Copper accumulation and acute toxicity in C6 glioma cells after application of copper oxide nanoparticles

The rat C6 glioma cell-line is frequently used as an experimental model for glial tumors. To investigate the potential use of copper oxide nanoparticles (CuO-NPs) as a therapeutic drug for glioma treatment, the consequences of an application of CuO-NPs on the cellular copper content and cell viability of C6 glioma cells was investigated. CuO-NPs were synthesized by a wet-chemical method and were coated with dimercaptosuccinic acid and bovine serum albumin to improve colloidal stability in physiological media. Application of these protein-coated nanoparticles (pCuO-NPs) to C6 cells caused a strong time-, concentration- and temperaturedependent copper accumulation. This cellular copper accumulation was accompanied by severe toxicity as indicated by the loss in cellular MTT-reduction capacity, the loss in cellular LDH activity, and by an increase in the number of propidium iodide-positive cells. Toxicity of pCuO-NPs to C6 cells was only observed for incubation conditions that increased the specific cellular copper contents above 20 nmol copper per mg protein. Despite severe toxicity, no obvious formation of reactive oxygen species was found in pCuO-NP-treated C6 cells. Unexpectedly, C6 glioma cells were less vulnerable to pCuO-NPs than cultured primary brain astrocytes. Both cellular copper accumulation and pCuO-NP-induced toxicity in C6 cells were prevented by application of cell membrane-permeable and -impermeable copper chelators, but not by frequently used endocytosis inhibitors. These data suggest that uptake of copper ions liberated extracellularly from the pCuO-NPs, rather than uptake of intact pCuO-NPs, leads to the observed toxicity of pCuO-NP-treated glioma cells.