Kubota Kohei, Onishi Kohei, Sawaki Kazuaki, Li Tianshu, Mitsuoka Kaoru, Sato Takaaki, Takeoka Shinji
Cooperative Major in Advanced Biomedical Sciences, Graduate School of Advanced Sciences and Engineering, Waseda University (TWIns), Tokyo, Japan.
Formulation Research and Phramaceutical Process Group, CMC R&D Center, Kyowa Hakko Kirin Co., Ltd, Shizuoka, Japan.
Int J Nanomedicine. 2017 Jul 19;12:5121-5133. doi: 10.2147/IJN.S136426. eCollection 2017.
Two lipid-based nanoformulations have been used to date in clinical studies: lipoplexes and lipid nanoparticles (LNPs). In this study, we prepared small interfering RNA (siRNA)-loaded carriers using lipid components of the same composition to form molecular assemblies of differing structures, and evaluated the impact of structure on cellular uptake and immune stimulation. Lipoplexes are electrostatic complexes formed by mixing preformed cationic lipid liposomes with anionic siRNA in an aqueous environment, whereas LNPs are nanoparticles embedding siRNA prepared by mixing an alcoholic lipid solution with an aqueous siRNA solution in one step. Although the physicochemical properties of lipoplexes and LNPs were similar except for small increases in apparent size of lipoplexes and zeta potential of LNPs, siRNA uptake efficiency of LNPs was significantly higher than that of lipoplexes. Furthermore, in the case of LNPs, both siRNA and lipid were effectively incorporated into cells in a co-assembled state; however, in the case of lipoplexes, the amount of siRNA internalized into cells was small in comparison with lipid. siRNAs in lipoplexes were thought to be more likely to localize on the particle surface and thereby undergo dissociation into the medium. Inflammatory cytokine responses also appeared to differ between lipoplexes and LNPs. For tumor necrosis factor-α, release was mainly caused by siRNA. On the other hand, the release of interleukin-1β was mainly due to the cationic nature of particles. LNPs released lower amounts of tumor necrosis factor-α and interleukin-1β than lipoplexes and were thus considered to be better tolerated with respect to cytokine release. In conclusion, siRNA-loaded nanoformulations effect their cellular uptake and immune stimulation in a manner that depends on the structure of the molecular assembly; therefore, nanoformulations should be optimized before extending studies into the in vivo environment.
迄今为止，已有两种基于脂质的纳米制剂用于临床研究：脂质体复合物和脂质纳米颗粒（LNP）。在本研究中，我们使用相同组成的脂质成分制备了负载小干扰RNA（siRNA）的载体，以形成不同结构的分子聚集体，并评估结构对细胞摄取和免疫刺激的影响。脂质体复合物是在水性环境中将预先形成的阳离子脂质体与阴离子siRNA混合形成的静电复合物，而LNP是通过将醇脂质溶液与水性siRNA溶液一步混合制备的包埋siRNA的纳米颗粒。尽管脂质体复合物和LNP的物理化学性质相似，只是脂质体复合物的表观尺寸和LNP的zeta电位略有增加，但LNP对siRNA的摄取效率明显高于脂质体复合物。此外，在LNP的情况下，siRNA和脂质都以共组装状态有效地掺入细胞中；然而，在脂质体复合物的情况下，与脂质相比，内化到细胞中的siRNA量较少。脂质体复合物中的siRNA被认为更有可能定位在颗粒表面，从而解离到培养基中。脂质体复合物和LNP之间的炎性细胞因子反应似乎也有所不同。对于肿瘤坏死因子-α，释放主要由siRNA引起。另一方面，白细胞介素-1β的释放主要归因于颗粒的阳离子性质。LNP释放的肿瘤坏死因子-α和白细胞介素-1β的量低于脂质体复合物，因此在细胞因子释放方面被认为具有更好的耐受性。总之，负载siRNA的纳米制剂以依赖于分子聚集体结构的方式影响其细胞摄取和免疫刺激；因此，在将研究扩展到体内环境之前，应优化纳米制剂。
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