Delivery of Shaped siRNA Nanoparticles by Retrograde Intrabiliary Infusion

描述(申请人提供)：肝脏是RNA干扰介导的基因敲除治疗最重要的靶点之一，但安全有效的非病毒肝脏靶向基因敲除策略尚未出现。这主要是由于缺乏大小和形状可控的递送载体，以及在生理介质和胞内隔室中具有高度胶体和络合物稳定性的载体，同时保持胞浆中siRNA的有效释放。这项研究的总体目标是开发一种有效的方法来促进siRNA的可控缩合，并通过与聚阳离子-聚乙二醇共聚物组装和改变溶剂极性来制备形状可调的siRNA致密纳米颗粒，并通过逆行胆道内输注检验siRNA纳米颗粒的形状显著影响其在体外和体内的细胞摄取、生物分布和基因敲除效率的假设。这项研究基于我们最近的发现，即通过将siRNA与聚阳离子-聚乙二醇共聚物缩合并调整溶剂极性，可以制备出形状不同的siRNA致密纳米颗粒(球形、棒状和蠕虫状)；胆道内输注是将siRNA纳米颗粒输送到肝脏的有效给药途径。利用这一探索性拨款，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在胞浆中的有效释放； (2)研究siRNA胶束纳米粒体外形态依赖的细胞摄取和基因敲除效率，并通过胆道内注射在大鼠肝脏中有效地传递和高基因敲除效率。本研究将为控制RNA致密纳米颗粒的形状提供一种可行的技术，证明胆道内输注对肝脏靶向递送siRNA纳米颗粒的有效性，并揭示纳米颗粒介导的基因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靶向RNA疗法的给药策略提供关键见解。 与公共卫生相关：这项研究将提供一种能够控制RNA致密纳米颗粒形状和大小的技术，这种纳米颗粒模仿自然病毒颗粒，并证明胆道内输注对肝脏靶向递送siRNA纳米颗粒的有效性。它将揭示siRNA纳米颗粒的形状如何影响其纳米颗粒在肝脏中的转运和基因敲除效率，并为设计更有效的RNA治疗策略提供关键见解，用于治疗各种肝脏特异性疾病。

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.