Abstract
Supramolecular aggregates containing cationic lipids have been widely used as transfection mediators due to their ability to interact with negatively charged DNA molecules and biological membranes. First steps of the process leading to transfection are partly electrostatic, partly hydrophobic interactions of liposomes/lipoplexes with cell and/or endosomal membrane. Negatively charged compounds of biological membranes, namely glycolipids, glycoproteins and phosphatidylserine (PS), are responsible for such events as adsorption, hemifusion, fusion, poration and destabilization of natural membranes upon contact with cationic liposomes/lipoplexes. The present communication describes the dependence of interaction of cationic liposomes with natural and artificial membranes on the negative charge of the target membrane, charges which in most cases were generated by charging the PS content or its exposure. The model for the target membranes were liposomes of variable content of PS or PG (phosphatidylglycerol) and erythrocyte membranes in which the PS and other anionic compound content/exposure was modified in several ways. Membranes of increased anionic phospholipid content displayed increased fusion with DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane) liposomes, while erythrocyte membranes partly depleted of glycocalix, its sialic acid, in particular, showed a decreased fusion ability. The role of the anionic component is also supported by the fact that erythrocyte membrane inside-out vesicles fused easily with cationic liposomes. The data obtained on erythrocyte ghosts of normal and disrupted asymmetry, in particular, those obtained in the presence of Ca2+, indicate the role of lipid flip-flop movement catalyzed by scramblase. The ATP-depletion of erythrocytes also induced an increased sensitivity to hemoglobin leakage upon interactions with DOTAP liposomes. Calcein leakage from anionic liposomes incubated with DOTAP liposomes was also dependent on surface charge of the target membranes. In all experiments with the asymmetric membranes the fusion level markedly increased with an increase of temperature, which supports the role of membrane lipid mobility. The decrease in positive charge by binding of plasmid DNA and the increase in ionic strength decreased the ability of DOTAP liposomes/lipoplexes to fuse with erythrocyte ghosts. Lower pH promotes fusion between erythrocyte ghosts and DOTAP liposomes and lipoplexes. The obtained results indicate that electrostatic interactions together with increased mobility of membrane lipids and susceptibility to form structures of negative curvature play a major role in the fusion of DOTAP liposomes with natural and artificial membranes.
Similar content being viewed by others
References
Abe A., Miyanohara A., Friedman T. 1998. Enhanced gene transfer with fusogenic liposomes containing vesicular stomatitis virus G glycoprotein. J. Virol. 72:6159–6163
Almofti M.R., Harashima H., Shinohara Y., Almofti A., Baba Y., Kiwada H. 2003. Cationic liposome-mediated gene delivery: Biophysical study and mechanism of internalization. Arch. Biochem. Biophys. 410:246–253
Bailey A.L., Cullis P.R. 1994. Modulation of membrane fusion by asymmetric transbilayer distributions of amino lipids. Biochemistry 33:12573–12580
Bailey A.L., Cullis P.R. 1997. Membrane fusion with cationic liposomes: effects of target membrane lipid composition. Biochemistry 36:1628–1634
Baldwin J.M., O’Reilly R., Whitney M., Lucy J.A. 1990. Surface exposure of phosphatidylserine is associated with the swelling and osmotically-induced fusion of human erythrocytes in the presence of Ca2+. Biochim. Biophys. Acta 1028:14–20
Baumann M., Sowers A.E. 1996. Membrane skeleton involvement in cell fusion kinetics: a parameter that correlates with erythrocyte osmotic fragility. Biophys. J. 71:336–340
Bhattacharya S., Mandal S.S. 1998. Evidence of interlipidic ion-pairing in anion-induced DNA release from cationic amphiphile-DNA complexes. Mechanistic implications in transfection. Biochemistry 37:7764–7777
Cevc G., Richardsen H. 1999. Lipid vesicles and membrane fusion. Adv. Drug Deliv. Rev. 38:207–232
Clague M.J., Cherry R.J. 1989. A comparative study of band 3 aggregation in erythrocyte membranes by mellitin and other cationic agents. Biochim. Biophys. Acta 980:93–99
Clamme J.P., Bernacchi S., Vuilleumier C., Duportail G., Mély Y. 2000. Gene transfer by cationic surfactants is essentially limited by the trapping of the surfactant/DNA complexes onto the cell membrane: a fluorescence investigation. Biochim. Biophys. Acta 1467:347–361
Connor J., Gillum K., Schroit A.J. 1990. Maintance of lipid asymmetry in red blood cells and ghosts: effect of divalent cations and serum albumin on the transbilayer distribution of phosphatidylserine. Biochim. Biophys. Acta 1025:82–86
Devaux P.F. 1991. Static and dynamic lipid asymmetry in cell membranes, Biochemistry 30:1163–1173
Dubois M., Gilles K., Hamilton J.K., Rebers P.A., Smith F. 1956. Colorimetric method for the determination of sugars and related substances. Anal. Chem. 28:350–356
Duguid J.G., Li C., Shi M., Logan M.J., Alila H., Rolland A., Tomlinson E., Sparrow J.T., Smith L.C. 1998. A physicochemical approach for predicting the effectiveness of peptide-based gene delivery systems for use in plasmid-based gene therapy. Biophys. J. 74:2802–2814
Duzgunes N., Simoes S., Konopka K., Rossi J.J., Pedroso de Lima M.C. 2001. Delivery of novel macromolecular drugs against HIV-1. Expert. Opin. Biol. Ther. 1:949–70
Eastman S.J., Hope M.J., Wong K.F., Cullis P.R. 1992. Influence of phospholipid asymmetry on fusion between large unilamellar vesicles. Biochemistry 31:4262–4268
Fadok V.A., de Cathelineau A., Daleke D.L., Henson P.M., Bratton D.L. 2001. Loss of phospholipid asymmetry and surface exposure of phosphatidylserine is required for phagocytosis of apoptotic cells by macrophages and fibroblasts. J. Biol. Chem. 276:1071–1077
Friend D.S., Papahadjopoulos D., Debs R.J. 1996. Endocytosis and intracellular processing accompanying transfection mediated by cationic liposomes. Biochim. Biophys. Acta 1278:41–50
Fuller N., Benatti C.R., Rand R.P. 2003. Curvature and bending constants for phosphatidylserine-containing membranes. Biophys. J. 85:1667–74
Girão da Cruz T., Simoes S., Pires P., Nir S., Pedroso de Lima M. 2001. Kinetic analysis of the initial steps involved in lipoplex-cell interactions: effect of various factors that influence transfection activity. Biochim. Biophys. Acta 1510:136–151
Hafez I.M., Cullis P.R. 2001. Roles of lipid polimorphism in intracellular delivery. Adv. Drug Deliv. Rev. 47:139–148
Hägerstrand H., Danieluk M., Bobrowska-Hägerstrand M., Pector V., Ruysschaert J-M., Kralj-Iglič V., Iglič A. 1999. Liposomes composed of a double-chain cationic amphiphile (vectamidine) induce their own encapsulation into human erythrocytes. Biochim. Biophys. Acta 1421:125–130
Harvie P., Wong F.M.P., Bally M.B. 1998. Characterization of lipid DNA interactions. I. Destabilization of bound lipids and DNA dissociation. Biophys. J. 75:1040–1051
Hui S.W., Langner M., Zhao Y.L., Ross P., Hurley E., Chan K. 1996. The role of helper lipids in cationic liposome-mediated gene transfer. Biophys. J. 71:590–599
Hyde S.C., Southern K.W., Gileadi U., Fitzjohn E.M., Mofford K.A., Waddell B.E., Gooi H.C., Goddard C.A., Hannavy K., Smyth S.E., Egan J.J., Sorgi F.L., Huang L., Cuthbert A.W., Evans M.J., Colledge W.H., Higgins C.F., Webb A.K., Gill D.R. 2000. Repeated administration of DNA/liposomes to the nasal epithelium of patients with cystic fibrosis. Gene Ther. 7:1156–1165
Koltover I., Salditt T., Radler J.O., Säfinya C.R. 1998. An inverted hexagonal phase of cationic liposomes-DNA complexes related to DNA release and delivery. Science 281:78–81
Kono K., Henmi A., Takagishi T. 1999. Temperature-controlled interaction of thermosensitive polymer-modified cationic liposomes with negatively charged phospholipid membranes. Biochim. Biophys. Acta 1421:183–197
Koulov A.V., Vares L., Mahim J., Smith B.D. 2002. Cationic triple-chain amphiphiles facilitate vesicle fusion compared to double-chain or single-chain analogues. Biochim. Biophys. Acta 1564:459–465
Li L., Hui S. 1997. The effect of lipid molecular packing stress on cationic liposome-induced rabbit erythrocyte fusion. Biochim. Biophys. Acta 1323:105–116
Liu F. Huang L. 2002. Development of non-viral vectors for systemic gene delivery. J. Control. Release 78:259–266
Lutz H.U., Liu S.C., Palek J. 1977. Release of spectrin free vesicles from human erythrocytes during ATP depletion. J. Cell. Biol. 73:548–60
Manno S., Takakuwa Y., Mohandas N. 2002. Identification of a functional role for lipid asymmetry in biological membranes: Phosphatidylserine – skeletal protein interactions modulate membrane stability. Proc. Natl. Acad. Sci. USA 99:1943–1948
May S., Harries D., Ben-Shaul A. 2000. The phase behavior of cationic lipid-DNA complexes. Biophys. J. 78:1681–1697
Meidan V.M., Cohen J.S., Amariglio N., Hirsch-Lerner D., Barenholz Y. 2000. Interaction of oligonucleotides with cationic lipids: the relationship between electrostatics, hydration and state of aggregation. Biochim. Biophys. Acta 1464:251–261
Miyata T., Yamamoto S., Sakamoto K., Morishita R., Kaneda Y. 2001. Novel immunotherapy for peritoneal dissemination of murine colon cancer with macrophage inflammatory protein-1beta mediated by a tumor-specific vector, HVJ cationic liposomes. Cancer Gene Ther. 8:852–60
Nabel G.J., Gordon D., Bishop D.K., Nickoloff B.J., Yang Z.Y., Aruga A., Cameron M.J., Nabel E.G., Chang A.E. 1996. Immune response in human melanoma after transfer of an allogeneic class I major histocompatibility complex gene with DNA-liposomes complexes. Proc. Natl. Acad. Sci. USA 93:15388–15393
Nakanishi M., Noguchi A. 2001. Confocal and probe microscopy to study gene transfection mediated by cationic liposomes with a cholesterol derivative. Adv. Drug Deliv. Rev. 52:197–201
Noguchi A., Furuno T., Kawaura C., Nakanishi M. 1998. Membrane fusion plays important role in gene transfection mediated by cationic liposomes. FEBS Lett. 433:169–173
Oberle V., Bakowsky U., Zuhorn I.S., Hoekstra D. 2000. Lipoplex formation under equilibrium conditions reveals a three-step mechanism. Biophys. J. 79:1447–1454
El Ouahabi A., Thiry M., Pector V., Fuks R., Ruysschaert J.-M., Vandenbranden M. 1997. The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids. FEBS Lett. 414:187–192
Pantazatos D.P., MacDonald R.C. 1999. Directly observed membrane fusion between oppositely charged phospholipid bilayers. J. Membrane Biol. 170:27–38
Pantazatos D.P., Pantazatos S.P., MacDonald R.C. 1999. Bilayer mixing, fusion, and lysis following the interaction of populations of cationic and anionic phospholipid bilayer vesicles. J. Membrane Biol. 194:129–139
Pantazatos S.P., MacDonald R.C. 2003. Real-time observation of lipoplex formation and interaction with anionic bilayer vesicles. J Membrane Biol. 191:99–112
Pedroso de Lima M.C., Simões S., Pires P., Gaspar R., Slepushkin V., Düzgünes N. 1999. Gene delivery mediated by cationic liposomes: from biophysical aspects to enhancement of transfection. Mol. Membrane Biol. 16:103–109
Peterson G.L. 1977. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83:346–356
Pires P., Simoes S., Nir S., Gaspar R., Düzgünes N., Pedroso de Lima M.C. 1999. Interaction of cationic liposomes and their DNA complexes with monocytic leukemia cells. Biochim. Biophys. Acta 1418:71–84
Rouse R.J., Seifried W., Mistry S.K., Goins W.F., Glorioso J.C. 2000. Herpes simplex virus-enhanced cationic lipid/DNA-mediated transfection. Biotechniques 29:810–814
Rouser G., Siakatos A., Fleischer S. 1966. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids 1:85–86
Schewe M., Muller P., Korte T., Herman A. 1992. The role of phospholipid asymmetry in calcium-phosphate-induced fusion of human erythrocytes. J. Biol. Chem. 267:5910–5915
Schreier H., Gagne L., Bock T., Erdos G.W., Druzgala P., Conary J.T., Muller B.W. 1997. Physicochemical properties and in vitro toxicity of cationic liposome cDNA complexes. Pharm. Acta Helv. 72:215–23
Senior J.H., Trimble K.R., Maskiewicz R. 1991. Interaction of positively-charged liposomes with blood; implications for their application in vivo. Biochim. Biophys. Acta 1070:173–179
Shichiri M., Tanaka A., Hirata Y. 2003. Intravenous gene therapy for familial hypercholesterolemia using ligand-facilitated transfer of a liposome: LDL receptor gene complex. Gene Ther. 1:827–31
Simberg D., Danino D., Talmon Y., Minsky A., Ferrari M.E., Wheeler C.J., Barenholz Y. 2001. Phase behavior, DNA ordering, and size instability of cationic lipoplexes. J. Biol. Chem. 276:47453–47459
Stamatatos L., Leventis R., Zuckermann M.J., Silvius J.R. 1988. Interaction of cationic lipid vesicles with negatively charged phospholipid vesicles and biological membranes. Biochemistry 27:3917–3925
Stegmann T., Legendre J.-Y. 1997. Gene transfer mediated by cationic lipids: lack of correlation between lipid mixing and transfection. Biochim. Biophys. Acta 1325:71–79
Struck D., Hoekstra D., Pagano R. 1981. Use of resonance energy transfer to monitor membrane fusion. Biochemistry 20:4093–4198
Subramanian M., Holopainen J.M., Paukku T., Eriksson O., Huhtaniemi I., Kinnunen P.K. 2000. Characterization of three novel cationic lipids as liposomal complexes with DNA. Biochim. Biophys. Acta 1466:289–305
Tarahovsky Y.S., Koynova R., MacDonald R.C. 2004. DNA release from lopoplexes by anionic lipids: correlation with lipid mesomorphism, interfacial curvature, and membrane fusion. Biophys. J. 87:1054–64
Wasan E.K., Harvie P., Edwards K., Karlsson G., Bally M.B. 1999. A multi-step assay to model structural changes in cationic lipoplexes used for in vitro transfection. Biochim. Biophys. Acta 1461:27–46
Wattiaux R., Jadot M., Warnier-Pirotte M.T., Wattiaux-De Coninck S. 1997. Cationic lipids destabilize lysosomal membrane in vitro. FEES Lett. 417:199–202
Williamson P., Schlegel R.A. 2002. Transbilayer phospholipid movement and the clearance of apoptotic cells. Biochim. Biophys. Acta 1585:53–63
van der Woude I., Visser H.W., ter Beest M.B., Waganaar A., Ruiters M.H., Engberts J.B., Hoekstra D. 1995. Parameters influencing the introduction of plasmid DNA into cells by the use of synthetic amphiphiles as a carrier system. Biochim. Biophys. Acta 1240:34–40
Wrobel I., Collins D. 1995. Fusion of cationic liposomes with mammalian cells occurs after endocytosis. Biochim. Biophys. Acta 1235:296–304
Xu Y., Szoka F.C. Jr. 1996. Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry 35:5616–5623
Zelpati O., Szoka F.C. Jr. 1996. Mechanism of oligonucleotide release of cationic liposomes. Proc. Natl. Acad. Sci. USA 93:11493–11498
Zou Y., Zong G., Ling Y.H., Perez-Soler R. 2000. Development of cationic liposomes formulations for intratracheal gene therapy of early lung cancer. Cancer Gene Ther. 7:683–696
Zuhorn I.S., Hoekstra D. 2002. On the mechanism of cationic amphiphile-mediated transfection. To fuse or not to fuse: is that a question ? J. Membrane Biol. 189:167–179
Zuidam N.J., Barenholz Y. 1997. Electrostatic parameters of cationic liposomes commonly used for gene delivery as determined by 4-heptadecyl-7-hydroxycoumarin. Biochim. Biophys. Acta 1329:211–222
Acknowledgments
The study was supported by grant No. 3P04B01325 from the State Committee for Scientific Research (KBN), Poland.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Stebelska, K., Dubielecka, P. & Sikorski, A. The Effect of PS Content on the Ability of Natural Membranes to Fuse with Positively Charged Liposomes and Lipoplexes. J Membrane Biol 206, 203–214 (2005). https://doi.org/10.1007/s00232-005-0793-0
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/s00232-005-0793-0