Differential cytotoxic effects of graphene and graphene oxide on skin keratinocytes

Marco Pelin,Verónica León,Cristina Martín,Aurelia Tubaro,Alejandro Criado,Silvio Sosa,Laura Fusco,Ester Vázquez,Maurizio Prato

doi:10.1038/srep40572

Abstract

Impressive properties make graphene-based materials (GBMs) promising tools for nanoelectronics and biomedicine. However, safety concerns need to be cleared before mass production of GBMs starts. As skin, together with lungs, displays the highest exposure to GBMs, it is of fundamental importance to understand what happens when GBMs get in contact with skin cells. The present study was carried out on HaCaT keratinocytes, an in vitro model of skin toxicity, on which the effects of four GBMs were evaluated: a few layer graphene, prepared by ball-milling treatment (FLG), and three samples of graphene oxide (GOs, a research-grade GO1, and two commercial GOs, GO2 and GO3). Even though no significant effects were observed after 24 h, after 72 h the less oxidized compound (FLG) was the less cytotoxic, inducing mitochondrial and plasma-membrane damages with EC50s of 62.8 μg/mL (WST-8 assay) and 45.5 μg/mL (propidium iodide uptake), respectively. By contrast, the largest and most oxidized compound, GO3, was the most cytotoxic, inducing mitochondrial and plasma-membrane damages with EC50s of 5.4 and 2.9 μg/mL, respectively. These results suggest that only high concentrations and long exposure times to FLG and GOs could impair mitochondrial activity associated with plasma membrane damage, suggesting low cytotoxic effects at the skin level.

Highlights

Considering the potential cutaneous effect of these carbon nanomaterials, we evaluated the in vitro effects of GBMs on human skin HaCaT keratinocytes, a spontaneously immortalized non-tumor cell line, widely used as a first-round screening to evaluate the toxicity of several compounds at the skin level[40]
Even though it is very difficult to predict a possible human exposure since no industrial-scale adoption of graphene has taken place, so far[62], these results demonstrate significant cellular damage induced by FLG and Graphene oxide (GO) only at high concentrations (>30 μg/mL and >1 μg/mL for FLG and GOs, respectively) on skin keratinocytes after an exposure time as long as 72 h, with variable potencies depending on GBMs oxidation state
It has been reported that single-wall carbon nanotubes (SWCNTs) significantly reduce viability of HEK keratinocytes at ng/ mL concentrations already after 24 h63, whereas the GBMs tested in this study displayed weak cytotoxic effects at μg/mL concentrations

Summary

Introduction

Considering the potential cutaneous effect of these carbon nanomaterials, we evaluated the in vitro effects of GBMs on human skin HaCaT keratinocytes, a spontaneously immortalized non-tumor cell line, widely used as a first-round screening to evaluate the toxicity of several compounds at the skin level[40]. This in vitro model was recently used to investigate the biocompatibility against normal skin cells of GBMs as anti-cancer therapy[41]. We selected and characterized four different materials: two research grade materials (few layer graphene, FLG, and graphene oxide 1, GO1) and two commercial GOs, prepared using two different starting materials (carbon nanofibers, GO2 and graphite, GO3) with notable differences in the amount of defects and oxygen content

Methods

Results

Conclusion