Rational Design of a Double-Locked Photoacoustic Probe for Precise In Vivo Imaging of Cathepsin B in Atherosclerotic Plaques

Atherosclerotic plaque rupture is a significant cause of acute cardiovascular events such as heart attack and stroke, triggered by the decomposition of fiber caps induced by cysteine cathepsin. However, the accurate measurement of cathepsin B (CTB) activity in plaques is challenging due to the low specificity and insufficient penetration depth of available atherosclerosis-associated cathepsin fluorescent probes, hampering reliable assessment of plaque vulnerability. To address these limitations, we added both lipophilic alkyl chain and hydrophilic CTB substrate to the hemicyanine scaffold to develop a lipid-unlocked CTB responsive probe (L-CRP) that uses lipids and CTB as two keys to unlock photoacoustic (PA) signals for measuring CTB activity in lipophilic environments. Such properties allow L-CRP for the reliable imaging of specific CTB activities in foam cells and atherosclerotic plaques while keeping in silence toward CTB in lipid-deficient environments, such as M1-type macrophages and LPS-induced inflammatory lesions. Moreover, the activatable PA signals of L-CRP exhibit a deeper tissue penetration ability (>1.0 cm) than current CTB probes based on near-infrared fluorescent imaging (∼0.3 cm), suitable for atherosclerosis imaging in living mice. In atherosclerotic mice, L-CRP dynamically reports intraplaque CTB levels, which is well-correlated with the plaque vulnerability characteristics such as fiber cap thickness, macrophage recruitment, and necrotic core size, thus enabling risk stratification of atherosclerotic mice complicated with pneumonia. Moreover, L-CRP successfully identifies atherosclerotic plaques in excised human artery tissues, promising for auxiliary diagnosis of plaque vulnerability in clinical application.