Delivery of Shaped siRNA Nanoparticles by Retrograde Intrabiliary Infusion

描述（由申请人提供）：肝脏是RNA干扰介导的基因敲减疗法的最重要靶点之一，但尚未出现安全有效的非病毒肝脏靶向基因敲减策略。这主要是由于缺乏具有良好控制的尺寸和形状以及在生理介质和内吞隔室中的高胶体和复合物稳定性同时维持siRNA在胞质溶胶中的有效释放的递送载体。本研究的总体目标是开发一种有效的方法来促进siRNA的受控缩合，并通过与聚阳离子-PEG共聚物和不同溶剂极性组装来制备具有可调形状的siRNA致密纳米颗粒，并测试siRNA纳米颗粒的形状显著影响其细胞摄取的假设，通过逆行胆管内输注进行体外和体内的生物分布和基因敲除效率。本研究建立在我们最近的发现基础上，即通过将siRNA与聚阳离子-PEG共聚物缩合并通过调节溶剂极性可以制备具有不同形状（球形、棒状和蠕虫状）的siRNA压实纳米颗粒;并且胆管内输注是将siRNA纳米颗粒递送到肝脏的有效给药途径。通过这项探索性资助，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，以实现在细胞外环境中的高稳定性，同时保持siRNA在胞质溶胶中的有效释放; 和（2）研究siRNA胶束纳米颗粒的体外形状依赖性细胞摄取和敲低效率，并证明通过胆内输注在大鼠肝脏中成形siRNA纳米颗粒的有效递送和高基因敲低效率。这项研究将提供一种控制RNA压缩纳米颗粒形状的技术，证明胆管内输注用于siRNA纳米颗粒肝脏靶向递送的有效性，并揭示纳米颗粒介导的基因敲除在肝脏和体内生物分布中的形状依赖性。它将为设计更有效的肝脏靶向RNA治疗策略提供关键见解。 公共卫生相关性：这项研究将提供一种使能技术，用于控制模仿天然病毒颗粒的RNAcompacting纳米颗粒的形状和大小，并证明胆管内输注用于siRNA纳米颗粒的肝脏靶向递送的有效性。它将揭示siRNA纳米颗粒形状如何影响其在肝脏中的纳米颗粒转运和基因敲低效率，并为设计更有效的RNA治疗剂递送策略以治疗各种肝脏特异性疾病提供关键见解。

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. PUBLIC HEALTH RELEVANCE: This study will provide an enabling technology for controlling the shape and size of RNAcompacting nanoparticles that mimic natural virus particles and demonstrate the effectiveness of intrabiliary infusion for liver-targeted delivery of siRNA nanoparticles. It will reveal how siRNA nanoparticle shape influences their nanoparticle transport and gene knockdown efficiency in the liver and offer key insights into designing more efficient delivery strategies for RNA therapeutics for treating a variety of liver-specific disease.