Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape
Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape
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DOI：
10.1038/nbt.2612
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发表时间：
2013-07-01
影响因子：
46.9
通讯作者：
Zerial, Marino
中科院分区：
文献类型：
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作者：
Gilleron, Jerome;Querbes, William;Zerial, Marino
短干扰RNA（siRNA）的递送仍然是RNA干扰（RNAi）疗法开发中的关键挑战。更好地理解siRNA的细胞摄取、细胞内转运和内体释放的机制可以对递送方法的改进做出重要贡献。在这里，我们监测的摄取脂质纳米粒子（LNP）加载可追踪的siRNA在不同类型的细胞在体外和小鼠肝脏中的定量荧光成像和电子显微镜。我们发现，LNP进入细胞的组成和诱导途径，在细胞类型特异性的方式，使用网格蛋白介导的内吞作用以及巨胞饮。通过直接检测与siRNA偶联的胶体金颗粒，我们估计siRNA从内体逃逸到胞质溶胶中的效率较低（1-2%），并且仅在LNP驻留在共享早期和晚期内体特征的特定区室中的有限时间窗期间发生。我们的研究结果提供了对LNP介导的siRNA递送的深入了解，可以指导下一代RNAi疗法的递送系统的开发。
Delivery of short interfering RNAs (siRNAs) remains a key challenge in the development of RNA interference (RNAi) therapeutics. A better understanding of the mechanisms of siRNA cellular uptake, intracellular transport and endosomal release could critically contribute to the improvement of delivery methods. Here we monitored the uptake of lipid nanoparticles (LNPs) loaded with traceable siRNAs in different cell types in vitro and in mouse liver by quantitative fluorescence imaging and electron microscopy. We found that LNPs enter cells by both constitutive and inducible pathways in a cell type-specific manner using clathrin-mediated endocytosis as well as macropinocytosis. By directly detecting colloidal-gold particles conjugated to siRNAs, we estimated that escape of siRNAs from endosomes into the cytosol occurs at low efficiency (1-2%) and only during a limited window of time when the LNPs reside in a specific compartment sharing early and late endosomal characteristics. Our results provide insights into LNP-mediated siRNA delivery that can guide development of the next generation of delivery systems for RNAi therapeutics.