Liposome Immobilization on Cross-linked Polymer Gel by In Situ Formation of Cleavable Covalent Bonds

Abstract

Immobilization of liposomes onto chemically modified Sephacryl gel particles by in situreaction between liposome-incorporated thiols and mercapto moieties on the gel to form disulfide linkages was investigated. For the immobilization, both the mercapto moieties and the incorporated thiol were essential. The immobilization occurred upon coincubation of the modified liposomes with the modified gel for 48 hours. Once immobilized, no spontaneous detachment of the immobilized liposomes was observed. The degree of immobilization depended on both the thiol content and the ratio of the liposomes to the gel partilces. In a typical immobilization with 25mol% 1-octanethiol, 82% of the liposomal phosphatidylcholine in the system was found to be associated with the gel. By decreasing the ratio of the liposomes to gel it was possible to bring the immobilization close to quantitative one. Among the three different thiols examined (1-octanethiol, 1-hexadecanethiol and thiocholesterol), the extent of the immobilization was slightly higher with thiocholesterol than the alkanethiols. The immobilized liposomes were detached from the gel with dithiothreitol. Approximately 60% of the fluorescent dextran derivative encapsulated in the liposomes was retained throughout the immobilization-detachment process. The gel left after the detachment remained active for immobilizing a fresh batch of thiol-liposomes.

Keywords

liposomes Sephacryl S-1000 disulfide linkages thiols FITC-dextran dithiothreitol hydrophobized gel

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References

Casey, R.P. (1984). Membrane Reconstitution of the Energy-conserving Enzymes of Oxidative Phosphorylation , Biochim. Biophys. Acta, 768: 319–347 .

Jain, M.K. and Zakim, D. (1987). The Spontaneous Incorporation of Proteins into Preformed Bilayers , Biochim. Biophys. Acta, 906: 33–68 .

Felnerova, D. , Viret, J.-F. , Glueck, R. and Moser, C. (2004). Liposomes and virosomes as delivery systems for antigens, nucleic acids and drugs , Curr. Opin. Biotechnol., 15: 518–529 .

Sandberg, M. , Lundahl, P. , Greijer, E. and Belew, M. (1987). Immobilization of Phospholipid Vesicles on Alkyl Derivatives of Agarose Gel Beads , Biochim. Biophys. Acta, 924: 185–192 .

Yang, Q. , Wallsten, M. and Lundahl, P. (1988). Immobilization of Phospholipid Vesicles and Protein-lipid Vesicles Containing Red Cell Membrane Proteins on Octylderivatives of Large-pore Gels , Biochim. Biophys. Acta, 938: 243–256 .

Lundahl, P. and Yang, Q. (1991). Liposome chromatography: liposomes immobilized in gel beads as a stationary phase for aqueous column chromatography , J. Chromatogr., 544: 283–304 .

Yang, Q. , Liu, X.-Y. , Yoshimoto, M. , Kuboi, R. and Miyake, J. (1999). Covalent Immobilization of Unilamellar Liposomes in Gel Beads for Chromatography , J. Anal. Biochem., 268: 354–362 .

Lundahl, P. , Zeng, C.-M. , Haegglund, C.L. , Gottschalk, I. and Greijer, E. (1999). Chromatographic Approaches to Liposomes, Proteoliposomes and Biomembrane Vesicles , J. Chromatogr. B, 720: 103–120 .

Liu, X.-Y. , Nakamura, C. , Yang, Q. , Kamo, N. and Miyake, J. (2002). Immobilized Liposome Chromatography to Study Drug-membrane Inteactions: Correlations with Drug Absorption in Humans , J. Chromatogr. A, 961: 113–118 .

10.

Yang, Q. , Liu, X.-Y. , Ajiki, S. , Hara, M. , Lundahl, P. and Miyake, J. (1998). Avidin-biotin Immobilization of Unilamellar Liposomes in Gel Beads for Chromatographic Analysis of Drug-membrane Partitioning , J. Chromatogr. B, 707: 131–141 .

11.

Liu, X.-Y. , Yang, Q. , Hara, M. , Nakamura, C. and Miyake, J. (2001). A Novel Chromatographic Solid Support with Immobilized Unilamellar Liposomes for Model Analysis of Solute-membrane Interaction: Comparison with Analysis Using Immobilized Artificial Membranes and Free Lipsomal Membranes , Mater. Sci. Eng., C, 17: 119–126 .

12.

Yoshimoto, M. , Kuboi, R. , Yang, Q. and Miyake, J. (1998). Immobilized Liposome Chromatography for Studies of Protein-membrane Interactions and Refolding of Denatured Bovine Carbonic Anhydrase , J. Chromatogr. B, 712: 59–71 .

13.

Khaleque, Md. A. , Oho, T. , Okumura, Y. and Mitani, M. (2000). Controlled Detachment of Immobilized Liposomes on Polymer Gel Support , Chem. Lett., 1402–1403 .

14.

Khaleque, Md. A. , Okumura, Y. , Yabushita, S. and Mitani, M. (2004). Detachable Immobilization of Liposomes on Polymer Gel Particles , Coll. and Surf. B, 37: 35–42 .

15.

Khaleque, Md. A. , Okumura, Y. , Yabushita, S. and Mitani, M. (2003). Liposome Immobilization on Polymer Gel Particles by In Situ Formation of Covalent Linkages , Chem. Lett., 32: 416–417 .

16.

Grassetti, D.R. and Nurray Jr, J.F. (1967). Determination of Sulfhydryl Groups with 2,2 or 4,4 -dithiodipyridine , Arch. Biochem. Biophys., 119: 41–49 .

17.

MacDonald, R.C. , MacDonald, R.I. , Menco, B.P. , Takeshita, K. , Subbarao, N.K. and Hu, L. (1991). Small-volume Extrusion Apparatus for Preparation of Large, Unilamellar Vesicles , Biochim. Biophys. Acta, 1061: 297–303 .

18.

Cleland, W.W. (1964). Dithiothreitol, a New Protective Reagent for SH Groups , Biochemistry, 3: 480–482 .