Toxicity, Uptake Kinetics and Efficacy of New Transfection Reagents: Increase of Oligonucleotide Uptake

Abstract

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-β₁ (TGF-β₁) 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 μg/ml of the liposome Cytofectin GSV^® (CF) and the dendrimer SuperFect^® (SF) reduced mitochondrial activity of haEC after ≥4 h transfection whereas viability of haSMC was not influenced. DAC-30^® showed significant toxic effects on haEC and haSMC at each dose after ≥4 h and Lipofectin^® (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-β₁ mRNA expression and synthesis after transfection for 3–5 h using 0.1–1.0 μM 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.

Cloning of a Novel Retinoid-inducible Serine Carboxypeptidase from Vascular Smooth Muscle Cells

“…exuberant cell growth and subverted cellular differentiation), numerous studies have been undertaken to demonstrate the utility of retinoids in conferring a desirable SMC or vascular wall phenotype. For example, retinoids can antagonize growth factor-stimulated SMC proliferation in vitro (7)(8)(9)(10)(11)(12), inhibit SMC migration (12,13), and promote a more differentiated SMC phenotype (12, 14 -16). In a series of complementary in vivo studies, retinoids were shown to minimize vascular narrowing following injury to the vessel wall (16 -22).…”

Discovery of a Novel Control Element within the 5′-Untranslated Region of the Vascular Endothelial Growth Factor

“…Transfection with the S 3 oligonucleotide resulted in no significant effect as compared with the effect on the control samples, which was the same in regard to protein concentration. Similarly, transfection with vehicle (cytofectin) alone produced a slight decrease (5%) in VEGF mRNA equivalent to that found for the protein reduction and may be reflective of the slight cytotoxic effect known to be associated with cytofectin (21).…”

“…The injection technique used has been described previously (21). Briefly, the conjunctiva was cut close to the limbus to expose the sclera, which was then punctured with a 30-guage needle.…”

Dendrimer delivery of an anti-VEGF oligonucleotide into the eye: a long-term study into inhibition of laser-induced CNV, distribution, uptake and toxicity

“…Other studies have shown dendrimers to possess toxic side effects both in vitro and in vivo at high doses, but the effects were minimized by optimizing the dose, chemistry and DNA:dendrimer ratio. [22][23][24] Although not yet tested in human trials, no inflammatory response to the injected dendrimer or to the dendrimer plus ODN-1 was observed in our study at any time using ophthalmological examination. In addition, further examination using immunohistochemistry could not detect an increase in the presence of macrophages, which are related to an inflammatory response mediated by toxicity.…”