Pulmonary Gene Silencing in Transgenic EGFP Mice Using Aerosolised Chitosan/siRNA Nanoparticles
Pulmonary Gene Silencing in Transgenic EGFP Mice Using Aerosolised Chitosan/siRNA Nanoparticles
复制标题
DOI：
10.1007/s11095-010-0255-y
复制
发表时间：
2010-12-01
影响因子：
3.7
通讯作者：
Howard, Kenneth A.
中科院分区：
文献类型：
--
作者：
Nielsen, Ebbe J. B.;Nielsen, Jan M.;Howard, Kenneth A.
这项工作描述了用于改善小鼠肺部 siRNA 递送和基因沉默的壳聚糖纳米粒子雾化制剂的生产和应用。使用雾化导管气动形成雾化壳聚糖/siRNA 纳米粒子，并通过激光衍射确定尺寸。通过流式细胞术在表达 EGFP 内源性的 H1299 细胞系中评估气雾化和非气雾化制剂的体外沉默。使用流式细胞术方法，通过组织学检测 Cy3 标记的 siRNA 和转基因 EGFP 小鼠肺部的基因沉默，采用导管的非侵入性气管内插入来研究纳米颗粒沉积。流式细胞术分析表明，在表达 EGFP 的 H1299 细胞中，气雾化之前（68%）和雾化之后（62%）基因沉默效率的变化极小。使用少量 siRNA，对小鼠进行气管内导管给药，导致纳米颗粒沉积在整个肺部的肺泡和细支气管区域。服用雾化纳米颗粒制剂的转基因 EGFP 小鼠表现出显着的 EGFP 基因沉默（与错配组相比减少 68%）。这项工作为 RNAi 疗法的有效肺部递送和基因沉默提供了技术平台，有可能用于呼吸系统疾病治疗的临床前研究。
This work describes the production and application of an aerosolised formulation of chitosan nanoparticles for improved pulmonary siRNA delivery and gene silencing in mice.Aerosolised chitosan/siRNA nanoparticles were pneumatically formed using a nebulising catheter and sized by laser diffraction. In vitro silencing of aerosolised and non-aerosolised formulations was evaluated in an EGFP endogenous-expressing H1299 cell line by flow cytometry. Non-invasive intratracheal insertion of the catheter was used to study nanoparticle deposition by histological detection of Cy3-labeled siRNA and gene silencing in transgenic EGFP mouse lungs using a flow cytometric method.Flow cytometric analysis demonstrated minimal alteration in gene silencing efficiency before (68%) and after (62%) aerosolisation in EGFP-expressing H1299 cells. Intratracheal catheter administration in mice resulted in nanoparticle deposition throughout the entire lung in both alveoli and bronchiolar regions using low amounts of siRNA. Transgenic EGFP mice dosed with the aerosolised nanoparticle formulation showed significant EGFP gene silencing (68% reduction compared to mismatch group).This work provides a technology platform for effective pulmonary delivery and gene silencing of RNAi therapeutics with potential use in preclinical studies of respiratory disease treatment.