Endocytic mechanisms and toxicity of a functionalized fullerene in human cells

Derivatized fullerenes could be used in biomedical applications and be suitable vectors for drug delivery due to their small size, large surface area and solubility. However, the interactions of derivatized fullerenes with biological systems and cells are not well understood. A water-soluble fullerene-substituted phenylalanine (Bucky amino acid, Baa) poly-lysine derivative with a FITC label (Baa-Lys(FITC)-(Lys)8-OH) was characterized by dynamic light scattering, transmission electron microscopy with negative staining, gel electrophoresis, zeta-potential, and UV/vis spectroscopy. Viability assays depicted the cytotoxicity was time, concentration and assay dependent. A decrease in ATP and glutathione at the high concentrations suggests that reactive oxygen species may be involved. Baa-Lys(FITC)-(Lys)8-OH was present near the cell membrane at 15 min and entered into the cytoplasm by 30 min but did not localize in the lysosomes. Endocytic inhibitors were used to investigate the uptake mechanism. These results showed that the endocytic pathways could be mediated by caveolae/lipid rafts and cytoskeletal components. A scavenger receptor inhibitor completely blocked the uptake of Baa-Lys(FITC)-(Lys)8-OH, suggesting a specific endocytic pathway was strongly involved in Baa-Lys(FITC)-(Lys)8-OH cellular uptake.