A Tandem-Responsive Probe for Dual-Enzyme Photoacoustic Imaging of Atherosclerotic Plaque in Vivo

Atherosclerosis often involves dysregulated enzymatic activities, but conventional probes lack specificity due to off-target activation. To address the critical issue of off-target activation plaguing conventional single-enzyme probes, we developed an innovative tandem-responsive probe (TRP) based on a hemicyanine scaffold. This probe features a novel sequential activation mechanism by neutrophil elastase (NE) and γ-glutamyl transpeptidase (GGT), ensuring precise detection of the diseased microenvironment with minimal false positives. TRP exhibited selective near-infrared fluorescence (740 nm) and photoacoustic (710 nm) signal enhancement only upon dual-enzyme activation, with no response to single enzymes or interferents. In vitro studies demonstrated linear NE detection (0–2.5 U/L, R 2 = 0.998) and superior tissue penetration (1 cm) via photoacoustic imaging. Cellular experiments confirmed TRP’s strict NE and GGT-dependency in cells, unlike the GGT-responsive control probe. In atherosclerotic mice with pneumonia comorbidity, TRP specifically localized to inflammatory sites while avoiding systemic GGT activation. Histopathology validated pneumonia-induced plaque destabilization. With excellent biosafety and dual-enzyme specificity, TRP represents a promising tool for precise inflammation imaging in deep tissues.