Faculty of Pharmaceutical Sciences , Hokkaido University , Kita-12, Nishi-6 , Kita-ku, Sapporo 060-0812 , Japan.
Mol Pharm. 2018 Jun 4;15(6):2142-2150. doi: 10.1021/acs.molpharmaceut.7b01166. Epub 2018 Apr 30.
Introducing siRNA into human immune cells by an artificial delivery system continues to be a challenging issue. We previously developed a multifunctional envelope-type nanodevice (MEND) containing the YSK12-C4, a fusogenic cationic lipid, (YSK12-MEND) and succeeded in the efficient delivery of siRNA into human immune cell lines. Significant cytotoxicity, however, was observed at siRNA doses needed for gene silencing in NK-92 cells. NK-92 cells, a unique natural killer (NK) cell line, would be applicable for use in clinical NK therapy. Thus, reducing the cytotoxicity of the YSK12-MEND in NK-92 cells would strengthen the efficacy of NK-92 cell-based therapy. The amount of the YSK12-C4 in the MEND needed to be reduced to reduce the cytotoxicity, because the cytotoxicity was directly associated with the YSK12-C4. In the present study, we decreased the total amount of lipid, including the YSK12-C4, by introducing a core formed by electrostatic interactions of siRNA with a polycation (protamine) (siRNA core), which led to a decrease in cytotoxicity in NK-92 cells. We prepared a YSK12-MEND containing an siRNA core (YSK12-MEND/core) at charge ratios (CR: YSK12-C4/siRNA) of 10, 5, 3, and 2.5 and compared the YSK12-MEND/core with that for a YSK12-MEND (CR16.9). Cell viability was increased by more than 2 times at a CR5 or less. On the other hand, the YSK12-MEND/core (CR5) maintained the same gene silencing efficiency (60%) as the YSK12-MEND. Interestingly, the cellular uptake efficiency and hemolytic activity of the YSK12-MEND/core (CR5) was reduced compared to that for the YSK12-MEND. In calculating the silencing activity per cellular uptake efficiency and hemolytic activity, the value for the YSK12-MEND/core (CR5) was more than 2 times as high as that of the YSK12-MEND. The fact indicates that after endosomal escape, the process can be enhanced by using a YSK12-MEND/core (CR5). Thus, introducing an siRNA core into lipid nanoparticles can be a potent strategy for decreasing cytotoxicity without an appreciable loss of gene silencing activity in NK-92 cells.
将 siRNA 通过人工递药系统导入人体免疫细胞仍然是一个具有挑战性的问题。我们之前开发了一种含有融合阳离子脂质体 YSK12-C4（YSK12-MEND）的多功能包膜型纳米器件（MEND），并成功将 siRNA 高效递送至人免疫细胞系。然而，在 NK-92 细胞中沉默基因所需的 siRNA 剂量下观察到明显的细胞毒性。NK-92 细胞是一种独特的自然杀伤（NK）细胞系，可应用于临床 NK 治疗。因此，降低 YSK12-MEND 在 NK-92 细胞中的细胞毒性将增强基于 NK-92 细胞的治疗效果。为了降低细胞毒性，需要减少 MEND 中的 YSK12-C4 量，因为细胞毒性与 YSK12-C4 直接相关。在本研究中，我们通过引入由 siRNA 与聚阳离子（鱼精蛋白）静电相互作用形成的核心（siRNA 核心）来减少包括 YSK12-C4 在内的脂质总量，从而降低了 NK-92 细胞的细胞毒性。我们制备了一种含有 siRNA 核心（YSK12-MEND/core）的 YSK12-MEND，其电荷比（CR：YSK12-C4/siRNA）为 10、5、3 和 2.5，并将 YSK12-MEND/core 与 YSK12-MEND（CR16.9）进行了比较。在 CR 为 5 或更低时，细胞活力增加了两倍以上。另一方面，YSK12-MEND/core（CR5）保持与 YSK12-MEND 相同的基因沉默效率（60%）。有趣的是，与 YSK12-MEND 相比，YSK12-MEND/core（CR5）的细胞摄取效率和溶血活性降低。在计算每细胞摄取效率和溶血活性的沉默活性时，YSK12-MEND/core（CR5）的值是 YSK12-MEND 的两倍以上。这一事实表明，在内涵体逃逸后，通过使用 YSK12-MEND/core（CR5）可以增强该过程。因此，在脂质纳米颗粒中引入 siRNA 核心可以是一种降低 NK-92 细胞细胞毒性而不明显损失基因沉默活性的有效策略。
