Greco Chad T, Epps Thomas H, Sullivan Millicent O
Department of Chemical and Biomolecular Engineering and ‡Department of Materials Science and Engineering, University of Delaware, Newark, Delaware 19716, United States.
Department of Chemical and Biomolecular Engineering and Department of Materials Science and Engineering, University of Delaware, Newark, Delaware 19716, United States.
ACS Biomater Sci Eng. 2016 Sep 12;2(9):1582-1594. doi: 10.1021/acsbiomaterials.6b00336. Epub 2016 Aug 24.
Current siRNA delivery systems lack the ability to precisely tune siRNA release and maximize gene silencing in a spatiotemporal manner. Herein, we investigate photoresponsive block copolymer solution assemblies, for which stimuli-triggered changes in polymer structure altered nanocarrier stability and defined siRNA activity. Uniquely, our biomaterials design enabled the development and validation of a simple kinetic model that accurately predicted the extent of intracellular nanocarrier disassembly and silencing. Moreover, our constructs showed that maximal gene silencing could be achieved using concentrations of siRNA 5-fold lower than typical formulations due to the ability to rapidly release sufficient amounts of siRNA to saturate the cellular RISC machinery. The ability of our nanocarriers to remain dormant prior to phototriggered siRNA release allowed for the generation of cell patterns in gene expression with spatial control on cellular length scales and no detectable off-target effects. Furthermore, precisely tuned changes in nanocarrier structure enabled the modulation of protein and mRNA knockdown levels in murine fibroblasts and terminally differentiated human primary cells. These advances lead to increased precision, potency, and utility relative to other recent spatiotemporally controlled nucleic acid delivery vehicles reported in the literature. Moreover, the combination of experimental examination and kinetic modeling described herein should be applicable to a host of systems for which temporal control over nucleic acid delivery is a critical parameter in influencing cellular responses.
目前的小干扰RNA（siRNA）递送系统缺乏以时空方式精确调节siRNA释放并最大化基因沉默的能力。在此，我们研究了光响应性嵌段共聚物溶液组装体，其聚合物结构的刺激触发变化改变了纳米载体的稳定性并决定了siRNA的活性。独特的是，我们的生物材料设计使得能够开发和验证一个简单的动力学模型，该模型准确预测了细胞内纳米载体的拆解程度和沉默效果。此外，我们构建的载体表明，由于能够快速释放足够量的siRNA以使细胞RNA诱导沉默复合体（RISC）机制饱和，使用比典型制剂低5倍的siRNA浓度即可实现最大程度的基因沉默。我们的纳米载体在光触发siRNA释放之前保持休眠的能力，使得能够在细胞长度尺度上对基因表达进行空间控制从而生成细胞模式，并且没有可检测到的脱靶效应。此外，纳米载体结构的精确调节变化能够调节小鼠成纤维细胞和终末分化的人原代细胞中的蛋白质和mRNA敲低水平。相对于文献中报道的其他近期时空控制核酸递送载体，这些进展提高了精确性、效力和实用性。此外，本文所述的实验研究和动力学建模的结合应适用于许多系统，对于这些系统而言，核酸递送的时间控制是影响细胞反应的关键参数。
