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Preparation of placenta-tropic mRNA lipid nanoparticles for pregnancy disorders

Nature Protocols (2026)Cite this article

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Abstract

Lipid nanoparticles (LNPs) have garnered tremendous enthusiasm in preclinical and clinical settings for the delivery of nucleic acids such as mRNA. With applications in protein replacement therapies, vaccines and gene editing, mRNA LNPs have only recently been explored in the context of pregnancy disorders. There is a significant need for the design of novel therapeutic technologies such as mRNA LNPs to treat obstetric disorders like pre-eclampsia that are associated with placental pathology and detrimental effects on maternal and fetal health. Here, we present a step-by-step procedure for the preparation and evaluation of placenta-tropic mRNA LNPs for researchers from varied disciplines to explore their application in treating pregnancy disorders. In this Protocol, we describe steps for synthesizing and purifying the key ionizable lipid excipient of the placenta-tropic LNP formulation (4 d) before preparing mRNA LNPs using microfluidic mixing (1 d). Then, we detail in vitro mechanistic evaluations of the effect of protein adsorption on LNP-mediated mRNA transfection to placental trophoblasts (3 d). Finally, we outline methods for isolating reproductive tissues from time-dated pregnant mice to assess in vivo LNP biodistribution and mRNA transfection to the murine placenta (16 d). Compared to alternative LNP formulation procedures, this Protocol focuses on delivering mRNA LNPs to the placenta with a workflow that can be applied for a range of obstetric disorders. This Protocol seeks to increase interdisciplinary work at the interface of nanomedicine, gene modulation and reproductive health.

Key points

  • The procedure covers synthesis and purification of the key ionizable lipid excipient, preparation of mRNA LNPs by microfluidic mixing, assessment of the effect of protein adsorption on LNP transfection to placental trophoblast cells and evaluation of LNP biodistribution and mRNA expression in the placentas of time-dated pregnant mice.

  • The mRNA LNP formulation presented here has demonstrated applications in treating pre-eclampsia in murine models of the condition and has the potential to be expanded for the treatment of other pregnancy disorders.

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Fig. 1: Delivering mRNA LNPs to the placenta upon systemic administration.
Fig. 2: Overview of the Protocol for formulating and evaluating placenta-tropic mRNA LNPs.
Fig. 3: Preparing placenta-tropic mRNA LNPs with the C14-494 ionizable lipid.
Fig. 4: Effect of protein adsorption on mRNA transfection to placental trophoblasts.
Fig. 5: Preparation of time-dated pregnancies and dissection of gestational day 16 placentas and fetuses.
Fig. 6: Evaluation of LNP biodistribution and mRNA transfection with the C14-494 placenta-tropic LNP in pregnant mice.

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Data availability

The data associated with this Protocol are provided in the article and Supplementary Information. Source data for Figs. 4 and 6 are provided with this paper. Source data are provided with this paper.

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Authors and Affiliations

  1. Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA

    Kelsey L. Swingle & Michael J. Mitchell

  2. Deparment of Bioengineering, Rice University, Houston, TX, USA

    Kelsey L. Swingle

  3. Penn Institute for RNA Innovation, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  4. Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  5. Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  6. Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  7. Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

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  1. Kelsey L. Swingle
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  2. Michael J. Mitchell
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Contributions

K.L.S. designed and conducted experiments, analyzed the data, wrote the original draft of the manuscript, and contributed to reviewing and editing the manuscript. M.J.M. supervised the experiments and overall study, was responsible for funding acquisition for the experiments, and contributed to reviewing and editing the manuscript.

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Correspondence to Michael J. Mitchell.

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Competing interests

K.L.S. and M.J.M. have filed a patent application based on this work. M.J.M. is an inventor on a patent related to this work filed by the Trustees of the University of Pennsylvania.

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Nature Protocols thanks Yusuke Sato and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references

Swingle, K. L. et al. Nature 637, 412–421 (2025): https://doi.org/10.1038/s41586-024-08291-2

Swingle, K. L. et al. J. Am. Chem. Soc. 145, 4691–4706 (2023): https://doi.org/10.1021/jacs.2c12893

Geisler, H. C. et al. J. Control. Release 371, 455–469 (2024): https://doi.org/10.1016/j.jconrel.2024.05.036

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Swingle, K.L., Mitchell, M.J. Preparation of placenta-tropic mRNA lipid nanoparticles for pregnancy disorders. Nat Protoc (2026). https://doi.org/10.1038/s41596-025-01325-7

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