A Bienzyme-Locked Activatable Photoacoustic Probe for Precise in Vivo Imaging of Drug-Induced Liver Injury

The efficient and accurate diagnosis of a drug-induced liver injury (DILI) is crucial for enabling timely clinical intervention. Here, we for the first time report the development of a novel bienzyme-locked activatable photoacoustic (PA) probe (SH-NAL) for precise in vivo imaging of DILI. This rationally designed probe integrates a sulfur-substituted hemicyanine derivative (SH-NH 2 ) as the PA reporter and a pseudopeptide moiety as the recognition unit. The PA signal activation mechanism relies on sequential enzymatic cleavage by two DILI-associated peptidases, achieving superior signal-to-background ratios compared with single-enzyme responsive probes (SH-NL for LAP and SH-NA for APN). PA imaging of DILI in vivo demonstrated a much higher activation efficiency for SH-NAL than for single-locked probes. Notably, SH-NAL enabled differential diagnosis of DILI from cirrhosis, hepatitis, and healthy liver tissue through quantitative PA signal analysis in both live animal imaging experiments and serological tests. This dual-enzyme activation strategy not only improves diagnostic specificity but also shows the potential to serve as a versatile platform for investigating DILI pathogenesis in clinical settings.