A novel anticancer theranostic prodrug, FDU-DB-NO, specifically activated by hypoxia for selective two-photon imaging hypoxia status, real-time tracking drug release, and solid tumor therapy was designed. The devised prodrug consists of an anticancer drug floxuridine (FDU), a fluorescence dye precursor 4'-(diethylamino)-1,1'-biphenyl-2-carboxylate (DB), and a hypoxic trigger 4-nitrobenzyl group. In normal cells, FDU-DB-NO is "locked". Whereas in tumor cells, the prodrug is "unlocked" by hypoxia and results in fluorescent dye 7-(diethylamino)coumarin (CM) generation along with FDU release. The amounts and rates of CM formation and FDU release were controlled by hypoxic status and increased with the decreasing of the O concentration. The hypoxic status, distribution of oxygen, and amount of FDU release in tumor cells, spheroids, and tumor tissue could be visualized by fluorescence. FDU-DB-NO showed high cytotoxicity against hypoxic MCF-7 and MCG-803 cell lines and no cytotoxicity against normoxic BRL-3A cells and exhibited effective inhibition on tumor growth of MCF-7-cell-inoculated xenograft nude mice. This strategy may provide a promising platform for selective two-photon imaging hypoxia status, real-time tracking drug release, and personalized solid tumor treatment.

A smart theranostic prodrug IMC-FDU-TZBC-NO 2 , releasing active drug on-demand based on hypoxiaactivated and indomethacin-mediated, for solid tumor imaging and efficient therapy was designed. This prodrug was constructed by conjugating chemotherapy drug 5-fluoro-2-deoxyuridine (FDU), targeting moiety indomethacin (IMC), and the hypoxic trigger 4-nitrobenzyl group to a fluorescent dye precursor, which was mediated by IMC and activated by NTR under hypoxic conditions. The fluorescent dye IMC-TZBCM was generated and FDU was released at the same time in tumor cells. The rates and amounts of FDU release and IMC-TZBCM generation were regulated by hypoxia status, and increased with increasing degree of hypoxia. Nevertheless, it is "locked" in normal cells. It combined the advantages of tumor targeting, diagnosis, and chemotherapy functions, showed excellent targeting ability to cancer cells, excellent stability in physiological conditions, high cellular uptake efficiency, and on-demand drug release behavior. The in vitro and in vivo assays demonstrated that IMC-FDU-TZBC-NO 2 exhibits enhanced anticancer potency and low side effects. The novel targeted theranostic prodrug activated by hypoxia shows a great potential in cancer therapy.