Toxicity, uptake kinetics and efficacy of new transfection reagents: Increase of oligonucleotide uptake

Human arterial smooth muscle cell (haSMC) proliferation is stimulated by pl atelet-derived growth factor (PDGF) release of human arterial endothelial c ells (haEC) whereas transforming growth factor-beta(1) (TGF-beta(1)) secret ion by haSMC promotes extracellular matrix formation. inhibitory concepts w ith antisense oligonucleotides (ASO) against those growth factors might be promising, requiring, however, sufficient transfection efficacy. Thus, toxi city and efficacy of new transfection reagents were examined. MTT tests sho wed that high doses >1.6 mu g/ml of the liposome Cytofectin GSV(R) (CF) and the dendrimer SuperFect(R) (SF) reduced mitochondrial activity of haEC aft er greater than or equal to 4 h transfection whereas viability of haSMC was not influenced. DAC-30(R) showed significant toxic effects on haEC and haS MC at each dose after greater than or equal to 4 h and Lipofectin(R) (LF) c aused complete detachment of haEC and haSMC in medium containing 10% serum. Uptake studies demonstrated that 'naked' ASO were not incorporated intrace llularly whereas transfection within CF or SF resulted in a strong cytoplas mic and nuclear labeling after 2-5 h. With DAC-30(R), only a slight cytopla smic fluorescence was found. SF caused an unexpected stimulation of endothe lial PDGF-AB synthesis. Thus, CF was favored for inhibition studies. ELISA, Western and Northern blotting showed a significant inhibition of endotheli al PDGF-B and smooth muscle TGF-beta(1) mRNA expression and synthesis after transfection for 3-5 h using 0.1-1.0 mu M ASO versus control aligonucleoti des. We conclude that Cytofectin GSV(R) is superior to the other transfecti on reagents, predominantly at haEC, showing an improved efficacy and less t oxicity than the classical liposome Lipofectin(R). Cytofectin GSV(R) might offer a promising tool for antisense strategies in the treatment of vascula r disorders. Copyright (C) 2000 S. Karger AG, Basel.