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

Nature Biomedical Engineering volume 1, Article number: 0039 (2017) Cite this article

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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.

Intracellular delivery, the introduction of exogenous materials into cells, is essential to many studies in basic biology and biomedical research1,2. A multitude of techniques exists for cell transfection, including biological, chemical and physical methods3. Biological and chemical methods usually rely on carriers, such as viruses, vesicles, peptides or nanoparticles4–7. Physical methods primarily use membrane-disruption techniques, such as microinjection, electroporation, laser optoporation and particle bombardment, for gene delivery8–13. Although electroporation has been widely used for DNA transfection since the early 1980s, the underlying mechanism of delivery is not completely understood in nucleated mammalian cells14–19. In the electroporation process, DNA molecules accumulate and interact with the electropermeabilized plasma membrane during the electric pulse to form aggregates. Afterwards, those DNA aggregates are internalized into the cytoplasm and subsequently expressed20–26. It is unlikely that DNA plasmids navigate through the viscous and crowded cytoplasm to reach the nucleus simply by diffusion27,28. Microtubule and actin networks have been proposed to play an important role in DNA transportation within the cytoplasm, and the timescale of such processes can be hours long depending on the cell type22. The lack of detailed mechanistic understanding and the complex nature of DNA transfer between the plasma membrane and nucleus limit our ability to enhance electroporation performance. Moreover, the strong fields used in current electroporation techniques can lead to significant damage or death to cells25,26. Nanostructure-based methods have demonstrated potential for effective gene transfection by penetrating DNA-loaded nanoneedles into the cell, or by diffusion or electrophoresis through a nanostraw29–31. However, such methods typically have relatively low throughput. Moreover, the nuclear envelope rupture is not well investigated. Hence, substantial interest remains in creating techniques that can quickly and directly deliver DNA to the nucleus in a large number of cells with controllable nuclear envelope damage.

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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.

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References

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Google Scholar 

  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. 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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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. 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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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.

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Author notes

  1. Xiaoyun Ding and Martin P. Stewart: These authors contributed equally to this work.

Authors and Affiliations

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

    James C. Weaver

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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.

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

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Competing interests

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

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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

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