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 causeof acutecardiovascular events such as heart attack and stroke, triggered bythe decomposition of fiber caps induced by cysteine cathepsin. However,the accurate measurement of cathepsin B (CTB) activity in plaquesis challenging due to the low specificity and insufficient penetrationdepth of available atherosclerosis-associated cathepsin fluorescentprobes, hampering reliable assessment of plaque vulnerability. Toaddress these limitations, we added both lipophilic alkyl chain andhydrophilic CTB substrate to the hemicyanine scaffold to develop alipid-unlocked CTB responsive probe (L-CRP) that uses lipids and CTBas two keys to unlock photoacoustic (PA) signals for measuring CTBactivity in lipophilic environments. Such properties allow L-CRP forthe reliable imaging of specific CTB activities in foam cells andatherosclerotic plaques while keeping in silence toward CTB in lipid-deficientenvironments, such as M1-type macrophages and LPS-induced inflammatorylesions. Moreover, the activatable PA signals of L-CRP exhibit a deepertissue penetration ability (>1.0 cm) than current CTB probes basedon near-infrared fluorescent imaging (& SIM;0.3 cm), suitable foratherosclerosis imaging in living mice. In atherosclerotic mice, L-CRPdynamically reports intraplaque CTB levels, which is well-correlatedwith the plaque vulnerability characteristics such as fiber cap thickness,macrophage recruitment, and necrotic core size, thus enabling riskstratification of atherosclerotic mice complicated with pneumonia.Moreover, L-CRP successfully identifies atherosclerotic plaques inexcised human artery tissues, promising for auxiliary diagnosis ofplaque vulnerability in clinical application.

Automated summary

Atherosclerotic plaque rupture, caused by the decomposition of fiber caps induced by cysteine cathepsin, is a significant cause of acute cardiovascular events. However, the accurate measurement of cathepsin B (CTB) activity in plaques is challenging due to the limitations of current fluorescent probes. To address this, a lipid-unlocked CTB responsive probe (L-CRP) was developed, which uses lipids and CTB as two keys to unlock photoacoustic signals for measuring CTB activity in lipophilic environments. L-CRP demonstrates reliable imaging of specific CTB activities in foam cells and atherosclerotic plaques, and exhibits deeper tissue penetration ability compared to current CTB probes. In atherosclerotic mice, L-CRP dynamically reports intraplaque CTB levels and enables risk stratification based on plaque vulnerability characteristics. L-CRP also successfully identifies atherosclerotic plaques in excised human artery tissues, showing promise for auxiliary diagnosis in clinical applications.