Cationic lipid polymerization as a novel approach for constructing new DNAdelivery agents

In vivo gene delivery mediated by cationic lipids is often compromised by a ggregation due to complexation with proteins in the blood. To improve the s tability of cationic lipid-DNA complexes, the present study aimed to develo p a novel approach in which a poly(cationic lipid) (PCL) is utilized to for m stable cationic polyplexes for gene transfection. Hydrogenation of the ac rylamide analogue of beta AE-DMRI, the polymerizable precursor of PCL, prov ided a monomeric lipid derivative (MHL) which was used for direct compariso n of corresponding lipoplex stability, toxicity, and transfection activity. Various formulations of cationic liposomes, such as MHL, MHL-cholesterol ( Chol), PCL, PCL-Chol, DOTAP-Chol, and commercially available lipofectamine were generated and examined in this study. The new poly(cationic lipid) did not display any significant toxicity to rat hepatocytes or Hep G(2) cells as indicated by an LDR leakage assay. Furthermore, PCL was significantly le ss toxic than MHL, DOTAP-Chol or lipofectamine. Suspensions of PCL were res istant to aggregation even after 24 h of exposure to solutions containing 5 0 and 100% fetal bovine serum (FBS). In contrast, suspensions of lipofectam ine extensively aggregated after 24 h of exposure to 50% FBS. To examine th e influence of lipid polymerization on gene transfer activity, liposome-med iated transfections of a luciferase vector (pGL(3)) were performed in Hep G (2) and Alexander cell lines. The luciferase activity of the PCL formulatio ns in Hep G(2) cells were similar to those of the MHL, DOTAP-Chol and lipof ectamine formulations, demonstrating that lipid polymerization does not com promise transfection activity. In comparison to the monomeric precursor MHL and to the industry transfection standards DOTAP and lipofectamine, the no vel poly(cationic lipid) exhibited the lowest cytotoxicity, was the most re sistant to serum-induced aggregation and had comparable transfection activi ty when coformulated with cholesterol. This novel polymerization approach f or the development of stable and active polyplexes may prove a valuable alt ernative for in vivo gene delivery.