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.

Generation of phospholipid vesicle-nanotube networks and transport of molecules therein

Nature Protocols volume 6, pages 791–805 (2011)Cite this article

Subjects

Abstract

We describe micromanipulation and microinjection procedures for the fabrication of soft-matter networks consisting of lipid bilayer nanotubes and surface-immobilized vesicles. These biomimetic membrane systems feature unique structural flexibility and expandability and, unlike solid-state microfluidic and nanofluidic devices prepared by top-down fabrication, they allow network designs with dynamic control over individual containers and interconnecting conduits. The fabrication is founded on self-assembly of phospholipid molecules, followed by micromanipulation operations, such as membrane electroporation and microinjection, to effect shape transformations of the membrane and create a series of interconnected compartments. Size and geometry of the network can be chosen according to its desired function. Membrane composition is controlled mainly during the self-assembly step, whereas the interior contents of individual containers is defined through a sequence of microneedle injections. Networks cannot be fabricated with other currently available methods of giant unilamellar vesicle preparation (large unilamellar vesicle fusion or electroformation). Described in detail are also three transport modes, which are suitable for moving water-soluble or membrane-bound small molecules, polymers, DNA, proteins and nanoparticles within the networks. The fabrication protocol requires ∼90 min, provided all necessary preparations are made in advance. The transport studies require an additional 60–120 min, depending on the transport regime.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: Schematic overview over the network generation procedure.
Figure 2: Optical differential interference contrast microscopy setup for nanotube-vesicle network fabrication.
Figure 3: Capillary needle and carbon fiber microelectrode preparation.
Figure 4: Electrophoresis electrode assemblies, each consisting of a 1-mm (OD) hydrophobized capillary needle with cross-linked polyacrylamide gel plug and internal Ag/AgCl electrode.
Figure 5: Lipid dehydration/hydration on no. 1 microscope cover slips and subsequent transfer.
Figure 6: Rehydration/swelling of lipid preparations.
Figure 7: Initial stage of network fabrication.
Figure 8: Stepwise fabrication of a linear network from a single GUV/MLV.
Figure 9: Stepwise fabrication of a branched network from a single GUV/MLV.
Figure 10: Confocal microscopy images showing diffusive transport of internalized 20-nm fluorescent latex nanoparticles between container vesicles through a nanotube.
Figure 11: Marangoni transport along a lipid nanotube in a two-vesicle network.
Figure 12: Electrophoretic transport of carboxylated nanoparticles along a lipid nanotube in a two-vesicle network.

References

  1. Belting, M. & Wittrup, A. Nanotubes, exosomes, and nucleic acid-binding peptides provide novel mechanisms of intercellular communication in eukaryotic cells: implications in health and disease. J. Cell Biol. 183, 1187–1191 (2008).

    Article  CAS  Google Scholar 

  2. Whitesides, G.M. The 'right' size in nanobiotechnology. Nat. Biotechnol. 21, 1161–1165 (2003).

    Article  CAS  Google Scholar 

  3. Davis, D.M. & Sowinski, S. Membrane nanotubes: dynamic long-distance connections between animal cells. Nat. Rev. Mol. Cell Biol. 9, 431–436 (2008).

    Article  CAS  Google Scholar 

  4. Veranic, P. et al. Different types of cell-to-cell connections mediated by nanotubular structures. Biophys. J. 95, 4416–4425 (2008).

    Article  CAS  Google Scholar 

  5. Hurtig, J., Chiu, D.T. & Onfelt, B. Intercellular nanotubes: insights from imaging studies and beyond. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2, 260–276 (2010).

    Article  CAS  Google Scholar 

  6. Onfelt, B. et al. Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria. J. Immunol. 177, 8476–8483 (2006).

    Article  Google Scholar 

  7. Gurke, S., Barroso, J.F.V. & Gerdes, H.H. The art of cellular communication: tunneling nanotubes bridge the divide. Histochem. Cell Biol. 129, 539–550 (2008).

    Article  CAS  Google Scholar 

  8. Lucas, W.J., Ham, L.K. & Kim, J.Y. Plasmodesmata—bridging the gap between neighboring plant cells. Trends Cell Biol. 19, 495–503 (2009).

    Article  CAS  Google Scholar 

  9. Lone, B. Computational nanotechnology in biomedical nanometrics and Nano-materials. J. Comput. Theor. Nanosci. 6, 2146–2151 (2009).

    Article  CAS  Google Scholar 

  10. Yum, K., Wang, N. & Yu, M.F. Nanoneedle: a multifunctional tool for biological studies in living cells. Nanoscale 2, 363–372 (2010).

    Article  CAS  Google Scholar 

  11. Wong, I.Y., Almquist, B.D. & Melosh, N.A. Dynamic actuation using nano-bio interfaces. Materials Today 13, 14–22 (2010).

    Article  CAS  Google Scholar 

  12. Shvedova, A.A., Kagan, V.E. & Fadeel, B. Close encounters of the small kind: adverse effects of man-made materials interfacing with the nano-cosmos of biological systems. Annu. Rev. Pharmacol. Toxicol. 50, 63–88 (2010).

    Article  CAS  Google Scholar 

  13. Fadeel, B., Kasemo, B., Malmsten, M. & Stromme, M. Nanomedicine: reshaping clinical practice. J. Intern. Med. 267, 2–8 (2010).

    Article  CAS  Google Scholar 

  14. Karlsson, A. et al. Molecular engineering—networks of nanotubes and containers. Nature 409, 150–152 (2001).

    Article  CAS  Google Scholar 

  15. Karlsson, M. et al. Biomimetic nanoscale reactors and networks. Annu. Rev. Phys. Chem. 55, 613–649 (2004).

    Article  CAS  Google Scholar 

  16. Karlsson, A. et al. Controlled initiation of enzymatic reactions in micrometer-sized biomimetic compartments. J. Phys. Chem. B 109, 1609–1617 (2005).

    Article  CAS  Google Scholar 

  17. Bauer, B., Davidson, M. & Orwar, O. Direct reconstitution of plasma membrane lipids and proteins in nanotube-vesicle networks. Langmuir 22, 9329–9332 (2006).

    Article  CAS  Google Scholar 

  18. Walde, P., Cosentino, K., Engel, H. & Stano, P. Giant vesicles: preparations and applications. Chem. Bio. Chem. 11, 848–865 (2010).

    Article  CAS  Google Scholar 

  19. Criado, M. & Keller, B.U. A membrane fusion strategy for single-channel recordings of membranes usually non-accessible to patch-clamp pipette electrodes. FEBS Lett. 224, 172–176 (1987).

    Article  CAS  Google Scholar 

  20. Karlsson, M. et al. Micropipet-assisted formation of microscopic networks of unilamellar lipid bilayer nanotubes and containers. Langmuir 17, 6754–6758 (2001).

    Article  CAS  Google Scholar 

  21. Lasic, D.D. Liposomes: From Physics to Applications (Elsevier Science, 1993).

  22. Luisi, P.L. & Walde, P. Giant Vesicles (Wiley, 1999).

  23. Barber, M.A. The pipette method in the isolation of single microorganisms and in the inoculation of substances into living cells. Phillipp. J. Sci. B. Trop. Med. 9, 307–360 (1914).

    Google Scholar 

  24. Taylor, C.V. An accurately controllable micropipette. Science 51, 617–618 (1920).

    Article  CAS  Google Scholar 

  25. Hamill, O.P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F.J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. Eur. J. Physiol. 391, 85–100 (1981).

    Article  CAS  Google Scholar 

  26. Neumann, E., Schaeferridder, 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 

  27. Lundqvist, J.A. et al. Altering the biochemical state of individual cultured cells and organelles with ultramicroelectrodes. Proc. Natl. Acad. Sci. USA 95, 10356–10360 (1998).

    Article  CAS  Google Scholar 

  28. Wang, M.Y., Orwar, O., Olofsson, J. & Weber, S.G. Single-cell electroporation. Anal. Bioanal. Chem. 397, 3235–3248 (2010).

    Article  CAS  Google Scholar 

  29. Karlsson, M. et al. Electroinjection of colloid particles and biopolymers into single unilamellar liposomes and cells for bioanalytical applications. Anal. Chem. 72, 5857–5862 (2000).

    Article  CAS  Google Scholar 

  30. Davidson, M. et al. Fluid mixing in growing microscale vesicles conjugated by surfactant nanotubes. J. Am. Chem. Soc. 127, 1251–1257 (2005).

    Article  CAS  Google Scholar 

  31. Sott, K. et al. Controlling enzymatic reactions by geometry in a biomimetic nanoscale network. Nano Lett. 6, 209–214 (2006).

    Article  CAS  Google Scholar 

  32. Lizana, L. & Konkoli, Z. Diffusive transport in networks built of containers and tubes. Phys. Rev. E 72, 026305 (2005).

    Article  CAS  Google Scholar 

  33. Lizana, L., Konkoli, Z. & Orwar, O. Tunable filtering of chemical signals in a simple nanoscale reaction-diffusion network. J. Phys. Chem. B 111, 6214–6219 (2007).

    Article  CAS  Google Scholar 

  34. Karlsson, R. et al. Moving-wall-driven flows in nanofluidic systems. Langmuir 18, 4186–4190 (2002).

    Article  CAS  Google Scholar 

  35. Tokarz, M. et al. Single-file electrophoretic transport and counting of individual DNA molecules in surfactant nanotubes. Proc. Natl. Acad. Sci. USA 102, 9127–9132 (2005).

    Article  CAS  Google Scholar 

  36. Tokarz, M., Hakonen, B., Dommersnes, P., Orwar, O. & Akerman, B. Electrophoretic transport of latex particles in lipid nanotubes. Langmuir 23, 7652–7658 (2007).

    Article  CAS  Google Scholar 

  37. Dommersnes, P.G., Orwar, O., Brochard-Wyart, F. & Joanny, J.F. Marangoni transport in lipid nanotubes. Europhys. Lett. 70, 271–277 (2005).

    Article  CAS  Google Scholar 

  38. Lobovkina, T., Dommersnes, P., Hurtig, J., Joanny, J.F. & Orwar, O. Zipper dynamics of surfactant nanotube junctions. Phys. Rev. Lett. 97, 188105/188101–188105/188104 (2006).

    Google Scholar 

  39. Evans, E. & Rawicz, W. Entropy-driven tension and bending elasticity in condensed-fluid membranes. Phys. Rev. Lett. 64, 2094–2097 (1990).

    Article  CAS  Google Scholar 

  40. Lobovkina, T., Dommersnes, P.G., Tiourine, S., Joanny, J.F. & Orwar, O. Shape optimization in lipid nanotube networks. Eur. Phys. J. E 26, 295–300 (2008).

    Article  CAS  Google Scholar 

  41. Lizana, L., Bauer, B. & Orwart, O. Controlling the rates of biochemical reactions and signaling networks by shape and volume changes. Proc. Natl. Acad. Sci. USA 105, 4099–4104 (2008).

    Article  CAS  Google Scholar 

  42. Lobovkina, T. et al. Mechanical tweezer action by self-tightening knots in surfactant nanotubes. Proc. Natl. Acad. Sci. USA 101, 7949–7953 (2004).

    Article  CAS  Google Scholar 

  43. Karlsson, M. et al. Formation of geometrically complex lipid nanotube-vesicle networks of higher order topologies. Proc. Natl. Acad. Sci. USA 99, 11573–11578 (2002).

    Article  CAS  Google Scholar 

  44. Onoue, Y. et al. A giant liposome for single-molecule observation of conformational changes in membrane proteins. Biochim. Biophys. Acta Biomemb. 1788, 1332–1340 (2009).

    Article  CAS  Google Scholar 

  45. Sparreboom, W., van den Berg, A. & Eijkel, J.C.T. Principles and applications of nanofluidic transport. Nat. Nanotechnol. 4, 713–720 (2009).

    Article  CAS  Google Scholar 

  46. Fernandes, P. Miniaturization in biocatalysis. Int. J. Mol. Sci. 11, 858–879 (2010).

    Article  CAS  Google Scholar 

  47. Lee, L.J. Polymer nanoengineering for biomedical applications. Ann. Biomed. Eng. 34, 75–88 (2006).

    Article  Google Scholar 

  48. Napoli, M., Eijkel, J.C.T. & Pennathur, S. Nanofluidic technology for biomolecule applications: a critical review. Lab Chip 10, 957–985 (2010).

    Article  CAS  Google Scholar 

  49. Sparreboom, W., van den Berg, A. & Eijkel, J.C.T. Transport in nanofluidic systems: a review of theory and applications. New J. Phys. 12, 015004 doi:015004 10.1088/1367-2630/12/1/015004 (2010).

    Article  Google Scholar 

  50. Charras, G.T. A short history of blebbing. J. Microsc. 231, 466–478 (2008).

    Article  CAS  Google Scholar 

  51. Chan, Y.H.M. & Boxer, S.G. Model membrane systems and their applications. Curr. Opin. Chem. Biol. 11, 581–587 (2007).

    Article  CAS  Google Scholar 

  52. Jelinek, R. & Silbert, L. Biomimetic approaches for studying membrane processes. Mol. BioSyst. 5, 811–818 (2009).

    Article  CAS  Google Scholar 

  53. Kirby, C. & Gregoriadis, G. Dehydration-rehydration vesicles—a simple method for high-yield drug entrapment in liposomes. Nat. Biotechnol. 2, 979–984 (1984).

    Article  CAS  Google Scholar 

  54. Kikuchi, H., Yamauchi, H. & Hirota, S. A spray-drying method for mass-production of liposomes. Chem. Pharma. Bull. 39, 1522–1527 (1991).

    Article  CAS  Google Scholar 

  55. Angelova, M.I. & Dimitrov, D.S. Liposome electroformation. Faraday Discuss. Chem. Soc. 81, 303–311 (1986).

    Article  CAS  Google Scholar 

  56. Kuribayashi, K., Tresset, G., Coquet, P., Fujita, H. & Takeuchi, S. Electroformation of giant liposomes in microfluidic channels. Measure. Sci. Technol. 17, 3121–3126 (2006).

    Article  CAS  Google Scholar 

  57. Estes, D.J. & Mayer, M. Electroformation of giant liposomes from spin-coated films of lipids. Colloids Surf. B Biointerfaces 42, 115–123 (2005).

    Article  CAS  Google Scholar 

  58. Estes, D.J. & Mayer, M. Giant liposomes in physiological buffer using electroformation in a flow chamber. Biochim. Biophys. Acta Biomemb. 1712, 152–160 (2005).

    Article  CAS  Google Scholar 

  59. Liu, H.Q. et al. Lipid nanotube formation from streptavidin-membrane binding. Langmuir 24, 3686–3689 (2008).

    Article  CAS  Google Scholar 

  60. Roux, A. et al. A minimal system allowing tubulation with molecular motors pulling on giant liposomes. Proc. Natl. Acad. Sci. USA 99, 5394–5399 (2002).

    Article  CAS  Google Scholar 

  61. Pascoal, P., Kosanic, D., Gjoni, M. & Vogel, H. Membrane nanotubes drawn by optical tweezers transmit electrical signals between mammalian cells over long distances. Lab Chip 10, 2235–2241 (2010).

    Article  CAS  Google Scholar 

  62. Mathivet, L., Cribier, S. & Devaux, P.F. Shape change and physical properties of giant phospholipid vesicles prepared in the presence of an AC electric field. Biophys. J. 70, 1112–1121 (1996).

    Article  CAS  Google Scholar 

  63. Perutkova, S., Kralj-Iglic, V., Frank, M. & Iglic, A. Mechanical stability of membrane nanotubular protrusions influenced by attachment of flexible rod-like proteins. J. Biomech. 43, 1612–1617 (2010).

    Article  Google Scholar 

  64. Davidson, M., Karlsson, M., Sinclair, J., Sott, K. & Orwar, O. Nanotube-vesicle networks with functionalized membranes and interiors. J. Am. Chem. Soc. 125, 374–378 (2003).

    Article  CAS  Google Scholar 

  65. Markstrom, M., Gunnarsson, A., Orwar, O. & Jesorka, A. Dynamic microcompartmentalization of giant unilamellar vesicles by sol gel transition and temperature induced shrinking/swelling of poly(N-isopropyl acrylamide). Soft Matter 3, 587–595 (2007).

    Article  Google Scholar 

  66. Karlsson, A. et al. Nanofluidic networks based on surfactant membrane technology. Anal. Chem. 75, 2529–2537 (2003).

    Article  CAS  Google Scholar 

  67. Lobovkina, T. et al. Pattern formation of different geometries and topologies as well as knotted structures in lipid nanotube networks. Biophys. J. 88, 208a (2005).

    Google Scholar 

  68. Konings, A.W.T. Lipid peroxidation in liposomes. in Liposome Technology Vol. 1 (ed. Gregoriadis, G.) 139–161 (CRC Press, 1984).

  69. Sott, K. et al. Micropipet writing technique for production of two-dimensional lipid bilayer nanotube-vesicle networks on functionalized and patterned surfaces. Langmuir 19, 3904–3910 (2003).

    Article  CAS  Google Scholar 

  70. Markstrom, M., Lizana, L., Orwar, O. & Jesorka, A. Thermoactuated diffusion control in soft matter nanofluidic devices. Langmuir 24, 5166–5171 (2008).

    Article  Google Scholar 

  71. Jesorka, A., Markstrom, M. & Orwar, O. Controlling the internal structure of giant unilamellar vesicles by means of reversible temperature dependent sol-gel transition of internalized poly (N-isopropyl acrylamide). Langmuir 21, 1230–1237 (2005).

    Article  CAS  Google Scholar 

  72. Jesorka, A. & Orwar, O. Complex nanotube-liposome networks. in Liposomes, Part F (Methods in Enzymology) Vol. 464 309–325 (Elsevier Academic, 2009).

  73. Lizana, L., Konkoli, Z., Bauer, B., Jesorka, A. & Orwar, O. Controlling chemistry by geometry in Nanoscale systems. Annu. Rev. Phys. Chem. 60, 449–468 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank all previous research group members and associates, in particular, former graduate students M. Karlsson, R. Karlsson, A. Karlsson, M. Davidson, K. Sott, M. Tokarz, J. Hurtig, M. Markström, B. Bauer and T. Lobovkina, who have contributed greatly to the development of these protocols during the time they completed their individual projects. We also thank P. Dommersnes for advice and discussion. Our research was and is supported by the Royal Swedish Academy of Sciences, the Swedish Research council (VR), the Swedish Foundation for Strategic Research, the Knut and Alice Wallenberg Foundation and the European Research Council.

Author information

Authors and Affiliations

  1. Department of Chemical and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden

    Aldo Jesorka, Natalia Stepanyants, Haijiang Zhang, Bodil Hakonen & Owe Orwar

  2. Department of Molecular Biology & Genetics, Bilkent University, Ankara, Turkey

    Bahanur Ortmen

Authors
  1. Aldo Jesorka
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Natalia Stepanyants
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Haijiang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bahanur Ortmen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bodil Hakonen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Owe Orwar
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.J. performed the experimental procedures for network generation, Marangoni transport and took part in manuscript preparation. N.S. and H.Z. performed experimental procedures for diffusive transport. B.O. carried out lipid preparation. B.H. performed electrophoretic transport and took part in the manuscript preparation. O.O. designed the original concept and took part in the manuscript preparation.

Corresponding author

Correspondence to Owe Orwar.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Jesorka, A., Stepanyants, N., Zhang, H. et al. Generation of phospholipid vesicle-nanotube networks and transport of molecules therein. Nat Protoc 6, 791–805 (2011). https://doi.org/10.1038/nprot.2011.321

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.321

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