title: Structure and gene silencing activities of monovalent and pentavalent cationic lipid vectors complexed with siRNA.
abstract: Small interfering RNAs (siRNAs) of 19-25 bp mediate the cleavage of complementary mRNA, leading to post-transcriptional gene silencing. We examined cationic lipid (CL)-mediated delivery of siRNA into mammalian cells and made comparisons to CL-based DNA delivery. The effect of lipid composition and headgroup charge on the biophysical and biological properties of CL-siRNA vectors was determined. X-ray diffraction revealed that CL-siRNA complexes exhibited lamellar and inverted hexagonal phases, qualitatively similar to CL-DNA complexes, but also formed other nonlamellar structures. Surprisingly, optimally formulated inverted hexagonal 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) CL-siRNA complexes exhibited high toxicity and much lower target-specific gene silencing than lamellar CL-siRNA complexes even though optimally formulated, inverted hexagonal CL-DNA complexes show high transfection efficiency in cell culture. We further found that efficient silencing required cationic lipid/nucleic acid molar charge ratios (rhochg) nearly an order of magnitude larger than those yielding efficiently transfecting CL-DNA complexes. This second unexpected finding has implications for cell toxicity. Multivalent lipids (MVLs) require a smaller number of cationic lipids at a given rhochg of the complex. Consistent with this observation, the pentavalent lipid MVL5 exhibited lower toxicity and superior silencing efficiency over a large range in both the lipid composition and rhochg when compared to monovalent DOTAP. Most importantly, MVL5 achieved much higher total knockdown of the target gene in CL-siRNA complex regimes where toxicity was low. This property of CL-siRNA complexes contrasts to CL-DNA complexes, where the optimized transfection efficiencies of multivalent and monovalent lipids are comparable.
authors: Materials Department, University of California, Santa Barbara, California 93106, USA.
authors: Nathan F
authors: NF
authors: Bouxsein
authors: Christopher S
authors: CS
authors: McAllister
authors: Kai K
authors: KK
authors: Ewert
authors: Charles E
authors: CE
authors: Samuel
authors: Cyrus R
authors: CR
authors: Safinya
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry* / Q000494:pharmacology; D020868:Gene Silencing / Q000187:drug effects / Q000502:physiology*; D007739:L Cells; D008081:Liposomes / Q000737:chemistry; D051379:Mice; D010714:Phosphatidylethanolamines / Q000737:chemistry* / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000737:chemistry* / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000494:pharmacology / Q000502:physiology*; D014961:X-Ray Diffraction
chemical_list: C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D005229:Fatty Acids, Monounsaturated; D008081:Liposomes; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D034741:RNA, Small Interfering; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane