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Tuning extracellular fluid viscosity to enhance transfection efficiency

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Abstract

Gene therapies and cellular programming rely on effective cell transfection. Despite continuous advancements in carrier development and transfection techniques to enhance efficiency, the biophysical parameter of extracellular fluid viscosity has been largely overlooked. Here we report a substantial impact of culture media viscosity on transfection efficiency of several delivery vehicles, including lipid nanoparticles, polyplexes, adeno-associated vectors and lentiviral vectors across a range of cell types. We observed substantially increased transfection efficiencies for lipid nanoparticles and polyplexes when the media viscosity matched that of biological fluids (2.0–4.0 centipoise (cP)). This enhancement correlates with higher levels of cellular uptake and improved endosomal escape. Moreover, cells cultured in optimized viscosity conditions exhibit a different profile of uptake pathways compared with those cultured at the standard viscosity of 0.8 cP. This discovery highlights the critical role of media viscosity in the transfection process and provides an additional method to optimize gene delivery and cell programming processes, potentially reducing production costs and increasing the accessibility of gene and cell therapies.

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Fig. 1: Extracellular-fluid-viscosity-dependent transfection efficiency mediated by mRNA LNPs.
Fig. 2: Media-viscosity-dependent transfection efficiency mediated by mLuc LNPs across various cell types.
Fig. 3: Effect of media viscosity on cellular uptake and endosomal escape efficiency of mRNA LNPs.
Fig. 4: Cell uptake pathways and actin remodeling/dynamics, NHE1-mediated swelling and RhoA-based contractility on endocytosis of mRNA LNPs at different viscosity levels in B16-F10 cells.
Fig. 5: Transfection/transduction efficiency of different nucleic acids and vehicles on HEK293T cells under different media viscosity conditions.
Fig. 6: Viscosity-enhanced transfection/transduction of viral production in HEK293F suspension cells and human PBMCs.

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Data availability

The main data supporting the results of this study are available within the Article and its Supplementary Information. The raw and analyzed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

The authors disclose support for the research described in this study from the National Institutes of Health (Grant numbers: R01 GM134542 to S.X.S. and K.K.; R01 CA257647 to K.K.). We thank J. Schneck from the Department of Pathology at the Johns Hopkins University School of Medicine for providing the human PBMCs as a gift and H. Bui from the Johns Hopkins University Integrated Imaging Center (IIC) for assistance with the flow cytometry assessments. Portions of the schematics in Figs. 3–6 and Supplementary Figs. 1, 2 and 13 were created with BioRender.com.

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Author notes

  1. These authors contributed equally: Jingyao Ma, Yining Zhu.

Authors and Affiliations

  1. Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA

    Jingyao Ma, Yining Zhu, Jiayuan Kong, Di Yu, Wu Han Toh, Milun Jain, Qin Ni, Zhuoxu Ge, Jinghan Lin, Joseph Choy, Leonardo Cheng, Konstantinos Konstantopoulos, Maximilian F. Konig, Sean X. Sun & Hai-Quan Mao

  2. Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD, USA

    Jingyao Ma, Jiayuan Kong, Jinghan Lin, Joseph Choy & Hai-Quan Mao

  3. Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Jingyao Ma, Yining Zhu, Jiayuan Kong, Di Yu, Wu Han Toh, Milun Jain, Jinghan Lin, Joseph Choy, Leonardo Cheng & Hai-Quan Mao

  4. Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Yining Zhu, Di Yu, Milun Jain, Leonardo Cheng, Konstantinos Konstantopoulos & Hai-Quan Mao

  5. Department of Computer Science, Johns Hopkins University, Baltimore, MD, USA

    Wu Han Toh

  6. Department of Biology, Johns Hopkins University, Baltimore, MD, USA

    Wu Han Toh

  7. Department of Mechanical Engineering, Johns Hopkins University, Baltimore, MD, USA

    Qin Ni, Zhuoxu Ge & Sean X. Sun

  8. Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA

    Konstantinos Konstantopoulos

  9. Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Konstantinos Konstantopoulos

  10. Division of Rheumatology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Maximilian F. Konig

  11. Ludwig Center for Cancer Genetics and Therapeutics, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Maximilian F. Konig

  12. Center for Cell Dynamics, School of Medicine, Johns Hopkins University, Baltimore, MD, USA

    Sean X. Sun

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  1. Jingyao Ma
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Contributions

H.-Q.M., S.X.S., Y.Z. and J.M. conceived and designed this study. J.M., Y.Z., J.K., D.Y., W.H.T., M.J. and J.L. performed the experiments. J.M., Y.Z., J.K., D.Y., W.H.T., M.J., Q.N., Z.G., J.C., K.K., M.F.K., S.X.S. and H.-Q.M. contributed to the data analysis and interpretation. J.M., Y.Z. and H.-Q.M. wrote the manuscript with input from all other authors. H.-Q.M. and S.X.S. secured the funding and supervised this study.

Corresponding authors

Correspondence to Sean X. Sun or Hai-Quan Mao.

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Competing interests

H.-Q.M., S.X.S., J.M., Y.Z., Q.N. and Z.G. are co-inventors of a patent application (PCT/US2024/039036, filed in July 2024) covering the compositions and transfection methods described in this paper, filed through and managed by Johns Hopkins Office of Technology Ventures. The other authors declare no competing interests.

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Nature Chemical Engineering thanks Michael Mitchell, Suzie Pun and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Ma, J., Zhu, Y., Kong, J. et al. Tuning extracellular fluid viscosity to enhance transfection efficiency. Nat Chem Eng (2024). https://doi.org/10.1038/s44286-024-00116-3

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