Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, Wisconsin 53706, USA.
Langmuir. 2011 Jun 21;27(12):7868-76. doi: 10.1021/la200815t. Epub 2011 May 16.
We report an approach to the design of degradable polyelectrolyte-based films for the controlled release of siRNA from surfaces. Our approach is based on stepwise, layer-by-layer assembly of multilayered polyelectrolyte films (or "polyelectrolyte multilayers", PEMs) using siRNA and a hydrolytically degradable poly(β-amino ester) (polymer 1). Fabrication of films using siRNA sequences for green fluorescent protein (GFP) or firefly luciferase resulted in linear growth of ultrathin films (∼50 nm thick) that promoted the surface-mediated release of siRNA upon incubation in physiologically relevant media. Physicochemical characterization of these siRNA-containing films revealed large differences in film growth profiles, physical erosion profiles, and siRNA release profiles as compared to PEMs fabricated using polymer 1 and larger plasmid DNA constructs. For example, whereas films fabricated using plasmid DNA erode gradually and release DNA over a period of ∼48 h, films fabricated using siRNA released ∼65% of incorporated siRNA within the first hour of incubation, prior to the onset of any observed film erosion. This initial burst of release was followed by a second, slower phase of release (accompanied by gradual film erosion) over the next 23 h. These differences in release profiles and other behaviors likely result, at least in part, from large differences in the sizes of siRNA and plasmid DNA. Finally, we demonstrate that the siRNA in these films is released in a form that remains intact, functional, and able to silence targeted protein expression upon administration to mammalian cells in vitro. The results of this investigation provide a platform for the design of thin films and coatings that could be used to localize the release of siRNA from surfaces in a variety of fundamental and applied contexts (e.g., for development of new research tools or approaches to delivery from film-coated implants and other devices).
我们报告了一种设计基于聚电解质的可降解薄膜的方法,用于从表面控制 siRNA 的释放。我们的方法是基于使用 siRNA 和一种可水解的聚(β-氨基酯)(聚合物 1)逐步、层层组装多层聚电解质薄膜(或“聚电解质多层”,PEMs)。使用绿色荧光蛋白(GFP)或萤火虫荧光素酶的 siRNA 序列制造薄膜导致超薄薄膜(约 50nm 厚)的线性生长,在生理相关的介质中孵育时促进 siRNA 的表面介导释放。这些包含 siRNA 的薄膜的物理化学特性表明,与使用聚合物 1 和更大的质粒 DNA 构建体制造的 PEMs 相比,薄膜的生长曲线、物理侵蚀曲线和 siRNA 释放曲线有很大差异。例如,尽管使用质粒 DNA 制造的薄膜逐渐侵蚀并在约 48 小时内释放 DNA,但使用 siRNA 制造的薄膜在观察到任何薄膜侵蚀之前的第一个小时内释放了约 65%的掺入的 siRNA。这种初始释放爆发之后是第二个较慢的释放阶段(伴随着逐渐的薄膜侵蚀),持续 23 小时。这些释放曲线和其他行为的差异可能至少部分归因于 siRNA 和质粒 DNA 大小的巨大差异。最后,我们证明这些薄膜中的 siRNA 以完整的、功能的形式释放,并在体外给药给哺乳动物细胞时能够沉默靶向蛋白的表达。这项研究的结果为设计薄膜和涂层提供了一个平台,可用于在各种基础和应用背景下(例如,用于开发新的研究工具或从涂层植入物和其他设备释放的方法)从表面局部释放 siRNA。