Enzyme-activatable hemicyanine-based probe for spatiotemporal detection of aminopeptidase N in colitis

The development of novel molecular probes for real-time monitoring of aminopeptidase N (APN) activity is crucial for understanding inflammatory bowel disease (IBD) progression. Here, we present a new probe, HDAPN, a near-infrared (NIR) activatable fluorescent probe based on a hemicyanine scaffold, designed for selective APN detection. Structural characterization confirmed the molecular integrity of the probe, while spectroscopic studies demonstrated its exceptional photostability and response. HDAPN exhibits a remarkable "turn-on" response (720 nm emission) upon APN-mediated cleavage, with outstanding sensitivity (LOD = 0.82 ng/mL) and linearity under physiological conditions. The probe showed minimal cross-reactivity with interferents (e.g., ROS, metal ions, and other enzymes) and successfully detected LPS-induced APN upregulation in RAW264.7 macrophages. In DSS-induced colitis models, HDAPN enabled non-invasive visualization of APN activity, with fluorescence intensity correlating with disease severity (2-fold increase in severe colitis). Comprehensive toxicity evaluations confirmed its biosafety in vitro and in vivo . These findings highlight the potential of HDAPN as a robust tool for studying APN-associated pathophysiology and therapeutic development.