Abstract
Water-soluble germanium nanoparticles (GeNP) are fabricated by reduction method which emits blue-green fluorescence. Surface of this GeNP is coated by allylamine that allows the particles to present in great physical stability and is suitable for bioconjugation. The cytotoxicity of GeNP was analyzed in this study. The fabricated water-soluble GeNP generated significant cytotoxicity was analyzed by MTT, chronogenic and propidium exclusion assay. Flow cytometric analysis revealed that GeNP did not cause the blockage of cell cycle progression and the appearance of sub-G1 fraction. Furthermore, neither DNA ladder nor caspase 3 activity was increased by GeNP treatment. Administration of necrosis inhibitor, necrostatin-1, attenuated the GeNP-induced cell damage. These results demonstrated that GeNP caused necrotic cell death in CHO K1 cells. The mechanism involved in the cell death was subsequently investigated. Intracellular calcium content was estimated after GeNP treatment. Elevation of the calcium content occurred 12 hours later and this effect was diminished by calcium cheletor, BAPTA-AM. Relative oxygen species (ROS) increased 15 hours after giving GeNP. Addition of N-acetylcysteine (NAC), an antioxidant, decreased the ROS level and cell damage. Reduction of mitochondrial membrane potential (MMP) was observed 21 hours after GeNP treatment. The reduction can be attenuated by the administration of cyclosporine A (CsA), an inhibitor of mitochondrial membrane transition pore. Using BAPTA-AM, NAC and CsA respectively to examine the relationship of the signaling factors, a pathway for the GeNP-induced necrotic cell death can be established. The water-soluble GeNP stimulates the release of intracellular calcium storage which leads to the elevation of ROS production. The ROS then causes the reduction of MMP and subsequently results in necrotic cell death.