The toxicity of graphene oxides: dependence on the oxidative methods used

Graphene, a class of two-dimensional carbon nanomaterial, has attracted extensive interest in recent years, with a significant amount of research focusing on graphene oxides (GOs). They have been primed as potential candidates for biomedical applications such as cell labeling and drug delivery, thus the toxicity and behavior of graphene oxides in biological systems are fundamental issues that need urgent attention. The production of GO is generally achieved through a top-down route, which includes the usage of concentrated H₂SO₄ along with: 1) concentrated nitric acid and KClO₃ oxidant (Hoffmann); 2) fuming nitric acid and KClO₃ oxidant (Staudenmaier); 3) concentrated phosphoric acid with KMnO₄ (Tour); or 4) sodium nitrate for in-situ production of nitric acid in the presence of KMnO₄ (Hummers). It has been widely assumed that the properties of these four GOs produced by using the above different methods are roughly similar, so the methods have been used interchangeably. However, several studies have reported that the toxicity of graphene-related nanomaterials in biological systems may be influenced by their physiochemical properties, such as surface functional groups and structural defects. In addition, considering how GOs are increasingly used in the field of biomedicine, we are interested to see how the oxygen content/functional groups of GOs can impact their toxicological profiles. Since in-vitro testing is a common first step in assessing the health risks related with engineered nanomaterials, the cytotoxicity of the GOs prepared by the four different oxidative treatments was investigated by measuring the mitochondrial activity in adherent lung epithelial cells (A549) by using commercially available viability assays. The dose-response data was generated by using two assays, the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and the water-soluble tetrazolium salt (WST-8). From the viability data, it is evident that there is a strong dose-dependent cytotoxic response resulting from the four GO nanomaterials tested after a 24 h exposure, and it is suggested that there is a correlation between the amounts of oxygen content/functional groups of GOs with their toxicological behavior towards the A549 cells.