The graphene family has captured the interest and the imagination of an increasing number of scientists working in different fields, ranging from composites to flexible electronics. In the area of biomedical applications, graphene is especially involved in drug delivery, biosensing and tissue engineering, with strong contributions to the whole nanomedicine area. Besides the interesting results obtained so far and the evident success, there are still many problems to solve, on the way to the manufacturing of biomedical devices, including the lack of standardization in the production of the graphene family members. Control of lateral size, aggregation state (single vs. few layers) and oxidation state (unmodified graphene vs. oxidized graphenes) is essential for the translation of this material into clinical assays. In this Tutorial Review we critically describe the latest developments of the graphene family materials into the biomedical field. We analyze graphene-based devices starting from graphene synthetic strategies, functionalization and processibility protocols up to the final in vitro and in vivo applications. We also address the toxicological impact and the limitations in translating graphene materials into advanced clinical tools. Finally, new trends and guidelines for future developments are presented.

The extraordinary physicochemical properties of graphene-based nanomaterials (GBNs) make them promising tools in nanotechnology and biomedicine. Considering the skin contact as one of the most feasible exposure routes to GBNs, the mechanism of toxicity of two GBNs (few-layer-graphene, FLG, and graphene oxide, GO) towards human HaCaT skin keratinocytes was investigated. Both materials induced a significant mitochondrial membrane depolarization: 72 h cell exposure to 100 μg mL⁻¹ FLG or GO increased mitochondrial depolarization by 44% and 56%, respectively, while the positive control valinomycin (0.1 μg mL⁻¹) increased mitochondrial depolarization by 48%. Since the effect was not prevented by cyclosporine-A, it appears to be unrelated to mitochondrial transition pore opening. By contrast, it seems to be mediated by reactive oxygen species (ROS) production: FLG and GO induced time- and concentration-dependent cellular ROS production, significant already at the concentration of 0.4 μg mL⁻¹ after 24 h exposure. Among a panel of specific inhibitors of the major ROS-producing enzymes, diphenyliodonium, rotenone and allopurinol significantly reverted or even abolished FLG- or GO-induced ROS production. Intriguingly, the same inhibitors also significantly reduced FLG- or GO-induced mitochondrial depolarization and cytotoxicity. This study shows that FLG and GO induce a cytotoxic effect due to a sustained mitochondrial depolarization. This seems to be mediated by a significant cellular ROS production, caused by the activation of flavoprotein-based oxidative enzymes, such as NADH dehydrogenase and xanthine oxidase.

In tissue engineering strategies, the design of scaffolds based on nanostructures is a subject undergoing intense research: nanomaterials may affect the scaffolds properties, including their ability to interact with cells favouring cell growth and improving tissue performance. Hydrogels are synthetic materials widely used to obtain realistic tissue constructs, as they resemble living tissues. Here, different hydrogels with varying content of graphene, are synthesised by in situ radical polymerization of acrylamide in aqueous graphene dispersions. Hydrogels are characterised focusing on the contribution of the nanomaterial to the polymer network. Our results suggest that graphene is not a mere embedded nanomaterial within the hydrogels, rather it represents an intrinsic component of these networks, with a specific role in the emergence of these structures. Moreover, a hybrid hydrogel with a graphene concentration of only 0.2 mg mL⁻¹ is used to support the growth of cultured brain cells and the development of synaptic activity, in view of exploiting these novel materials to engineer the neural interface of brain devices of the future. The main conclusion of this work is that graphene plays an important role in improving the biocompatibility of polyacrylamide hydrogels, allowing neuronal adhesion.