NTR-Activated NIR-I Photosensitizer for Ultrasensitive Hypoxia Tumor Imaging and Photodynamic Therapy

Abstract Nitroreductase (NTR), overexpressed in solid tumors, is a specific biomarker indicating tumor hypoxia. NTR-activatable photosensitizers are an effective approach for tumor theranostics. However, challenges such as slow response, imprecise tumor recognition, and weak photodynamic effects under hypoxic conditions limit their utility in cancer imaging and therapy. Using an iodinated xanthene-cyanine near-infrared fluorescent scaffold, we developed a series of NTR-activatable photosensitizers, IX-NO2-H, IX-NO2-F, and IX-NO2-CF3, incorporating 4-nitrophenyl as the NTR-responsive unit with different ortho-substituents. Upon NTR and NADH activation, the nitro group is reduced to an amino group, triggering the release of the photosensitizer through a self-immolative reaction. Spectral analyses and molecular docking experiments revealed that IX-NO2-CF3, based on 3-trifluoromethyl-4-nitrophenyl, exhibited the best performance, showing rapid NTR response (<7 min) and a substantial fluorescence increase (∼78-fold) at 718 nm. In addition, owing to its specific activation by endogenous NTR, IX-NO2-CF3 was successfully used to evaluate the degree of hypoxia in cancer cells and tumor-bearing mice while exhibiting excellent mitochondrial targeting ability (Mander’s overlap coefficients >0.9). Upon laser irradiation, significant generation of reactive oxygen species, including singlet oxygen (1O2) and superoxide anion (O2•−), from IX-NO2-CF3 post NTR activation induced cancer cell apoptosis and effectively suppressed tumor growth through NTR-activated type I and/or II photodynamic therapy under normoxic and severely hypoxic conditions. This study presents a novel strategy for highly sensitive tumor hypoxia imaging and an NTR-triggered photosensitizer for accurate cancer therapy (80% TGI) with minimal side effects.