Mn(3)O(4) Nanocrystal-Induced Eryptosis Features Ca(2+) Overload, ROS and RNS Accumulation, Calpain Activation, Recruitment of Caspases, and Changes in the Lipid Order of Cell Membranes.

Accumulating evidence suggests that manganese oxide nanoparticles (NPs) show multiple enzyme-mimicking antioxidant activities, which supports their potential in redox-targeting therapeutic strategies for diseases with impaired redox signaling. However, the systemic administration of any NP requires thorough hemocompatibility testing. In this study, we assessed the hemocompatibility of synthesized Mn(3)O(4) NPs, identifying their ability to induce spontaneous hemolysis and eryptosis or impair osmotic fragility. Concentrations of up to 20 mg/L were found to be safe for erythrocytes. Eryptosis assays were shown to be more sensitive than hemolysis and osmotic fragility as markers of hemocompatibility for Mn(3)O(4) NP testing. Flow cytometry- and confocal microscopy-based studies revealed that eryptosis induced by Mn(3)O(4) NPs was accompanied by Ca(2+) overload, altered redox homeostasis verified by enhanced intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS), and a decrease in the lipid order of cell membranes. Furthermore, Mn(3)O(4) NP-induced eryptosis was calpain- and caspase-dependent.