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Single-cell quantification and dose-response of cytosolic siRNA delivery

Author

Listed:
  • Hampus Hedlund

    (Lund University)

  • Hampus Rietz

    (Lund University)

  • Johanna M. Johansson

    (Lund University)

  • Hanna C. Eriksson

    (Lund University)

  • Wahed Zedan

    (Lund University)

  • Linfeng Huang

    (Duke Kunshan University)

  • Jonas Wallin

    (Lund University)

  • Anders Wittrup

    (Lund University
    Skane University Hospital, Oncology
    Wallenberg Center for Molecular Medicine)

Abstract

Endosomal escape and subsequent cytosolic delivery of small interfering RNA (siRNA) therapeutics is believed to be highly inefficient. Since it has not been possible to quantify cytosolic amounts of delivered siRNA at therapeutic doses, determining delivery bottlenecks and total efficiency has been difficult. Here, we present a confocal microscopy-based method to quantify cytosolic delivery of fluorescently labeled siRNA during lipid-mediated delivery. This method enables detection and quantification of sub-nanomolar cytosolic siRNA release amounts from individual release events with measures of quantitation confidence for each event. Single-cell kinetics of siRNA-mediated knockdown in cells expressing destabilized eGFP unveiled a dose-response relationship with respect to knockdown induction, depth and duration in the range from several hundred to thousands of cytosolic siRNA molecules. Accurate quantification of cytosolic siRNA, and the establishment of the intracellular dose-response relationships, will aid the development and characterization of novel delivery strategies for nucleic acid therapeutics.

Suggested Citation

  • Hampus Hedlund & Hampus Rietz & Johanna M. Johansson & Hanna C. Eriksson & Wahed Zedan & Linfeng Huang & Jonas Wallin & Anders Wittrup, 2023. "Single-cell quantification and dose-response of cytosolic siRNA delivery," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-36752-1
    DOI: 10.1038/s41467-023-36752-1
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    References listed on IDEAS

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    1. Laura I. FitzGerald & Luigi Aurelio & Moore Chen & Daniel Yuen & Joshua J. Rennick & Bim Graham & Angus P. R. Johnston, 2020. "A molecular sensor to quantify the localization of proteins, DNA and nanoparticles in cells," Nature Communications, Nature, vol. 11(1), pages 1-13, December.
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