Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Molecular machines open cell membranes

Nature volume 548, pages 567–572 (2017)Cite this article

Subjects

Abstract

Beyond the more common chemical delivery strategies, several physical techniques are used to open the lipid bilayers of cellular membranes1. These include using electric2 and magnetic3 fields, temperature4, ultrasound5 or light6 to introduce compounds into cells, to release molecular species from cells or to selectively induce programmed cell death (apoptosis) or uncontrolled cell death (necrosis). More recently, molecular motors and switches that can change their conformation in a controlled manner in response to external stimuli have been used to produce mechanical actions on tissue for biomedical applications7,8,9. Here we show that molecular machines can drill through cellular bilayers using their molecular-scale actuation, specifically nanomechanical action. Upon physical adsorption of the molecular motors onto lipid bilayers and subsequent activation of the motors using ultraviolet light, holes are drilled in the cell membranes. We designed molecular motors and complementary experimental protocols that use nanomechanical action to induce the diffusion of chemical species out of synthetic vesicles, to enhance the diffusion of traceable molecular machines into and within live cells, to induce necrosis and to introduce chemical species into live cells. We also show that, by using molecular machines that bear short peptide addends, nanomechanical action can selectively target specific cell-surface recognition sites. Beyond the in vitro applications demonstrated here, we expect that molecular machines could also be used in vivo, especially as their design progresses to allow two-photon, near-infrared and radio-frequency activation10.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Molecular motors for disruption of lipid bilayers through molecular mechanical action and control molecules.
Figure 2: NIH 3T3 cells in the presence of the fluorescent molecular machines 1 and 2.
Figure 3: The effects of nanomachines 3 and 4 and control molecule 5 on PC-3 cells upon UV activation.
Figure 4: Whole-cell patch-clamp studies of the dynamic effects of UV-induced molecular mechanical action of 3 on HEK293 cells.

Similar content being viewed by others

References

  1. Lakshmanan, S. et al. Physical energy for drug delivery; poration, concentration and activation. Adv. Drug Deliv. Rev. 71, 98–114 (2014)

    Article  CAS  Google Scholar 

  2. Chang, D. C. Cell poration and cell fusion using an oscillating electric field. Biophys. J. 56, 641–652 (1989)

    Article  CAS  ADS  Google Scholar 

  3. Liu, D., Wang, L., Wang, Z. & Cuschieri, A. Magnetoporation and magnetolysis of cancer cells via carbon nanotubes induced by rotating magnetic fields. Nano Lett. 12, 5117–5121 (2012)

    Article  CAS  ADS  Google Scholar 

  4. Ivanov, I. T. Spectrofluorometric and microcalorimetric study of the thermal poration relevant to the mechanism of thermohaemolysis. Int. J. Hyperthermia 15, 29–43 (1999)

    Article  CAS  Google Scholar 

  5. Tachibana, K., Uchida, T., Ogawa, K., Yamashita, N. & Tamura, K. Introduction of cell-membrane porosity by ultrasound. Lancet 353, 1409 (1999)

    Article  CAS  Google Scholar 

  6. Waleed, M. et al. Single-cell optoporation and transfection using femtosecond laser and optical tweezers. Biomed. Opt. Express 4, 1533–1547 (2013)

    Article  ADS  Google Scholar 

  7. Barber, D. M. et al. Optical control of neuronal activity using a light-operated GIRK channel opener (LOGO). Chem. Sci. 7, 2347–2352 (2016)

    Article  CAS  Google Scholar 

  8. Broichhagen, J. et al. Optical control of insulin secretion using an Incretin switch. Angew. Chem. Int. Ed. 54, 15565–15569 (2015)

    Article  CAS  Google Scholar 

  9. Watson, M. A. & Cockroft, S. L. Man-made molecular machines: membrane bound. Chem. Soc. Rev. 45, 6118–6129 (2016)

    Article  CAS  Google Scholar 

  10. Xu, T., Gao, W., Xu, L.-P., Zhang, X. & Wang, S. Fuel-free synthetic micro-/nanomachines. Adv. Mater. 29, 1603250 (2017)

    Article  Google Scholar 

  11. Haywood-Reid, P. L., Zipf, D. R. & Springer, W. R. Quantification of integrin subunits on human prostatic cell lines—comparison of nontumorigenic and tumorigenic lines. Prostate 31, 1–8 (1997)

    Article  CAS  Google Scholar 

  12. Collins, A. T., Berry, P. A., Hyde, C., Stower, M. J. & Maitland, N. J. Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res. 65, 10946–10951 (2005)

    Article  CAS  Google Scholar 

  13. Huang, C. W., Li, Z. & Conti, P. S. In vivo near-infrared fluorescence imaging of integrin α2β1 in prostate cancer with cell-penetrating-peptide–conjugated DGEA Probe. J. Nucl. Med. 52, 1979–1986 (2011)

    Article  CAS  Google Scholar 

  14. Mandelin, J. et al. Selection and identification of ligand peptides targeting a model of castrate-resistant osteogenic prostate cancer and their receptors. Proc. Natl Acad. Sci. USA 112, 3776–3781 (2015)

    CAS  PubMed  ADS  Google Scholar 

  15. García-López, V. et al. Unimolecular submersible nanomachines. Synthesis, actuation and monitoring. Nano Lett. 15, 8229–8239 (2015)

    Article  ADS  Google Scholar 

  16. Pal, R. Phase modulation nanoscopy: a simple approach to enhanced optical resolution. Faraday Discuss. 177, 507–515 (2015)

    Article  CAS  ADS  Google Scholar 

  17. Butler, S. J. et al. EuroTracker dyes: design, synthesis, structure and photophysical properties of very bright europium complexes and their use in bioassays and cellular optical imaging. Dalton Trans. 44, 4791–4803 (2015)

    Article  CAS  Google Scholar 

  18. Kessner, S., Krause, A., Rothe, U. & Bendas, G. Investigation of the cellular uptake of E-Selectin-targeted immunoliposomes by activated human endothelial cells. Biochim. Biophys. Acta 1514, 177–190 (2001)

    Article  CAS  Google Scholar 

  19. Dunn, W. A., Hubbard, A. L. & Aronson, N. N. Jr. Low temperature selectively inhibits fusion between pinocytic vesicles and lysosomes during heterophagy of 125I-asialofetuin by the perfused rat liver. J. Biol. Chem. 255, 5971–5978 (1980)

    CAS  PubMed  Google Scholar 

  20. Purcell, E. M. Life at low Reynolds number. Am. J. Phys. 45, 3–11 (1977)

    Article  ADS  Google Scholar 

  21. Lecoeur, H. Nuclear apoptosis detection by flow cytometry: influence of endegenous endonucleases. Exp. Cell Res. 277, 1–14 (2002)

    Article  CAS  Google Scholar 

  22. Elmore, S. Apoptosis: a review of programmed cell death. Toxicol. Pathol. 35, 495–516 (2007)

    Article  CAS  Google Scholar 

  23. Barros, L. F. et al. Apoptotic and necrotic blebs in epithelial cells display similar neck diameters but different kinase dependency. Cell Death Differ. 10, 687–697 (2003)

    Article  CAS  Google Scholar 

  24. Chen, J., Kistemaker, J. C. M., Robertus, J. & Feringa, B. L. Molecular stirrers in action. J. Am. Chem. Soc. 136, 14924–14932 (2014)

    Article  CAS  Google Scholar 

  25. Sandre, O., Moreaux, L. & Brochard-Wyart, F. Dynamics of transient pores in stretched vesicles. Proc. Natl Acad. Sci. USA 96, 10591–10596 (1999)

    Article  CAS  ADS  Google Scholar 

  26. Li, F., Chan, C. U. & Ohl, C. D. Yield strength of human erythrocyte membranes to impulsive stretching. Biophys. J. 105, 872–879 (2013)

    Article  CAS  ADS  Google Scholar 

  27. Evans, E. & Smith, B. A. Kinetics of hole nucleation in biomembrane rupture. New J. Phys. 13, 095010 (2011)

    Article  ADS  Google Scholar 

  28. Shigematsu, T., Koshiyama, K. & Wada, S. Effects of stretching speed on mechanical rupture of phospholipid/cholesterol bilayers: molecular dynamics simulation. Sci. Rep. 5, 15369 (2015)

    Article  CAS  ADS  Google Scholar 

  29. Bennett, W. F. D., Sapay, N. & Tieleman, D. P. Atomistic simulations of pore formation and closure in lipid bilayers. Biophys. J. 106, 210–219 (2014)

    Article  CAS  ADS  Google Scholar 

  30. Wang, X., Shindel, M. S., Wang, S.-W. & Ragan, R. Elucidating driving forces for liposome rupture: external perturbations and chemical affinity. Langmuir 28, 7417–7427 (2012)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

G.W. acknowledges North Carolina State University start-up funds and the FRPD Award. R.P. acknowledges support from the Royal Society and BSI Durham. J.M.T. acknowledges support from the National Science Foundation (CHE-1007483). Imaging for this project was supported by the Integrated Microscopy Core at Baylor College of Medicine with funding from NIH (DK56338 and CA125123), CPRIT (RP150578), the Dan L. Duncan Comprehensive Cancer Center and the John S. Dunn Gulf Coast Consortium for Chemical Genomics. We thank R. S. Gunasekera for his assistance with imaging and A. Marti for the use of his high-performance liquid chromatography (HPLC) system for the separation of 7–10. W. Sikkema prepared Fig. 1a.

Author information

Authors and Affiliations

  1. Department of Chemistry, Rice University, Houston, 77005, Texas, USA

    Víctor García-López, Lizanne G. Nilewski, Amir Aliyan, Anatoly B. Kolomeisky & James M. Tour

  2. Smalley-Curl Institute and NanoCarbon Center, Rice University, Houston, 77005, Texas, USA

    Víctor García-López, Lizanne G. Nilewski & James M. Tour

  3. Department of Chemistry, North Carolina State University, Raleigh, 27695, North Carolina, USA

    Fang Chen & Gufeng Wang

  4. Department of Electrical and Computer Engineering, Rice University, Houston, 77005, Texas, USA

    Guillaume Duret & Jacob T. Robinson

  5. Department of Chemistry, Durham University, South Road, DH1 3LE, Durham, UK

    Robert Pal

  6. Department of Materials Science and NanoEngineering, Rice University, 77005, Houston, Texas, USA

    James M. Tour

Authors
  1. Víctor García-López
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lizanne G. Nilewski
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Guillaume Duret
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Amir Aliyan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Anatoly B. Kolomeisky
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jacob T. Robinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Gufeng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Robert Pal
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. James M. Tour
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

The idea to use the nanomachines to open membranes was suggested by J.M.T., then further discussed with G.W. and R.P. V.G.-L. carried out the synthesis of 1–13 and 16 and L.G.N. carried out the synthesis of 14, both under the supervision of J.M.T. F.C. performed the experiments on lipid vesicles under the supervision of G.W. R.P. carried out all of the experiments on live cells. A.A. and V.G.-L. carried out the HPLC separation of 7–10. A.B.K. and G.W. developed the theory. G.D. performed the patch-clamp work under the supervision of J.T.R.; G.D. and J.T.R. also prepared the text for that section. V.G.-L., G.W., R.P. and J.M.T. wrote the manuscript. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Jacob T. Robinson, Gufeng Wang, Robert Pal or James M. Tour.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks R. D. Astumian, S. Cockroft and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Discussion, Supplementary Figures 1-26, Supplementary Tables 1-2, a Supplementary Video figure and additional references (PDF 7096 kb)

Reporting Summary (PDF 69 kb)

Supplementary Data

This file contains data for Supplementary Figures 3 and 5-12. (ZIP 119 kb)

When 1 was introduced to cells, it localized on the cell surface in pit-like localization patterns

Red-blue-green (RGB) fluorescent and transmission overlay video for NIH 3T3 cells bearing Nanomachine 1 (red, Cloading 500 nM/1 h, λex 633 nm, λem 650-700 nm, 1 mW) and LysoTrackerGreen (green, Cloading 200 nM/5 min, λex 488 nm, λem 500-530 nm, 0.2 mW) depicting natural homeostatic intracellular lysosomal movement. In the absence of UV activation the clusters of nanomachine 1 remains stationary and cell-membrane-bound with subsequent cell starvation and shriveling that leads to apoptotic cell death. (AVI 2407 kb)

When 1 was introduced to cells, it localized on the cell surface.

RGB fluorescent and transmission overlay video for NIH 3T3 cells bearing nanomachine 1 (red, Cloading 500 nM/1 h, λex 633 nm, λem 650-700 nm, 1 mW) and LysoTrackerGreen (green, Cloading 200 nM/5 min, λex 488 nm, λem 500-530 nm, 0.2 mW) showing that after cells died via natural starvation induced apoptosis in the absence of UV activation (confirmed by the absence of both blebbing and lysosomal rupture associated with necrosis), natural homeostatic lysosomal movement can be seen but more importantly the membrane bound nanomachine 1 clusters are inactivated and did not cross the cell membrane and cannot be detected in the cytosol. (AVI 636 kb)

NIH 3T3 cells in the presence of the nanomachines 1 with UV activation.

Upon UV-induced motor activation for 150 s (355 nm), 1, introduced by Method A, was found to cross the cell membrane, and it was internalized into cells in a time-dependent manner, displaying fast accelerated intracellular motion. Fluorescent and transmission overlay video for unwashed NIH 3T3 cells incubated with nanomachine 1 (red, Cloading 500 nM/1, λex 633 nm, λem 650-700 nm, 1 mW) depicting the uptake via membrane penetration of the cell surface formed 1 clusters in the presence of UV activation. (AVI 222 kb)

NIH 3T3 cells in the presence of the nanomachines 1 with UV activation.

RGB fluorescent and transmission overlay video for NIH 3T3 cells introduced to nanomachine 1 (red, Cloading 500 nM, λex 633 nm, λem 640-700 nm, 1 mW) depicting UV-induced necrosis (no acceleration compared to control experiment) characterized by mitochondrial autofluorescence disappearance due to mitochondrial fragmentation. Final stages of necrosis are characterized by blebbing observable at later time-points. That is also accompanied with rapid formation and movement of cytosol internalized 1 clusters which, upon UV activation, burst and contribute to detectable evenly distributed cytosolic cy5 fluorescence. (AVI 7222 kb)

NIH 3T3 cells in the presence of the nanomachines 1 with UV activation.

The same as Supplementary Video 4, but zoomed into the highlighted area on the Video 4 image surrounded by the dashed line. (AVI 2565 kb)

Natural homeostatic cellular organelle movement or 1 in NIH 3T3 cells in the absence of UV-nanomechanical activation.

Fluorescent and transmission overlay video for NIH 3T3 cells in situ loaded nanomachine 1 (red, Cloading 500 nM, λex 633 nm, λem 650-700 nm, 1 mW) depicting the uptake and subsequent clusters formation of 1 in the absence of UV activation followed by at 460 s continuous UV activation (λactivation 355 nm, 12 mW). (AVI 1364 kb)

Small aggregates of 1 inside the cytoplasm prior to UV activation.

Fluorescent and transmission overlay video for washed NIH 3T3 cells incubated with 1 (red, Cloading 500 nM/1, λex 633 nm, λem 650-700 nm, 1 mW) depicting the accumulated 1 clusters being quasi-stationery in the absence of UV activation. (AVI 681 kb)

Small aggregates of 1 inside the cytoplasm upon UV activation.

Fluorescent and transmission overlay video for washed NIH 3T3 cells incubated with 1 (red, Cloading 500 nM/1, λex 633 nm, λem 650-700 nm, 1 mW, λactivation 355 nm, 12 mW) depicting the erratic intracellular movement of the accumulated 1 clusters in the presence of UV activation. This is imaged as shown in Supplementary Video 7 but Supplementary Video 7 had no UV activation. As a further explanation, using nanomachine 1 according to Method B and no UV activation, once the cell membrane adhesion of the small aggregates is observed microscopically, allowing parent cell natural proliferation and division, well-defined and detectable cell membrane-bound clusters of 1 are still present, with detectable cy5 fluorescence in the surrounding culture media. Similar observations were made with nanomachine 2 with traceable BODIPY fluorescence in the daughter cells with predominantly mitochondrial localization with some detectable amount revealed with co-staining in the lysosomes and cytosol, further confirming non-toxicity. In the absence of UV activation, and disruptive cell membrane opening during natural cellular homeostatic division, no traceable amount 2 is detected in the surrounding cell culture media. Both verification experiments have been carried out by employing Method B (2 h nanomachine incubation followed by wash and 48 h subsequent incubation without nanomachines). (AVI 299 kb)

PowerPoint slides

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

García-López, V., Chen, F., Nilewski, L. et al. Molecular machines open cell membranes. Nature 548, 567–572 (2017). https://doi.org/10.1038/nature23657

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature23657

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research