Erythrotoxicity of iron-doped cerium oxide nanoparticles is mediated by eryptosis

Iron doping has been reported to significantly modify the properties of metal oxide nanoparticles (NPs), including their interaction with cells and therefore toxicity. The present study explores the ability of Fe³ ⁺-doped CeO₂ NPs with the varying content of Fe³⁺ ions (3, 5, and 10 at%) to stimulate eryptosis, a controllable cell death pathway of mature erythrocytes, as an attempt to shed light on hemocompatibility of iron-doped CeO₂ NPs. Overall erythrotoxicity of iron-doped CeO₂ NPs was evaluated by investigating their ability to trigger spontaneous hemolysis and affect osmotic fragility of rat erythrocytes. Eryptosis of erythrocytes exposed to iron-doped and non-doped CeO₂ NPs for 24 h was evaluated by the state-of-the-art flow cytometry-based annexin V staining. Mechanisms involved in iron-doped CeO₂ NP-induced eryptosis were evaluated by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and caspase-3 assays, as well as the fluorescent O1O (2-(2'-hydroxy-phenyl)-5-phenyl-1,3-oxazole), NR12S, Fluo-3 AM, BODIPY™ 581/591 C11, and BioTracker Far-red Fe²⁺ Live Cell imaging probes. Dose-dependent effects of iron-doped CeO₂ NPs on hemolysis, osmotic fragility, and eryptosis were revealed. Eryptosis triggered by iron-doped CeO₂ NPs was found to be oxidative stress-mediated, cation channel-driven, and caspase-dependent. Oxidative stress and alterations of lipid membranes demonstrated by the tested NPs could be attributable to their direct •OH generation and peroxidase-like activity, as well as Fe²⁺-mediated Fenton reaction at the surface of Fe³⁺-doped CeO₂ NPs (presumably due to Fe³⁺/Fe²⁺ redox cycling). Importantly, non-doped CeO₂ NPs did not promote eryptosis. Nor they stimulated generation of ROS and Ca²⁺ influx. The presence of iron was found to mediate phosphatidylserine externalization, caspase activation, and changes in lipid membranes of erythrocytes. Notably, Fe³⁺-doped CeO₂ NP-induced eryptosis was independent of p38 MAPK and CK1α. Iron-doped CeO₂ NPs trigger eryptosis, an effect mediated by iron. Iron doping is a promising modification of CeO₂ NPs, which can modulate their toxicity and widen their pharmaceutical profile.