of graphene and graphene oxide. Chem. Res. Toxicol. 2014;27:159–168. doi: 10.1021/tx400385x. PubMed DOI

Wörle-Knirsch J.M., Pulskamp K., Krug H.F. Oops they did it again! Carbon nanotubes hoax scientists in viability assays. Nano Lett. 2006;6:1261–1268. doi: 10.1021/nl060177c. PubMed DOI

Chang 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. Toxicol. Lett. 2011;200:201–210. doi: 10.1016/j.toxlet.2010.11.016. PubMed DOI

Li Y., Liu Y., Fu Y., Wei T., Le Guyader L., Gao G., Liu R.S., Chang Y.Z., Chen C. The triggering of apoptosis in macrophages by pristine graphene through the MAPK and TGF-beta signaling pathways. Biomaterials. 2012;33:402–411. doi: 10.1016/j.biomaterials.2011.09.091. PubMed DOI

Zhang Y., Ali S.F., Dervishi E., Xu Y., Li Z., Casciano D., Biris A.S. Cytotoxicity effects of graphene and single-wall carbon nanotubes in neural phaeochromocytoma-derived PC12 cells. ACS Nano. 2010;4:3181–3186. doi: 10.1021/nn1007176. PubMed DOI

Kvakova M., Stroffekova K., Stofilova J., Girman V., Bomba A., Antalik M. Toxicological evaluation of fluorescent 11-mercaptoundecanoic gold nanoclusters as promising label-free bioimaging probes in different cancer cell lines. Toxicol. In Vitro. 2021;73:105140. doi: 10.1016/j.tiv.2021.105140. PubMed DOI

Xie B., Yi J., Peng J., Zhang X., Lei L., Zhao D., Lei Z., Nie H. Characterization of synergistic anti-tumor effects of doxorubicin and p53 via graphene oxide-polyethyleneimine nanocarriers. J. Mater. Sci. Technol. 2017;33:807–814. doi: 10.1016/j.jmst.2017.05.005. DOI

González-Ballesteros N., Diego-González L., Lastra-Valdor M., Grimaldi M., Cavazza A., Bigi F., Rodríguez-Argülles M.C., Simón-Vazquez R. Saccorhiza polyschides used to synthesize gold and silver nanoparticles with enhanced antiproliferative and immunostimulant activity. Mater. Sci. Eng. C. 2021;123:111960. doi: 10.1016/j.msec.2021.111960. PubMed DOI

Zha M.X., Cai Z.C., Zhu B.J., Zhang Z.Q. The apoptosis effect on liver cancer cells of gold nanoparticles modified with litholic acid. Nanoscale Res. Lett. 2018;13:304. doi: 10.1186/s11671-018-2653-8. PubMed DOI PMC

Razaghi M., Ramazani A., Khoobi M., Mortezazadeh T., Aksoy E.A., Kücükkilinc T.T. Highly fluorinated graphene oxide nanosheets for anticancer linoleic-curcumin conjugate delivery and T2-Weighted magnetic resonance imaging: In vitro and in vivo studies. J. Drug Deliv. Sci. Technol. 2020;60:101967. doi: 10.1016/j.jddst.2020.101967. DOI

Gao H., Hammer T., Zhang X., He W., Xu G., Wang J. Quantifying respiratory tract deposition of airborne graphene nanoplatelets: The impact of plate-like shape and folded structure. Nanoimpact. 2021;21:100292. doi: 10.1016/j.impact.2021.100292. PubMed DOI

Shin J.H., Han S.G., Kim J.K., Kim B.W., Hwang J.H., Lee J.S., Lee J.H., Baek J.E., Kim T.G., Kim K.S., et al. 5-day repeated inhalation and 28-day post-exposure study of graphene. Nanotoxicology. 2015;9:1023–1031. doi: 10.3109/17435390.2014.998306. PubMed DOI

Kanakia S., Toussaint J., Chowdhury S.M., Tembulkar T., Lee S., Jiang Y.P., Lin R.Z., Shroyer K.R., Moore W., Sitharaman B. Dose ranging, expanded acute toxicity and safety pharmacology studies for intravenously administered functionalized graphene nanoparticle formulations. Biomaterials. 2014;35:7022–7031. doi: 10.1016/j.biomaterials.2014.04.066. PubMed DOI PMC

Fu C., Liu T., Li L., Liu H., Liang Q., Meng X. Effects of graphene oxide on the development of offspring mice in lactation period. Biomaterials. 2015;40:23–31. doi: 10.1016/j.biomaterials.2014.11.014. PubMed DOI

Wang A., Pu K., Dong B., Liu Y., Zhang L., Zhang Z., Duan W., Zhu Y. Role of surface charge and oxidative stress in cytotoxicity and genotoxicity of graphene oxide towards human lung fibroblast cells. J. Appl. Toxicol. 2013;33:1156–1164. doi: 10.1002/jat.2877. PubMed DOI

Li J., Zhang X., Jiang J., Wang Y., Jiang H., Zhang J., Nie X., Liu B. Systematic assessment of the toxicity and potential mechanism of graphene derivatives in vitro and in vivo. Toxicol. Sci. 2019;167:269–281. doi: 10.1093/toxsci/kfy235. PubMed DOI

Demirel E., Karaca E., Durmaz Y.Y. Effective PEGylation method to improve biokompatibility of graphene derivatives. Eur. Polym. J. 2020;124:109504. doi: 10.1016/j.eurpolymj.2020.109504. DOI

Mbeh D.A., Akhavan O., Javanbakth T., Mahmoudi M., Yahia L. Cytotoxicity of protein corona-graphene oxide nanoribbons on human epithelial cells. Appl. Surf. Sci. 2014;320:596–601. doi: 10.1016/j.apsusc.2014.09.155. DOI

Pinto A.M., Moreira S., Goncalves I.C., Gama F.M., Mendes A.M., Magalhaes F.D. Biocompatibility of poly(lactic acid) with incorporated graphene-based materials. Colloids Surf. B. 2013;104:229–238. doi: 10.1016/j.colsurfb.2012.12.006. PubMed DOI

Go W., Qiu J., Liu J., Liu H. Graphene microfiber as a scaffold for regulation of neural stem cells differentiation. Sci. Rep. 2017;7:5678. doi: 10.1038/s41598-017-06051-z. PubMed DOI PMC

Kim J.K., Shin J.H., Lee J.S., Hwang J.H., Lee J.H., Baek J.E., Kim T.G., Kim B.W., Kim J.S., Lee G.H., et al. 28-day inhalation toxicity of graphene nanoplatelets in Sprague-Dawley rats. Nanotoxicology. 2016;10:891–901. doi: 10.3109/17435390.2015.1133865. PubMed DOI

Yang K., Gong H., Shi X., Wan J., Zhang Y., Liu Z. In vivo distribution and toxikology of functionalized nano-graphene oxide in mice after oral and intraperitoneal administration. Biomaterials. 2013;34:2787–2795. doi: 10.1016/j.biomaterials.2013.01.001. PubMed DOI