DDS Research Group, Formulation Research Laboratory, Pharmaceutical Science and Technology, Medicine Development Center, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan.
DDS Research Group, Formulation Research Laboratory, Pharmaceutical Science and Technology, Medicine Development Center, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan.
Int J Pharm. 2016 Aug 20;510(1):350-8. doi: 10.1016/j.ijpharm.2016.06.124. Epub 2016 Jun 29.
Lipid nanoparticles (LNPs) represent the most advanced platform for the systemic delivery of siRNA. We have previously reported the discovery of novel ionizable lipids with asymmetric lipid tails, enabling potent gene-silencing activity in hepatocytes in vivo; however, the structure and delivery mechanism had not been elucidated. Here, we report the structure, activity and uptake mechanism of LNPs with an asymmetric ionizable lipid. Zeta potential and hemolytic activity of LNPs showed that LNPs were neutral at the pH of the blood compartment but become increasingly charged and fusogenic in the acidic endosomal compartment. (31)P NMR experiments indicated that the siRNA was less mobile inside particles, presumably because of an electrostatic interaction with an ionizable lipid. The role of Apolipoprotein E (apoE) was studied using recombinant human apoE both in vitro and in vivo. A comparative study in wild-type and apoE-deficient mice revealed that apoE significantly influenced the in vivo biodistribution of LNPs and enhanced the cellular uptake. Pretreatment of mice with siRNA targeting low-density lipoprotein receptor (LDLR) impaired gene-silencing of the following siRNA treatment, demonstrating that in vivo activity of LNPs is dependent on LDLR. Our studies on the detailed mechanism should lead to the creation of more sophisticated LNP-based RNAi therapeutics.
脂质纳米粒 (LNPs) 代表了用于系统递送 siRNA 的最先进平台。我们之前曾报道过发现具有不对称脂质尾部的新型可离子化脂质，使它们能够在体内肝细胞中发挥强大的基因沉默活性；然而，其结构和递药机制尚未阐明。在这里，我们报告了具有不对称可离子化脂质的 LNPs 的结构、活性和摄取机制。LNPs 的 ζ 电位和溶血活性表明，LNPs 在血液腔室的 pH 值下呈中性，但在酸性内涵体腔室中变得越来越带电荷和融合。（31）P NMR 实验表明，siRNA 在颗粒内的流动性较差，可能是因为与可离子化脂质发生静电相互作用。使用重组人载脂蛋白 E (apoE) 在体外和体内研究了载脂蛋白 E 的作用。在野生型和载脂蛋白 E 缺陷型小鼠中的比较研究表明，载脂蛋白 E 显著影响 LNPs 的体内分布，并增强细胞摄取。用靶向低密度脂蛋白受体 (LDLR) 的 siRNA 预处理小鼠会损害随后的 siRNA 处理的基因沉默，表明 LNPs 的体内活性依赖于 LDLR。我们对详细机制的研究应导致更复杂的基于 LNP 的 RNAi 治疗方法的创建。
