Meneksedag-Erol Deniz, Tang Tian, Uludağ Hasan
‡Department of Mechanical Engineering, Faculty of Engineering, University of Alberta, Alberta, Canada.
⊥Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Alberta, Canada.
J Phys Chem B. 2015 Apr 30;119(17):5475-86. doi: 10.1021/acs.jpcb.5b00415. Epub 2015 Apr 16.
Delivery of small interfering RNA (siRNA) for silencing of aberrantly expressed genes is a promising therapy for the treatment of various genetic disorders. Polymeric carriers have been used in the design of efficient delivery systems to generate nanoscale siRNA polyplexes. Despite the great amount of research pursued on siRNA therapeutics, the underlying mechanisms of polyplex dissociation in cytosol are still unclear. The fate of siRNA polyplexes during intracellular stages of delivery and how the endogenous molecules may affect the integrity of polyplexes remains to be explored. In this study, we have focused on miRNA-21 as a representative anionic endogenous molecule and performed gel electrophoresis mobility shift assays, particle size and zeta (ζ)-potential analyses, and a series of all-atom molecular dynamics simulations to elucidate the effect of miRNA on siRNA-PEI polyplexes. We report a slightly better binding to PEI by miRNA than that of siRNA, and speculated that miRNA may disrupt the integrity of preformed siRNA-PEI polyplexes. In contrast to our initial speculation, however, introduction of miRNA to a preformed siRNA-PEI polyplex revealed formation of a miRNA layer surrounding the polyplex through interactions with PEI. The resulting structure is a ternary siRNA-PEI-miRNA complex, where the experimentally determined ζ-potential was found to decrease as a function of miRNA added.
递送小干扰RNA（siRNA）以沉默异常表达的基因是治疗各种遗传疾病的一种有前景的疗法。聚合物载体已被用于设计高效递送系统，以生成纳米级siRNA多聚体。尽管对siRNA疗法进行了大量研究，但多聚体在细胞质中解离的潜在机制仍不清楚。siRNA多聚体在细胞内递送阶段的命运以及内源性分子如何影响多聚体的完整性仍有待探索。在本研究中，我们聚焦于miRNA-21作为代表性的阴离子内源性分子，并进行了凝胶电泳迁移率变动分析、粒径和zeta（ζ）电位分析以及一系列全原子分子动力学模拟，以阐明miRNA对siRNA-PEI多聚体的影响。我们报告miRNA与PEI的结合略优于siRNA，并推测miRNA可能破坏预先形成的siRNA-PEI多聚体的完整性。然而，与我们最初的推测相反，将miRNA引入预先形成的siRNA-PEI多聚体后发现，通过与PEI的相互作用，在多聚体周围形成了一层miRNA。最终形成的结构是三元siRNA-PEI-miRNA复合物，实验测定的ζ电位随着添加的miRNA的增加而降低。
