Abstract
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.
Key points
This protocol represents one possible approach for the formulation, characterization and evaluation of mRNA lipid nanoparticles.
It assesses key parameters such as size, polydispersity index and zeta potential, as well as mRNA concentration, encapsulation efficiency and stability. In vitro evaluations include protein expression, cell uptake and mechanism investigations, while assessment of intracellular and secreted protein expression levels, biodistribution and additional tolerability studies are carried out in vivo.
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Fig. 1: Overview of the protocol for formulating and evaluating mRNA LNPs.
Fig. 2: Considerations for the formulation and characterization of mRNA LNPs.
Fig. 3: Outline of in vitro evaluation methods for mRNA LNPs.
Fig. 4: Outline of mechanism investigation methods for mRNA LNPs.
Fig. 5: LNP formulation and characterization methods.
Fig. 6: In vitro evaluation methods of mRNA LNPs.
Fig. 7: Mechanism investigation methods.
Fig. 8: In vivo evaluation methods of mRNA LNPs.
Data availability
The datasets generated and/or analyzed during the current study are provided in Supplementary Information, source data for Figs. 5–8 and statistical source data for Supplementary Figs. 1, 5, 6, 7, 11 and 12. Any additional data required for research purposes are available from the corresponding authors upon request. Source data are provided with this paper.
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Acknowledgements
This work was supported by an NIH National Institute of Biomedical Imaging and Bioengineering award (1R21EB034942-01). This work was also supported by the NC Translational and Clinical Sciences (NC TraCS) Institute, which is supported by the NIH National Center for Advancing Translational Sciences (NCATS) award 1K12TR004416-01. This work was also supported by the National Institute of General Medicine Sciences (NIGMS) award 1R35GM157060-01. We also thank C. Pham, M. Evangelista and R. Sellers in the Pathology Services Core (PSC) for expert technical assistance with Histopathology and Digital Pathology. The PSC is supported in part by an NCI Center Core Support Grant (5P30CA016080-42). All animal studies were approved by the UNC Institutional Animal Care and Use Committee, were consistent with local, state and federal regulations as applicable and were supported within the UNC Lineberger ASC at the University of North Carolina at Chapel Hill which is supported in part by an NCI Center Core Support Grant (CA16086) to the UNC Lineberger Comprehensive Cancer Center. Flow cytometry was performed at UNC Flow Cytometry Core Facility (RRID: SCR_019170). Microscopy was performed at the UNC Neuroscience Microscopy Core (RRID: SCR_019060), supported, in part, by funding from the NIH-NINDS Neuroscience Center Support Grant P30 NS045892 and the NIH-NICHD Intellectual and Developmental Disabilities Research Center Support Grant P50 HD103573. Research reported in this publication was supported in part by the North Carolina Biotech Center institutional support grant 2017-IDG-1025 and by the National Institutes of Health 1UM2AI30836-01. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Yutian Ma & Owen S. Fenton
Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Rachel VanKeulen-Miller
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Yutian Ma
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2. Rachel VanKeulen-Miller
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Y.M. designed and conducted experiments, analyzed the data and contributed to writing and editing the manuscript. R.V.-M. contributed to writing and editing the manuscript. O.S.F. supervised all the experiments, wrote and edited the manuscript and managed the project. All authors contributed to the development of the protocol and improved the manuscript.
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Correspondence to Owen S. Fenton.
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The University of North Carolina at Chapel Hill has filed some patents on data related to this work, with the authors named as inventors.
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Nature Protocols thanks Yizhou Dong and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references
Ma, Y. & Fenton, O. S. J. Am. Chem. Soc. 145, 19800–19811 (2023): https://doi.org/10.1021/jacs.3c05574
Ma, Y. & Fenton, O. S. J. Am. Chem. Soc. 145, 11375–11386 (2023): https://doi.org/10.1021/jacs.3c02584
Ma, Y. & Fenton, O. S. Adv. Therap. 6, 2200305 (2023): https://doi.org/10.1002/adtp.202200305
Supplementary information
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Supplementary Figs. 1–13 and Tables 1 and 2.
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Supplementary Data 1
Source data for Supplementary Figs. 1, 5, 6a–c, 7b, 11 and 12.
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Ma, Y., VanKeulen-Miller, R. & Fenton, O.S. mRNA lipid nanoparticle formulation, characterization and evaluation. Nat Protoc (2025). https://doi.org/10.1038/s41596-024-01134-4
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Received:10 May 2024
Accepted:12 December 2024
Published:11 March 2025
DOI:https://doi.org/10.1038/s41596-024-01134-4
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