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


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          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-β<sub>1</sub> (TGF-β<sub>1</sub>) 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<sup>®</sup> (CF) and the dendrimer SuperFect<sup>®</sup> (SF) reduced mitochondrial activity of haEC after ≥4 h transfection whereas viability of haSMC was not influenced. DAC-30<sup>®</sup> showed significant toxic effects on haEC and haSMC at each dose after ≥4 h and Lipofectin<sup>®</sup> (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<sup>®</sup>, 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-β<sub>1</sub> mRNA expression and synthesis after transfection for 3–5 h using 0.1–1.0 μ M ASO versus control oligonucleotides. We conclude that Cytofectin GSV<sup>®</sup> is superior to the other transfection reagents, predominantly at haEC, showing an improved efficacy and less toxicity than the classical liposome Lipofectin<sup>®</sup>. Cytofectin GSV<sup>®</sup> might offer a promising tool for antisense strategies in the treatment of vascular disorders.

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          Rapid colorimetric assay for cell growth and survival

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            Cytoplasmic domains of the interleukin-2 receptor beta and gamma chains mediate the signal for T-cell proliferation.

            The interleukin-2 receptor (IL-2R) consists of three distinct chains (alpha, beta, gamma) which bind IL-2 and generate a proliferative signal in T cells. To define the mechanism of receptor activation, chimaeric receptors were constructed from the intracellular region of either IL-2R beta or IL-2R gamma and the extracellular region of c-kit, a receptor tyrosine kinase that homodimerizes on binding stem cell factor (SCF). We report here that binding of SCF to the beta-chain chimaera induced proliferation of the pro-B-cell line BA/F3, but not T cells. But in T cells expressing both the beta- and gamma-chain chimaeras, SCF induced proliferation and tyrosine phosphorylation characteristic of the native IL-2R signal. Chimaeric IL-2 receptor beta and gamma chains constructed with the heterodimeric extracellular regions of the granulocyte-macrophage colony stimulating factor receptor (GM-CSFR) also provided the IL-2R signal. Thus, heterodimerization of the cytoplasmic domains of IL-2R beta and -gamma appears necessary and sufficient for signalling in T cells.
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              A serum-resistant cytofectin for cellular delivery of antisense oligodeoxynucleotides and plasmid DNA.

              Development of antisense technology has focused in part on creating improved methods for delivering oligodeoxynucleotides (ODNs) to cells. In this report, we describe a cationic lipid that, when formulated with the fusogenic lipid dioleoylphosphatidyliethanolamine, greatly improves the cellular uptake properties of antisense ODNs, as well as plasmid DNA. This lipid formulation, termed GS 2888 cytofectin, (i) efficiently transfects ODNs and plasmids into many cell types in the presence or absence of 10% serum in the medium, (ii) uses a 4- to 10-fold lower concentration of the agent as compared to the commercially available Lipofectin liposome, and (iii) is > or = 20-fold more effective at eliciting antisense effects in the presence of serum when compared to Lipofectin. Here we show antisense effects using GS 2888 cytofectin together with C-5 propynyl pyrimidine phosphorothioate ODNs in which we achieve inhibition of gene expression using low nanomolar concentrations of ODN. This agent expands the utility of antisense ODNs for their use in understanding gene function and offers the potential for its use in DNA delivery applications in vivo.

                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                August 2000
                14 August 2000
                : 37
                : 4
                : 221-234
                aMedical Clinic III, Department of Cardiology and bDepartment of Surgery, Tübingen, Germany
                25737 J Vasc Res 2000;37:221–234
                © 2000 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 5, References: 57, Pages: 14
                Internet Discussion Forum

                General medicine,Neurology,Cardiovascular Medicine,Internal medicine,Nephrology
                Growth factors,Endothelial cells,Transfection,Liposome,Restenosis,Antisense oligonucleotides,Smooth muscle cells


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