Multi-organ photoacoustic imaging of Cathepsin B dysregulation reveals hepatic-to-aortic progression in atherosclerosis

Atherosclerosis is a chronic inflammatory disease of the arterial wall and a leading cause of cardiovascular morbidity worldwide. We report the development of a sulfur-substituted hemicyanine-based photoacoustic probe (S-HD-CTB) for sensitive detection of cathepsin B (CTB) activity in atherosclerosis. The probe utilizes a valine-citrulline dipeptide linker that undergoes CTB-specific cleavage, triggering an intramolecular charge transfer mechanism that enhances both fluorescence and photoacoustic signals. Sulfur substitution provides 2.6-fold greater photoacoustic signal intensity compared to oxygen-substituted analogs. Using ApoE-/- mouse models (Healthy, Early AS, Late AS), simultaneous imaging of hepatic and aortic CTB activity revealed a hepatic-to-aortic temporal sequence of protease dysregulation during atherosclerotic progression. Remarkably, despite massive CD68+ macrophage infiltration in Late AS aortas, conventional CTB immunofluorescence remained weak, whereas S-HD-CTB generated robust signals-demonstrating superior sensitivity of activity-based imaging over antibody-based detection. Serum assays confirmed stepwise increases in circulating CTB activity correlating with disease stage. With excellent biocompatibility, S-HD-CTB offers a valuable tool for staging atherosclerosis and monitoring inflammatory protease activity.