title: Analysis and optimization of the cationic lipid component of a lipid/peptide vector formulation for enhanced transfection in vitro and in vivo.
abstract: We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K16GACRRETAWACG) and Lipofectin, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.
authors.affiliation: Wolfson Centre for Gene Therapy of Childhood Disease, UCL Institute of Child Health, London, UK.
authors.forename: Michele
authors.initials: M
authors.lastname: Writer
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Hurley
authors.forename: Supti
authors.initials: S
authors.lastname: Sarkar
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Copeman
authors.forename: John B
authors.initials: JB
authors.lastname: Wong
authors.forename: Marianne
authors.initials: M
authors.lastname: Odlyha
authors.forename: M
authors.initials: M
authors.lastname: Jayne Lawrence
authors.forename: Alethea B
authors.initials: AB
authors.lastname: Tabor
authors.forename: Robin J
authors.initials: RJ
authors.lastname: McAnulty
authors.forename: Parviz
authors.initials: P
authors.lastname: Ayazi Shamlou
authors.forename: Helen C
authors.initials: HC
authors.lastname: Hailes
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Hart
mesh_terms: D000818:Animals; D002151:Calorimetry; D002412:Cations / Q000032:analysis / Q000737:chemistry; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D005452:Fluoresceins; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008055:Lipids / Q000032:analysis* / Q000737:chemistry* / Q000633:toxicity; D008081:Liposomes; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D015394:Molecular Structure; D010455:Peptides / Q000008:administration & dosage* / Q000737:chemistry*; D014162:Transfection / Q000295:instrumentation* / Q000379:methods*
chemical_list: D002412:Cations; D005452:Fluoresceins; D008055:Lipids; D008081:Liposomes; D010455:Peptides; D004247:DNA; C007740:fluorexon