184. Novel Cationic Multivalent Lipids Show Improved Delivery of siRNA and DNA
Authors: Nathan F. Bouxsein, Kai K. Ewert, Christopher S. McAllister, Heather M. Evans, Charles E. Samuel, Cyrus R. Safinya
Journal: Molecular Therapy (2006)

Complexes of nucleic acids (Nas) with cationic lipids (CLs) are promising delivery vectors with numerous potential applications, particularly in human therapeutics. As synthetic vectors, CL-NA complexes can carry Nas of arbitrary size and are safer and easier to produce than viruses. Our work aims at the design of optimized lipid vectors through an improved understanding of the mechanism of transfection and how it is affected by NA type and size, CL-NA complex structure, lipid structure and lipid composition[1]. We have synthesized multivalent cationic lipids (MVLs) with systematically varied headgroup charge (+2 up to +16) and investigated siRNA and DNA complexes of these MVLs and 1,2-Dioleoyl-sn-glycero-3- phosphocholine (DOPC), comparing both their structure and in vitro transfection properties to analogous complexes made from 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) / 1,2- Diloleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and DOTAP / DOPC. Efficient delivery of siRNAs to cells in culture requires large CL to siRNA molar charge ratios (ρchg). This large ρchg is a direct result of complex instability created by the short length of the siRNA molecule. In certain cases, the amount of CL necessary to meet the ρchg requirement results in toxic side effects. Our results suggest that this toxicity is related to the number of cationic lipids present in the delivery vector, rather than on the number of charges. We found that MVL-siRNA complexes are more stable, show improved silencing efficiencies and are less toxic when compared with the analogous DOTAP-siRNA complexes. Also, MVLs have the ability to incorporate large amounts of DOPC without extensive loss of efficient silencing. Structurally, CL-siRNA complexes behave similar to CL-DNA complexes. However, the inverted hexagonal phase formed with DOTAP / DOPE lipids and siRNA does not show improved silencing efficiency over lamellar DOTAP / DOPC complexes, even at low contents of DOTAP in the membrane. DNA complexes of a newly synthesized, highly charged (16+) lipid, MVLBG2, with a dendritic headgroup and DOPC exhibit the well-known lamellar phase at 90 mole-% DOPC[2]. At higher contents of dendritic lipid, however, two novel non-lamellar phases are observed by synchrotron x-ray diffraction. The structure of one of these phases, present in a narrow range of composition around 25 mole-% MVLBG2, has been solved. In this novel dual lattice structure, termed HIC, hexagonally arranged tubular lipid micelles are surrounded by DNA rods forming a substructure with honeycomb symmetry. Complexes in the HIC phase efficiently transfect mouse and human cells in culture. Their transfection efficiency, as well as that of the lamellar complexes containing only 10 mole-% dendritic lipid reaches and surpasses that of optimized DOTAP/DOPC complexes. In particular, complexes containing MVLBG2 are significantly more transfectant over the entire composition range in a mouse embryonic fibroblast, a cell line that is empirically hard to trasfect. [1] Expert Opin. Biol. Ther. (2005) 5(1): 33–53 [2] Ewert KK, JACS (2006) In Print.