Delivery of Shaped siRNA Nanoparticles by Retrograde Intrabiliary Infusion

描述（由申请人提供）：肝脏是RNA干扰介导的基因敲低疗法最重要的靶点之一，但一种安全有效的非病毒性肝脏靶向基因敲低策略尚未出现。这主要是由于缺乏具有良好控制大小和形状的递送载体，以及在生理介质和内吞室中具有高胶体和复杂稳定性，同时保持细胞质中siRNA的有效释放。本研究的总体目标是开发一种有效的方法来促进siRNA的可控冷凝，并通过聚阳离子- peg共聚物和不同溶剂极性的组装来制备具有可调形状的siRNA压缩纳米颗粒，并通过胆内逆行输注来验证siRNA纳米颗粒的形状显著影响其细胞摄取、生物分布和基因敲除效率的假设。本研究是基于我们最近的发现，通过用聚阳离子- peg共聚物凝聚siRNA和调整溶剂极性，可以制备出具有不同形状（球形、棒状和蠕虫状）的siRNA压缩纳米颗粒；胆道内输注是将siRNA纳米颗粒递送到肝脏的有效给药途径。利用这一探索性资助，我们计划(1)确定控制共聚物/siRNA纳米颗粒形状和大小的关键参数，并优化方案，以在细胞外环境中实现高稳定性，同时保持siRNA在细胞质溶胶中的有效释放；

DESCRIPTION (provided by applicant): The liver represents one of the most important targets for RNA interference-mediated gene knockdown therapies, yet a safe and efficacious non-viral liver-targeted gene knockdown strategy has yet to emerge. This is primarily due to the lack of delivery vehicles with well controlled size and shape, and high colloidal and complex stability in physiological media and in endocytic compartments while maintaining efficient release of siRNA in the cytosol. The overall objective of this proposed study is to develop an efficient method to facilitate controlled condensation of siRNA and prepare siRNA-compacting nanoparticles with tunable shapes by assembling with polycation-PEG copolymers and varying solvent polarity, and to test the hypothesis that the shape of siRNA nanoparticles significantly influences their cellular uptake, biodistribution and gene knockdown efficiency in vitro and in vivo through retrograde intrabiliary infusion. This study is built on our recent findings that siRNA-compacting nanoparticle with distinct shapes (spherical, rod-like and worm-like) can be prepared by condensing siRNA with polycation-PEG copolymers and by tuning solvent polarity; and the intrabiliary infusion is an effective administrative route for delivering siRNA nanoparticles to th liver. With this Exploratory Grant, we plan to (1) determine the key parameters that control the shape and size of copolymer/siRNA nanoparticles, and optimize protocol to achieve high stability in extracellular environment while maintaining efficient release of siRNA in the cytosol; and (2) investigate shape dependent cellular uptake and knockdown efficiency for siRNA micellar nanoparticles in vitro, and demonstrate efficient delivery and high gene knockdown efficiency of shaped siRNA nanoparticles in rat liver by intrabiliary infusion. This study will provide an enabling technology for controlling the shape of RNA-compacting nanoparticles, demonstrate the effectiveness of intrabiliary infusion for liver-targeted delivery of siRNA nanoparticles, and reveal the shape dependence in nanoparticle-mediated gene knockdown in the liver and in vivo biodistribution. It will provide key insights into designing more efficient delivery strategies for liver-targeted RNA therapeutics.