Cytotoxicity profile of pristine graphene on brain microvascular endothelial cells

Graphene-based nanomaterials hold the potential to be used in a wide variety of applications, including biomedical devices. Pristine graphene (PG) is an un-functionalized, defect-free type of graphene that could be used as a material for neural interfacing. However, the neurotoxic effects of PG, particularly to the blood-brain barrier (BBB), have not been fully studied. The BBB separates the brain tissue from the circulating substances in the blood and is essential to maintain the brain homeostasis. The principal components of the BBB are brain microvascular endothelial cells (BMVECs), which maintain a protectively low permeability due to the expression of tight junction proteins. Here we analyzed the effects of PG on BMVECs in an in vitro model of the BBB. BMVECs were treated with PG at 0, 10, 50 and 100 μg/mL for 24 hours and viability and functional analyses of BBB integrity were performed. PG increased lactate dehydrogenase release at 50 and 100 μg/mL, suggesting the induction of necrosis. Surprisingly, 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium (XTT) conversion was increased at 10 and 50 μg/mL. In contrast, XTT conversion was decreased at 100 μg/mL, suggesting the induction of cell death. In addition, 100 μg/mL PG increased DNA fragmentation, suggesting induction of apoptosis. At the same time, 50 and 100 μg/mL of PG increased the endothelial permeability, which corresponded with a decrease in the expression of the tight junction protein occludin at 100 μg/mL. In conclusion, these results suggest that PG negatively affects the viability and function of the BBB endothelial cells in vitro.