Centre for Molecular Simulation, Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada.
Nanoscale. 2019 Aug 1;11(30):14141-14146. doi: 10.1039/c9nr02297j.
Lipid nanoparticles (LNPs) composed of ionizable cationic lipids are currently the leading systems for siRNA delivery in liver disease, with the major limitation of low siRNA release efficacy into the cytoplasm. Ionizable cationic lipids are known to be of critical importance in LNP structure and stability, siRNA entrapment, and endosomal disruption. However, their distribution inside the LNPs and their exact role in cytoplasmic delivery remain unclear. A recent study [Kulkarni et al., On the formation and morphology of lipid nanoparticles containing ionizable cationic lipids and siRNA, ACS Nano, 2018, 12(5), 4787-4795] on LNP-siRNA systems containing the ionizable lipid DLin-KC2-DMA (also known as KC2 with an apparent pKa of ca. 6.7) suggested that neutral KC2 segregates from other components and forms an amorphous oil droplet in the core of LNPs. In this paper, we present evidence supporting the model proposed by Kulkarni et al. We studied KC2 segregation in the presence of POPC using molecular dynamics simulation, deuterium NMR, SAXS, and cryo-TEM experiments, and found that neutral KC2 has a high tendency to separate from POPC dispersions. KC2 confinement, upon raising the pH during the formulation process, could result in rearrangement of the internal structure of LNPs. As interactions between cationic KC2 and anionic endosomal lipids are thought to be a key factor in cargo release, KC2 confinement inside the LNP may be responsible for the observed low release efficacy.
由可离子化阳离子脂质组成的脂质纳米颗粒（LNPs）是目前用于肝脏疾病中 siRNA 递送的主要系统，其主要限制是将 siRNA 有效递送到细胞质中的能力较低。可离子化阳离子脂质对于 LNP 的结构和稳定性、siRNA 包封和内涵体破坏至关重要。然而，它们在 LNPs 内部的分布及其在细胞质递送中的确切作用仍不清楚。最近一项关于含有可离子化阳离子脂质 DLin-KC2-DMA（也称为 KC2，表观 pKa 约为 6.7）的 LNP-siRNA 系统的研究[Kulkarni 等人，关于含有可离子化阳离子脂质的脂质纳米颗粒的形成和形态，ACS Nano，2018，12（5），4787-4795]表明，中性 KC2 与其他成分分离并在 LNPs 的核心形成非晶态油滴。在本文中，我们提出了支持 Kulkarni 等人提出的模型的证据。我们使用分子动力学模拟、氘 NMR、SAXS 和冷冻 TEM 实验研究了在存在 POPC 的情况下 KC2 的分离情况，发现中性 KC2 有很高的倾向与 POPC 分散体分离。在配方过程中提高 pH 值时，KC2 的限制可能导致 LNPs 内部结构的重新排列。由于阳离子 KC2 与阴离子内涵体脂质之间的相互作用被认为是货物释放的关键因素，因此 KC2 限制在 LNP 内可能是观察到的低释放效率的原因。
