A Novel PSMA-Targeted Probe for NIRF-Guided Surgery and Photodynamic Therapy: Synthesis and Preclinical Validation.

ABSTRACT

A total of 20% to 50% of prostate cancer (PCa) patients leave the surgery room with positive tumour margins. The intraoperative combination of fluorescence guided surgery (FGS) and photodynamic therapy (PDT) may be very helpful for improving tumour margin delineation and cancer therapy. PSMA is a transmembrane protein overexpressed in 90−100% of PCa cells. The goal of this work is the development of a PSMA-targeted Near InfraRed Fluorescent probe to offer the surgeon a valuable intraoperative tool for allowing a complete tumour removal, implemented with the possibility of using PDT to kill the eventual not resected cancer cells. PSMA-617 binding motif was conjugated to IRDye700DX-NHS and the conjugation did not affect the photophysical characteristics of the fluorophore. The affinity of IRDye700DX-PSMA-617 towards PCa cells followed the order of their PSMA expression, i.e., PC3-PIP > LNCaP > PC3, PC3-FLU. NIRF imaging showed a significant PC3-PIP tumour uptake after the injection of 1 or 5 nmol with a maximum tumour-to-muscle ratio (ca. 60) observed for both doses 24 h post-injection. Importantly, urine, healthy prostate, and the bladder were not fluorescent at 24 h post-injection. Flow cytometry and confocal images highlighted a co-localization of PSMA+ cells with IRDye700DX-PSMA uptake. Very interestingly, ex vivo analysis on a tumour specimen highlighted a significant PSMA expression by tumour-associated macrophages, likely attributable to extracellular vesicles secreted by the PSMA(+) tumour cells. FGS proved that IRDye700DX-PSMA was able to easily delineate tumour margins. PDT experiments showed a concentration-dependent decrease in cell viability (from 75% at 10 nM to 12% at 500 nM), whereas controls did not show any cytotoxicity. PC3-PIP tumour-bearing mice subjected to photodynamic therapy showed a delayed tumour growth. In conclusion, a novel PSMA-targeted NIRF dye with dual imaging-PDT capabilities was synthesized and displayed superior specificity compared to other small PSMA targeted molecules.