Development of a PSMA and EphA2 dual-receptor-activated fluorescent probe for wash-free prostate cancer cell imaging

Prostate cancer (PCa) remains a leading cause of cancer-related mortality in men, underscoring the need for improved diagnostic tools. In this study, a novel dual-receptor-activated fluorescence probe (9) designed to simultaneously bind to prostate-specific membrane antigen (PSMA) and erythropoietin-producing hepatocellular receptor type A2 (EphA2), two key biomarkers in PCa, was developed and applied for wash-free cell imaging. The probe was synthesized by conjugating both PSMA- and EphA2-targeting ligands with a sulfonated benzothiazole environment-sensitive dye. 9 demonstrated good binding affinity (K_d = 34 nM for PSMA, 9.70 nM for EphA2) with significant fluorescence activation upon receptor binding (14.1-fold for PSMA; 8.6-fold for EphA2). Cell studies demonstrated 9 showed negligible cytotoxicity and good targeting specificity, which could enable convenient monitoring of the dynamic biological processes in PCa, such as ligand-receptor interactions. The study may also provide insights into the design and applications of multiple-receptor-activated fluorescence probes.