Comparative analysis of polymers for short interfering RNA delivery in vascular smooth muscle cells
Comparative analysis of polymers for short interfering RNA delivery in vascular smooth muscle cells
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DOI：
10.1016/j.jss.2015.07.025
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发表时间：
2015-11-01
影响因子：
2.2
通讯作者：
Mountain, Deidra J. H.
中科院分区：
文献类型：
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作者：
Bools, Lindsay M.;Fisher, Richard K.;Mountain, Deidra J. H.
使用短干扰RNA（siRNA）降解细胞质中的信使RNA并瞬时减弱细胞内蛋白质显示出抑制血管发病机制的希望。然而，治疗应用的关键障碍是安全有效的递送系统。生物可降解聚合物是一种很有前途的遗传物质替代分子载体。在这里，我们的目的是进行比较分析的聚（B-氨基酯）（PBAE）和聚乙烯亚胺（PEI）聚合物在其功效的血管平滑肌细胞转染使用siRNA对甘油醛3-磷酸脱氢酶（GAPDH）管家基因作为我们的测试target.Methods：人主动脉平滑肌细胞（HASMC）在体外转染聚合物结合GAPDH或阴性对照（NC）siRNA。测试增加siRNA：聚合物比率以获得最佳转染效率。DharmaFECT 2化学转染复合物用于比较分析。采用活/死双重染色法检测细胞存活率，采用定量聚合酶链反应（PCR）检测GAPDH基因沉默。结果：PEI介导的沉默率最高的是9 mL聚合物：220 pmol/mL siRNA偶联物（16 +/-2%vs NC; n = 6）。用1.95 μ L聚合物：100 pmol/mL siRNA偶联物可以实现相当的PBAE介导的沉默（相对于NC，10 +/-1%表达; n = 5）。使用PEI的转染导致与其他方法等同的沉默，但在24小时聚合物暴露时效率较低且细胞毒性增加。PEI暴露时间减少到4小时导致类似的沉默效果（21 +/- 9%的表达与NC，n = 6），具有改善的毒性profile.Conclusions：聚合物生物缀合物转染HASMCs的方式类似于化学复合物，具有可比的细胞毒性和沉默效率。PEI生物缀合物表现出与PBAE生物缀合物等同的沉默，尽管在所需的聚合物浓度方面效率较低。考虑到测定的聚合物之间的成本效益差异，以及PEI在短时间暴露内以改善的毒性特征转染HASMC的能力，该研究表明PEI生物缀合物是用于血管组织的潜在转染剂。未来的研究将扩大这种基因治疗方法，以验证旨在减弱平滑肌细胞增殖，粘附和迁移的基因特异性抑制剂的递送。这些研究将为我们未来的实验计划奠定框架，以扩大这种基因治疗方法在血管疾病动物模型中的体内转染。(C)2015爱思唯尔公司All rights reserved.
The use of short interfering RNA (siRNA) to degrade messenger RNA in the cell cytoplasm and transiently attenuate intracellular proteins shows promise in the inhibition of vascular pathogenesis. However, a critical obstacle for therapeutic application is a safe and effective delivery system. Biodegradable polymers are promising alternative molecular carriers for genetic material. Here, we aim to perform a comparative analysis of poly(B-amino ester) (PBAE) and polyethylenimine (PEI) polymers in their efficacy for vascular smooth muscle cell transfection using siRNA against the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene as our test target.Methods: Human aortic smooth muscle cells (HASMC) were transfected in vitro with polymers conjugated to GAPDH or negative control (NC) siRNAs. Increasing siRNA: polymer ratios were tested for optimal transfection efficiency. DharmaFECT2 chemical transfection complexes were used for comparative analysis. Live/dead dual stain was used to measure cell viability, and GAPDH gene silencing was measured by quantitative polymerase chain reaction normalized to 18S.Results: The highest rate of PEI-mediated silencing was achieved with a 9mL polymer: 220 pmol/mL siRNA conjugate (16 +/- 2% expression versus NC; n = 6). Comparable PBAEmediated silencing could be achieved with a 1.95 mu L polymer: 100 pmol/mL siRNA conjugate (10 +/- 1% expression versus NC; n = 5). Transfection using PEIs resulted in silencing equivalent to other methods but with less efficiency and increased cell toxicity at 24h polymer exposure. Decreasing PEI exposure time to 4 h resulted in similar silencing efficacy (21 +/- 9% expression versus NC, n = 6) with an improved toxicity profile.Conclusions: Polymeric bioconjugates transfected HASMCs in a manner similar to chemical complexes, with comparable cell toxicity and silencing efficiency. PEI bioconjugates demonstrated silencing equivalent to PBAE bioconjugates, although less efficient in terms of required polymer concentrations. Given the cost-to-benefit difference between the assayed polymers, and PEI's ability to transfect HASMCs within a short duration of exposure with an improved toxicity profile, this study shows that PEI bioconjugates are a potential transfection agent for vascular tissue. Future studies will expand on this method of gene therapy to validate delivery of gene-specific inhibitors aimed at attenuating smooth muscle cell proliferation, adhesion, and migration. These studies will lay the framework for our future experimental plans to expand on this method of gene therapy for in vivo transfection in animal models of vascular disease. (C) 2015 Elsevier Inc. All rights reserved.