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Elucidating lipid nanoparticle properties and structure through biophysical analyses

Nature Biotechnology (2025)Cite this article

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An Author Correction to this article was published on 22 January 2026

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Abstract

Designing lipid nanoparticle (LNP) delivery systems with specific targeting, potency and minimal side effects is crucial for their clinical use. However, traditional characterization methods, such as dynamic light scattering, cannot accurately quantify physicochemical properties of LNPs and how these are influenced by the lipid composition and mixing method. Here, we structurally characterize polydisperse LNP formulations by applying emerging solution-based biophysical methods that have higher resolution and provide biophysical data beyond size and polydispersity. These techniques include sedimentation velocity analytical ultracentrifugation, field-flow fractionation followed by multiangle light scattering and size-exclusion chromatography in line with synchrotron small-angle X-ray scattering. We show that LNPs have intrinsic polydispersity in size, RNA loading and shape, which depend on both the formulation technique and the lipid composition. Lastly, we predict LNP transfection in vitro and in vivo by examining the relationship between mRNA translation and physicochemical characteristics. Solution-based biophysical methods will be essential for determining LNP structure–function relationships, facilitating the creation of new design rules for LNPs.

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Fig. 1: Traditional physicochemical characterization of LNPs.
Fig. 2: Analysis of LNPs by DLS, AUC and FFF–MALS.
Fig. 3: LNP batches contain discrete subspecies as identified by SEC–SAXS.
Fig. 4: SEC–SAXS profiles have low degree of similarity and show that LNPs adopt nonspherical morphologies.
Fig. 5: LNP transfection in biological models and correlation with physicochemical parameters.
Fig. 6: Analysis of LNP toxicity and endosomal escape.

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Data availability

All data supporting the findings of this study are available within the paper and Supplementary Information. The SV-AUC, FFF–MALS and SEC–SAXS raw and analyzed data are available from Zenodo (https://doi.org/10.5281/zenodo.17042311)60. Source data are provided with this paper.

Code availability

No original code was generated for this study.

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Acknowledgements

The SV-AUC experiments were performed at the Johnson Foundation Biophysical and Structural Biology Core Facility (University of Pennsylvania). The LiX beamline is part of the Center for Biomolecular Structure (CBMS), which is primarily supported by the Department of Energy Office of Biological and Environmental Research (KP1605010). As part of NSLS-II, a national user facility at Brookhaven National Laboratory, work performed at the CBMS is supported in part by the US Department of Energy, Office of Science, Office of Basic Energy Sciences Program under contract number DE-SC0012704. Additionally, we thank E. Cento, Z. Chen, M. A. Eldabbas and E. Maddox of the Human Immunology Core and the Division of Transfusion Medicine and Therapeutic Pathology at the Perelman School of Medicine (University of Pennsylvania) for providing deidentified CD4+ and CD8+ T cells that were purified from healthy donor apheresis using StemCell RosetteSep kits. Cryo-EM imaging was provided by the Beckman Center for Cryo-EM at the University of Pennsylvania Perelman School of Medicine. M.J.M. acknowledges support from a Burroughs Wellcome Fund Career Award at the Scientific Interface, an American Cancer Society Research Scholar Grant (RSG-22-122-01-ET) and a US National Science Foundation CAREER Award (CBET-2145491).

Author information

Authors and Affiliations

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

    Marshall S. Padilla, Sarah J. Shepherd, Andrew R. Hanna, Ryann A. Joseph, Hannah M. Yamagata, Adele S. Ricciardi, Kaitlin Mrksich, David Issadore & Michael J. Mitchell

  2. Waters Corporation, Goleta, CA, USA

    Martin Kurnik, Xujun Zhang & Michelle Chen

  3. National Synchrotron Light Source II, Brookhaven National Laboratory, Upton, NY, USA

    James Byrnes

  4. Department of Surgery, University of Pennsylvania Health System, Philadelphia, PA, USA

    Adele S. Ricciardi

  5. Center for Fetal Research, Division of General, Thoracic and Fetal Surgery, Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Adele S. Ricciardi

  6. Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA, USA

    David Issadore

  7. Department of Electrical and Systems Engineering, University of Pennsylvania, Philadelphia, PA, USA

    David Issadore

  8. Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Kushol Gupta

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

    Michael J. Mitchell

  10. Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  11. Penn Institute for RNA Innovation, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

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

    Michael J. Mitchell

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

    Michael J. Mitchell

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

    Michael J. Mitchell

Authors
  1. Marshall S. Padilla
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  2. Sarah J. Shepherd
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  14. Michael J. Mitchell
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Contributions

M.S.P., S.J.S., M.K., X.Z., M.C., J.B., K.G. and M.J.M. conceptualized and designed the experiments. M.S.P., S.J.S., A.H., M.K., X.Z., J.B., H.M.Y., A.R., R.A.J. and K.M. performed the experiments. M.S.P., S.J.S., M.K., X.Z., M.C., J.B. and K.G. analyzed the data. M.S.P., D.I., K.G. and M.J.M. wrote and edited the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Kushol Gupta or Michael J. Mitchell.

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Nature Biotechnology thanks Yizhou Dong, Guangjun Nie and Wei Tao for their contribution to the peer review of this work.

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Extended data

Extended Data Table 1 Physicochemical parameters of LNPs obtained by FFF-MALS
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Padilla, M.S., Shepherd, S.J., Hanna, A.R. et al. Elucidating lipid nanoparticle properties and structure through biophysical analyses. Nat Biotechnol (2025). https://doi.org/10.1038/s41587-025-02855-x

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