Distal Cationic Poly(Ethylene Glycol) Lipid Conjugates in Large Unilamellar Vesicles Prepared by Extrusion Enhance Liposomal Cellular Uptake

Abstract

Cationic poly(ethylene glycol)‐lipid conjugates (CPLs), a class of lipid designed to enhance the interaction of liposomes with cells, possess the following architectural features: 1) a hydrophobic lipid anchor of distearoylphosphatidylethanolamine (DSPE); 2) a hydrophilic spacer of poly(ethylene glycol); and 3) a cationic head group prepared with 0, 1, 3, or 7 lysine residues located at the distal end of the PEG chain, giving rise to CPL possessing 1, 2, 4, or 8 positive charges, respectively (CPL1 to CPL8). Previously we have described the synthesis of CPL, have characterized the postinsertion of CPL into PEG‐containing LUVs and SPLP (stabilized plasmid‐lipid particles), have shown significant increases in the binding of CPL‐LUV to cells, and have observed dramatically enhanced transfection (up to a million‐fold) of cells with CPL‐SPLP in the presence of calcium [Chen et al. (2000) Bioconjugate Chem. 11, 433–437; Fenske et al. (2001) Biochim. Biophys. Acta 1512, 259–272; Palmer et al. (2003) Biochim. Biophys. Acta 1611, 204–216]. In the present study, we examine a variety of CPL properties (such as polarity and CMC) and characterize CPL‐vesicular systems formed by extrusion and examine their interaction with cells. While CPL polarity was observed to increase dramatically with increasing charge number, CMC values were all found to be low, in the range of other PEGylated lipids, and exhibited only a small increase, going from CPL1 (1.3 µM) to CPL8 (2 µM). The CPLs were almost quantitatively incorporated into large unilamellar vesicles (LUVs) prepared by the extrusion method and were evenly distributed across the lipid bilayer. Lower levels of incorporation were obtained when CPLs were incubated with preformed liposomes (DSPC/Chol, 55:45) at 60°C. The binding of CPL‐LUVs to BHK cells in vitro was found to be dependent on the distal charge density of the CPL rather than total surface charge. Liposomes possessing CPL4 or CPL8 were observed to bind efficiently to cell surfaces and enhance cellular uptake in BHK cells (as observed with both lipid and aqueous content markers), whereas those possessing CPL1 or CPL2 exhibited little or no binding. These results suggest new directions for the design of liposomal systems capable of in vivo delivery of both conventional and genetic (plasmid and antisense) drugs.