title: Characterization of endotoxin and cationic liposome interaction.
abstract: The objective of this study was to characterize the interaction of endotoxin with cationic liposomes used in nonviral gene delivery. Endotoxin-cationic liposome interaction was characterized using fluorescent anisotropy, and the Limulus amebocyte lysate (LAL) assay. Cellular toxicity of endotoxin-cationic liposome complex was examined using a dimethylthiazol diphenyltetrazolium bromide (MTT) assay. The effect of endotoxin on the lipid-DNA complex and subsequent transfection into COS-1 cells was also examined. A competitive interaction occurred between fluoroscein isothiocyanate (FITC)-labeled endotoxin and plasmid DNA for binding dioleoyl glycero trimethylammonium propane:dioleoyl glycero phosphoethanolamine (DOTAP:DOPE) liposomes using fluorescent anisotropy techniques. The LAL assay demonstrated no change in endotoxin activity upon interaction with liposomes. No loss of COS cell viability was detected via the MTT assay during a 5-hr exposure to endotoxin. Transient transfection studies indicate that increasing levels of endotoxin lowered activity more than 90% at 50,000 endotoxin units (EU)/ml. Endotoxin and cationic liposomes interact mainly by an electrostatic attraction. Endotoxin contamination can potentially impact transfection efficiency via competition with plasmid DNA for cationic liposome binding by increasing transfection variability at 50 EU/ml, a concentration of endotoxin contamination that can occur with small-scale plasmid preparations used for in vitro cell transfections, but would not be expected with typical GLP or GMP preparations used in clinical studies.
authors.affiliation: Department of Pharmaceutics, University of Florida, Gainesville 32610, USA. spoxon@ufl.edu
authors.forename: S W
authors.initials: SW
authors.lastname: Poxon
authors.forename: J A
authors.initials: JA
authors.lastname: Hughes
mesh_terms: D000818:Animals; D019556:COS Cells; D004731:Endotoxins / Q000737:chemistry*; D005454:Fluorescence Polarization; D015316:Genetic Therapy; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry*; D010957:Plasmids; D055672:Static Electricity; D014162:Transfection
chemical_list: D004731:Endotoxins; D008055:Lipids; D008081:Liposomes