Comparative in vitro study of single and four layer graphene oxide nanoflakes - Cytotoxicity and cellular uptake.

In recent years, graphene and its derivatives have been extensively investigated because of their unique properties, which can be used in many fields including biomedical applications. Therefore, detailed biological study is required. In the current paper the detailed toxicological studies on single and four layer graphene oxide (GO) nanoflakes is presented. The morphology and size of the nanomaterials were characterized via atomic force microscopy. Cytotoxicity, proliferation and internalization study were performed using various methods, including optical, confocal and Raman microscopy imaging, flow cytometry analysis, colorimetric and luminescent cell assays. Our first findings undeniably show that the nanomaterials' functionalization has a considerable impact on their behavior in a biological environment. The cytotoxicity assay confirmed comparable, dose dependent cytotoxicity of single and four layers GO flakes. The differences between these two nanomaterials became more distinct during cell proliferation study and ROS detection. Namely, markedly stronger inhibition of cell proliferation and higher ROS generation by one-layer GO-PEG than four-layer GO-PEG were observed. Cell imaging revealed efficient internalization of the both GO nanoflakes in a time dependent manner. These findings emphasize the role of number of layer and functionalization in GO toxicological characteristics and may provide helpful information for their further biomedical applications.