Nuclear delivery of doxorubicin via folate-targeted liposomes with bypass of multidrug-resistance efflux pump

Folic acid, attached to polyethyleneglycol-derivatized, distearoyl-phosphat idylethanolamine, was used to target irt vitro liposomes to folate receptor (FR)-overexpressing tumor cells, Confocal fluorescence microscopic observa tions demonstrated binding and subsequent internalization of rhodamine-labe led liposomes by a high FR-expressing, murine lung carcinoma line (M109-HiF R cells), with inhibition by free folic acid. Additional experiments tracki ng doxorubicin (DOX) fluorescence with DOS-loaded, folate-targeted liposome s (FTLs) indicate that liposomal DOX is rapidly internalized, released in t he cytoplasmic compartment, and, shortly thereafter, detected in the nucleu s, the entire process lasting 1-2 h, FR-mediated cell uptake of targeted Li posomal DOX into a multidrug-resistant subline of M109-HiFR cells (M109R-Hi FR) was unaffected by P-glycoprotein-mediated drug efflux, in sharp contras t to uptake of free DOX, based on verapamil-blockade experiments with quant itation of cell-associated DOX and flow cytometry analysis, Delivery of DOX by FTLs to M109R-HiFR cells increased continuously with time of exposure, reaching higher drug concentrations in whole cells and nuclei compared with exposure to free DOS. The ill vitro cytotoxic activity obtained with DOX-l oaded FTLs was 10-fold greater than that of the nontargeted liposome formul ation, but was not improved over that of free DOX despite the higher cellul ar drug levels obtained with the targeted liposomes in M109R-HiFR cells. Ho wever, if M109R-HiFR cells were exposed to drugs in vitro and tested in an in vivo adoptive assay for tumor growth in syngeneic mice along a 5-week ti me span, FTL DOX was significantly more tumor inhibitory than free DOT. It is suggested that the biological activity of liposomal DOX released inside the cellular compartment is reduced in vitro due to the aggregated state of DOX, resulting from the liposome drug-loading process, and requires a long period of time and/or an irt vivo environment for full expression.