An Atelocollagen Coating for Efficient Local Gene Silencing by Using Small Interfering RNA

Abstract

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. 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.