Tuning Lipophilicity to Develop an MMP-Independent Fluorescent Probe for Visualizing Mitochondrial Peroxynitrite in Drug-Induced Liver Injury

Mitochondrial peroxynitrite (ONOO – ), a highly reactive nitrogen species, is critically involved in oxidative-stress-induced cell damage, playing vital roles in the pathogenesis of numerous diseases. However, the real-time detection of ONOO – in mitochondria is still challenging due to the reliance of existing probes on mitochondrial membrane potential (MMP), often disrupted under disease conditions. Therefore, we attempted to develop and investigate an MMP-independent, near-infrared fluorescent probe ( Mito-ONOO – ). To achieve this, a series of fluorophores Flu-(1–5) was rationally constructed by introducing alkyl chains of varying lengths (C2, C4, C8, C12, and C16) to fine-tune their lipophilicity and mitochondrial retention potential. Flu-(1–5) were initially screened for their lipophilicity and mitochondria-targeting capability under ONOO – -stimulated stress. Flu-5 was finally selected for its optimal combination of notable lipophilicity and mitochondrial retention to construct the Mito-ONOO – . Upon evaluation, Mito-ONOO – displayed a reputable Stokes shift (88 nm), fast response (around 1 min), and excellent mitochondrial localization potential (PC = 0.96). In addition, the designed probe demonstrated excellent selectivity and sensitivity, enabling precise visualization of mitochondrial ONOO – in living HepG2 cells. Furthermore, the probe was successfully employed to monitor ONOO – dynamics during a drug-induced liver injury (DILI) mice model. Collectively, the findings of this study underscore the potential of Mito-ONOO – as a powerful tool for exploring mitochondrial redox homeostasis and its perturbation under pathological conditions.