Article

High-throughput nuclear delivery and rapid expression of DNA via mechanical and electrical cell-membrane disruption

  • Nature Biomedical Engineering 1, Article number: 0039 (2017)
  • doi:10.1038/s41551-017-0039
  • Download Citation
Received:
Accepted:
Published online:

Abstract

Nuclear transfection of DNA into mammalian cells is challenging yet critical for many biological and medical studies. Here, by combining cell squeezing and electric-field-driven transport in a device that integrates microfluidic channels with constrictions and microelectrodes, we demonstrate nuclear delivery of plasmid DNA within 1 h after treatment—the most rapid DNA expression in a high-throughput setting (up to millions of cells per minute per device). Passing cells at high speed through microfluidic constrictions smaller than the cell diameter mechanically disrupts the cell membrane, allowing a subsequent electric field to further disrupt the nuclear envelope and drive DNA molecules into the cytoplasm and nucleus. By tracking the localization of the endosomal sorting complex required for transport III protein CHMP4B (charged multivesicular body protein 4B), we show that the integrity of the nuclear envelope is recovered within 15 minutes of treatment. We also provide insight into subcellular delivery by comparing the performance of the disruption-and-field-enhanced method with those of conventional chemical, electroporation and manual-injection systems.

  • Subscribe to Nature Biomedical Engineering for full access:

    $99

    Subscribe

  • Purchase article full text and PDF:

    $32

    Buy now

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. 1.

    et al. Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 15, 541–555 (2014).

  2. 2.

    et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N. Engl. J. Med. 371, 1507–1517 (2014).

  3. 3.

    et al. In vitro and ex vivo strategies for intracellular delivery. Nature 538, 183–192 (2016).

  4. 4.

    & Progress and prospects: immune responses to viral vectors. Gene Ther. 17, 295–304 (2010).

  5. 5.

    , & Production of retrovirus and adenovirus vectors for gene therapy: a comparative study using microcarrier and stationary cell culture. Biotechnol. Progr. 18, 617–622 (2002).

  6. 6.

    et al. Liposome mediated gene transfer into the rat oesophagus. Gut 41, 549–556 (1997).

  7. 7.

    et al. Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery. Nat. Nanotech. 7, 389–393 (2012).

  8. 8.

    & Biolistic transfection of neuronal cultures using a hand-held gene gun. Nat. Protoc. 1, 977–981 (2006).

  9. 9.

    Gene therapy progress and prospects: electroporation and other physical methods. Gene Ther. 11, 1363–1369 (2004).

  10. 10.

    , , & Physical methods for intracellular delivery: practical aspects from laboratory use to industrial-scale processing. J. Lab. Autom. 19, 1–18 (2014).

  11. 11.

    High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell 22, 479–488 (1980).

  12. 12.

    , , & Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Laboratory, 2003).

  13. 13.

    & The mechanism of naked DNA uptake and expression. Adv Genet. 54, 3–20 (2005).

  14. 14.

    , , & Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J. 1, 841–845 (1982).

  15. 15.

    et al. What is (still not) known of the mechanism by which electroporation mediates gene transfer and expression in cells and tissues. Mol. Biotechnol. 41, 286–295 (2009).

  16. 16.

    , , & Membrane electroporation: the absolute rate equation and nanosecond time scale pore creation. Phys. Rev. E 74, 021904 (2006).

  17. 17.

    , , , & Electrically induced DNA uptake by cells is a fast process involving DNA electrophoresis. Biophys. J. 60, 804–811 (1991).

  18. 18.

    , , , & A brief overview of electroporation pulse strength–duration space: a region where additional intracellular effects are expected. Bioelectrochemistry 87, 236–243 (2012).

  19. 19.

    , & (eds) Electroporation and Electrofusion in Cell Biology (Springer Science & Business Media, 2013).

  20. 20.

    , & Direct visualization at the single-cell level of electrically mediated gene delivery. Proc. Natl Acad. Sci. USA 99, 1292–1297 (2002).

  21. 21.

    et al. Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells. Proc. Natl Acad. Sci. USA 108, 10443–10447 (2011).

  22. 22.

    et al. Intracellular tracking of single-plasmid DNA particles after delivery by electroporation. Mol. Ther. 21, 2217–2226 (2013).

  23. 23.

    et al. Nanochannel electroporation delivers precise amounts of biomolecules into living cells. Nat. Nanotech. 6, 747–754 (2011).

  24. 24.

    , & Mechanisms of cell membrane electropermeabilization: a minireview of our present (lack of ?) knowledge. Biochim. Biophys. Acta 1724, 270–280 (2005).

  25. 25.

    , , , & Electroporation-based technologies for medicine: principles, applications, and challenges. Annu. Rev. Biomed. Eng. 16, 295–320 (2014).

  26. 26.

    & Microfluidic electroporation for cellular analysis and delivery. Lab Chip 13, 3803–3821 (2013).

  27. 27.

    , Verkman, A. & Lukacs, G. Intracellular routing of plasmid DNA during non-viral gene transfer. Adv. Drug Deliv. Rev. 57, 755–767 (2005).

  28. 28.

    , , , & Plasmid DNA entry into post-mitotic nuclei of primary rat myotubes. Proc. Natl Acad. Sci. USA 92, 4572–4576 (1995).

  29. 29.

    et al. Vertical silicon nanowires as a universal platform for delivering biomolecules into living cells. Proc. Natl Acad. Sci. USA 107, 1870–1875 (2010).

  30. 30.

    et al. Nanostraw–electroporation system for highly efficient intracellular delivery and transfection. ACS Nano 7, 4351–4358 (2013).

  31. 31.

    et al. Poking cells for efficient vector-free intracellular delivery. Nat. Commun. 5, 4466 (2014).

  32. 32.

    et al. A vector-free microfluidic platform for intracellular delivery. Proc. Natl Acad. Sci. USA 110, 2082–2087 (2013).

  33. 33.

    et al. Nonendocytic delivery of functional engineered nanoparticles into the cytoplasm of live cells using a novel, high-throughput microfluidic device. Nano Lett. 12, 6322–6327 (2012).

  34. 34.

    et al. Live-cell protein labelling with nanometre precision by cell squeezing. Nat. Commun. 7, 10372 (2016).

  35. 35.

    et al. Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines. Sci. Rep. 5, 10276 (2015).

  36. 36.

    , et al. ESCRT machinery is required for plasma membrane repair. Science 343, 1247136 (2014).

  37. 37.

    et al. ESCRT III repairs nuclear envelope ruptures during cell migration to limit DNA damage and cell death. Science 352, 359–362 (2016).

  38. 38.

    et al. Nuclear envelope rupture and repair during cancer cell migration. Science 352, 353–358 (2016).

  39. 39.

    , , , & ESCRT-III controls nuclear envelope reformation. Nature 522, 236–239 (2015).

  40. 40.

    , , & Membrane reserves and hypotonic cell swelling. J. Membr. Biol. 214, 43–56 (2006).

  41. 41.

    et al. A laminar flow electroporation system for efficient DNA and siRNA delivery. Anal. Chem. 83, 5881–5887 (2011).

  42. 42.

    , , & Modeling a conventional electroporation pulse train: decreased pore number, cumulative calcium transport and an example of electrosensitization. IEEE Trans. Biomed. Eng. 63, 571–580 (2016).

  43. 43.

    et al. BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat. Methods 5, 409–415 (2008).

Download references

Acknowledgements

We thank P. Qi from the Division of Comparative Medicine at Massachusetts Institute of Technology (MIT) for performing the microinjection, Q. Chen from Richard Sherwood Lab at Brigham and Women’s Hospital for providing mouse embryonic stem cells, and X. Yin from the Jeff Karp Lab at Brigham and Women’s Hospital, Harvard Medical School for providing human embryonic stem cells. We thank I. Poser from Tony Hyman Lab at Max Planck Institute of Molecular Cell Biology and Genetics for supplying the CHMP4B-GFP HeLa cells. The assistance and expertise of G. Paradis and personnel in the flow cytometry core at the Koch Institute and the Microsystem Technology Laboratory at MIT are highly acknowledged. This research was supported by National Institutes of Health (R01GM101420-01A1), and device fabrication was performed at the Microsystem Technology Laboratory at MIT. M.P.S. was supported by the Swiss National Science Foundation through the advanced postdoc mobility fellowship P300P3_151179. M.P.S. acknowledges support from a Keith Murdoch Fellowship via the American Australian Association, a Life Sciences Research Foundation Fellowship sponsored by Good Ventures, and a Broadnext10 Catalytic Steps funding gift from the Broad Institute.

Author information

Author notes

    • Xiaoyun Ding
    •  & Martin P. Stewart

    These authors contributed equally to this work.

Affiliations

  1. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Xiaoyun Ding
    • , Martin P. Stewart
    • , Armon Sharei
    • , Robert S. Langer
    •  & Klavs F. Jensen
  2. The David Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Xiaoyun Ding
    • , Martin P. Stewart
    • , Armon Sharei
    •  & Robert S. Langer
  3. Harvard–MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • James C. Weaver

Authors

  1. Search for Xiaoyun Ding in:

  2. Search for Martin P. Stewart in:

  3. Search for Armon Sharei in:

  4. Search for James C. Weaver in:

  5. Search for Robert S. Langer in:

  6. Search for Klavs F. Jensen in:

Contributions

X.D., M.P.S., A.S., R.S.L. and K.F.J. designed the research, X.D. and M.P.S. performed the experiments and X.D. fabricated the devices. X.D., M.P.S., A.S., J.C.W., R.S.L. and K.F.J. analysed the data. X.D., M.P.S., A.S., J.C.W., R.S.L. and K.F.J. wrote the article.

Competing interests

A.S., R.S.L. and K.F.J. have a financial interest in SQZ Biotechnologies.

Corresponding authors

Correspondence to Robert S. Langer or Klavs F. Jensen.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary figures