Reducing the Cytotoxicity of Lipid Nanoparticles Associated with a Fusogenic Cationic Lipid in a Natural Killer Cell Line by Introducing a Polycation-Based siRNA Core
Reducing the Cytotoxicity of Lipid Nanoparticles Associated with a Fusogenic Cationic Lipid in a Natural Killer Cell Line by Introducing a Polycation-Based siRNA Core
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DOI：
10.1021/acs.molpharmaceut.7b01166
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发表时间：
2018-06-01
影响因子：
4.9
通讯作者：
Harashima, Hideyoshi
中科院分区：
文献类型：
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作者：
Nakamura, Takashi;Yamada, Koharu;Harashima, Hideyoshi
通过人工递送系统将siRNA引入人类免疫细胞仍然是一个具有挑战性的问题。我们先前开发了一种含有YSK12-C4（一种融合阳离子脂质）的多功能包膜型纳米器件（MEND）（YSK12-MEND），并成功地将siRNA有效递送到人类免疫细胞系中。然而，在NK-92细胞中基因沉默所需的siRNA剂量下观察到显著的细胞毒性。NK-92细胞是一种独特的自然杀伤（NK）细胞系，可用于临床NK治疗。因此，降低YSK 12-MEND在NK-92细胞中的细胞毒性将增强基于NK-92细胞的疗法的功效。需要减少MEND中YSK 12-C4的量以降低细胞毒性，因为细胞毒性与YSK 12-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）的静电相互作用形成的核心（siRNA核心）来减少脂质（包括YSK 12-C4）的总量，这导致NK-92细胞中的细胞毒性降低。我们以10、5、3和2.5的电荷比（CR：YSK 12-C4/siRNA）制备了含有siRNA核心的YSK 12-MEND（YSK 12-MEND/核心），并将YSK 12-MEND/核心与YSK 12-MEND（CR 16.9）进行了比较。另一方面，YSK 12-MEND/core（CR5）保持了与YSK 12-MEND相同的基因沉默效率（60%）。有趣的是，与YSK 12-MEND相比，YSK 12-MEND/核心（CR5）的细胞摄取效率和溶血活性降低。在计算沉默活性/细胞摄取效率和溶血活性时，YSK 12-MEND/核心（CR5）的值是YSK 12-MEND的2倍多。事实表明，在内体逃逸后，该过程可以通过使用YSK 12-MEND/核心（CR5）来增强。因此，将siRNA核心引入脂质纳米颗粒可以是用于降低细胞毒性而不明显损失NK-92细胞中的基因沉默活性的有效策略。
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.