Ma Yutian, VanKeulen-Miller Rachel, Fenton Owen S
Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Nat Protoc. 2025 Mar 11. doi: 10.1038/s41596-024-01134-4.
mRNA-based therapies have emerged as a cutting-edge approach for diverse therapeutic applications. However, substantial barriers exist that hinder scientists from entering this research field, including the technical complexity and multiple potential workflows available for formulating and evaluating mRNA lipid nanoparticles (LNPs). Here we present an easy-to-follow and step-by-step guide for mRNA LNP formulation, characterization and in vitro and in vivo evaluation that could lower these barriers, facilitating entry for scientists in academia, industry and clinical settings into this research space. In this protocol, we detail steps for formulating representative mRNA LNPs (0.5 d) and characterizing key parameters (1-6 d) such as size, polydispersity index, zeta potential, mRNA concentration, mRNA encapsulation efficiency and stability. Then, we describe in vitro evaluations (3-6 d), such as protein expression, cell uptake and mechanism investigations (3-5 d), including endosomal escape, as well as in vivo delivery evaluation (2-3 d) encompassing intracellular and secreted protein expression levels, biodistribution and additional tolerability studies (1-2 weeks). Unlike some alternative protocols that may focus on discrete aspects of the workflow-such as formulation, characterization or evaluation-our protocol instead aims to integrate each of these aspects into a simplified, singular workflow applicable across multiple types of mRNA LNP formulations. In describing these procedures, we wish to disseminate one potential workflow for mRNA LNP production and evaluation, with the ultimate goal of furthering innovation, collaboration and the translational advancement of mRNA LNPs.
基于信使核糖核酸（mRNA）的疗法已成为用于多种治疗应用的前沿方法。然而，存在重大障碍阻碍科学家进入这一研究领域，包括技术复杂性以及用于制备和评估mRNA脂质纳米颗粒（LNP）的多种潜在工作流程。在此，我们提供一份易于遵循的、循序渐进的指南，用于mRNA LNP的制备、表征以及体外和体内评估，这可以降低这些障碍，便于学术界、工业界和临床环境中的科学家进入这一研究领域。在本方案中，我们详细介绍了制备代表性mRNA LNP（0.5天）以及表征关键参数（1 - 6天）的步骤，这些参数包括粒径、多分散指数、zeta电位、mRNA浓度、mRNA包封效率和稳定性。然后，我们描述了体外评估（3 - 6天），如蛋白质表达、细胞摄取和机制研究（3 - 5天），包括内体逃逸，以及体内递送评估（2 - 3天），涵盖细胞内和分泌蛋白表达水平、生物分布以及额外的耐受性研究（1 - 2周）。与一些可能侧重于工作流程离散方面（如制备、表征或评估）的替代方案不同，我们的方案旨在将这些方面整合到一个适用于多种类型mRNA LNP制剂的简化、单一工作流程中。在描述这些程序时，我们希望传播一种mRNA LNP生产和评估的潜在工作流程，最终目标是推动mRNA LNP的创新、合作和转化进展。
