The Journal of Membrane Biology

, Volume 194, Issue 2, pp 129–139 | Cite as

Bilayer Mixing, Fusion, and Lysis Following the Interaction of Populations of Cationic and Anionic Phospholipid Bilayer Vesicles

  • D. P. Pantazatos
  • S. P. Pantazatos
  • R. C. MacDonaldEmail author


Cationic, O-alkylphosphatidylcholines, recently developed as DNA transfection agents, form bilayers indistinguishable from those of natural phospholipids and undergo fusion with anionic bilayers. Membrane merging (lipid mixing), contents release, and contents mixing between populations of positive vesicles containing O-ethylphosphatidylcholine (EDOPC) and negative vesicles containing dioleolylphosphatidylglycerol (DOPG) have been determined with standard fluorometric vesicle-population assays. Surface-charge densities were varied from zero to full charge. All interactions depended critically on surface-charge density, as expected from the adhesion-condensation mechanism. Membrane mixing ranged from zero to 100%, with significant mixing (>10 <70%) occurring between cationic vesicles that were fully charged and anionic vesicles that had fractional surface charges as low as 0.1. Such mixing with membranes as weakly charged as cell membranes should be relevant to transfection with cationic lipids. Unexpectedly, lipid mixing was higher at high than at low ionic strength when one lipid dispersion was prepared from EDOPC plus DOPG (in different proportions), especially when the other vesicles were of EDOPC; this may somehow be a consequence of the ability of the former mixture to assume non-lamellar phases. Leakage of aqueous contents was also a strong function of charge, with fully charged vesicles releasing essentially all of their contents less than 1 min after mixing. EDOPC was more active in this regard than was DOPG, which probably reflects stronger intermolecular interactions of DOPG. Fusion, as measured by contents mixing, exhibited maximal values of 10% at intermediate surface charge. Reduced fusion at higher charge is attributed to multiple vesicle interactions leading to rupture. The existence of previously published data on individual interactions of vesicles of the same composition made it possible for the first time to compare pairwise with population interactions, confirming the likelihood of population studies to overestimate rupture and hemifusion and underestimate true vesicle fusion.


Cationic lipid EDOPC Hemifusion 



This research was supported by National Institute of Health grants GM57305 and GM52329. We are very grateful to Rumiana Koynova for carrying out the light-scattering measurement to characterize vesicle sizes. We would also like to acknowledge Reviewer #1 for several suggestions that led to an improved manuscript.


  1. 1.
    Anzai, K., Masumi, M., Kawasaki, K., Kirino, Y. 1993Frequent fusion of liposomes to a positively charged planar bilayer without calcium ions.J. Biochem.114487491PubMedGoogle Scholar
  2. 2.
    Ashley, G.W., Shida, M.M., Qiu, R., Lahiri, M.K., Levisay, P.C., Jones, R.D., Baker, K.A., MacDonald, R.C. 1996Phosphatidylcholinium compounds: A new class of phospholipids with transfection activity and unusual physical properties.Biophys. J.70A88Google Scholar
  3. 3.
    Bailey, A.L., Cullis, P.R. 1997Membrane fusion with cationic liposomes: effects of target membrane lipid composition.Biochemistry3616281634CrossRefPubMedGoogle Scholar
  4. 4.
    Breisblatt, W., Ohki, S. 1976Fusion in phospholipid spherical membranes. II. Effect of cholesterol, divalent ions and pH.J. Membrane Biol.29127146Google Scholar
  5. 5.
    Düzgüneş, N., Allen, T.M., Fedor, J., Papahadjopoulos, D. 1987Lipid mixing during membrane aggregation and fusion: why fusion assays disagree.Biochemistry2684358442PubMedGoogle Scholar
  6. 6.
    Düzgüneş, N., Goldstein, J.A., Friend, D.S., Felgner, P.L. 1989Fusion of liposomes containing a novel cationic lipid, N-[2,3-(Dioleyloxy)propyl]-N,N,N-trimethylammonium: induction by multivalent anions and asymmetric fusion with acidic phospholipid vesicles.Biochemistry2891799184PubMedGoogle Scholar
  7. 7.
    Düzgüneş, N., Wilschut, J. 1993Fusion assays monitoring intermixing of aqueous contents.Methods Enzymol220314PubMedGoogle Scholar
  8. 8.
    Evans, E.A., Parsegian, V.A. 1983Energetics of membrane deformation and adhesion in cell and vesicle aggregation.Ann. N. Y. Acad. Sci.4161333PubMedGoogle Scholar
  9. 9.
    Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M., Danielsen, M. 1987Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.Proc. Natl. Acad. Sci. USA8474137417PubMedGoogle Scholar
  10. 10.
    Gao, X., Huang, L. 1991A novel cationic liposome reagent for efficient transfection of mammalian cells.Biochem. Biophys. Res. Commun.179280285PubMedGoogle Scholar
  11. 11.
    Garcia, R.A., Pantazatos, S.P., Pantazatos, D.P., MacDonald, R.C. 2001Cholesterol stabilizes hemifused phospholipid bilayer vesicles.Biochim. Biophys. Acta1511264270PubMedGoogle Scholar
  12. 12.
    Gershon, H., Ghirlando, R., Guttman, S.B., Minsky, A. 1993Mode of formation and structural features of DNA-cationic liposome complexes used for transfection.Biochemistry3271437151PubMedGoogle Scholar
  13. 13.
    Hoekstra, D., Düzgüneş, N. 1993Lipid mixing assays to determine fusion in liposome systems.Methods Enzymol2201532PubMedGoogle Scholar
  14. 14.
    Huang, L., Hung, M.-C., Wagner, E. 1999Nonviral Vectors for Gene Therapy.Academic PressSan DiegoGoogle Scholar
  15. 15.
    Kachar, B., Fuller, N., Rand, R.P. 1986Morphological responses to calcium-induced interaction of phosphatidylserine-containing vesicles.Biophys. J.50779788PubMedGoogle Scholar
  16. 16.
    Kendall, D.A., MacDonald, R.C. 1983Characterization of a fluorescence assay to monitor changes in the aqueous volume of lipid vesicles.Anal. Biochem.1342633PubMedGoogle Scholar
  17. 17.
    Kendall, D.A., MacDonald, R.C. 1982A fluorescence assay to monitor vesicle fusion and lysis.J. Biol. Chem.2571389213895PubMedGoogle Scholar
  18. 18.
    Kennedy, M.T., Pozharski, E.V., Rakhmanova, V.A., MacDonald, R.C. 2000Factors governing the assembly of cationic phospholipid-DNA complexes.Biophys. J.7816201633PubMedGoogle Scholar
  19. 19.
    Keren-Zur, M., Beigel, M., Loyter, A. 1989Induction of fusion in aggregated and nonaggregated liposomes bearing cationic detergents.Biochim. Biophys. Acta983253258CrossRefPubMedGoogle Scholar
  20. 20.
    Kozlov, M.M., Markin, V.S. 1984On the theory of membrane fusion. The adhesion-condensation mechanism.Gen. Physiol. Biophys.5379402Google Scholar
  21. 21.
    Leventis, R., Silvius, J.R. 1990Interactions of mammalian cells with lipid dispersions containing novel metabolizable cationic amphiphiles.Biochim. Biophys. Acta1023124132CrossRefPubMedGoogle Scholar
  22. 22.
    MacDonald, R.C. 1988Mechanisms of membrane fusion in acidic lipid-cation systems.Ohki, S.Doyle, D.Flanagan, T.D.Hui, S.W.Mayhew, E. eds. Molecular Mechanisms of Membrane Fusion.Plenum PressN.Y.101112Google Scholar
  23. 23.
    MacDonald, R.C., Ashley, G.W., Shida, M.M., Rakhmanova, V.A., Tarahovsky, Y.S., Pantazatos, D.P., Kennedy, M.T., Pozharski, E.V., Baker, K.A., Jones, R.D., Rosenzweig, H.S., Choi, K.L., Qiu, R., McIntosh, T.J. 1999Physical and biological properties of cationic triesters of phosphatidylcholine.Biophys. J.7726122629PubMedGoogle Scholar
  24. 24.
    MacDonald, R.C., Rakhmanova, V.A., Choi, K.L., Rosenzweig, H.S., Lahiri, M.K. 1999O-Ethylphosphatidylcholine: A metabolizable cationic phospholipid which is a serum-compatible DNA transfection Agent.J. Pharm. Sci.88896904CrossRefPubMedGoogle Scholar
  25. 25.
    McLaughlin, S. 1977Electrostatic potentials at membrane-solution interfaces.Curr. Top. Membr. Transp.971144Google Scholar
  26. 26.
    Menger, F.M., Keiper, J.S. 1997Electrostatic layering of giant vesicles.Angew. Chem., Int. Ed. Eng.3624892491Google Scholar
  27. 27.
    Menger, F.M., Keiper, J.S., Lee, S.J. 1997Adhesion of giant liposomes as observed by light microscopy.Langmuir1346144620CrossRefGoogle Scholar
  28. 28.
    Oku, N., Scheerer, J.F., MacDonald, R.C. 1982Preparation of giant liposomes.Biochim. Biophys. Acta.692384388CrossRefPubMedGoogle Scholar
  29. 29.
    Pantazatos, D.P., MacDonald, R.C. 1999Directly observed membrane fusion between oppositely charged phospholipid bilayers.J. Membrane. Biol1702738CrossRefGoogle Scholar
  30. 30.
    Pantazatos, S.P., MacDonald, R.C. 2003Real-time observation of lipoplex formation and interaction with anionic bilayer vesicles.J. Membrane Biol.191112CrossRefGoogle Scholar
  31. 31.
    Papahadjopoulos, D., Nir, S., Düzgüneş, N. 1990Molecular mechanisms of calcium-induced membrane fusion.J. Bioenerg. Biomembr.22157179PubMedGoogle Scholar
  32. 32.
    Papahadjopoulos, D., Vail, W.J., Pangborn, W.A., Poste, G. 1976Studies on membrane fusion. II. Induction of fusion in pure phospholipid membranes by calcium ions and other divalent metals.Biochim. Biophys. Acta.448265283CrossRefPubMedGoogle Scholar
  33. 33.
    Parsegian, V.A., Rand, R.P. 1983Membrane interaction and deformation.Ann. N. Y. Acad. Sci.416112Google Scholar
  34. 34.
    Siegel, D.P. 1984Inverted micellar structures in bilayer membranes. Formation rates and half-lives.Biophys. J.45399420PubMedGoogle Scholar
  35. 35.
    Silvius, J.R., Leventis, R., Brown, P.M., Zuckermann, M. 1987Novel fluorescent phospholipids for assays of lipid mixing between membranes.Biochemistry2642794287PubMedGoogle Scholar
  36. 36.
    Stamatatos, L., Leventis, R., Zuckermann, M.J., Silvius, J.R. 1988Interactions of cationic lipid vesicles with negatively charged phospholipid vesicles and biological membranes.Biochemistry2739173925PubMedGoogle Scholar
  37. 37.
    Struck, D.K., Hoekstra, D., Pagano, R.E. 1981Use of resonance energy transfer to monitor membrane fusion.Biochemistry2040934099PubMedGoogle Scholar
  38. 38.
    Tarahovsky, Y.S., Arsenault, A.L., MacDonald, R.C., McIntosh, T.J., Epand, R.M. 2000Electrostatic control of phospholipid polymorphism.Biophys. J.7931933200PubMedGoogle Scholar
  39. 39.
    Watts, A., Harlos, K., Maschke, W., Marsh, D. 1978Control of structure and fluidity of phosphatidylglycerol bilayers by pH titration.Biochim. Biophys. Acta5106374CrossRefPubMedGoogle Scholar
  40. 40.
    Wilschut, J., Düzgüneş, N., Hong, K., Hoekstra, D., Papahadjopoulos, D. 1983Retention of aqueous contents during divalent cation-induced fusion of phospholipid vesicles.Biochim. Biophys. Acta734309318CrossRefGoogle Scholar
  41. 41.
    Wilschut, J., Hoekstra, D. 1986Membrane fusion: lipid vesicles as a model system.Chem. Phys. Lipids40145166CrossRefPubMedGoogle Scholar
  42. 42.
    Wilschut, J., Papahadjopoulos, D. 1979Ca2+-induced fusion of phospholipid vesicles monitored by mixing of aqueous contents.Nature281690692PubMedGoogle Scholar
  43. 43.
    Xu, Y., Szoka Jr., F.C. 1996Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection.Biochemistry3556165623Google Scholar
  44. 44.
    Zabner, J., Fasbender, A.J., Moninger, T., Poellinger, K.A., Welsh, M.J. 1995Cellular and molecular barriers to gene transfer by a cationic lipid.J. Biol. Chem.2701899719007CrossRefPubMedGoogle Scholar
  45. 45.
    Zschornig, O., Arnold, K., Richter, W., Ohki, S. 1992Dextran sulfate-dependent fusion of liposomes containing cationic stearylamine.Chem. Phys. Lipids631522CrossRefPubMedGoogle Scholar
  46. 46.
    Zuhorn, I.S., Hoekstra, D. 2002On the mechanism of cationic amphiphile-mediated transfection. To fuse or not to fuse: is that the question?J. Membrane Biol.189167179CrossRefGoogle Scholar

Copyright information

© Springer-Verlag New York Inc. 2003

Authors and Affiliations

  • D. P. Pantazatos
    • 1
  • S. P. Pantazatos
    • 1
  • R. C. MacDonald
    • 1
    Email author
  1. 1.Department of Biochemistry, Molecular Biology and Cell BiologyNorthwestern University, Evanston, IL 60208-3500USA

Personalised recommendations