An activatable Hemicyanine-based fluorescent probe for specific detection and imaging of aminopeptidase N in diabetic wound healing

Aminopeptidase N (APN) plays crucial roles in wound healing and disease progression, yet tools for precise in vivo monitoring remain limited. We developed APN-activatable fluorescent probe, AFP, a hemicyanine-based probe that exhibits remarkable specificity and sensitivity toward APN through an enzymatic cleavage-mediated fluorescence turn-on mechanism. AFP demonstrates a bathochromic shift upon APN activation (λabs = 705 nm), with an increasing fluorescence response. The probe maintains excellent stability against reactive species and shows negligible cytotoxicity (<5% viability loss at 25 μM). In diabetic wound models, AFP reliably distinguished impaired healing through differential APN activity patterns, correlating with histopathological and biochemical markers of disease severity. Systemic biosafety evaluations confirmed no organ toxicity, supporting AFP's potential for clinical translation in wound management and beyond.