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Toxicity, uptake kinetics and efficacy of new transfection reagents: increase of oligonucleotide uptake.
Human arterial smooth muscle cell (haSMC) proliferation is stimulated by platelet-derived growth factor (PDGF) release of human arterial endothelial cells (haEC) whereas transforming growth factor-beta(1) (TGF-beta(1)) secretion by haSMC promotes extracellular matrix formation. Inhibitory concepts with antisense oligonucleotides (ASO) against those growth factors might be promising, requiring, however, sufficient transfection efficacy. Thus, toxicity and efficacy of new transfection reagents were examined. MTT tests showed that high doses >1.6 microg/ml of the liposome Cytofectin GSV((R)) (CF) and the dendrimer SuperFect (SF) reduced mitochondrial activity of haEC after > or =4 h transfection whereas viability of haSMC was not influenced. DAC-30((R)) showed significant toxic effects on haEC and haSMC at each dose after > or =4 h and Lipofectin((R)) (LF) caused complete detachment of haEC and haSMC in medium containing 10% serum. Uptake studies demonstrated that 'naked' ASO were not incorporated intracellularly whereas transfection within CF or SF resulted in a strong cytoplasmic and nuclear labeling after 2-5 h. With DAC-30, only a slight cytoplasmic fluorescence was found. SF caused an unexpected stimulation of endothelial PDGF-AB synthesis. Thus, CF was favored for inhibition studies. ELISA, Western and Northern blotting showed a significant inhibition of endothelial PDGF-B and smooth muscle TGF-beta(1) mRNA expression and synthesis after transfection for 3-5 h using 0.1-1.0 microM ASO versus control oligonucleotides. We conclude that Cytofectin GSV is superior to the other transfection reagents, predominantly at haEC, showing an improved efficacy and less toxicity than the classical liposome Lipofectin. Cytofectin GSV might offer a promising tool for antisense strategies in the treatment of vascular disorders.[1]References
- Toxicity, uptake kinetics and efficacy of new transfection reagents: increase of oligonucleotide uptake. Axel, D.I., Spyridopoulos, I., Riessen, R., Runge, H., Viebahn, R., Karsch, K.R. J. Vasc. Res. (2000) [Pubmed]
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