Ionizable lipids are widely recognized as the crucial component of lipid nanoparticles (LNPs). They enable mRNA encapsulation, shield it from enzymatic degradation, facilitate cellular uptake, and foster its cytosolic release for subsequent translation into proteins. In addition, PEGylated lipids are added to stabilize the particles in storage and in vivo. In this study, we investigate the potency of LNPs prepared using commonly adopted ionizable and pegylated lipids in vitro (using HEK293 cells) and in vivo (mouse studies) to consider the impact of structure on potency. LNPs were prepared using a fixed molar ratio of DSPC: Cholesterol: ionizable/cationic lipid: PEG lipid (10:38.5:50:1.5 mol%). All LNP formulations exhibited similar critical quality attributes (CQAs), including particle size <100 nm, low PDI (<0.2), near-neutral zeta potential, and high encapsulation efficiency (>90%). However, the potency of these LNPs, as measured by in vitro mRNA expression and in vivo expression following intramuscular injection in mice varied significantly. LNPs formulated with SM-102 exhibited the highest expression in vitro, whilst in vivo SM-102 and ALC-0315 LNPs showed significantly higher mRNA expression than DLin-MC3-DMA (MC3), DODAP and DOTAP LNPs. We also investigated the effect of PEG lipid choice (ALC-0159, DMG-PEG2k, and DSPE-PEG2k), which did not impact LNP CQAs, nor their clearance from the injection site. However, PEG lipid choice significantly influenced mRNA expression with the incorporation of DSPE-PEG2k reducing expression. This work contributes valuable insights to the evolving landscape of mRNA research, emphasizing that CQAs are a marker of the quality of the LNP production process, but not discriminatory regarding LNP potency. Similarly, standard in vitro studies do not provide insights into in vivo potency. These results further emphasize the intricacies of formulation design and the importance of bridging gaps between experimental outcomes in different settings.

中文翻译：

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探索常用的可电离和聚乙二醇化脂质对 mRNA-LNP 的影响：体外和临床前相结合的视角


可电离脂质被广泛认为是脂质纳米颗粒 （LNP） 的关键成分。它们能够封装 mRNA，保护其免受酶降解，促进细胞摄取，并促进其胞质释放，以便随后翻译成蛋白质。此外，添加聚乙二醇化脂质以稳定储存和体内的颗粒。在这项研究中，我们研究了使用普遍采用的可电离和聚乙二醇化脂质在体外（使用 HEK293 细胞）和体内（小鼠研究）制备的 LNP 的效力，以考虑结构对效力的影响。使用 DSPC：胆固醇：可电离/阳离子脂质：PEG 脂质 （10：38.5：50：1.5 mol%） 的固定摩尔比制备 LNP。所有 LNP 配方都表现出相似的关键质量属性 （CQA），包括粒径 <100 nm、低 PDI （<0.2）、近中性 zeta 电位和高包封效率 （>90%）。然而，通过小鼠肌肉注射后的体外 mRNA 表达和体内表达来衡量的这些 LNP 的效力差异很大。用 SM-102 配制的 LNP 在体外表现出最高的表达，而体内 SM-102 和 ALC-0315 LNP 的 mRNA 表达显著高于 DLin-MC3-DMA （MC3）、DODAP 和 DOTAP LNP。我们还研究了 PEG 脂质选择 （ALC-0159 、 DMG-PEG2k 和 DSPE-PEG2k） 的影响，它不会影响 LNP CQA 及其从注射部位的清除。然而，PEG 脂质选择显着影响 mRNA 表达，掺入 DSPE-PEG2k 降低表达。 这项工作为 mRNA 研究不断发展的前景提供了宝贵的见解，强调 CQA 是 LNP 生产过程质量的标志，但在 LNP 效力方面没有歧视性。同样，标准的体外研究也没有提供对体内效力的见解。这些结果进一步强调了配方设计的复杂性以及弥合不同环境中实验结果之间差距的重要性。