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.

Nonadditive Effects of Repetitive Administration of Lipoplexes in Immunocompetent Mice

“…In order to reduce the potential immune response to the plasmid used in our experiments, we have minimized the number of CpG motifs. Also, the lipoplexes employed here are comprised of naturally-occurring lipids (i.e., phosphatidylcholine, cholesterol, sphingosine) which greatly reduce their toxicity and potential immunogenicity, as compared to other gene delivery vehicles[20]. Consistent with the absence of accelerated blood clearance, the cytokine/chemokine response to repeat administration of our formulation is comparable to that for saline, with the exception of KC (Figure 4, Table I).…”

“…In addition to PEGylation, it is important to recognize that the DNA in delivery systems can also elicit an immune response that could potentially contribute to accelerated blood clearance [20, 24, 43]. In order to reduce the potential immune response to the plasmid used in our experiments, we have minimized the number of CpG motifs.…”

“…Sphingosine, cholesterol, and 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC) were purchased from Avanti Polar Lipids (Alabaster, AL) and used to prepare liposomes at a 3:2:5 mole ratio (respectively) as previously described[20]. Liposomes were then mixed with a modified (CMV removed, ROSA26 added, based upon Watcharanurak et al[24]) pSelect-LucSh (Invivogen, San Diego, CA) plasmid encoding luciferase at a charge ratio of 0.5[19, 20].…”

“…Liposomes were then mixed with a modified (CMV removed, ROSA26 added, based upon Watcharanurak et al[24]) pSelect-LucSh (Invivogen, San Diego, CA) plasmid encoding luciferase at a charge ratio of 0.5[19, 20]. These modifications to the plasmid have been shown to prolong expression for weeks to months[24].…”

“…In addition to the lipoplex formulation described above, mice were treated with phosphate buffered saline (negative control), lipoplexes formulated with 5% DSPE-PEG2000 (Avanti), CpG-containing plasmid (Valentis, Inc.), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP; Avanti) instead of sphingosine, or Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as previously described [19, 20, 22, 25]. In these studies, a single mouse was treated with each formulation as described above, and blood was collected in Eppendorf tubes 2 h after the second injection of lipoplexes.…”