Aminopeptidase N-Triggered Electron Rearrangement of Fluorescein for Covalent Fluorescent Labeling and Image-Guided Orthotopic Bladder Cancer Resection

Abstract

Bladder cancer is the most common type of malignant tumor in the urinary system, which occurs in the bladder mucosa. Due to the lack of precise diagnostic tools, bladder cancer has a high recurrence rate. Fluorescence cystoscopy is recognized as a promising tool for improving the detection rate of bladder cancer, but its clinical application is hindered due to the scarcity of fluorescent agents. To address this issue, an aminopeptidase N (APN)-activated fluorescent agent with specific recognition and labeling abilities for bladder cancer has been developed. The fluorescent agent, namely, Flu-FAPN, exhibited high sensitivity and specificity toward APN under physiological conditions. The fluorescent intensity increased more than 37-fold after reacting with APN, and the detection limit is 0.10 ng/mL for APN. In addition, the hydrolysis of alanine catalyzed by APN could initiate the intramolecular electron rearrangement process, which subsequently contributed to the formation of protein-fluorescein adduct and thereby achieved covalent labeling of bladder cancer cells. Cell experiments conducted in cancer and normal cells demonstrate that Flu-FAPN has low cytotoxicity and high specificity for bladder cancer cells with an obvious fluorescence signal, which could be retained over 80% even in fixed cells. Finally, Flu-FAPN was successfully applied for the fluorescence-image-guided resection of tumor tissues in mice with orthotopic bladder cancer. We hope this covalent labeling-based agent can provide a promising tool for surgery navigation and accelerate the clinical application of fluorescence cystoscopy, thereby improving the cure rate of bladder cancer.