Thermodynamics of Lipid Interactions with Cell-Penetrating Peptides

  • Reto Sauder
  • Joachim Seelig
  • André Ziegler
Part of the Methods in Molecular Biology book series (MIMB, volume 683)


Cationic peptides are efficiently taken up by biological cells through different pathways, which can be exploited for delivery of intracellular drugs. For example, their endocytosis is known since 1967, and this typically produces entrapment of the peptides in endocytotic vesicles. The resulting peptide (and cargo) degradation in lysosomes is of little therapeutic interest. Beside endocytosis (and various subtypes thereof), cationic cell-penetrating peptides (CPPs) may also gain access to cytosol and nucleus of livings cells. This process is known since 1988, but it is poorly understood whether the cytosolic CPP appearance requires an active cellular machinery with membrane proteins and signaling molecules, or whether this translocation occurs by passive diffusion and thus can be mimicked with model membranes devoid of proteins or glycans. In the present chapter, protocols are presented that allow for testing the membrane binding and disturbance of CPPs on model membranes with special focus on particular CPP properties. Protocols include vesicle preparation, lipid quantification, and analysis of membrane leakage, lipid polymorphism (31P NMR), and membrane binding (isothermal titration calorimetry). Using these protocols, a major difference among CPPs is observed: At low micromolar concentration, nonamphipathic CPPs, such as nona-arginine (WR9) and penetratin, have only a poor affinity for model membranes with a lipid composition typical of eukaryotic membranes. No membrane leakage is induced by these compounds at low micromolar concentration. In contrast, their amphipathic derivatives, such as acylated WR9 (C14, C16, C18) or amphipathic penetratin mutant p2AL (Drin et al., Biochemistry 40:1824–1834, 2001), bind and disturb lipid model membranes already at low micromolar peptide concentration. This suggests that the mechanism for cytosolic CPP delivery (and potential toxicity) differs among CPPs despite their common name.

Key words

Cell membrane Drug delivery Liposomes Membrane anchor Protein transport 



This work was supported by the Swiss National Science Founda tion (SNF) Grant # 31.107793.


  1. 1.
    Morad, N., Ryser, H.J. and Shen, W.C. (1984) Binding sites and endocytosis of heparin and polylysine are changed when the two molecules are given as a complex to Chinese hamster ovary cells. Biochim. Biophys. Acta. 801, 117–126.PubMedGoogle Scholar
  2. 2.
    Ryser, H.J. (1967) A membrane effect of basic polymers dependent on molecular size. Nature 215, 934–936.PubMedCrossRefGoogle Scholar
  3. 3.
    Kaplan, I.M., Wadia, J.S. and Dowdy, S.F. (2005) Cationic TAT peptide transduction domain enters cells by macropinocytosis. J. Control. Release 102, 247–253.PubMedCrossRefGoogle Scholar
  4. 4.
    Belting, M., Mani, K., Jonsson, M., Cheng, F., Sandgren, S., Jonsson, S., Ding, K., Delcros, J.G. and Fransson, L.A. (2003) Glypican-1 is a vehicle for polyamine uptake in mammalian cells: a pivital role for nitrosothiol-derived nitric oxide. J. Biol. Chem. 278, 47181–47189.PubMedCrossRefGoogle Scholar
  5. 5.
    Lundberg, M., Wikstrom, S. and Johansson, M. (2003) Cell surface adherence and endocytosis of protein transduction domains. Mol. Ther. 8, 143–150.PubMedCrossRefGoogle Scholar
  6. 6.
    Kopatz, I., Remy, J.S. and Behr, J.P. (2004) A model for non-viral gene delivery: through syndecan adhesion molecules and powered by actin. J Gene Med 6, 769–776.PubMedCrossRefGoogle Scholar
  7. 7.
    Zenke, M., Steinlein, P., Wagner, E., Cotten, M., Beug, H. and Birnstiel, M.L. (1990) Receptor-mediated endocytosis of transferrin-polycation conjugates: an efficient way to introduce DNA into hematopoietic cells. Proc. Natl. Acad. Sci. USA 87, 3655–3659.PubMedCrossRefGoogle Scholar
  8. 8.
    Frankel, A.D. and Pabo, C.O. (1988) Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55, 1189–1193.PubMedCrossRefGoogle Scholar
  9. 9.
    Green, M. and Loewenstein, P.M. (1988) Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell 55, 1179–1188.PubMedCrossRefGoogle Scholar
  10. 10.
    Marinova, Z., Vukojevic, V., Surcheva, S., Yakovleva, T., Cebers, G., Pasikova, N., Usynin, I., Hugonin, L., Fang, W., Hallberg, M., Hirschberg, D., Bergman, T., Langel, Ü., Häuser, K.F., Pramanik, A., Aldrich, J.V., Gräslund, A., Terenius, L. and Bakalkin, G. (2005) Translocation of dynorphin neuropeptides across the plasma membrane. A putative mechanism of signal transmission. J. Biol. Chem. 280, 26360–26370.PubMedCrossRefGoogle Scholar
  11. 11.
    Wadia, J.S., Stan, R.V. and Dowdy, S.F. (2004) Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat. Med. 10, 310–315.PubMedCrossRefGoogle Scholar
  12. 12.
    Fretz, M.M., Penning, N.A., Al-Taei, S., Futaki, S., Takeuchi, T., Nakase, I., Storm, G. and Jones, A.T. (2007) Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells. Biochem. J. 403, 335–342.PubMedCrossRefGoogle Scholar
  13. 13.
    Zaro, J.L., Rajapaksa, T.E., Okamoto, C.T. and Shen, W.C. (2006) Membrane transduction of oligoarginine in HeLa cells is not mediated by macropinocytosis. Mol. Pharm. 3, 181–186.PubMedCrossRefGoogle Scholar
  14. 14.
    Vives, E., Brodin, P. and Lebleu, B. (1997) A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 272, 16010–16017.PubMedCrossRefGoogle Scholar
  15. 15.
    Mitchell, D.J., Kim, D.T., Steinman, L., Fathman, C.G. and Rothbard, J.B. (2000) Polyarginine enters cells more efficiently than other polycationic homopolymers. J. Pept. Res. 56, 318–325.PubMedCrossRefGoogle Scholar
  16. 16.
    Thoren, P.E., Persson, D., Isakson, P., Goksor, M., Onfelt, A. and Norden, B. (2003) Uptake of analogs of penetratin, Tat(48–60) and oligoarginine in live cells. Biochem. Biophys. Res. Commun. 307, 100–107.PubMedCrossRefGoogle Scholar
  17. 17.
    Mano, M., Henriques, A., Paiva, A., Prieto, M., Gavilanes, F., Simoes, S. and Pedroso de Lima, M.C. (2006) Cellular uptake of S413-PV peptide occurs upon conformational changes induced by peptide-membrane interactions. Biochim. Biophys. Acta 1758, 336–346.PubMedCrossRefGoogle Scholar
  18. 18.
    Tunnemann, G., Ter-Avetisyan, G., Martin, R.M., Stockl, M., Herrmann, A. and Cardoso, M.C. (2008) Live-cell analysis of cell penetration ability and toxicity of oligo-arginines. J. Pept. Sci. 14, 469–476.PubMedCrossRefGoogle Scholar
  19. 19.
    Ter-Avetisyan, G., Tunnemann, G., Nowak, D., Nitschke, M., Herrmann, A., Drab, M. and Cardoso, M.C. (2009) Cell entry of arginine-rich peptides is independent of endocytosis. J. Biol. Chem. 284, 3370–3378.PubMedCrossRefGoogle Scholar
  20. 20.
    Fischer, R., Fotin-Mleczek, M., Hufnagel, H. and Brock, R. (2005) Break on through to the other side-biophysics and cell biology shed light on cell-penetrating peptides. Chembiochem. 6, 2126–2142.PubMedCrossRefGoogle Scholar
  21. 21.
    Ziegler, A., Nervi, P., Durrenberger, M. and Seelig, J. (2005) The cationic cell-penetrating peptide CPP(TAT) derived from the HIV-1 protein TAT is rapidly transported into living fibroblasts: optical, biophysical, and metabolic evidence. Biochemistry 44, 138–148.PubMedCrossRefGoogle Scholar
  22. 22.
    Geueke, B., Namoto, K., Agarkova, I., Perriard, J.C., Kohler, H.P. and Seebach, D. (2005) Bacterial cell penetration by beta3-oligohomoarginines: indications for passive transfer through the lipid bilayer. Chembiochem. 6, 982–985.PubMedCrossRefGoogle Scholar
  23. 23.
    Nekhotiaeva, N., Elmquist, A., Rajarao, G.K., Hällbrink, M., Langel, Ü. and Good, L. (2004) Cell entry and antimicrobial properties of eukaryotic cell-penetrating peptides. FASEB J. 18, 394–396.PubMedGoogle Scholar
  24. 24.
    Holm, T., Netzereab, S., Hansen, M., Langel, Ü. and Hällbrink, M. (2005) Uptake of cell-penetrating peptides in yeasts. FEBS Lett. 579, 5217–5222.PubMedCrossRefGoogle Scholar
  25. 25.
    Glaeser, R.M. and Jap, B.K. (1984) The “Born Energy” Problem in Bacte riorhodopsin. Biophys. J. 45, 95–97.PubMedCrossRefGoogle Scholar
  26. 26.
    Nishihara, M., Perret, F., Takeuchi, T., Futaki, S., Lazar, A.N., Coleman, A.W., Sakai, N. and Matile, S. (2005) Arginine magic with new counterions up the sleeve. Org. Biomol. Chem. 3, 1659–1669.PubMedCrossRefGoogle Scholar
  27. 27.
    Richard, J.P., Melikov, K., Vives, E., Ramos, C., Verbeure, B., Gait, M.J., Chernomordik, L.V. and Lebleu, B. (2003) Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. J. Biol. Chem. 278, 585–590.PubMedCrossRefGoogle Scholar
  28. 28.
    Ziegler, A. (2008) Thermodynamic studies and binding mechanisms of cell-penetrating peptides with lipids and glycosaminoglycans. Adv. Drug Deliv. Rev. 60, 580–597.PubMedCrossRefGoogle Scholar
  29. 29.
    Scheller, A., Oehlke, J., Wiesner, B., Dathe, M., Krause, E., Beyermann, M., Melzig, M. and Bienert, M. (1999) Structural requirements for cellular uptake of alpha-helical amphipathic peptides. J. Pept. Sci. 5, 185–194.PubMedCrossRefGoogle Scholar
  30. 30.
    Bechinger, B. and Lohner, K. (2006) Detergent-like actions of linear amphipathic cationic antimicrobial peptides. Biochim. Biophys. Acta 1758, 1529–1539.PubMedCrossRefGoogle Scholar
  31. 31.
    Takeshima, K., Chikushi, A., Lee, K.K., Yonehara, S. and Matsuzaki, K. (2003) Translocation of analogues of the antimicrobial peptides magainin and buforin across human cell membranes. J. Biol. Chem. 278, 1310–1315.PubMedCrossRefGoogle Scholar
  32. 32.
    Shai, Y. (1999) Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim. Biophys. Acta 1462, 55–70.PubMedCrossRefGoogle Scholar
  33. 33.
    Deshayes, S., Plenat, T., Aldrian-Herrada, G., Divita, G., Le Grimellec, C. and Heitz, F. (2004) Primary amphipathic cell-penetrating peptides: structural requirements and interactions with model membranes. Biochemistry 43, 7698–7706.PubMedCrossRefGoogle Scholar
  34. 34.
    Cho, Y.W., Kim, J.D. and Park, K. (2003) Polycation gene delivery systems: escape from endosomes to cytosol. J. Pharm. Pharmacol. 55, 721–734.PubMedCrossRefGoogle Scholar
  35. 35.
    Subbarao, N.K., Parente, R.A., Szoka, F.C., Jr., Nadasdi, L. and Pongracz, K. (1987) pH-dependent bilayer destabilization by an amphipathic peptide. Biochemistry 26, 2964–2972.PubMedCrossRefGoogle Scholar
  36. 36.
    Jones, S.W., Christison, R., Bundell, K., Voyce, C.J., Brockbank, S.M., Newham, P. and Lindsay, M.A. (2005) Characterisation of cell-penetrating peptide-mediated peptide delivery. Br. J. Pharmacol. 145, 1093–1102.PubMedCrossRefGoogle Scholar
  37. 37.
    Saar, K., Lindgren, M., Hansen, M., Eiriksdottir, E., Jiang, Y., Rosenthal-Aizman, K., Sassian, M. and Langel, Ü. (2005) Cell-penetrating peptides: a comparative membrane toxicity study. Anal. Biochem. 345, 55–65.PubMedCrossRefGoogle Scholar
  38. 38.
    Macdonald, P.M., Crowell, K.J., Franzin, C.M., Mitrakos, P. and Semchyschyn, D.J. (1998) Polyelectrolyte-induced domains in lipid bilayer membranes: the deuterium NMR perspective. Biochem. Cell. Biol. 76, 452–464.PubMedCrossRefGoogle Scholar
  39. 39.
    Tiriveedhi, V. and Butko, P. (2007) A fluorescence spectroscopy study on the interactions of the TAT-PTD peptide with model lipid membranes. Biochemistry 46, 3888–3895.PubMedCrossRefGoogle Scholar
  40. 40.
    Roux, M., Neumann, J.M., Bloom, M. and Devaux, P.F. (1988) 2H and 31P NMR study of pentalysine interaction with headgroup deuterated phosphatidylcholine and phosphatidylserine. Eur. Biophys. J. 16, 267–273.PubMedCrossRefGoogle Scholar
  41. 41.
    Esbjorner, E.K., Lincoln, P. and Norden, B. (2007) Counterion-mediated membrane penetration: Cationic cell-penetrating peptides overcome Born energy barrier by ion-pairing with phospholipids. Biochim. Biophys. Acta 1768, 1550–1558.PubMedCrossRefGoogle Scholar
  42. 42.
    Sakai, N., Takeuchi, T., Futaki, S. and Matile, S. (2005) Direct observation of anion-mediated translocation of fluorescent oligoarginine carriers into and across bulk liquid and anionic bilayer membranes. Chembiochem. 6, 114–122.PubMedCrossRefGoogle Scholar
  43. 43.
    Henriques, S.T., Costa, J. and Castanho, M.A. (2005) Translocation of beta-galactosidase mediated by the cell-penetrating peptide pep-1 into lipid vesicles and human HeLa cells is driven by membrane electrostatic potential. Biochemistry 44, 10189–10198.PubMedCrossRefGoogle Scholar
  44. 44.
    Afonin, S., Frey, A., Bayerl, S., Fischer, D., Wadhwani, P., Weinkauf, S. and Ulrich, A.S. (2006) The cell-penetrating peptide TAT(48–60) induces a non-lamellar phase in DMPC membranes. Chemphyschem. 7, 2134–2142.PubMedCrossRefGoogle Scholar
  45. 45.
    Thoren, P.E., Persson, D., Karlsson, M. and Norden, B. (2000) The antennapedia peptide penetratin translocates across lipid bilayers – the first direct observation. FEBS Lett. 482, 265–268.PubMedCrossRefGoogle Scholar
  46. 46.
    Thoren, P.E., Persson, D., Esbjorner, E.K., Goksor, M., Lincoln, P. and Norden, B. (2004) Membrane binding and translocation of cell-penetrating peptides. Biochemistry 43, 3471–3489.PubMedCrossRefGoogle Scholar
  47. 47.
    Futaki, S., Ohashi, W., Suzuki, T., Niwa, M., Tanaka, S., Ueda, K., Harashima, H. and Sugiura, Y. (2001) Stearylated arginine-rich peptides: a new class of transfection systems. Bioconjug. Chem. 12, 1005–1011.PubMedCrossRefGoogle Scholar
  48. 48.
    Drin, G., Demene, H., Temsamani, J. and Brasseur, R. (2001) Translocation of the pAntp peptide and its amphipathic analogue AP-2AL. Biochemistry 40, 1824–1834.PubMedCrossRefGoogle Scholar
  49. 49.
    Schindler, H. (1979) Exchange and interactions between lipid layers at the surface of a liposome solution. Biochim. Biophys. Acta 555, 316–336.PubMedCrossRefGoogle Scholar
  50. 50.
    Qiu, R. and MacDonald, R.C. (1994) A metastable state of high surface activity produced by sonication of phospholipids. Biochim. Biophys. Acta. 1191, 343–353.PubMedCrossRefGoogle Scholar
  51. 51.
    Smith, R. and Tanford, C. (1972) Critical micelle concentration of L-alpha-dipalmi toylphosphatidylcholine in water and water/methanol solutions. J. Mol. Biol. 67, 75–83.PubMedCrossRefGoogle Scholar
  52. 52.
    Altenbach, C. and Seelig, J. (1984) Ca-2+ Binding to phosphatidylcholine bilayers as studied by deuterium magnetic-resonance – evidence for the formation of a Ca-2+ complex with 2 phospholipid molecules. Biochemistry 23, 3913–3920.PubMedCrossRefGoogle Scholar
  53. 53.
    Lewis, B.A. and Engelman, D.M. (1983) Lipid bilayer thickness varies linearly with acyl chain length in fluid phosphatidylcholine vesicles. J. Mol. Biol. 166, 211–217.PubMedCrossRefGoogle Scholar
  54. 54.
    Santaren, J.F., Rico, M., Guilleme, J. and Ribera, A. (1982) Thermal and 13C-NMR study of the dynamic structure of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and 1-oleyl-2-palmitoyl-sn-glycero-3-phosphocholine in aqueous dispersions. Biochim. Biophys. Acta 687, 231–237.PubMedCrossRefGoogle Scholar
  55. 55.
    Cullis, P.R., Hope, M.J. and Tilcock, C.P. (1986) Lipid polymorphism and the roles of lipids in membranes. Chem. Phys. Lipids 40, 127–144.PubMedCrossRefGoogle Scholar
  56. 56.
    Langner, M. and Hui, S.W. (1993) Dithio nite penetration through phospholipid bilayers as a measure of defects in lipid molecular packing. Chem. Phys. Lipids 65, 23–30.PubMedCrossRefGoogle Scholar
  57. 57.
    Fabrie, C.H., de Kruijff, B. and de Gier, J. (1990) Protection by sugars against phase transition-induced leak in hydrated dimyristoylphosphatidylcholine liposomes. Biochim. Biophys. Acta 1024, 380–384.PubMedCrossRefGoogle Scholar
  58. 58.
    Volodkin, D., Mohwald, H., Voegel, J.C. and Ball, V. (2007) Coating of negatively charged liposomes by polylysine: drug release study. J. Control. Release 117, 111–120.PubMedCrossRefGoogle Scholar
  59. 59.
    Marsh, D. (1996) Intrinsic curvature in normal and inverted lipid structures and in membranes. Biophys. J. 70, 2248–2255.PubMedCrossRefGoogle Scholar
  60. 60.
    Felgner, J.H., Kumar, R., Sridhar, C.N., Wheeler, C.J., Tsai, Y.J., Border, R., Ramsey, P., Martin, M. and Felgner, P.L. (1994) Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J. Biol. Chem. 269, 2550–2561.PubMedGoogle Scholar
  61. 61.
    Prochiantz, A. (1996) Getting hydrophilic compounds into cells: lessons from homeopeptides. Curr. Opin. Neurobiol. 6, 629–634.PubMedCrossRefGoogle Scholar
  62. 62.
    Szoka, F. and Papahadjopoulos, D. (1980) Comparative properties and methods of preparation of lipid vesicles (liposomes). Annu. Rev. Biophys. Biol. 9, 467–508.CrossRefGoogle Scholar
  63. 63.
    Fischer, A., Oberholzer, T. and Luisi, P.L. (2000) Giant vesicles as models to study the interactions between membranes and proteins. Biochim. Biophys. Acta 1467, 177–188.PubMedCrossRefGoogle Scholar
  64. 64.
    Elorza, B., Elorza, M.A., Sainz, M.C. and Chantres, J.R. (1993) Analysis of the particle size distribution and internal volume of liposomal preparations. J. Pharm. Sci. 82, 1160–1163.PubMedCrossRefGoogle Scholar
  65. 65.
    Seelig, A. (1987) Local anesthetics and pressure: a comparison of dibucaine binding to lipid monolayers and bilayers. Biochim. Biophys. Acta 899, 196–204.PubMedCrossRefGoogle Scholar
  66. 66.
    Herbig, M.E., Fromm, U., Leuenberger, J., Krauss, U., Beck-Sickinger, A.G. and Merkle, H.P. (2005) Bilayer interaction and localization of cell penetrating peptides with model membranes: a comparative study of a human calcitonin (hCT)-derived peptide with pVEC and pAntp(43–58). Biochim. Biophys. Acta 1712, 197–211.PubMedCrossRefGoogle Scholar
  67. 67.
    Michaelson, D.M., Horwitz, A.F. and Klein, M.P. (1973) Transbilayer asymmetry and surface homogeneity of mixed phospholipids in cosonicated vesicles. Biochemistry 12, 2637–2645.PubMedCrossRefGoogle Scholar
  68. 68.
    Wieprecht, T., Apostolov, O., Beyermann, M. and Seelig, J. (2000) Membrane binding and pore formation of the antibacterial peptide PGLa: thermodynamic and mechanistic aspects. Biochemistry 39, 442–452.PubMedCrossRefGoogle Scholar
  69. 69.
    Ruocco, M.J. and Shipley, G.G. (1982) Characterization of the sub-transition of hydrated dipalmitoylphosphatidylcholine bilayers – kinetic, hydration and structural study. Biochim. Biophys. Acta 691, 309–320.CrossRefGoogle Scholar
  70. 70.
    Zhou, Z., Sayer, B.G., Hughes, D.W., Stark, R.E. and Epand, R.M. (1999) Studies of phospholipid hydration by high-resolution magic-angle spinning nuclear magnetic resonance. Biophys. J. 76, 387–399.PubMedCrossRefGoogle Scholar
  71. 71.
    Newman, G.C. and Huang, C. (1975) Structural studies on phophatidylcholine-cholesterol mixed vesicles. Biochemistry 14, 3363–3370.PubMedCrossRefGoogle Scholar
  72. 72.
    Bangham, A.D., Standish, M.M. and Watkins, J.C. (1965) Diffusion of univalent ions across lamellae of swollen phospholipids. J. Mol. Biol. 13, 238–252.PubMedCrossRefGoogle Scholar
  73. 73.
    Huang, C. (1969) Studies on phosphatidylcholine vesicles. Formation and physical characteristics. Biochemistry 8, 344–352.PubMedCrossRefGoogle Scholar
  74. 74.
    Hope, M.J., Bally, M.B., Webb, G. and Cullis, P.R. (1985) Production of large unilamellar vesicles by a rapid extrusion procedure – characterization of size distribution, trapped volume and ability to maintain a membrane-potential. Biochim. Biophys. Acta 812, 55–65.CrossRefGoogle Scholar
  75. 75.
    Kaasgaard, T., Mouritsen, O.G. and Jorgensen, K. (2003) Freeze/thaw effects on lipid-bilayer vesicles investigated by differential scanning calorimetry. Biochim. Biophys. Acta. 1615, 77–83.PubMedCrossRefGoogle Scholar
  76. 76.
    Traikia, M., Warschawski, D.E., Recouvreur, M., Cartaud, J. and Devaux, P.F. (2000) Formation of unilamellar vesicles by repe titive freeze-thaw cycles: characterization by electron microscopy and 31P-nuclear magnetic resonance. Eur. Biophys. J. 29, 184–195.PubMedCrossRefGoogle Scholar
  77. 77.
    Larrabee, A.L. (1979) Time-dependent changes in the size distribution of distearoyl phosphatidylcholine vesicles. Biochemistry 18, 3321–3326.PubMedCrossRefGoogle Scholar
  78. 78.
    Suurkuusk, J., Lentz, B.R., Barenholz, Y., Biltonen, R.L. and Thompson, T.E. (1976) Calorimetric and fluorescent-probe study of gel-liquid crystalline phase-transition in small, single-lamellar dipalmitoylphosphatidylcholine vesicles. Biochemistry 15, 1393–1401.PubMedCrossRefGoogle Scholar
  79. 79.
    Mayer, L.D., Hope, M.J. and Cullis, P.R. (1986) Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim. Biophys. Acta 858, 161–168.PubMedCrossRefGoogle Scholar
  80. 80.
    Grit, M. and Crommelin, D.J.A. (1992) The effect of aging on the physical stability of liposome dispersions. Chem. Phys. Lipids 62, 113–122.PubMedCrossRefGoogle Scholar
  81. 81.
    Lasic, D.D. (1988) The mechanism of vesicle formation. Biochem. J. 256, 1–11.PubMedGoogle Scholar
  82. 82.
    Petersen, N.O. and Chan, S.I. (1978) Effects of thermal prephase transition and salts on coagulation and flocculation of phosphatidylcholine bilayer vesicles. Biochim. Biophys. Acta 509, 111–128.PubMedCrossRefGoogle Scholar
  83. 83.
    Lichtenberg, D., Freire, E., Schmidt, C.F., Barenholz, Y., Felgner, P.L. and Thompson, T.E. (1981) Effect of surface curvature on stability, thermodynamic behavior, and osmotic activity of dipalmitoylphosphatidylcholine single lamellar vesicles. Biochemistry 20, 3462–3467.PubMedCrossRefGoogle Scholar
  84. 84.
    Winterhalter, M. and Lasic, D.D. (1993) Liposome stability and formation – experimental parameters and theories on the size distribution. Chem. Phys. Lipids 64, 35–43.PubMedCrossRefGoogle Scholar
  85. 85.
    Maulucci, G., De Spirito, M., Arcovito, G., Boffi, F., Castellano, A.C. and Briganti, G. (2005) Particle size distribution in DMPC vesicles solutions undergoing different sonication times. Biophys. J. 88, 3545–3550.PubMedCrossRefGoogle Scholar
  86. 86.
    Pereira-Lachataignerais, J., Pons, R., Panizza, P., Courbin, L., Rouch, J. and Lopez, O. (2006) Study and formation of vesicle systems with low polydispersity index by ultrasound method. Chem. Phys. Lipids 140, 88–97.PubMedCrossRefGoogle Scholar
  87. 87.
    Hauser, H.O. (1971) Effect of ultrasonic irradiation on chemical structure of egg lecithin. Biochem. Bioph. Res. Commun. 45, 1049–1055.CrossRefGoogle Scholar
  88. 88.
    Woodbury, D.J., Richardson, E.S., Grigg, A.W., Welling, R.D. and Knudson, B.H. (2006) Reducing liposome size with ultrasound: bimodal size distributions. J. Liposome Res. 16, 57–80.PubMedCrossRefGoogle Scholar
  89. 89.
    Frimer, A.A., Strul, G., Buch, J. and Gottlieb, H.E. (1996) Can superoxide organic chemistry be observed within the liposomal bilayer? Free Radic. Biol. Med. 20, 843–852.PubMedCrossRefGoogle Scholar
  90. 90.
    Andrews, S.B., Hoffman, R.M. and Borison, A. (1975) Variations of size and distribution in suspensions of sonicated phospholipid bilayers. Biochem. Biophys. Res. Commun. 65, 913–920.PubMedCrossRefGoogle Scholar
  91. 91.
    Yamaguchi, T., Nomura, M., Matsuoka, T. and Koda, S. (2009) Effects of frequency and power of ultrasound on the size reduction of liposome. Chem. Phys. Lipids 160, 58–62.PubMedCrossRefGoogle Scholar
  92. 92.
    Martin, F.J. and MacDonald, R.C. (1976) Phospholipid exchange between bilayer membrane vesicles. Biochemistry 15, 321–327.PubMedCrossRefGoogle Scholar
  93. 93.
    McCulloch, A. (2003) Chloroform in the environment: occurrence, sources, sinks and effects. Chemosphere 50, 1291–1308.PubMedCrossRefGoogle Scholar
  94. 94.
    Constan, A.A., Wong, B.A., Everitt, J.I. and Butterworth, B.E. (2002) Chloroform inhalation exposure conditions necessary to initiate liver toxicity in female B6C3F1 mice. Toxicol. Sci. 66, 201–208.PubMedCrossRefGoogle Scholar
  95. 95.
    Blixt, Y., Valeur, A. and Everitt, E. (1990) Cultivation of HeLa cells with fetal bovine serum or Ultroser G: effects on the plasma membrane constitution. In Vitro Cell Dev. Biol. 26, 691–700.PubMedCrossRefGoogle Scholar
  96. 96.
    White, D.A. (1973) Phospholipid composition of mammalian tissue. In: Ansell, G.B., Hawthorne, J.A., and Dawson, R.M.C. (editors), 2nd ed., Form and function of phospholipids, Elsevier, Amsterdam, pp. 441–482.Google Scholar
  97. 97.
    Epand, R.M. and Epand, R.F. (2009) Lipid domains in bacterial membranes and the action of antimicrobial agents. Biochim. Biophys. Acta 1788, 289–294.PubMedCrossRefGoogle Scholar
  98. 98.
    Beining, P.R., Huff, E., Prescott, B. and Theodore, T.S. (1975) Characterization of the lipids of mesosomal vesicles and plasma membranes from Staphylococcus aureus. J. Bacteriol. 121, 137–143.PubMedGoogle Scholar
  99. 99.
    Devaux, P.F. (1991) Static and dynamic lipid asymmetry in cell membranes. Biochemistry 30, 1163–1173.PubMedCrossRefGoogle Scholar
  100. 100.
    Martin, S.J., Reutelingsperger, C.P., McGahon, A.J., Rader, J.A., van Schie, R.C., LaFace, D.M. and Green, D.R. (1995) Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med. 182, 1545–1556.PubMedCrossRefGoogle Scholar
  101. 101.
    Ziegler, A., Blatter, X.L., Seelig, A. and Seelig, J. (2003) Protein transduction domains of HIV-1 and SIV TAT interact with charged lipid vesicles. Binding mechanism and thermodynamic analysis. Biochemistry 42, 9185–9194.PubMedCrossRefGoogle Scholar
  102. 102.
    Persson, D., Thoren, P.E., Lincoln, P. and Norden, B. (2004) Vesicle membrane interactions of penetratin analogues. Biochemistry 43, 11045–11055.PubMedCrossRefGoogle Scholar
  103. 103.
    Hristova, K. and Needham, D. (1994) The influence of polymer-grafted lipids on the physical-properties of lipid bilayers – a theoretical-study. J. Colloid Interface Sci. 168, 302–314.CrossRefGoogle Scholar
  104. 104.
    Tirosh, O., Barenholz, Y., Katzhendler, J. and Priev, A. (1998) Hydration of polyethylene glycol-grafted liposomes. Biophys. J. 74, 1371–1379.PubMedCrossRefGoogle Scholar
  105. 105.
    Garbuzenko, O., Barenholz, Y. and Priev, A. (2005) Effect of grafted PEG on liposome size and on compressibility and packing of lipid bilayer. Chem. Phys. Lipids 135, 117–129.PubMedCrossRefGoogle Scholar
  106. 106.
    Allende, D., Simon, S.A. and McIntosh, T.J. (2005) Melittin-induced bilayer leakage depends on lipid material properties: evidence for toroidal pores. Biophys. J. 88, 1828–1837.PubMedCrossRefGoogle Scholar
  107. 107.
    Kaasgaard, T., Mouritsen, O.G. and Jorgensen, K. (2001) Screening effect of PEG on avidin binding to liposome surface receptors. Int. J. Pharm. 214, 63–65.PubMedCrossRefGoogle Scholar
  108. 108.
    Itaya, K. and Ui, M. (1966) A new micromethod for the colorimetric determination of inorganic phosphate. Clin. Chim. Acta 14, 361–366.PubMedCrossRefGoogle Scholar
  109. 109.
    Baykov, A.A., Evtushenko, O.A. and Avaeva, S.M. (1988) A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay. Anal. Biochem. 171, 266–270.PubMedCrossRefGoogle Scholar
  110. 110.
    Vemuri, S. (2005) Comparison of assays for determination of peptide content for lyophi lized thymalfasin. J. Pept. Res. 65, 433–439.PubMedCrossRefGoogle Scholar
  111. 111.
    Edelhoch, H. (1967) Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6, 1948–1954.PubMedCrossRefGoogle Scholar
  112. 112.
    Ziegler, A. and Seelig, J. (2008) Binding and clustering of glycosaminoglycans: a common property of mono- and multivalent cell-penetrating compounds. Biophys. J. 94, 2142–2149.PubMedCrossRefGoogle Scholar
  113. 113.
    Schiffer, M. and Edmundson, A.B. (1967) Use of helical wheels to represent the structures of proteins and to identify segments with helical potential. Biophys. J. 7, 121–135.PubMedCrossRefGoogle Scholar
  114. 114.
    Iritani, N. and Miyahara, T. (1973) Deter mination of dissociation-constants of calcein by potentiometric method. Jpn. Anal. 22, 174–178.Google Scholar
  115. 115.
    Wallach, D.F.H., Surgenor, D.M., Soderberg, J. and Delano, E. (1959) Preparation and properties of 3, 6-dihydroxy-2, 4-bis-(N, N′-di-(carboxymethyl)-aminomethyl) fluoran – utilization for the ultramicrodetermination of calcium. Anal. Chem. 31, 456–460.CrossRefGoogle Scholar
  116. 116.
    Niesman, M.R., Khoobehi, B. and Peyman, G.A. (1992) Encapsulation of sodium fluorescein for dye release studies. Invest. Ophthalmol. Vis. Sci. 33, 2113–2119.PubMedGoogle Scholar
  117. 117.
    Garcia, M.A., Paje, S.E., Villegas, M.A. and Llopis, J. (2002) Preparation and characterisation of calcein-doped thin coatings. Appl. Phys. A Mater. 74, 83–88.CrossRefGoogle Scholar
  118. 118.
    Aschi, M., D’Archivio, A.A., Fontana, A. and Formiglio, A. (2008) Physicochemical properties of fluorescent probes: experimental and computational determination of the overlapping pKa values of carboxyfluorescein. J. Org. Chem. 73, 3411–3417.PubMedCrossRefGoogle Scholar
  119. 119.
    Rothbard, J.B., Jessop, T.C., Lewis, R.S., Murray, B.A. and Wender, P.A. (2004) Role of membrane potential and hydrogen bonding in the mechanism of translocation of guanidinium-rich peptides into cells. J. Am. Chem. Soc. 126, 9506–9507.PubMedCrossRefGoogle Scholar
  120. 120.
    Chen, R.F. and Knutson, J.R. (1988) Mechanism of fluorescence concentration quenching of carboxyfluorescein in liposomes: energy transfer to nonfluorescent dimers. Anal. Biochem. 172, 61–77.PubMedCrossRefGoogle Scholar
  121. 121.
    Gavino, V.C., Miller, J.S., Dillman, J.M., Milo, G.E. and Cornwell, D.G. (1981) Polyunsaturated fatty acid accumulation in the lipids of cultured fibroblasts and smooth muscle cells. J. Lipid Res. 22, 57–62.PubMedGoogle Scholar
  122. 122.
    Seelig, J. (1978) P-31 Nuclear magnetic-resonance and head group structure of phospholipids in membranes. Biochim. Biophys. Acta 515, 105–140.PubMedGoogle Scholar
  123. 123.
    Soubias, O. and Gawrisch, K. (2007) Nuclear magnetic resonance investigation of oriented lipid membranes. Methods Mol. Biol. 400, 77–88.Google Scholar
  124. 124.
    Seelig, J. (2004) Thermodynamics of lipid-peptide interactions. Biochim. Biophys. Acta. 1666, 40–50.PubMedGoogle Scholar
  125. 125.
    Persson, D., Thoren, P.E., Herner, M., Lincoln, P. and Norden, B. (2003) Appli cation of a novel analysis to measure the binding of the membrane-translocating peptide penetratin to negatively charged liposomes. Biochemistry 42, 421–429.PubMedCrossRefGoogle Scholar
  126. 126.
    Beschiaschvili, G. and Seelig, J. (1990) Peptide binding to lipid bilayers. Binding isotherms and zeta-potential of a cyclic somatostatin analogue. Biochemistry 29, 10995–11000.PubMedCrossRefGoogle Scholar
  127. 127.
    Franzin, C.M. and Macdonald, P.M. (2001) Polylysine-induced 2H NMR-observable domains in phosphatidylserine/phosphatidylcholine lipid bilayers. Biophys. J. 81, 3346–3362.PubMedCrossRefGoogle Scholar
  128. 128.
    Macdonald, P.M., Crowell, K.J., Franzin, C.M., Mitrakos, P. and Semchyschyn, D. (2000) 2H NMR and polyelectrolyte-induced domains in lipid bilayers. Solid State Nucl. Magn. Reson. 16, 21–36.PubMedCrossRefGoogle Scholar
  129. 129.
    Dennison, S.R., Baker, R.D., Nicholl, I.D. and Phoenix, D.A. (2007) Interactions of cell penetrating peptide Tat with model membranes: a biophysical study. Biochem. Biophys. Res. Commun. 363, 178–182.PubMedCrossRefGoogle Scholar
  130. 130.
    Baker, B.M. and Murphy, K.P. (1996) Evalua tion of linked protonation effects in protein binding reactions using iso thermal titration calorimetry. Biophys. J. 71, 2049–2055.PubMedCrossRefGoogle Scholar
  131. 131.
    Yi, D., Guoming, L., Gao, L. and Wei, L. (2007) Interaction of arginine oligomer with model membrane. Biochem. Biophys. Res. Commun. 359, 1024–1029.PubMedCrossRefGoogle Scholar
  132. 132.
    Kramer, S.D. and Wunderli-Allenspach, H. (2003) No entry for TAT(44–57) into liposomes and intact MDCK cells: novel approach to study membrane permeation of cell-penetrating peptides. Biochim. Biophys. Acta. 1609, 161–169.PubMedCrossRefGoogle Scholar
  133. 133.
    Thoren, P.E., Persson, D., Lincoln, P. and Norden, B. (2005) Membrane destabilizing properties of cell-penetrating peptides. Biophys. Chem. 114, 169–179.PubMedCrossRefGoogle Scholar
  134. 134.
    Lamaziere, A., Burlina, F., Wolf, C., Chassaing, G., Trugnan, G. and Ayala-Sanmartin, J. (2007) Non-metabolic membrane tubulation and permeability induced by bioactive peptides. PLoS One 2, e201.PubMedCrossRefGoogle Scholar
  135. 135.
    Fuchs, S.M. and Raines, R.T. (2004) Pathway for polyarginine entry into mammalian cells. Biochemistry 43, 2438–2444.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Sciecne+Business Media, LLC 2011

Authors and Affiliations

  • Reto Sauder
    • 1
  • Joachim Seelig
    • 2
  • André Ziegler
    • 2
  1. 1.Department of Biophysical ChemistryBiozentrum of the University of BaselBaselSwitzerland
  2. 2.Department of Biophysical ChemistryBiozentrum of the University of BaselBaselSwitzerland

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