Liposomal gene therapy vectors that penetrate cells by endocytosis must escape an endosomal compartment in order to enter the target cell's nucleus. Because such endosomal compartments are generally acidic in nature, pH-sensitive
liposomes have been designed that are stable at extracellular pH ( approximately pH 7.4) but fusogenic at endosomal pH values ( approximately pH 5). We report here the use of a novel
folate-targeted, pH-sensitive, anionic liposomal vector that mediates the efficient delivery of
DNA into
folate receptor-bearing cells and discharges the
DNA into the cytoplasm. N-Citraconyl-
dioleoylphosphatidylethanolamine (C-DOPE), a derivative of dioleylphosphatidylethanolamine (DOPE) that hydrolyzes rapidly at pH 5 to yield DOPE, was synthesized and incorporated with DOPE and
folate-polyethyleneglycol-DOPE into
liposomes. The resulting
liposomes were stable at neutral pH but fusogenic at pH 5.
Folate-labeled gene transfer vectors were prepared by compacting plasmid
DNA with
polylysine at a 1:0.75 (w/w) ratio and complexing the condensed cationic plasmid with the above anionic
liposomes. Association of the
polylysine-
DNA with the
liposomes was confirmed by
sucrose gradient centrifugation, where migration of the
folate-labeled vectors was midway between that of the free
liposomes and condensed
polylysine-
DNA. Transfection of cultured
cancer cells with the pH-sensitive liposomal vectors was found to be significantly more efficient than transfection with DOPE-
cholesterol hemisuccinate-based vectors, the more commonly used pH-dependent, liposomal transfection formulation. Optimization studies revealed that inclusion of only 3% C-DOPE and 0.1%
folate-derivatized DOPE yielded the highest transfection activity. Nearly quantitative competition with free
folic acid as well as direct correlation of transfection efficiency with
folate receptor density for several different cell lines further documented that vector uptake was mediated by
folate receptor endocytosis. Taken together, these data argue that C-DOPE warrants further consideration as a pH sensitive component of
lipid-based gene delivery formulations.