Real-Time Monitoring Ferroptosis-Mediated Drug-Induced Liver Injury Using a Near-Infrared Mitochondrial Viscosity Probe

Abstract Drug-induced liver injury (DILI) is a frequent clinical condition in which hepatocyte ferroptosis serves as a core pathological mechanism, leading to critical alterations in the mitochondrial microenvironment, including viscosity. However, visualizing mitochondrial viscosity changes in real time during ferroptosis-driven DILI remains technically challenging. Herein, we report XT-TPE, a mitochondrion-targeted near-infrared (NIR) “turn-on” viscosity probe based on a tetraphenylethylene (TPE)-xanthene hybrid. This probe gives a remarkable 129-fold fluorescence increase in high-viscosity media with an outstanding linear relationship (R2 > 0.999). The probe displays low cytotoxicity toward hepatocytes, excellent mitochondrial colocalization (Pearson’s coefficient: 0.916), and enables sensitive monitoring of viscosity fluctuations during ferroptosis-mediated DILI both in vitro and in vivo. Using this probe, we screened 14 redox-active compounds and identified N-acetylcysteine (NAC) and ferrostatin-1 (Fer-1) as strong inhibitors of ferroptosis-driven DILI. In acetaminophen-induced DILI mouse models, XT-TPE enables noninvasive hepatic viscosity imaging and confirms the therapeutic efficacy of NAC and Fer-1. This work provides a reliable molecular tool for ferroptosis-related viscosity imaging and establishes a new strategy for anti-DILI drug screening.