Development and Application of a Hemicyanine Dye-Based Probe for Real-Time Detection of Aminopeptidase N Activity in Foam Cells and Serum

Detecting aminopeptidase N (APN) activity in foam cells is crucial for understanding atherosclerosis (AS) progression, but it presents challenges in achieving high specificity and sensitivity to accurately monitor enzyme activity within complex cellular environments. In this investigation, a novel probe, which is specifically targeted at APN and constructed utilizing hemicyanine dyes (HD), has been successfully developed. The probe named AP, in which HD was integrated with APN-specific sequences, exhibited high sensitivity and selectivity when employed in the detection of APN carried out in solution, cellular, and animal models. The probe has undergone significant fluorescence changes upon APN interaction, enabling detection of molecular interactions and enzyme kinetics. Fluorescence analysis showed a linear correlation between fluorescence intensity and APN concentration. In vitro tests confirmed the probe's specificity without interference from other agents. The probe has detected APN activity in RAW264.7-derived foam cells and monitored serum APN in ApoE−/− mouse models of AS. Cytotoxicity studies showed low toxicity, supporting its biocompatibility. These findings suggest that AP is a promising tool for studying APN-related processes and advancing clinical diagnostics and tissue imaging.