Mn₃O₄ Nanocrystal-Induced Eryptosis Features Ca²⁺ Overload, ROS and RNS Accumulation, Calpain Activation, Recruitment...

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 Mn₃O₄ 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 Mn₃O₄ NP testing. Flow cytometry- and confocal microscopy-based studies revealed that eryptosis induced by Mn₃O₄ NPs was accompanied by Ca²⁺ 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, Mn₃O₄ NP-induced eryptosis was calpain- and caspase-dependent.