Novel Heteroleptic Iridium(III) Complexes Containing COUBPY Ligands for Effective Photoinduction of Ferroptosis for Cancer Therapy

Ferroptosis, a recently described form of regulated, nonapoptotic cell death mechanism, presents significant potential for cancer treatment, particularly when combined with photodynamic therapy (PDT). In this study, we report the synthesis and biological evaluation of a series of Ir-COUBPY complexes as novel photosensitizers (PSs) for effective cancer phototherapy. These complexes exhibit high stability under both dark and light conditions and are capable of photogenerating Type I and Type II reactive oxygen species (ROS), as well as photo-oxidizing NADH. Electron paramagnetic resonance (EPR) spectroscopy provided direct evidence of light-induced superoxide and singlet oxygen generation, confirming dual ROS pathways. Moreover, the Ir-COUBPY complexes preferentially accumulated in the mitochondria of cancer cells, leading to the photogeneration of hydroxyl radicals and hydrogen peroxide. Photocytotoxicity studies on HeLa and A375 cancer cells underscored the role of the COUBPY ligand in enhancing PDT efficiency upon irradiation with both green and red light. Among the Ir-COUBPY complexes, the most effective PS, <b>Ir4a</b>, was encapsulated in polyurethane-polyurea hybrid nanocapsules (<b>NC-Ir4a</b>), resulting in a significant increase in phototoxic index values (e.g., from 64 to 179.6 in A375 cells). Mechanistic studies confirmed ferroptosis as the primary cell death pathway induced by <b>Ir4a</b>, supported by light-dependent lipid peroxidation, glutathione oxidation and depletion, intracellular ATP photodepletion, and the viability-restoring effect of Fer-1. These effects were more pronounced upon nanoencapsulation. Photobiological studies with 3D tumor spheroids of A375 cells further confirmed higher cellular uptake of <b>NC-Ir4a</b>, contributing to improved phototoxic efficiency. Overall, these findings highlight the potential of coumarin-based COUBPY ligands in the design of new Ir-(III)-based PSs that can be activated with light within the phototherapeutic window, operating through nonconventional cell death mechanisms such as ferroptosis.