Increase of doxorubicin sensitivity for folate receptor positive cells when given as the prodrug N‐(phenylacetyl) doxorubicin in combination with folate‐conjugated PGA

Folate receptor (FR) has been proposed as a promising target for tumor drug targeting. The aim of this study was to increase the chemo‐sensitivity of FR‐positive cells to doxorubicin by folate‐directed enzyme prodrug therapy (FDEPT). Folate conjugated penicillin‐G amidase was prepared and its ability to hydrolyze N‐(phenylacetyl) doxorubicin was measured by HPLC. Fluorescence and confocal image analysis revealed that Folate‐PGA can be specifically delivered into FR‐positive HeLa and SKOV3 tumor cells. In vitro cytotoxity assays, IC50 was reduced with N‐(phenylacetyl) doxorubicin versus doxorubicin for HeLa (3.1‐fold reduction; p < 0.001) and SKOV3 (3.3‐fold reduction; p < 0.001) when Folate‐PGA was specifically bound to the cells. Complete activation was confirmed in HeLa and SKOV3 cells pretreated with free folic acid (1 mM), where the combination of N‐(phenylacetyl) doxorubicin with Folate‐PGA did not show any significant cell toxicity to the IC50 of doxorubicin. Pharmacokinetic clearance and biodistribution studies in vivo showed that 125I‐Folate‐PGA was cleared from blood within 24 h and had significantly higher tumor uptake compared to 125I‐PGA (p < 0.05). These results demonstrate that the FDEPT approach may be a potential promising strategy to improve chemotherapy‐resistant cancers therapeutic ratio and warranted future studies.