An Extrinsic Enzyme-Activatable Fluorescent Probe for High-Contrast Tumor Imaging

Abstract

Malignant tumors pose a major threat to human health, and the effectiveness of treatment largely depends on accurate early diagnosis and complete resection. Although fluorescence imaging offers high sensitivity and enables real-time visualization for intraoperative navigation, conventional fluorescent dyes are limited by photobleaching, poor targeting, and low signal-to-noise (S/N) ratios. These limitations result in false-negative or false-positive outcomes, thereby compromising the precision of surgery and the validity of subsequent treatment decisions. Herein, we developed a fluorescent probe, TMN-CPG, which is specifically activated by carboxypeptidase G2 (CPG2), an enzyme notable for prodrug activation in antibody-directed enzyme prodrug therapy (ADEPT). Upon CPG2-mediated hydrolysis of the TMN-CPG amide bond, the highly fluorescent TMN-NH2 is released and subsequently internalized by cancer cells, resulting in tumor-specific fluorescence. In a 4T1 tumor-bearing mouse model, this extrinsic enzyme-activated fluorescence system produced high-contrast tumor imaging. These results indicate that this external enzyme-activatable probe can minimize nonspecific activation, thereby improving the accuracy of tumor imaging.