title: Converting an alcohol to an amine in a cationic lipid dramatically alters the co-lipid requirement, cellular transfection activity and the ultrastructure of DNA-cytofectin complexes.
abstract: Cytofectins are positively charged lipophilic molecules that readily form complexes with DNA and other anionic polynucleotides. Normally, cytofectins are combined with an activity-augmenting phospholipid such as dioleoylphosphatidylethanolamine (DOPE), and a film of dried, mixed lipid is prepared and hydrated to form cationic liposomes. The liposome solution is then mixed with a plasmid DNA solution to afford cytofectin-DNA complexes which, when presented to living cells, are internalized and the transgene is expressed. One of the most potent cytofectins, dimyristoyl Rosenthal inhibitor ether (DMRIE), is presently being used to deliver transcriptionally active DNA into human tumor tissues. Here we report the remarkable consequences of replacing the alcohol moiety of DMRIE with a primary amine. The resulting cytofectin, called beta-aminoethyl-DMRIE (betaAE-DMRIE), promoted high level transfection over a broad range of DNA and cationic lipid concentrations. A comparison of in vitro transfection activity between DMRIE and betaAE-DMRIE in 10 cell types revealed that betaAE-DMRIE was more active than DMRIE, and that betaAE-DMRIE, unlike DMRIE, was maximally effective in the absence of colipid. The consequences of the alcohol-to-amine conversion on the structure of the cytofectin/DNA complex was also examined by Atomic Force Microscopy. Strikingly dissimilar images were found for plasmid DNA alone and for the plasmid complexes of betaAE-DMRIE and DMRIE/DOPE.
authors.affiliation: Vical Incorporated, San Diego, CA 92121, USA. carlatvicl@aol.com
authors.forename: C J
authors.initials: CJ
authors.lastname: Wheeler
authors.forename: L
authors.initials: L
authors.lastname: Sukhu
authors.forename: G
authors.initials: G
authors.lastname: Yang
authors.forename: Y
authors.initials: Y
authors.lastname: Tsai
authors.forename: C
authors.initials: C
authors.lastname: Bustamente
authors.forename: P
authors.initials: P
authors.lastname: Felgner
authors.forename: J
authors.initials: J
authors.lastname: Norman
authors.forename: M
authors.initials: M
authors.lastname: Manthorpe
mesh_terms: D000438:Alcohols; D000588:Amines; D000818:Animals; D002460:Cell Line; D004247:DNA / Q000008:administration & dosage* / Q000378:metabolism / Q000648:ultrastructure; D004337:Drug Carriers; D005798:Genes, Bacterial; D005805:Genes, MHC Class I; D015793:HLA-B7 Antigen / Q000096:biosynthesis; D006801:Humans; D008055:Lipids; D008081:Liposomes; D010714:Phosphatidylethanolamines; D010957:Plasmids; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D014162:Transfection / Q000379:methods*; D014407:Tumor Cells, Cultured; D001613:beta 2-Microglobulin / Q000096:biosynthesis; D001616:beta-Galactosidase / Q000096:biosynthesis
chemical_list: D000438:Alcohols; D000588:Amines; D004337:Drug Carriers; D015793:HLA-B7 Antigen; D008055:Lipids; D008081:Liposomes; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins; D001613:beta 2-Microglobulin; C087276:(3-dimyristyloxypropyl)(dimethyl)(hydroxyethyl)ammonium; C020888:1,2-dielaidoylphosphatidylethanolamine; D004247:DNA; D001616:beta-Galactosidase