A Novel Fluorescent Gemcitabine Prodrug That Follows a Nucleoside Transporter‐Independent Internalization and Bears Enhanced Therapeutic Efficacy With Respect to Gemcitabine

The multiplexity of cancer has rendered it the second leading cause of mortality worldwide and theragnostic prodrugs have gained popularity in recent years as a means of treatment. Theragnostic prodrugs enable the simultaneous diagnosis and therapy of tumors via high‐precision real‐time drug release monitoring. Herein, we report the development of the small theragnostic prodrug GF, based on the nucleoside anticancer agent gemcitabine and the fluorescent dye 5(6)‐carboxyfluorescein. We have successfully demonstrated its efficient internalization in tumor cells, showing localization throughout both the early and late endocytic pathways. Its mechanism of cell internalization was evaluated, confirming its independence from nucleoside transporters. Its cellular localization via confocal microscopy revealed a clathrin‐mediated endocytosis mechanism, distinguishing it from analogous compounds studied previously. Furthermore, GF exhibited stability across various pH values and in human blood plasma. Subsequently, its in vitro cytotoxicity was assessed in three human cancer cell lines (A549, U87 and T98). Additionally, its pharmacokinetic profile in mice was investigated and the consequent drug release was monitored. Finally, its in vivo visualization was accomplished in zebrafish xenotransplantation models and its in vivo efficacy was evaluated in A549 xenografts. The results unveiled an intriguing efficacy profile, positioning GF as a compelling candidate warranting further investigation. A fluorescent prodrug, named GF, based on gemcitabine and 5(6)‐carboxyfluorescein was developed in a single step. GF follows a nucleoside transporter‐independent endocytosis internalization mechanism, surpassing a key resistance mechanism against gemcitabine. Its pharmacokinetic profile and drug release efficiency were evaluated in mice, and eventually its visualization was achieved in zebrafish and its tumor inhibition was monitored in A549 xenografted mice.