Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, 69 N Eagleville Rd, Unit 3092, Storrs, Connecticut 06269, USA.
Pharm Res. 2013 Apr;30(4):1161-75. doi: 10.1007/s11095-012-0952-9. Epub 2012 Dec 13.
To investigate cellular uptake pathways of novel anionic siRNA-lipoplexes as a function of formulation composition.
Anionic formulations with anionic lipid/Ca(2+)/siRNA ratio of 1.3/2.5/1 (AF1) and 1.3/0.3/1 (AF2) were utilized. Uptake mechanisms were investigated using uptake inhibition and co-localization approaches in breast cancer cells. Actin-mediated uptake was investigated using actin polymerization and rearrangement assays. Silencing efficiency and endosomal escaping capability of lipoplexes were evaluated. The cationic formulation Lipofectamine-2000 was used as a control.
Anionic lipoplexes entered the breast cancer cells via endocytosis specifically via macropinocytosis or via both macropinocytosis and HSPG (heparin sulfate proteoglycans) pathways, depending on the Ca(2+)/siRNA ratio. Additionally, uptake of these lipoplexes was both microtubule and actin dependent. The control cationic lipid-siRNA complexes (Lipofectamine-2000) were internalized via both endocytic (phagocytosis, HSPG) and non-endocytic (membrane fusion) pathways. Their uptake was microtubule independent but actin dependent. Silencing efficiency of the AF2 formulation was negligible mainly due to poor endosomal release (rate-limiting step).
Formulation composition significantly influences the internalization mechanism of anionic lipoplexes. Uptake mechanism together with formulation bioactivity helped in identification of the rate-limiting steps to efficient siRNA delivery. Such studies are extremely useful for formulation optimization to achieve enhanced intracellular delivery of nucleic acids.
研究新型阴离子 siRNA-脂质体复合物作为配方组成的功能的细胞摄取途径。
使用带负电荷的脂质/Ca(2+)/siRNA 比为 1.3/2.5/1（AF1）和 1.3/0.3/1（AF2）的阴离子配方。通过在乳腺癌细胞中采用摄取抑制和共定位方法研究摄取机制。通过肌动蛋白聚合和重排测定研究肌动蛋白介导的摄取。评估脂质体的沉默效率和内体逃逸能力。阳离子配方 Lipofectamine-2000 用作对照。
阴离子脂质体通过内吞作用进入乳腺癌细胞，具体途径是巨胞饮作用或巨胞饮作用和 HSPG（硫酸乙酰肝素蛋白聚糖）途径，具体取决于 Ca(2+)/siRNA 比。此外，这些脂质体的摄取既依赖微管又依赖肌动蛋白。对照阳离子脂质-siRNA 复合物（Lipofectamine-2000）通过内吞作用（吞噬作用、HSPG）和非内吞作用（膜融合）途径被内化。它们的摄取与微管无关，但与肌动蛋白有关。AF2 配方的沉默效率可以忽略不计，主要是由于内体释放不佳（限速步骤）。
配方组成显着影响阴离子脂质体的内化机制。摄取机制以及配方生物活性有助于确定有效 siRNA 递送至体内的限速步骤。这些研究对于实现核酸的细胞内有效递送至体内非常有用。
