Riboflavin–Ceria Nanoparticles Induce ROS-Driven Mitochondrial Dysfunction in Breast Cancer

Abstract Cerium oxide nanoparticles (CeO2 NPs) are redox-active nanomaterials with emerging potential in cancer therapy, although their limited cytotoxicity and insufficient mechanistic understanding hinder clinical application. Here, we developed riboflavin-functionalized CeO2 nanoparticles (Rf-CeO2) to enhance photodynamic anticancer efficacy via controlled reactive oxygen species (ROS) generation and mitochondrial targeting. Rf conjugation improves nanoparticle stability and enables photodynamic activation under UV irradiation leading to elevated intracellular oxidative stress. In MDA-MB-231 breast cancer cells, Rf-CeO2 exhibits significantly enhanced cytotoxicity compared to pristine CeO2, inducing dose-dependent cell death and apoptosis. This effect is markedly potentiated by UV exposure, consistent with increased ROS-mediated oxidative stress. Proteomic analysis identifies 60 differentially expressed proteins, revealing disruptions of metabolic pathways, protein synthesis, and DNA repair. Notably, citrate synthase (CS), a key enzyme in the tricarboxylic acid cycle, is significantly downregulated and associated with poor prognosis in breast cancer. Functional validation using Seahorse Mito Stress and TMRM assays confirms mitochondrial dysfunction, including impaired oxidative phosphorylation, reduced ATP production, and loss of membrane potential. Overall, these results demonstrate that Rf-CeO2 acts as an effective photodynamic nanoplatform that induces cancer cell death through synergistic ROS generation and mitochondrial metabolic disruption. This study provides new proteomic insights into ceria-based nanotherapeutics and highlights citrate synthase as a potential therapeutic target for breast cancer.