Center for Controlled Chemical Delivery (CCCD), Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112, United States.
J Control Release. 2010 Feb 25;142(1):61-9. doi: 10.1016/j.jconrel.2009.09.021. Epub 2009 Oct 7.
This study was designed to assess the in vitro gene expression efficiency and therapeutic effectiveness of polymer mediated transfection of primary myoblasts. Autologous primary myoblast transplantation may improve the function of infarcted myocardium via myogenesis. In addition, primary myoblasts can carry exogenous angiogenic genes that encode angiogenic factors to promote therapeutic angiogenesis. Viral vectors have limited clinical application due to the induction of inflammatory reactions, tumorigenic mutations and genome integration. To overcome these problems, two new biodegradable poly(disulfide amine)s, poly(cystaminebisacryamide-diaminohexane) [poly(CBA-DAH)] and poly(cystaminebisacryamide-diaminohexane-arginine) [poly(CBA-DAH-R)], were synthesized as polymer carriers for gene delivery. In this study, primary myoblasts were isolated and purified from rat skeletal muscles. Based on an optimized polymer mediated transfection procedure using a luciferase assay and confocal microscopy, these two poly(disulfide amine)s induced up to 16-fold higher luciferase expression and much higher green fluorescence protein expression than branched poy(ethylenimine) (bPEI, 25kDa) in primary myoblasts. By flow cytometry, poly(CBA-DAH) and poly(CBA-DAH-R) promote rates of cellular uptake of florescence-labeled polymer/pDNA complexes of 97% and 99%, respectively, which are rates higher than that of bPEI 25kDa (87%). Both poly(disulfide amine)s were much less cytotoxic than bPEI 25kDa. The in vitro time-course and co-culture experiments verified that polymer engineered primary myoblasts have the ability to stimulate endothelial proliferation. These data confirmed that poly(disulfide amine)s are the safe and feasible polymeric gene carriers to transfect VEGF(165) into primary myoblasts. Polymer engineered primary myoblasts have potential for therapeutic application in the treatment of ischemic heart diseases.
这项研究旨在评估聚合物介导的原代肌母细胞转染的体外基因表达效率和治疗效果。自体原代肌母细胞移植可以通过成肌作用改善梗死心肌的功能。此外，原代肌母细胞可以携带编码血管生成因子的外源性血管生成基因，以促进治疗性血管生成。由于诱导炎症反应、致瘤性突变和基因组整合，病毒载体的临床应用受到限制。为了克服这些问题，两种新型可生物降解的聚（二硫代氨基甲酸盐），聚（半胱氨酸双丙烯酰胺-二氨基己烷）[聚（CBA-DAH）]和聚（半胱氨酸双丙烯酰胺-二氨基己烷-精氨酸）[聚（CBA-DAH-R）]，被合成作为基因传递的聚合物载体。在这项研究中，原代肌母细胞从大鼠骨骼肌中分离和纯化。基于使用荧光素酶测定法和共聚焦显微镜优化的聚合物介导的转染程序，这两种聚（二硫代氨基甲酸盐）在原代肌母细胞中诱导的荧光素酶表达比分支聚（乙基亚胺）（bPEI，25kDa）高 16 倍，绿色荧光蛋白表达高得多。通过流式细胞术，聚（CBA-DAH）和聚（CBA-DAH-R）分别促进荧光标记的聚合物 / pDNA 复合物的细胞摄取率达到 97%和 99%，高于 bPEI 25kDa（87%）的摄取率。这两种聚（二硫代氨基甲酸盐）的细胞毒性均明显低于 bPEI 25kDa。体外时程和共培养实验证实，聚合物工程化的原代肌母细胞具有刺激内皮细胞增殖的能力。这些数据证实，聚（二硫代氨基甲酸盐）是安全可行的聚合物基因载体，可将 VEGF（165）转染到原代肌母细胞中。聚合物工程化的原代肌母细胞在治疗缺血性心脏病方面具有潜在的治疗应用价值。
