In vitro toxicity screening of graphene nanomaterials

Graphene-family nanomaterials (GFNs) with their unique physicochemical properties (e.g. electrical and thermal conductivity, strong mechanical strength, high surface area)[1, 2], are raising great research interests in different fields of nanotechnology, such as nanoelectronics, energy and sensors technologies and biomedicine[2, 3]. The widespread applications of graphene and its derivatives, referred as the graphene-family nanomaterials (GFNs), are increasing the concerns about their safety for human health, resulting from occupational, consumer and environmental exposures[4, 5]. However, the research towards the toxicity of GFNs is limited and controversial, and a proper toxicological risk assessment is still needed [1, 6]. Within the scope of the PLATOX project, funded by the FP7-SIINN ERANET on Nanosafety, commercially available graphene nanomaterials were selected (comprising single layer graphene (#1, #2) and graphene oxide (#5), multilayer (#4, #7) and carboxyl graphene (#3) and graphite oxide nanoplatelets (#6)) and their in vitro toxicity was assessed in RAW 264.7 macrophages (0-50 μg/cm2) by evaluating their effects on both cell viability and cell cycle dynamics. Cell viability was investigated using lactate dehydrogenase (LDH) release and AlamarBlue (AB) assays. Cell-cycle dynamics was analyzed by flow cytometry. Carboxyl graphene (#3) and the single layer graphenes (#1, #2) markedly impaired metabolic activity of macrophages. LDH release, although increased for all nanomaterials after 48h exposure was a less sensitive endpoint. The benchmark dose 30 (BMD30) of each GFN was calculated from the results obtained in the two assays (Table 1). The AB results thereby showed that the toxicity of GFNs increases with time of exposure. Concerning the effect of GFN on cell cycle dynamics of RAW 264.7 cells, the results showed that the single layer graphenes and graphene nanoplatelets (#4, #7) induced an increase in the % of sub-G0/G1population, indicating DNA damage and an increase in the G0/G1 full peak coefficient of variation, pointing to putative clastogenicity. In conclusion, these findings highlight the impor-tance of comparative in vitro toxicity screening for GFNs, with the ultimate goal of establishing a toxicological ranking.

References 

[1] Horváth, L., et al., Carbon 64 (2013), p. 45-60;

[2] Chatterjee, N., Eom, H.-J. and Choi, J., Biomaterials 35 (2014), p. 1109-1127; 

[3] Goenka, S., V. Sant, and S. Sant, Journal of Controlled Release 173 (2014), p. 75-88; 

[4] Sanchez, V.C., et al., Chemical Research in Toxicology 25 (2012), p. 15-34; 

[5] Park, E.-J., et al., Archives of Toxicology 89 (2015), p. 1557-1568; [6] Seabra, A.B., et al., Chemical Research in Toxicology 27 (2014), p. 159-168