mRNA-carrying lipid nanoparticles that induce lysosomal rupture activate NLRP3 inflammasome and reduce mRNA transfection efficiency
mRNA-carrying lipid nanoparticles that induce lysosomal rupture activate NLRP3 inflammasome and reduce mRNA transfection efficiency
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DOI：
10.1039/d2bm00883a
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发表时间：
2022-08-09
影响因子：
6.6
通讯作者：
Kulkarni, Ashish
中科院分区：
文献类型：
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作者：
Forster, James, III;Nandi, Dipika;Kulkarni, Ashish
在过去几年中，在设计更高效、更有效的 mRNA 脂质纳米颗粒疫苗方面取得了无数的进展，最终导致针对 COVID-19 的有效 mRNA 疫苗的快速开发。然而，尽管取得了这些进步和材料方法，但仍然缺乏对 mRNA 脂质纳米颗粒的免疫原性的了解。因此，需要采取更加机械化、设计驱动的方法来确定哪些生物物理特征，特别是与脂质成分的变化相关的生物物理特征，驱动纳米颗粒的免疫原性。在这里，我们合成了一组六种 mRNA 脂质纳米颗粒制剂，改变不同脂质成分的浓度，并系统地研究了它们对 NLRP3 炎症小体激活的影响；控制各种炎症反应的关键细胞内蛋白质复合物。最初的实验旨在通过 IL-1 beta ELISA 确定纳米颗粒激活 NLRP3 炎症小体的差异，结果揭示了具有高浓度可电离脂质 DLin-MC3-DMA 的纳米颗粒与高阳离子脂质 DPTAP 和低胆固醇浓度串联，诱导了 NLRP3 炎症小体的最大激活。通过测量指示 NLRP3 复合物组装的 ASC 斑点以及指示复合物激活的切割的 Gasdermin-D 和 caspase-1 表达，进一步证实了这些结果。我们还发现这些激活谱在机械上主要与溶酶体破裂相关，溶酶体破裂是由可电离脂质的延迟膜破坏能力直到溶酶体阶段引起的，以及线粒体活性氧（ROS）的产生和一些颗粒的钙流入引起的。因此，我们报告说，每种脂质类型（最显着的是可电离脂质、阳离子脂质和胆固醇）的特定综合作用是关键的 mRNA 脂质纳米颗粒特征，它改变制剂的内/溶酶体破裂能力，并以溶酶体破裂依赖性方式激活 NLRP3 炎症小体。这些结果提供了对用于激活分子水平免疫反应的mRNA脂质纳米颗粒相关分子模式的更具体的理解，并为未来的mRNA递送方法提供了新的脂质成分设计考虑因素。
In the last several years, countless developments have been made to engineer more efficient and potent mRNA lipid nanoparticle vaccines, culminating in the rapid development of effective mRNA vaccines against COVID-19. However, despite these advancements and materials approaches, there is still a lack of understanding of the resultant immunogenicity of mRNA lipid nanoparticles. Therefore, a more mechanistic, design-driven approach needs to be taken to determine which biophysical characteristics, especially related to changes in lipid compositions, drive nanoparticle immunogenicity. Here, we synthesized a panel of six mRNA lipid nanoparticle formulations, varying the concentrations of different lipid components and systematically studied their effect on NLRP3 inflammasome activation; a key intracellular protein complex that controls various inflammatory responses. Initial experiments aimed to determine differences in nanoparticle activation of NLRP3 inflammasomes by IL-1 beta ELISA, which unveiled that nanoparticles with high concentrations of ionizable lipid DLin-MC3-DMA in tandem with high cationic lipid DPTAP and low cholesterol concentration induced the greatest activation of the NLRP3 inflammasome. These results were further corroborated by the measurement of ASC specks indicative of NLRP3 complex assembly, as well as cleaved gasdermin-D and caspase-1 expression indicating complex activation. We also uncovered these activation profiles to be mechanistically correlated primarily with lysosomal rupturing caused by the delayed membrane disruption capabilities of ionizable lipids until the lysosomal stage, as well as by mitochondrial reactive oxygen species (ROS) production and calcium influx for some of the particles. Therefore, we report that the specific, combined effects of each lipid type, most notably ionizable, cationic lipids, and cholesterol, is a crucial mRNA lipid nanoparticle characteristic that varies the endo/lysosomal rupture capabilities of the formulation and activate NLRP3 inflammasomes in a lysosomal rupture dependent manner. These results provide a more concrete understanding of mRNA lipid Nanoparticle-Associated Molecular Patterns for the activation of molecular-level immune responses and provide new lipid composition design considerations for future mRNA-delivery approaches.