An enhanced bioluminescent probe for in vivo imaging of drug-induced apoptosis via acetylcholinesterase targeting.

Acetylcholinesterase has emerged as a compelling biomarker for optical imaging of tissue apoptosis. Existing AChE-targeted imaging approaches rely heavily on fluorescent probes, whose performance is often hindered by autofluorescence, light scattering, phototoxicity, photobleaching, and limited in vivo accuracy. Bioluminescence imaging, an intrinsic light-emitting modality that requires no external excitation, overcomes these limitations while offering superior sensitivity and applicability in living systems. However, no bioluminescent (BL) probes responsive to endogenous AChE have been reported to date. Herein, we report the design and synthesis of three AChE-activatable BL probes (AhBL-1, AhBL-2, and AhBL-3) by incorporating N, N-dimethyl ester groups into the D-luciferin scaffold. Among them, AhBL-3 demonstrated the highest AChE specificity, with a detection limit of 0.384 mU·mL and a cellular detection threshold as low as 152 cells. In cisplatin-induced apoptotic tumor tissues, AhBL-3 enabled real-time visualization of intracellular AChE dynamics with excellent biocompatibility and imaging performance. This study presents the first AChE-responsive BL probe platform, providing a robust tool for noninvasive apoptosis imaging in biomedical and pharmaceutical research.