Intracellular Nitroreductase‐Triggered “On” and “Enhanced” Photoacoustic Signals for Sensitive Imaging of Tumor Hypoxia

Current molecular photoacoustic (PA) probes are designed with either stimulus‐turned “on” or assembly‐enhanced signals to trace biological analytes/events. PA probes based on the nature‐derived click reaction between 2‐cyano‐6‐aminobenzothiazole (CBT) and cysteine (Cys) (i.e., CBT‐Cys click reaction) possess both “turn‐on” and “enhanced” PA signals; and thus, should have higher sensitivity. Nevertheless, such PA probes, particularly those for sensitive imaging of tumor hypoxia, remain scarce. Herein, a PA probe NI‐Cys(StBu)‐Dap(IR780)‐CBT (NI‐C‐CBT) is rationally designed, which after being internalized by hypoxic tumor cells, is cleaved by nitroreductase under the reduction condition to yield cyclic dimer C‐CBT‐Dimer to turn the PA signal “ON” and subsequently assembled into nanoparticles C‐CBT‐NPs with additionally enhanced PA signal (“Enhanced”). NI‐C‐CBT exhibits 1.7‐fold “ON” and 3.2‐fold overall “Enhanced” PA signals in vitro. Moreover, it provides 1.9‐fold and 2.8‐fold overall enhanced PA signals for tumor hypoxia imaging in HeLa cells and HeLa tumor‐bearing mice, respectively. This strategy is expected to be widely applied to design more “smart” PA probes for sensitive imaging of important biological events in vivo in near future. A PA probe NI‐C‐CBT, after being internalized by hypoxic tumor cells, is cleaved by nitroreductase under reduction condition to yield cyclic dimer C‐CBT‐Dimer to turn the PA signal “ON,” and subsequently assembled into nanoparticles C‐CBT‐NPs with additionally enhanced PA signal (“Enhanced”), enabling sensitive imaging of tumor hypoxia.