, Volume 8, Issue 1, pp 207–217 | Cite as

Easy and Fast Preparation of Large and Giant Vesicles from Highly Confined Thin Lipid Films Deposited at the Air–Water Interface

  • Valter Bavastrello
  • Adriano Caliari
  • Isabella Pesce
  • Luis A. Bagatolli
  • Martin M. Hanczyc


Lipid vesicles are supramolecular structures of great interest for industrial and research applications. They can be used simply to compartmentalize solutions and pack active molecules in femtoliter-scale volumes or as highly sophisticated drug delivery vehicles and dynamic cell-size bioreactors. For these reasons, many methods for the production of vesicles have been developed, and some of them present several drawbacks, such as long working times and the requirement of specific equipment to perform the technique. In this work, we present a method to produce vesicles from highly confined lipid films at the air–water interface. The procedure involves two simple steps: the formation of the thin lipid film at the air–water interface and then brief sonication (10 s). These films are obtained by depositing different aliquots of lipid organic solutions at the air–liquid interface of round-bottom Eppendorfs tubes. The morphology of the highly confined lipid thin films was studied by optical microscopy noting the formation of non-uniform depositions at the air–liquid interface, with the presence of thicker portions close to the container sidewall. Post-sonication, the presence of vesicles composed of 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) was confirmed using the complementary techniques of fluorescence microscopy and flow cytometry. The size distribution investigations carried out by flow cytometry revealed the optimal concentrations to favor the formation of giant vesicles (GVs). Furthermore, we investigated aqueous phase encapsulation by adding calcein or green fluorescent protein (GFP) to the aqueous phase then characterized by fluorescence microscopy and flow cytometry. We demonstrate a fast and easy method for producing vesicles including GVs on demand.


Giant vesicles Langmuir films Phospholipids Encapsulation Protocol Flow cytometry Microscopy 



We would like to thank Prof Tetsuya Yomo for informative discussions. LAB wants to thank Program Prometeo, SENESCYT, Ecuador, for support.

Funding Information

V.B and M.M.H. were financially supported in part by the European Commission FP7 Future and Emerging Technologies Proactive (EVOBLISS 611640).

Supplementary material

12668_2017_464_MOESM1_ESM.doc (11.4 mb)
ESM 1 (DOC 11711 kb)


  1. 1.
    Whitesides, G. M., & Grzybowski, B. (2002). Self-assembly at all scales. Science, 295, 2418–2421.CrossRefGoogle Scholar
  2. 2.
    Chiruvolu, S., Walker, S., Israelachvili, J., Schmitt, F. J., Leckband, D., & Zasadzinski, J. A. (1994). Higher-order self-assembly of vesicles by site-specific binding. Science, 264, 1753–1756.CrossRefGoogle Scholar
  3. 3.
    Sunami, T., Sato, K., Matsuura, T., Tsukada, K., Urabe, I., & Yomo, T. (2006). Femtoliter compartment in liposomes for in vitro selection of proteins. Analytical Biochemistry, 357, 128–136.CrossRefGoogle Scholar
  4. 4.
    Jung, S.-Y., Liu, Y., & Collier, C. P. (2008). Fast mixing and reaction initiation control of single-enzyme kinetics in confined volumes. Langmuir, 24, 4439–4442.CrossRefGoogle Scholar
  5. 5.
    Deng, Y., Wang, Y., Holtz, B., Li, J., Traaseth, N., Veglia, G., Stottrup, B. J., Elde, R., Pei, D., Guo, A., & Zhu, X.-Y. (2008). Fluidic and air-stable supported lipid bilayer and cell-mimicking microarrays. Journal of the American Chemical Society, 130, 6267–6271.CrossRefGoogle Scholar
  6. 6.
    Bagatolli, L. A. (2009). Membranes and fluorescence microscopy. In Reviews in fluorescence 2007 (pp. 33–51). New York: Springer.CrossRefGoogle Scholar
  7. 7.
    Yang, P., Dimova, R. (2011). Nanoparticle synthesis in vesicle microreactors. In Biomimetic based applications, INTECH Open Access Publisher.Google Scholar
  8. 8.
    Wesołowska, O., Michalak, K., Maniewska, J., & Hendrich, A. B. (2009). Giant unilamellar vesicles—a perfect tool to visualize phase separation and lipid rafts in model systems. Acta Biochimica Polonica, 56, 33–39.Google Scholar
  9. 9.
    Dua, J. S., Rana, A. C., & Bhandari, A. K. (2012). Liposome: methods of preparation and applications. International Journal Pharmaceutical Studies Research, 3, 14–20.Google Scholar
  10. 10.
    Manley, S., Gordon, V. D., et al. (2008). Current Protocols in Cell Biology, 40, 24.3:24.3.1–24.324.3.13.Google Scholar
  11. 11.
    Rodriguez, N., Pincet, F., & Cribier, S. (2005). Giant vesicles formed by gentle hydration and electroformation: a comparison by fluorescence microscopy. Colloids and Surfaces, B: Biointerfaces, 42, 125–130.CrossRefGoogle Scholar
  12. 12.
    Pautot, S., Frisken, B. J., & Weitz, D. A. (2003). Production of unilamellar vesicles using an inverted emulsion. Langmuir, 19, 2870–2879.CrossRefGoogle Scholar
  13. 13.
    Hadorn, M., Boenzli, E., Sørensen, K. T., De Lucrezia, D., Hanczyc, M. M., & Yomo, T. (2013). Defined DNA-mediated assemblies of gene-expressing giant unilamellar vesicles. Langmuir, 29, 15309–15319.CrossRefGoogle Scholar
  14. 14.
    Walde, P., Cosentino, K., Engel, H., & Stano, P. (2010). Giant vesicles: preparations and applications. Chembiochem, 11, 848–865.CrossRefGoogle Scholar
  15. 15.
    Langmuir, I. (1917). Constitution and fundamental properties of solids and liquids. II. Liquids. Journal of the American Chemical Society, 39, 1848–1906.CrossRefGoogle Scholar
  16. 16.
    Kaganer, V. M., Mohwald, H., & Dutta, P. (1999). Structure and phase transitions in Langmuir monolayers. Reviews of Modern Physics, 71, 779–819.CrossRefGoogle Scholar
  17. 17.
    Pichot, R., Watson, R. L., & Norton, I. T. (2013). Phospholipids at the interface: current trends and challenges. International Journal of Molecular Sciences, 14, 11767–11794.CrossRefGoogle Scholar
  18. 18.
    Lee, K. Y. C. (2008). Collapse mechanisms of Langmuir monolayers. Annual Review of Physical Chemistry, 59, 771–791.CrossRefGoogle Scholar
  19. 19.
    Gopal, A., & Lee, K. Y. C. (2001). Morphology and collapse transitions in binary phospholipid monolayers. The Journal of Physical Chemistry. B, 105, 10348–10354.CrossRefGoogle Scholar
  20. 20.
    Dua, J. S., Rana, A. C., & Bhandari, A. K. (2012). Liposome: methods of preparation and applications. IJPSR, 3, 14–20.Google Scholar
  21. 21.
    Vácha, R., Jurkiewicz, P., Petrov, M., Berkowitz, M. L., Böckmann, R. A., Barucha-Kraszewska, J., Hof, M., & Jungwirth, P. (2010). Mechanism of interaction of monovalent ions with Phosphatidylcholine lipid membranes. The Journal of Physical Chemistry. B, 114, 9504–9509.CrossRefGoogle Scholar
  22. 22.
    Sackett, D. L., & Wolff, J. (1987). Nile red as a polarity-sensitive fluorescent probe of hydrophobic protein surfaces. Analytical Biochemistry, 167, 228–234.CrossRefGoogle Scholar
  23. 23.
    Dutta, A. K., Kamada, K., & Ohta, K. (1996). Spectroscopic studies of nile red in organic solvents and polymers. Journal of Photochemistry and Photobiology, A: Chemistry, 93, 57–64.CrossRefGoogle Scholar
  24. 24.
    Greenspan, P., Mayer, E. P., & Fowler, S. D. (1985). Nile Red: a selective fluorescent stain for intracellular lipid droplets. The Journal of Cell Biology, 100, 965–973.CrossRefGoogle Scholar
  25. 25.
    Fowler, S. D., & Greenspan, P. (1985). Application of Nile Red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O. The Journal of Histochemistry and Cytochemistry, 33, 833–836.CrossRefGoogle Scholar
  26. 26.
    Nishimura, K., Hosoi, T., Sunami, T., Toyota, T., Fujinami, M., Oguma, K., Matsuura, T., Suzuki, H., & Yomo, T. (2009). Population analysis of structural properties of giant liposomes by flow cytometry. Langmuir, 25, 10439–10443.CrossRefGoogle Scholar
  27. 27.
    Rugonyi, S., Smith, E. C., & Hall, S. B. (2004). Transformation diagrams for the collapse of a phospholipid monolayer. Langmuir, 20, 10100–10106.CrossRefGoogle Scholar
  28. 28.
    Smith, E. C., Crane, J. M., Laderas, T. G., & Hall, S. B. (2003). Metastability of a supercompressed fluid monolayer. Biophysical Journal, 85, 3048–3057.CrossRefGoogle Scholar
  29. 29.
    Schief, W. R., Antia, M., Discher, B. M., Hall, S. B., & Vogel, V. (2003). Liquid-crystalline collapse of pulmonary surfactant monolayers. Biophysical Journal, 84, 3792–3806.CrossRefGoogle Scholar
  30. 30.
    Baoukina, S., Monticelli, L., Risselada, H. J., Marrink, S. J., & Tieleman, D. P. (2008). The molecular mechanism of lipid monolayer collapse. PNAS, 105, 10803–10808.CrossRefGoogle Scholar
  31. 31.
    Finkelstein, A., & Cass, A. (1968). Permeability and electrical properties of thin lipid membranes. The Journal of General Physiology, 52, 145–172.CrossRefGoogle Scholar
  32. 32.
    Simonsen, A. C., & Bagatolli, L. A. (2004). Structure of spin-coated lipid films and domain formation in supported membranes formed by hydration. Langmuir, 20, 9720–9728.CrossRefGoogle Scholar
  33. 33.
    Fukuma, T., Higgins, M. J., & Jarvis, S. P. (2007). Direct imaging of individual intrinsic hydration layers on lipid bilayers at Ångstrom resolution. Biophysical Journal, 92, 3603–3609.CrossRefGoogle Scholar
  34. 34.
    Oku, N., Kendall, D. A., & MacDonald, R. C. (1982). A simple procedure for the determination of the trapped volume of liposomes. Biochimica et Biophysica Acta, 691, 332–340.CrossRefGoogle Scholar
  35. 35.
    Moscho, A., Orwar, O., Chiu, D. T., Modi, B. P., & Zare, R. N. (1996). Rapid preparation of giant unilamellar vesicles. Proceedings of the National Academy of Sciences of the United States of America, 93, 11443–11447.CrossRefGoogle Scholar
  36. 36.
    Hadorn, M., Boenzli, E., & Eggenberger Hotz, P. (2011). A quantitative analytical method to test for salt effects on giant unilamellar vesicles. Scientific Reports, 1, 168.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Valter Bavastrello
    • 1
  • Adriano Caliari
    • 1
  • Isabella Pesce
    • 1
  • Luis A. Bagatolli
    • 2
  • Martin M. Hanczyc
    • 1
    • 3
  1. 1.Centre for Integrative Biology (CIBIO)University of TrentoPovoItaly
  2. 2.Memphys-Center for Biomembrane Physics/Yachay EP and Yachay Tech UniversityUrcuquiEcuador
  3. 3.Chemical and Biological EngineeringUniversity of New MexicoAlbuquerqueUSA

Personalised recommendations