An Atelocollagen Coating for Efficient Local Gene Silencing by Using Small Interfering RNA.
An Atelocollagen Coating for Efficient Local Gene Silencing by Using Small Interfering RNA.
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DOI：
10.1016/j.omtn.2017.01.006
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发表时间：
2017-03-17
期刊：
影响因子：
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通讯作者：
Nolte A
中科院分区：
文献类型：
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作者：
Koenig O;Nothdurft D;Perle N;Neumann B;Behring A;Degenkolbe I;Walker T;Schlensak C;Wendel HP;Nolte A
在过去的几十年里，人们已经做出了许多努力来抵消支架植入动脉粥样硬化冠状动脉后的不良反应。在药物洗脱支架的新时代，人们注意到血管壁再生更好的突破。一种新的个性化方法是开发基因洗脱支架，有望改变参与再生的基因表达。我们研究了由聚合物端肽胶原（ATCOL）和细胞间粘附分子-1（ICAM-1）的特异性小干扰RNA（siRNA）组成的涂层系统，该系统发现于缺陷内皮细胞（EC）表面。我们证明了非常高的细胞活力，其中EA.hy926在0.008%或0.032%ATCOL层上生长。此外，血液相容性试验证明了该涂层的生物相容性。2天后，在ATCOL中固定5 μg siRNA时，EA.hy926的转染效率最高。荧光标记的siRNA的释放约为9天。通过流式细胞术分析ICAM-1的长期敲低，揭示了具有0.008%ATCOL和5 μg siICAM-1的涂层引起基因沉默长达8天。5′-RNA连接酶介导的cDNA末端快速扩增PCR（RLM-RACE-PCR）证明了我们建立的ATCOL基因沉默涂层的特异性，这意味着我们的涂层非常适合于进一步的体内研究。在此，我们希望证明我们的ATCOL非常适合支架植入后更好的动脉壁再生。
In the last decades, many efforts have been made to counteract adverse effects after stenting atherosclerotic coronary arteries. A breakthrough in better vascular wall regeneration was noted in the new era of drug-eluting stents. A novel personalized approach is the development of gene-eluting stents promising an alteration in gene expression involved in regeneration. We investigated a coating system consisting of the polymer atelocollagen (ATCOL) and a specific small interfering RNA (siRNA) for intercellular adhesion molecule-1 (ICAM-1) found on the surface of defective endothelial cells (ECs). We demonstrated very high cell viability, in which EA.hy926 grew on 0.008% or 0.032% ATCOL layers. Additionally, hemocompatibility assays proved the biocompatibility of this coating. The highest transfection efficiency with EA.hy926 was achieved with 5 μg siRNA immobilized in ATCOL after 2 days. The release of fluorescent-labeled siRNA was about 9 days. Long-term knockdown of ICAM-1 was analyzed by flow cytometry, revealing that the coating with 0.008% ATCOL and 5 μg siICAM-1 provoked gene silencing up to 8 days. 5′-RNA ligase-mediated rapid amplification of cDNA ends PCR (RLM-RACE-PCR) demonstrated the specificity of our established ATCOL gene-silencing coating, meaning that our coating is well suited for further investigations in in vivo studies. Herein, we would like to demonstrate that our ATCOL is well-suited for better artery wall regeneration after stent implantation.