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


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Device structure and working mechanism.
Figure 2: DNA transfection performance and expression dynamics depend on the applied electric field and methods.
Figure 3: Visualization of the delivery of fluorescence-labelled plasmid DNA to HeLa cells.
Figure 4: ESCRT-III recruitment for plasma membrane and nuclear envelope repair.


  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

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

    Article  Google Scholar 

  3. 3

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

    CAS  Article  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 5

    Wu, S. C., Huang, G. Y. L. & Liu, J. H. Production of retrovirus and adenovirus vectors for gene therapy: a comparative study using microcarrier and stationary cell culture. Biotechnol. Progr. 18, 617–622 (2002).

    CAS  Article  Google Scholar 

  6. 6

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

    CAS  Article  Google Scholar 

  7. 7

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

    CAS  Article  Google Scholar 

  8. 8

    O’Brien, J. A. & Lummis, S. C. R. Biolistic transfection of neuronal cultures using a hand-held gene gun. Nat. Protoc. 1, 977–981 (2006).

    Article  Google Scholar 

  9. 9

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

    CAS  Article  Google Scholar 

  10. 10

    Meacham, J. M., Durvasula, K., Degertekin, F. L. & Fedorov, A. G. Physical methods for intracellular delivery: practical aspects from laboratory use to industrial-scale processing. J. Lab. Autom. 19, 1–18 (2014).

    CAS  Article  Google Scholar 

  11. 11

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

    CAS  Article  Google Scholar 

  12. 12

    Nagy, A., Gertsenstein, M., Vintersten, K. & Behringer, R. Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Laboratory, 2003).

    Google Scholar 

  13. 13

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

    CAS  PubMed  Google Scholar 

  14. 14

    Neumann, E., Schaefer-Ridder, M., Wang, Y. & Hofschneider, P. H. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J. 1, 841–845 (1982).

    CAS  Article  Google Scholar 

  15. 15

    Escoffre, J.-M. 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).

    CAS  Article  Google Scholar 

  16. 16

    Vasilkoski, Z., Esser, A. T., Gowrishankar, T. R. & Weaver, J. C. Membrane electroporation: the absolute rate equation and nanosecond time scale pore creation. Phys. Rev. E 74, 021904 (2006).

    Article  Google Scholar 

  17. 17

    Klenchin, V. A., Sukharev, S., Serov, S. M., Chernomordik, L. V. & Chizmadzhev, Y. A. Electrically induced DNA uptake by cells is a fast process involving DNA electrophoresis. Biophys. J. 60, 804–811 (1991).

    CAS  Article  Google Scholar 

  18. 18

    Weaver, J. C., Smith, K. C., Esser, A. T., Son, R. S. & Gowrishankar, T. R. A brief overview of electroporation pulse strength–duration space: a region where additional intracellular effects are expected. Bioelectrochemistry 87, 236–243 (2012).

    CAS  Article  Google Scholar 

  19. 19

    Jordan, C. A., Neumann, E. & Sowers, A. E. (eds) Electroporation and Electrofusion in Cell Biology (Springer Science & Business Media, 2013).

    Google Scholar 

  20. 20

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

    CAS  Article  Google Scholar 

  21. 21

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

    CAS  Article  Google Scholar 

  22. 22

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

    CAS  Article  Google Scholar 

  23. 23

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

    CAS  Article  Google Scholar 

  24. 24

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

    CAS  Article  Google Scholar 

  25. 25

    Yarmush, M. L., Golberg, A., Serša, G., Kotnik, T. & Miklavčič, D. Electroporation-based technologies for medicine: principles, applications, and challenges. Annu. Rev. Biomed. Eng. 16, 295–320 (2014).

    CAS  Article  Google Scholar 

  26. 26

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

    CAS  Article  Google Scholar 

  27. 27

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

    CAS  Article  Google Scholar 

  28. 28

    Dowty, M. E., Williams, P., Zhang, G., Hangstrom, J. E. & Wolff, J. A. Plasmid DNA entry into post-mitotic nuclei of primary rat myotubes. Proc. Natl Acad. Sci. USA 92, 4572–4576 (1995).

    CAS  Article  Google Scholar 

  29. 29

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

    CAS  Article  Google Scholar 

  30. 30

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

    CAS  Article  Google Scholar 

  31. 31

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

    CAS  Article  Google Scholar 

  32. 32

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

    CAS  Article  Google Scholar 

  33. 33

    Lee, J. 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).

    CAS  Article  Google Scholar 

  34. 34

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

    CAS  Article  Google Scholar 

  35. 35

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

    Article  Google Scholar 

  36. 36

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

    Article  Google Scholar 

  37. 37

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

    CAS  Article  Google Scholar 

  38. 38

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

    CAS  Article  Google Scholar 

  39. 39

    Olmos, Y., Hodgson, L., Mantell, J., Verkade, P. & Carlton, J. G. ESCRT-III controls nuclear envelope reformation. Nature 522, 236–239 (2015).

    CAS  Article  Google Scholar 

  40. 40

    Groulx, N., Boudreault, F., Orlov, S. N. & Grygorczyk, R. Membrane reserves and hypotonic cell swelling. J. Membr. Biol. 214, 43–56 (2006).

    CAS  Article  Google Scholar 

  41. 41

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

    CAS  Article  Google Scholar 

  42. 42

    Son, R. S., Gowrishankar, T. R., Smith, K. C. & Weaver, J. C. 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).

    Article  Google Scholar 

  43. 43

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

    CAS  Article  Google Scholar 

Download references


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




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.

Corresponding authors

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

Ethics declarations

Competing interests

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

Supplementary information

Supplementary Information

Supplementary figures (PDF 1026 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Ding, X., Stewart, M., Sharei, A. et al. High-throughput nuclear delivery and rapid expression of DNA via mechanical and electrical cell-membrane disruption. Nat Biomed Eng 1, 0039 (2017). https://doi.org/10.1038/s41551-017-0039

Download citation

Further reading