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Preparation of targeted lipid nanoparticles for precision nucleic acid delivery

Nature Protocols (2026)Cite this article

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Abstract

Intravenous administration of lipid nanoparticles for the delivery of nucleic acid therapeutics remains constrained by passive uptake mechanisms in the liver, often necessitating high doses to achieve meaningful transfection in specific cells of interest. Targeted LNPs (tLNPs) can overcome these challenges by (i) enabling receptor-mediated endocytosis in difficult-to-transfect cells, thereby reducing passive clearance; (ii) increasing the proportion of LNPs reaching their intended target; and (iii) enabling comparable protein expression at lower doses. Here, we provide a step-by-step guide for formulating tLNPs functionalized with whole antibodies or antibody fragments using traditional laboratory equipment. We outline procedures for antibody preparation and labeling (0.5–1 d), antibody–LNP conjugation (1–2 d), tLNP purification and characterization (1 d) and in vivo and ex vivo targeting evaluation (3–4 d). To demonstrate the versatility of this protocol, we validate in vivo targeting to two mouse tissues: we show that anti-platelet endothelial cell adhesion molecule 1 antibody conjugation to lung-tropic LNPs enhances lung transfection by five times compared to nontargeted LNPs, and anti-epidermal growth factor receptor antibody conjugation to liver-tropic LNPs enhances liver transfection by 20 times. We also demonstrate ex vivo targeting to primary human T cells, where anti-CD5 antibody conjugation to LNPs boosts uptake by 4.5 times and significantly increases mRNA transfection. Importantly, this modular strategy is compatible with any LNP formulation or antibody. In outlining these procedures, we seek to deliver a robust and reproducible workflow for the manufacturing of tLNPs, with the ultimate goal of advancing their therapeutic potential and facilitating clinical translation.

Key points

  • Strain-promoted azide–alkyne cycloaddition (SPAAC) chemistry is used to covalently conjugate dibenzocyclooctyne (DBCO)-labeled antibodies or antibody fragments to LNPs containing azide. After antibody labeling and LNP conjugation, tLNPs are purified by size exclusion chromatography and characterized ex vivo and in vivo.

  • This protocol offers a versatile approach to precisely target nucleic acid-containing LNPs to cell types or tissues, as evidenced by in vivo evaluation of liver- and lung-targeting LNPs and ex vivo evaluation of human primary T cell-targeting LNPs.

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Fig. 1: Key advantages of tLNPs.
Fig. 2: Overview of the protocol to formulate and evaluate tLNPs.
Fig. 3: Antibody labeling and tLNP formulation.
Fig. 4: tLNP purification and characterization.
Fig. 5: In vivo tLNP targeting and mRNA delivery to the liver and lungs.
Fig. 6: Ex vivo tLNP targeting and mRNA delivery to primary human T cells.

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Data availability

The data associated with this protocol are provided in the article and Supplementary Information and as Source Data. Source data are provided with this paper.

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Authors and Affiliations

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

    Hannah C. Geisler, Elisa Battistini, Ajay S. Thatte, Marshall S. Padilla & Michael J. Mitchell

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

    Michael J. Mitchell

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

    Michael J. Mitchell

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

    Michael J. Mitchell

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

    Michael J. Mitchell

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

    Michael J. Mitchell

Authors
  1. Hannah C. Geisler
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  2. Elisa Battistini
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  3. Ajay S. Thatte
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  4. Marshall S. Padilla
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Contributions

H.C.G. designed and conducted experiments, analyzed the data, wrote the original draft of the manuscript, and contributed to reviewing and editing the manuscript. E.B., A.S.T. and M.S.P. contributed to data collection and reviewing and editing of the manuscript. M.J.M. supervised the experiments and overall study, and contributed to reviewing and editing the manuscript.

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Correspondence to Michael J. Mitchell.

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Competing interests

M.J.M. is an inventor on patent applications describing lipid nanoparticle technology related to this work filed by the Trustees of the University of Pennsylvania.

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

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Key references

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

Zhao, G. et al. Proc. Natl Acad. Sci. USA 121, e2314747121 (2024): https://doi.org/10.1073/pnas.2314747121

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Geisler, H.C., Battistini, E., Thatte, A.S. et al. Preparation of targeted lipid nanoparticles for precision nucleic acid delivery. Nat Protoc (2026). https://doi.org/10.1038/s41596-025-01330-w

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