Cationic poly(ethyleneglycol) lipids incorporated into pre-formed vesiclesenhance binding and uptake to BHK cells

This paper describes a new method for enhancing the interaction of liposome s with cells. A novel class of cationic poly(ethyleneglycol) (PEG)-lipid (C PL) conjugates have been characterized for their ability to insert into pre -formed vesicles and enhance in vitro cellular binding and uptake of neutra l and sterically-stabilized liposomes. The CPLs, which consist of a distear oylphosphatidylethanolamine (DSPE) anchor, a fluorescent dansyl moiety, a h eterobifunctional PEG polymer (M-r 3400), and a cationic headgroup composed of lysine derivatives, have been described previously [Bioconjug. Chem. 11 (2000) 433]. Five separate CPL, possessing 1-4 positive charges in the hea dgroup (referred to as CPL1-CPL4, respectively), were incubated (as micella r solutions) in the presence of neutral or sterically-stabilized cationic l arge unilamellar vesicles (LUVs), and were found to insert into the externa l leaflet of the LUVs in a manner dependent on temperature, time, CPL/lipid ratio, and LW composition. For CPL/lipid molar ratios less than or equal t o 0.1, optimal insertion levels of approximately 70% of initial CPL were ob tained following 3 h at 60 degreesC. The insertion of CPL resulted in aggre gation of the LUVs, as assessed by fluorescence microscopy, which could be prevented by the presence of 40 mM Ca2+. The effect of CPL-insertion on the binding of LUVs to cells was examined by fluorescence microscopy and quant ified by measuring the ratio of rhodamine fluorescence to protein concentra tion. Neither control LUVs or LUVs containing CPL2 displayed significant up take by BHK cells. However, a 3-fold increase in binding was observed for L UVs possessing CPL3, while for CPL4-LUVs values as high as 10-fold were ach ieved. Interestingly, the increase in lipid uptake did not correlate with t otal surface charge, but rather with increased positive charge density loca lized at the CPL distal headgroups. These results suggest that incorporatio n of CPLs into existing liposomal drug delivery systems may lead to signifi cant improvements in intracellular delivery of therapeutic agents. (C) 2001 Elsevier Science B.V. All rights reserved.