ROS and Caspase-3 Dual-Activated Photoacoustic Probe for Enhanced Imaging of Myocardial Ischemia-Reperfusion Injury

Myocardial ischemia-reperfusion (MI/R) injury continues to be a major clinical concern, but effective imaging approaches for the accurate detection of MI/R-induced damage remain scarce. Herein, we rationally design a dual-activated photoacoustic (PA) probe, AcDEVDFFG-Hcy-BOH ( P1 ), which responds to two hallmark biomarkers of MI/R injury: reactive oxygen species (ROS) and caspase-3. P1 comprises four functional domains: a hydrophilic DEVD peptide (caspase-3 substrate and hydrophilicity enhancer), a diphenylalanine (FF) motif (self-assembling unit), hemicyanine (PA chromophore), and a H 2 O 2 -responsive boronic acid moiety (caging the PA signal of hemicyanine). In the MI/R pathological microenvironment with elevated ROS and caspase-3, P1 first undergoes H 2 O 2 -triggered boronic acid cleavage to uncage hemicyanine, followed by caspase-3-mediated DEVD hydrolysis. This dual activation of P1 drives the formation of self-assembled nanoparticles, leading to significant fluorescence quenching and consequent PA signal enhancement. P1 showed 6.9-fold, 5.3-fold, and 4.8-fold PA signal enhancement relative to inactive P1 in vitro, in hypoxia/reoxygenation-induced cardiomyocytes, and in a murine MI/R injury model, respectively. In contrast, control probes lacking responsiveness to ROS ( P2 ) or caspase-3 ( P3 ) exhibited significantly weaker PA signals. This strategy enables specific and sensitive MI/R injury imaging, holding promise for enhanced cardiac pathology diagnostics.