Probing mitochondrial peroxynitrite biogenesis by a N-morpholinoarylimine-based iridium(III) complex in drug-induced liver cells

Peroxynitrite (ONOO⁻), a strong oxidizing agent, has an important function in the pathogenesis of various diseases, including cardiovascular, inflammatory and neurodegenerative diseases. Specifically, mitochondrial ONOO⁻ exacerbates liver injury by driving oxidative/nitrative stress and mitochondrial dysfunction, ultimately triggering dual apoptotic-necrotic hepatocyte death pathways. ONOO⁻ and its functions have been widely studied by fluorescence imaging probes, owing to their strong sensitivity, non-invasiveness, and real-time ability. However, existing probes are heavily constrained by interference from other reactive species. Herein, we describe a luminescent iridium(III) complex (1) with an N-morpholinoarylimine moiety as the recognition site for ONOO⁻ for imaging mitochondrial ONOO⁻. The probe shows high luminescence response to ONOO⁻ in aqueous buffer, with a luminescence enhancement of 27-fold at 100 μM ONOO⁻ and a limit of detection (LOD) of 0.65 μM, as well as high selectivity over other reactive species. Furthermore, the probe can sense both exogenous and endogenous mitochondrial ONOO⁻. Further experiments demonstrated it could visualize exogenous ONOO⁻ in 3D multicellular tumor spheroids (MCTSs) and unmask endogenous ONOO⁻ production through an NADPH oxidase 4 (NOX-4)-mediated pathway in drug-induced liver cells. This work demonstrates the potential of this strategy for developing imaging tools for probing the pathological roles of subcellar ONOO⁻ and diagnosing liver injury in the clinic. © 2025 The Authors