Abstract: AimTo demonstrate the specific killing of folate receptor (FR)-positive tumor cells can be achieved by folate-targeted penicillin-G amidase (PGA) combined with its prodrug substrate N-(phenylacetyl) doxorubicin (DOXP). MethodsFolic acid was covalently linked to PGA and folate content value was determined by quantitative UV spectrophotometry. The ability of folate conjugated PGA to hydrolyze DOXP was measured by RP-HPLC. Visual demonstration of uptake by FR(+) HeLa and SKOV3 cells was detected by using FITC labeled folate-PGA and a fluorescence microscopy. The cytotoxicity of DOXP towards the cells in the presence or absence of folate-PGA was assayed by using MTT method. ResultsThe folate-PGA has a specific activity of 29.8 U·mg^-1(protein). FR selectivity was confirmed by fluorescence microscopy. The combination of DOXP prodrug with folate-PGA generated higher cytotoxicity towards the FR(+) cells than free doxorubicin. The IC₅₀was 0.72 μmol·L^-1 for HeLa cells and 0.75 μmol·L^-1 for SKOV3 cells, respectively. Further, the enhanced cytotoxicity reduced greatly with the addition of free folic acid. ConclusionFolate conjugated PGA did not significantly compromise PGA catalytic activity and enabled binding prodrug-activating enzyme PGA to folate receptor expressing cells, and increased the sensitivity of the cells to doxorubicin followed by administration of its prodrug substrate.