title: Relating toxicity to transfection: using sphingosine to maintain prolonged expression in vitro.
abstract: Cationic reagents are commonly used to facilitate DNA delivery, and transfection experiments are typically initiated in cell culture where the optimal charge ratio is determined. While transfection rates are often enhanced at higher +/- charge ratios, the cellular toxicity associated with the greater amounts of cationic components at elevated charge ratios is often not considered. In addition, the prolonged effects of cationic lipid uptake on cell viability are not evident in a typical 24-48 h transfection experiment. In this study, we compare the transfection efficiency of cationic lipoplexes to effects on viability of cultured cells in both the short and long term (7 days). Our results indicate that, while minimal toxicity is evident 24 h after exposure to DOTAP-based lipoplexes, cell viability continues to decline and ultimately compromises reporter gene expression at longer times. Substitution of a naturally occurring cationic amphiphile, sphingosine, for DOTAP greatly reduces toxicity and allows high expression to be maintained over prolonged periods.
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado , Anschutz Medical Campus, 12850 East Montview Boulevard, Aurora, Colorado 80045, United States.
authors.forename: Jamie L
authors.initials: JL
authors.lastname: Betker
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Anchordoquy
mesh_terms: D002412:Cations / Q000737:chemistry; D002470:Cell Survival; D004247:DNA / Q000737:chemistry; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D061986:MCF-7 Cells; D010316:Particle Size; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D013110:Sphingosine / Q000737:chemistry*; D013997:Time Factors; D014162:Transfection
chemical_list: C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D002412:Cations; D005229:Fatty Acids, Monounsaturated; D008055:Lipids; D008081:Liposomes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; C044819:1,2-diarachidonoyl-glycero-3-phosphocholine; D004247:DNA; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane; D013110:Sphingosine
keywords: DOTAP; cationic lipid; sphingosine; toxicity; transfection; viability