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Relating toxicity to transfection: using sphingosine to maintain prolonged expression in vitro
Jamie L Betker1, Thomas J Anchordoquy
1Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado , Anschutz Medical Campus, 12850 East Montview Boulevard, Aurora, Colorado 80045, United States.
Molecular Pharmaceutics
|November 25, 2014
Summary
High charge ratios in DNA delivery increase transfection but cause toxicity. Sphingosine, a natural alternative to DOTAP, reduces toxicity and maintains gene expression over time.
Area of Science:
- Biochemistry
- Cell Biology
- Gene Delivery
Background:
- Cationic reagents are essential for DNA delivery and gene transfection.
- Optimal charge ratios are determined in cell culture, often overlooking toxicity.
- Prolonged effects of cationic lipid uptake on cell viability are not well understood.
Purpose of the Study:
- To compare the transfection efficiency of cationic lipoplexes with their effects on cell viability.
- To evaluate both short-term (24-48 h) and long-term (7 days) impacts.
- To assess the potential of sphingosine as a less toxic alternative to DOTAP.
Main Methods:
- Transfection experiments using DOTAP-based lipoplexes and sphingosine.
- Assessment of cell viability at 24 hours and 7 days post-transfection.
- Measurement of reporter gene expression over time.
Main Results:
- DOTAP-based lipoplexes showed minimal toxicity at 24 hours but significant decline in cell viability by 7 days.
- Declining cell viability negatively impacted reporter gene expression in the long term.
- Replacing DOTAP with sphingosine significantly reduced toxicity and maintained high gene expression over prolonged periods.
Conclusions:
- Standard transfection protocols may underestimate cationic reagent toxicity.
- Long-term cell viability is crucial for sustained gene expression.
- Sphingosine offers a promising, less toxic alternative for effective and prolonged gene delivery.
