Azoreductase-ResponsiveNIR Fluorescent Self-AssembledProbe for Hypoxia Imaging of Tumors, Hindlimb Ischemia, and UlcerativeColitis

Azoreductase (AzoR) has emerged as a key player in reductive metabolism under hypoxia and a potential biomarker for hypoxia-associated pathologies, such as cancer and ulcerative colitis (UC). However, current clinical practice lacks a reliable method for real-time AzoR monitoring to effectively guide personalized therapies and assess treatment responses. We address this challenge by developing an AzoR-activatable near-infrared (NIR) fluorescent self-assembled probe, NFK-(Azo)-DCM, which combines self-assembly properties with hypoxia-specific activation through a rationally designed azobenzene unit. This molecular architecture enables a selective chemical transformation─the conversion of azobenzene to an electron-donating amino group under AzoR overexpression in hypoxic tissues─which initiates a cascade of NIR fluorescence signal amplification. Confocal imaging revealed that NFK-(Azo)-DCM exhibited statistically significant (P < 0.01) fluorescence enhancement in LLC, 4T1, and HEK293 cells under hypoxic conditions. In LLC tumor-bearing mice, the probe produced a 3.5-fold increase in the NIR fluorescence signal specifically in hypoxic tumor regions compared to the negative controls, establishing its utility for tumor bioimaging. Most notably, in a murine hindlimb ischemia model, the probe achieved rapid and accurate detection of AzoR activity through its NIR emission, with a strong linear correlation between the signal intensity and ischemic severity (R2 > 0.9). The diagnostic versatility of NFK-(Azo)-DCM was further validated in both the acute and chronic UC models. These findings establish NFK-(Azo)-DCM as a dual-functional platform capable of noninvasive, real-time monitoring of hypoxia-related diseases, providing a promising method for advancing research in AzoR-associated pathologies.