近年來，本實驗室建立了對於Deinococcus radiodurans IR表面層的two-dimensional crystalline cell surface layer的結構以及對於其自組裝特性有了更進一步的研究與分析，同時亦可快速有效率的純化出此蛋白質( S-layer fraction 簡稱 SLF )；由於SLF可以抵抗強酸以及高滲透壓…等的壓迫之故，又基於目前已在臨床上大量應用的微脂體仍有許多缺點，因此本實驗室本著改善微脂體本身穩定性不足的這方面缺點，利用SLF與微脂體進行membrane interaction，開發出可以快速在微脂體表面進行膜融合的一種表面結晶蛋白，這個新合成的新型藥物載體優點不只在於利用正負電荷進行SLF與微脂體的貼附作用，甚至兩者已經藉由膜融合效應的作用讓兩者緊緊的結合在一起，因此不必再擔心穩定性方面的問題；在實驗的驗證方面，我們利用了confocal laser scanning microscopy和Zeta-Sizer 針對SLF以及SLL ( S-layer fused with liposome )的尺寸大小、形態以及表面電位的變化做了詳細的分析以及針對於耐酸、鹼以及在於高、低滲透壓做了比較，發現微脂體原本表面電位為+60~+100，SLF表面電位為+70~+100，而進行膜融合之後的SLL表面電位接近電中性；另一方面，微脂體在於酸鹼溶液以及高、低滲透壓溶液都與原本的型態以及尺寸大小都與原本 (pH7，等滲透壓)有明顯的不同，而SLF以及SLL在於不同環境下的型態與尺寸大小皆與原本相差不遠。最後我們也利用SLL以及微脂體包覆alcohol dehydrogense 置於5%、15%、40%、60%酒精溶液中，發現微脂體包覆前後的溶液並沒有明顯的變化，而SLL包覆後則發現部分酒精消失而產生乙醛，利用gas chromatography可以很明顯的推測出此結論，我們推測SLL表面的結晶蛋白提供了完整的基質可以連接其他的功能性分子以及做為一個通透性的channel，提升藥物輸送的控制以及釋放率。此外，SLL的螢光特性亦可當作生物感應器或是診斷用的定量標記 (quantitative makers )，亦增添了許多SLL的應用價值。

In recent years, our lab set up a condition for isolated subunits of the crystalline cell surface layer (S-layer) protein from Deinococcus radiodurans IR and purify this protein efficiently. Up to now, we developed a new method for high-yield preparation of S-layer fused with liposome (SLL) and could improve liposome instability. We used simple and convenient method to make SLF (S-layer fraction) fuse with liposome. Isolated subunits of the negatively charged SLF were fused with positively charged liposome not only they fuse together dependent on electric potential，but also they take advantage of lipid membrane fusion effect. We can observe SLF and SLL under confocal laser scanning microscopy and Zeta-Sizer .Furthermore，we had investigated and analyzed the stabilizing effect of SLL towards acid、base or osmotic solutions by Zeta-Sizer. We have demonstrated the SLL is more stable than plain liposome and SLL could coat alcohol dehydrogense and control it release by gas chromatography. The high stability of SLL and possibility for immobilizing biologically active molecules on the crystalline array may offer potential in various different SLL applications.

1.Thornley, M.J.,Horne,R.W. & Glauert,A.M.. 1965. The fine structure of Micrococcus radiodurans. Arch. Mikrobiol. 51: 267-289.
2.Sleytr, U.B., Kocur,M., Glauert, A.M. & Thornley M.J.1973. A study by freeze-etching of the fine structure of Micrococcus radiodurans.Arch.Mikrobiol.94: 77-87.
3.Baumeister,W. &Barth,M., Hegerl,R. 1986. Three-dimensional structure of the regular surface layer (HPI layer) of Deinococcus radiodurans . J. Mol. Biol.187:241-253.
4.Sleytr, U. B., & P. Messner. 1983. Crystalline surface layers on bacteria. Annu. Rev. Microbiol. 37:331-339.
5.Thornley, M. J.A. M. Glauert, & U. B. Sleytr. 1974. Structure and assembly of bacteria surface layers composed of regular arrays of subunits. Phil. Trans. R. Soc. Lond. B. 268:147-153.
6.Glauert, A. M. & M. J. Thornley. 1969. The topography of the bacterial cell wall. A. Rev. Microbiol. 23:159-198
7.Holt, S. C. & E. R. Leadbetter. 1969. Comparative ultrastructure of selected aerobic spore-forming bacteria: a freeze-etching study. Bact. Rev. 33:346-378
8.Sleytr, U. B., S. Margit, P. Dietmar, & S. Bernhard. 2001. Characterization and use of crystalline bacterial cell surface layers. Progress in Surface Science. 68:231-278.
9.Pum, D., A. Neubauer, E. Gyoervary, M. Sara, U. B. Sleytr. 2000. S-layer proteins as basic building blocks in a biomolecular construction kit. Nanotechnology. 11: 100-107
10.Glaurt, A. M., & M. J. Thornley. 1973. Self-assembly of a surface component of a bacterial outer membrane. John Innes Symp. 1: 297-305
11.Sleytr, U. B. 1976. Self-assembly of the hexagonally and tetragonally arranged subunits of bacterial surface layers and their reattachment to cell walls. J. Ultrastruct. Res. 55: 360-377
12.Baumeister, W. & Kubler, O. 1978. Topographic study of the cell surface of Micrococcus radiodurans. Proc. Natl. Acad. Sci. 75: 5525-5528
13.Reinhoudt, D. N. & M. Crego-Calama. 2002. Synthesis beyond the molecule. Science. 295: 2403-2407.
14.Chu, S., S. Cavaignac, J. Feutrier, B. M. Phipps, M. Kostrzynska, W. W. Kay & T. J. Trust. 1991. Structure of the tetragonal surface virulence array protein and gene of Aeromonas salmonicida. J. Biol .Chem. 266: 15258-15265
15.Sara, M. & U. B. Sleytr. 1987. Molecular sieving through S-layers of Bacillus stearothermophilus strains. J. Bacteriol. 169: 4092-4098.
16.Brechtel, E., M. Matuschek, A. Hellberg, E. M. Egelseer, R. Schmid, and H. Bahl. 1999. Cell wall of Thermoanaerobacterium thermosulfurigenes EM1: isolation of its components and attachment of the xylanase XynA. Arch. Microbiol. 171: 159-165
17.Engelhardt,H. & J. Peters. 1998. Structure research on surface layers: a focus on stability, surface layer homology domains, and surface layer-cell wall interaction. J. Struct. Biol. 124: 276-302.
18.Dietmar, P. & U. B. Sleytr. 1999. The application of bacterial S-layers in molecular nanotechnology. Nanotechnology. 17: 8-12.
19.Sleytr, U. B.,Gyorvary E. & Pum. D. 2003. Crystallization of S-layer protein lattices on surfaces and interfaces. Prog. Organ. Coat. 47: 279-287
20.Work, E. & H. Griffiths. 1968. Morphology and chemistry of cell walls of Micrococcus radiodurans. J. Bacteriol. 95: 641-657
21.Sleytr, U. B., M. Kocur, A. M. Gluert & M. J. Thornley. 1973. A study of freeze-etching of the fine structure of Micrococcus radiodurans. Arch. Mikrobiol. 94: 77-87
22.Lancy, JR. & R. G. E. Murray. 1978. The envelope of Micrococcus radiodurans.: isolation , purification, and preliminary, analysis of the cell wall layers. Can. J. Microbiol. 24: 162-176.
23.Thompson, B. G & R. G. E. Murray. 1982. The association of the surface array and the outer membrane of Deinococcus radioduans. Can. J. Microbiol. 28: 1081-1088.
24.U. B. Sleytr, E. Gyorvary & D. Pum. 2003. Crystallization of S-layer protein lattices on surface and interfaces. Progress in Organic Coatings. 47: 279-289.
25.A. S. Blawas & W. M. Reichert. 1998. Protein pattening. Biomaterials 19: 595-609.
26.S. Dieluweit, D. Pum & U. B. Sleytr. 1997. Formation of a gold superlattice on an S-layer with square lattice symmetry. Supramolecular Science. 5: 15-19
27.M. Sara & U. B. Sleytr. 1996. Biotechnology and biomimetic with crystalline bacterial cell surface layers (S-layers). Micron. 27:141-156.
28.Elizabeth, W & Hilary G. 1968. Morphology and chemistry of cell walls of Micrococcus radiodurans. J. Bacteriol. 95: 641-657.
29.Huang, Y. & Amderson, R. 2002. Enhanced immune protection by a liposome-encapsulated recombinant respiratory syncytial virus (RSV) vaccine using immunogenic lipids from Deinococcus radiodurans. Vaccine. 20: 1586-1592
30.Moussaoui, N., Cansell, M. & Denizot. A. 2002. Marinosomes, marine lipid based liposomes: physical characterization and potential application in cosmetics. Int. J.Pharmaceutics. 242: 361-365.
31.Hui, S. W., Kuhl, T. L. & Guo, Y. Q. & Israelachvili, J. 1999. Use of poly(ethylene glycol) to control cell aggregation and fusion . Coll. Surf. B: Biointerf. 14: 213-222.
32. A.D. Bangham, M.M. Standish, J.C. Watkins, J. Mol. Biol., 13, 238-252, 1965.
33. G.Gregoriadis, New Engl. J. Med., 295, 704-710; 765-770, 1976.
34. R.R.C. New, ed. Liposomes: a practical approach (1990), Oxford.
35. D.D. Lasic, Science & Medicine, 3, 34-43, 1996.
36. M.C.Woodle et al, Proc Int. Symp Control. Rel. Bioact. Mater., 77-78, CRS (1990).
37. S. Zalipsky et al, Bioconjugate Chem., 8, 111-118, 1997.
38. G. Gregoriadis, ed. Liposome Technology, 1, (1993) CRC.
39. H. Talsma and D.J.A. Crommelin, Pharmaceutical Technology, 96-106 (Oct. 1992)
40. J.P. Adler-Moore, R.T. Profitt, J. Liposome Res. 3., 429-450, 1993.
41. S. de Marie et al, J. Antimicrobial Chemotherapy, 33, 907-916, 1994.
42. A.S. Janoff et al, J. Liposome Res., 3, 451-471, 1993.
43. M.C. Woodle and G. Storm, eds. Long Circulating Liposomes: Old drugs, New Therapeutics, (1998) Springer-Verlag.
44. E.A. Forssen and M.E. Ross, J. Liposome Res. 4, 481-512, 1994.
45. W. Wang, J. Drug Targeting, 4, 195-232, 1996.
46. K.B. Choudhari et al, J. Microencapsulation, 11, 319-325, 1994.
47. A. Arien et al, Life Science, 53, 1279-1290, 1993
48. M. Fukunaga et al, Horm. metab. Res., 23, 166-167, 1991.
49. G. Storm et al, Adv. Drug Deliv. Rev., 24, 225-231, 1997.
50. F.H.D. Roerdink & A.M.Kroon eds., Drug Carrier Systems (1989), wiley.
51. F. Puisieux, P. Couvreur, J. Delattre & J.-P. Devissaguet, Liposomes : New Systems and New Trends in their Application (1995) Editions de Sante.
52. D.D. Lasic and N.S. Templeton Adv. Drug Deliv. Rev., 20, 221-226, 1996.
53. R.I. Mahato et al, Pharm. Res., 14, 853-859, 1997.
54. S. Li and L. Huang, J.Liposome Res., 8, 239-250, 1998.
55. P.A.M. Peeters et al, Biochim Bio phys. Acta., 981, 269-276, 1989.
56. G. Gregoriads et al, Eds., New-Generation Vaccines : The role of Basic Immunology (1993), Plenum.
57. B.A, Hart et al. FEBS Lett., 409, 91-95, 1997.
58. G. Gregoriads, Immunol. Today, 11, 89-97, 1990.
59. J.J. Bergers et al, J. Liposome Res., 6, 339-355, 1996.
60. N.K.Childers et al, Reg. Immunol., 3, 8-16, 1990.
61. A. L. Klibanov and L Huang, J. Liposome Res. 2, 321-334, 1992.
62. M.H. Vingehoeds et al. Immunomethods, 4, 259-272, 1994
63. Barenholz, Y. 2001. Liposome application: problems and prospects. Curr. Opin. Colloid Interfacial Sci. 6: 66-77.
64. Vemuri, S. & Rhodes, C. T. 1995. Preparation and characterization of liposomes as therapeutic delivery systems: a review. Pharm. Acta Helv. 70: 95-111.
65. Ishihara, K., Tsujino, R. & Hamada, M. 2002. Stabilized liposomes with phospholipids polymers and their interactions with blood cells. Coll. Surf. B: Biointerf. 25: 325-333.
66. U. B. Sleytr & Sara.M. 1995. Liposomes coated with crystalline bacterial cell surface protein (S-layer) as immobilization structures for macromolecules. Biochimica et Biophysica Acta , 1235: 263-269.
67. Margit Sara & Madar. C. 2000. S-layer-coated liposomes as a versatile system for entrapping and binding target molecules. Biochimica et Biophysica Acta , 1463 ,142-150.
68. Madar, C.,Kupcu, S., Sara. M & Sleytr, U. B. 1999. Stabilizing effect of an S-layer on liposomes towards thermal or mechanical stress. Biochem Biophys Acta. 1418: 106-116.
69. Schuster, B. P., Gufler. C, Pum, D & Sleytr, U. B. 2004. S-layer protein as supporting scaffoldings for functional lipid membranes. IEEE Trans. Nanobio. 3: 16-21.
70. Manuel, A., Coelho, N., Alessandra. G & Helmuth Mohwald. 2004. Nanocapsules with functionalized surfaces and walls. IEEE trans. Nanobio. 3: 3-5.
71. Manuela, V. & Maya. S. 2002. Designing of “intelligent” liposomes. J. Cell. Mol. Med. 6: 465-474.
72. Sprott. G. D., Dicaire, C. J., Gurnani, K. & Deschatelets, L. A. 2004. Liposome adjuvants prepared from the total polar lipids of Haloferax volcanii, Planococcus spp. and Bacillus firmus differ in ability to elicit and sustain immune response. Vaccine. 22: 2154-2162.
73. Nicola, I., Seta, K., Gerald, M. & Sigrid, K. 2004. A functional cheimaeric S-layer-enhanced green fluorescence protein to follow the uptake of S-layer-coated liposomes into eukaryotic cells. Biochem. J. 379: 441-448.
74. Ji, E. K., Ahn, D. J. & Kim, J. M. 2003. The fluorescence polydiacetylene liposome. Bull. Korean Chem. Soc. 24: 667-670.
75. Sleytr, U. B. & Messner, P., Pum. D. 1999. Crystalline bacterial cell surface layers (S-layers) : From supramolecular cell structure to biomimetics and nanotechnology. Angew. Chem. Int. Ed. 38: 1034-1054.
76. Ruysschaert, T. & Germain, M. 2004. Liposome-based nanocapsules. IEEE Trans. Nanobio. 3: 49-55.