本研究利用三種不同的離子對雙親分子為材料，分別為DeTMA-DS (decyltrimethylammonium-dodecylsulfate)、DeTMA-TS (decyltrimethylammonium-tetradecylsulfate) 和DTMA-DS (dodecyltrimethylammonium-dodecylsulfate)，在含有20%酒精的緩衝溶液(tris buffer)中以半自發的製程製備出類乙醇體陰陽離子液胞(ethosome-like catanionic vesicles)，探討在不同溫度下，膽固醇的添加對於液胞雙層膜流動性的影響，並進一步以水溶性螢光劑CF (5[6]-carboxyfluorescein)作為探測分子，探討膽固醇濃度對於包覆效率的影響，以及對應於雙層膜特性的溫度下，膽固醇對藥物的釋放行為。
由實驗結果顯示，在DeTMA-DS與DeTMA-TS的液胞系統，15oC~40oC之間添加膽固醇，均有助於降低雙層膜的流動性，而流動性的下降，使包覆螢光劑CF的包覆效率提升，釋放速率下降。另外，溫度的提升，增加了雙層膜的流動性，反映到包覆/釋放行為上，則可以看到包覆效率下降，釋放速率增加。但是對於DTMA-DS的液胞系統，卻有相反的影響，20oC~50oC之間添加膽固醇反而使雙層膜的流動性增加，溫度的提升，對於雙層膜的流動性沒有太大影響，然而因為在含有CF藥物時，無法形成穩定的液胞，所以無法觀察其包覆/釋放的行為。此外，在釋放行為中亦可以看到，溫度的提升，對於DeTMA-TS液胞系統之釋放速率的影響較DeTMA-DS液胞系統來的明顯，因為DeTMA-TS結構的不對稱性較高，所形成之雙層膜的空缺較多，所以溫度對於其流動性的影響較DeTMA-DS液胞系統的影響還大，因此DeTMA-TS的液胞系統有較高的釋放速率。以上結果可望提供此新型材料在藥物傳輸載體的應用能有更進一步的推進。

In this work, using three different ion-pair-amphiphiles (IPAs) as new raw materials (DeTMA-DS, DeTMA-TS, DTMA-DS) prepared ethosome-like catanionic vesicles containing 20% ethanol in buffer solution by a semispontaneous process to investigate the cholesterol effect on bilayer membrane fluidity at different temperatures. Furthermore, the ethosome-like catanionic vesicles were used to encapsulate 5[6]-carboxyfluorescein (CF) in order to study the encapsulation efficiency and the release behavior.
For DeTMA-DS and DeTMA-TS systems, the experiment results showed that the vesicular membrane fluidity decreased with the increase of cholesterol concentration at 15oC~40oC and increased with the increase of temperature. And the bilayer membrane fluidity corresponded to the encapsulation/release behavior, show that the lower fluidity can get higher encapsulation efficiency and slower release rate. On the other hand, for DTMA-DS systems, the vesicular membrane fluidity increased with the increase of cholesterol concentration at 20℃~50℃ and was no significant change with the increase of temperature. However, the CF molecules were unable to be encapsulated. As to the release rate for DeTMA-TS system was faster than the DeTMA-DS system due to the poorer symmetry for former.

[1] D.D. Lasic, The mechanism of vesicle formation, Biochemical Journal, 256 1-11, 1988.
[2] A. Jesorka, O. Orwar, Liposomes: technologies and analytical applications, Annual review of analytical chemistry, 1, 801-832, 2008.
[3] R.R.C.E. New, Liposomes: A practical approach, 1990.
[4] M.E. Rosoff, Vesicles, Marcel Dekker, New York, 1996.
[5] 陳炳宏, 馮思慎, 微脂粒在藥物輸送的應用, 中國化學工程學會會刊, 47, 68-84, 2000.
[6] I. Ahmad, M. Longenecker, J. Samuel, T.M. Allen, Antibody-targeted delivery of doxorubicin entrapped in sterically stabilized liposomes can eradicate lung cancer in mice, Cancer research, 53, 1484-1488, 1993.
[7] D.D. Lasic, Liposomes in gene delivery, 1997.
[8] M. Valero, M.M. Velázquez, Effect of the addition of water-soluble polymers on the interfacial properties of aerosol OT vesicles, Journal of colloid and interface science, 278, 465-471, 2004.
[9] J.I. Briz, M.M. Velázquez, Effect of water-soluble polymers on the morphology of aerosol OT vesicles, Journal of colloid and interface science, 247, 437-446, 2002.
[10] M.M. Velázquez, M. Valero, F. Ortega, J.B. Rodríguez González, Structure and size of spontaneously formed aggregates in aerosol OT/PEG mixtures: Effects of polymer size and composition, Journal of colloid and interface science, 316, 762-770, 2007.
[11] M.M. Velázquez, M. Valero, F. Ortega, Spontaneous vesicles modulated by polymers, Polymers, 3, 1255-1267, 2011.
[12] E.W. Kaler, A.K. Murthy, B.E. Rodriguez, J. Zasadzinski, Spontaneous vesicle formation in aqueous mixtures of single-tailed surfactants, Science, 245, 1371-1374, 1989.
[13] 鍾依玲, 陰/陽離子液胞自發性形成之探討, 國立成功大學化學工程學系碩士論文, 2002.
[14] C. Tondre, C. Caillet, Properties of the amphiphilic films in mixed cationic/anionic vesicles: a comprehensive view from a literature analysis, Advances in colloid and interface science, 93, 115-134, 2001.
[15] K.-C. Wu, Z.-L. Huang, Y.-M. Yang, C.-H. Chang, T.-H. Chou, Enhancement of catansome formation by means of cosolvent effect: Semi-spontaneous preparation method, Colloids and surfaces A: physicochemical and engineering aspects, 302, 599-607, 2007.
[16] 黃鉦琳, 帶電陰陽離子液胞的形成及其膠化之研究, 國立成功大學化學工程學系碩士論文, 2007.
[17] Y.-M. Yang, K.-C. Wu, Z.-L. Huang, C.-H. Chang, On the stability of liposomes and catansomes in aqueous alcohol solutions, Langmuir, 24 , 1695-1700, 2008.
[18] 柯政遠, 乙醇體及陰陽體的製備及其包覆/釋放行為之探討, 國立成功大學化學工程學系碩士論文, 2008.
[19] Y.-S. Liu, C.-F. Wen, Y.-M. Yang, Development of ethosome-like catanionic vesicles for dermal drug delivery, Journal of the taiwan institute of chemical engineers, 2012.
[20] V.P. Torchilin, Recent advances with liposomes as pharmaceutical carriers, Nature reviews drug discovery, 4, 145-160, 2005.
[21] M. Elsayed, O.Y. Abdallah, V.F. Naggar, N.M. Khalafallah, Lipid vesicles for skin delivery of drugs: reviewing three decades of research, International journal of pharmaceutics, 332, 1-16, 2007.
[22] G. Cevc, Lipid vesicles and other colloids as drug carriers on the skin, Advanced drug delivery reviews, 56, 675-711, 2004.
[23] G. Cevc, G. Blume, Lipid vesicles penetrate into intact skin owing to the transdermal osmotic gradients and hydration force, Biochimica et biophysica acta (BBA)-biomembranes, 1104, 226-232, 1992.
[24] E. Touitou, N. Dayan, L. Bergelson, B. Godin, M. Eliaz, Ethosomes—novel vesicular carriers for enhanced delivery: characterization and skin penetration properties, Journal of controlled release, 65, 403-418, 2000.
[25] W.R. Hargreaves, D.W. Deamer, Liposomes from ionic, single-chain amphiphiles, Biochemistry, 17, 3759-3768, 1978.
[26] H. Trommer, R. Neubert, Overcoming the stratum corneum: the modulation of skin penetration, Skin pharmacology and physiology, 19, 106-121, 2006.
[27] J.N. Israelachvili, Intermolecular and surface forces, 1992.
[28] R.J. Stokes, D.F. Evans, Fundamentals of interfacial engineering, 1996.
[29] H. Fukuda, K. Kawata, H. Okuda, S.L. Regen, Bilayer-forming ion pair amphiphiles from single-chain surfactants, Journal of the american chemical society, 112, 1635-1637, 1990.
[30] C.L. Chien, S.J. Yeh, Y.M. Yang, C.H. Chang, J. R. Maa, Formation and encapsulation of catanionic vesicles, Journal of the chinese colloid and interface society, 24, 31-45, 2002.
[31] J.A. Barry, K. Gawrisch, Direct NMR evidence for ethanol binding to the lipid-water interface of phospholipid bilayers, Biochemistry, 33, 8082-8088, 1994.
[32] M. Patra, E. Salonen, E. Terama, I. Vattulainen, R. Faller, B.W. Lee, J. Holopainen, M. Karttunen, Under the influence of alcohol: the effect of ethanol and methanol on lipid bilayers, Biophysical journal, 90, 1121-1135, 2006.

[33] H.I. Petrache, T. Zemb, L. Belloni, V.A. Parsegian, Salt screening and specific ion adsorption determine neutral-lipid membrane interactions, Proceedings of the national academy of sciences, 103, 7982-7987, 2006.
[34] A. McLaughlin, W.-K. Eng, G. Vaio, T. Wilson, S. McLaughlin, Dimethonium, a divalent cation that exerts only a screening effect on the electrostatic potential adjacent to negatively charged phospholipid bilayer membranes, The journal of membrane biology, 76, 183-193, 1983.
[35] E. Evans, D. Needham, Physical properties of surfactant bilayer membranes: thermal transitions, elasticity, rigidity, cohesion and colloidal interactions, Journal of physical chemistry, 91, 4219-4228, 1987.
[36] D. Grasso, K. Subramaniam, M. Butkus, K. Strevett, J. Bergendahl, A review of non-DLVO interactions in environmental colloidal systems, reviews in environmental science and biotechnology, 1, 17-38, 2002.
[37] J. Sabın, G. Prieto, J. Ruso, R. Hidalgo-Alvarez, F. Sarmiento, Size and stability of liposomes: A possible role of hydration and osmotic forces, The european physical journal E, 20, 401-408, 2006.
[38] M.F. Brown, R.L. Thurmond, S.W. Dodd, D. Otten, K. Beyer, Elastic deformation of membrane bilayers probed by deuterium NMR relaxation, Journal of the american chemical society, 124, 8471-8484, 2002.
[39] J.Y. Walz, E. Ruckenstein, Comparison of the van der waals and undulation interactions between uncharged lipid bilayers, The journal of physical chemistry B, 103 7461-7468, 1999.
[40] T.J. McIntosh, A.D. Magid, S.A. Simon, Cholesterol modifies the short-range repulsive interactions between phosphatidylcholine membranes, Biochemistry, 28, 17-25, 1989.
[41] G. Cevc, Hydration force and the interfacial structure of the polar surface, Journal of the Chemical Society, Faraday Transactions, 87, 2733-2739, 1991.
[42] X. Bin, J. Lipkowski, Electrochemical and PM-IRRAS studies of the effect of cholesterol on the properties of the headgroup region of a DMPC bilayer supported at a Au (111) electrode, The journal of physical chemistry B, 110, 26430-26441, 2006.
[43] T. Róg, M. Pasenkiewicz-Gierula, Cholesterol effects on the phospholipid condensation and packing in the bilayer: a molecular simulation study, FEBS letters, 502, 68-71, 2001.
[44] W.-C. Hung, M.-T. Lee, F.-Y. Chen, H.W. Huang, The condensing effect of cholesterol in lipid bilayers, Biophysical journal, 92, 3960-3967, 2007.
[45] 歐陽鐘汕, 劉寄星, 從肥皂泡到液晶生物膜, 牛頓,1995.
[46] G.F. Fuller, D. Shields, Molecular basis of medical cell biology, 1998.
[47] F. Severcan, Ü. Baykal, Ş. Süzer, FTIR studies of vitamin E-cholesterol-DPPC membrane interactions in CH2 region, Fresenius' journal of analytical chemistry, 355 415-417, 1996.
[48] S. Bhattacharya, S. Haldar, The effects of cholesterol inclusion on the vesicular membranes of cationic lipids, Biochimica et biophysica acta (BBA)-biomembranes, 1283, 21-30, 1996.
[49] S. Bhattacharya, S. Haldar, Interactions between cholesterol and lipids in bilayer membranes. Role of lipid headgroup and hydrocarbon chain–backbone linkage, Biochimica et biophysica acta (BBA)-biomembranes, 1467, 39-53, 2000.
[50] I. Fournier, J. Barwicz, M. Auger, P. Tancrède, The chain conformational order of ergosterol-or cholesterol-containing DPPC bilayers as modulated by Amphotericin B: a FTIR study, Chemistry and physics of lipids, 151, 41-50, 2008.
[51] D.A. Mannock, M.Y. Lee, R.N. Lewis, R.N. McElhaney, Comparative calorimetric and spectroscopic studies of the effects of cholesterol and epicholesterol on the thermotropic phase behaviour of dipalmitoylphosphatidylcholine bilayer membranes, Biochimica et biophysica acta (BBA)-biomembranes, 1778, 2191-2202, 2008.
[52] D.A. Mannock, R.N. Lewis, R.N. McElhaney, A calorimetric and spectroscopic comparison of the effects of ergosterol and cholesterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes, Biochimica et Biophysica Acta (BBA)-biomembranes, 1798, 376-388, 2010.
[53] C. Silva, F.J. Aranda, A. Ortiz, V. Martínez, M. Carvajal, J.A. Teruel, Molecular aspects of the interaction between plants sterols and DPPC bilayers: An experimental and theoretical approach, Journal of colloid and interface science, 358, 192-201, 2011.
[54] N. Yellin, I.W. Levin, Hydrocarbon chain disorder in lipid bilayers: Temperature dependent ram an spectra of 1, 2-diacyl phosphatidylcholine-water gels, Biochimica et biophysica acta (BBA)-lipids and lipid metabolism, 489, 177-190, 1977.
[55] R. Mendelsohn, M.A. Davies, H.F. Schuster, Z. Xu, R. Bittman, CD2 rocking modes as quantitative infrared probes of one-, two-, and three-bond conformational disorder in dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylcholine/cholesterol mixtures, Biochemistry, 30, 8558-8563, 1991.
[56] K. Weisz, G. Gröbner, C. Mayer, J. Stohrer, G. Kothe, Deuteron nuclear magnetic resonance study of the dynamic organization of phospholipid/cholesterol bilayer membranes: molecular properties and viscoelastic behavior, Biochemistry, 31, 1100-1112, 1992.
[57] S. Bucak, B.H. Robinson, A. Fontana, Kinetics of induced vesicle breakdown for cationic and catanionic systems, Langmuir, 18, 8288-8294, 2002.
[58] O. Lopez, A. de la Maza, L. Coderch, C. Lopez-Iglesias, E. Wehrli, J.L. Parra, Direct formation of mixed micelles in the solubilization of phospholipid liposomes by Triton X-100, FEBS lett, 426, 314-318, 1998.
[59] C. Caillet, M. Hebrant, C. Tondre, Sodium octyl sulfate/cetyltrimethylammonium bromide catanionic vesicles: Aggregate composition and probe encapsulation, Langmuir, 16, 9099-9102, 2000.
[60] K. Iga, N. Hamaguchi, Y. Igari, Y. Ogawa, H. Toguchi, T. Shimamoto, Heat-specific drug release of large unilamellar vesicle as hyperthermia-mediated targeting delivery, International journal of pharmaceutics, 57, 241-251, 1989.
[61] B. Elorza, M. Elorza, G. Frutos, J. Chantres, Characterization of 5-fluorouracil loaded liposomes prepared by reverse-phase evaporation or freezing-thawing extrusion methods: study of drug release, Biochimica et biophysica acta (BBA)-biomembranes, 1153, 135-142, 1993.
[62] N. Berger, A. Sachse, J. Bender, R. Schubert, M. Brandl, Filter extrusion of liposomes using different devices: comparison of liposome size, encapsulation efficiency, and process characteristics, International journal of pharmaceutics, 223, 55-68, 2001.
[63] M. Glavas-Dodov, E. Fredro-Kumbaradzi, K. Goracinova, M. Simonoska, S. Calis, S. Trajkovic-Jolevska, A. Hincal, The effects of lyophilization on the stability of liposomes containing 5-FU, International journal of pharmaceutics, 291, 79-86, 2005.
[64] X. Xu, M.A. Khan, D.J. Burgess, A quality by design (QbD) case study on liposomes containing hydrophilic API: I. Formulation, processing design and risk assessment, International journal of pharmaceutics, 419 , 52-59, 2011.
[65] B. Maherani, E. Arab-tehrany, A. Kheirolomoom, V. Reshetov, M.J. Stebe, M. Linder, Optimization and characterization of liposome formulation by mixture design, Analyst, 137, 773-786, 2012.
[66] A. Sharma, U.S. Sharma, Liposomes in drug delivery: progress and limitations, International journal of pharmaceutics, 154, 123-140, 1997.
[67] 邱文昱, 陰陽離子液胞包覆油/水溶性藥物之行為探討, 成功大學化學工程學系碩博士班學位論文, 2012.
[68] P.J. Ryan, M.A. Davis, D.L. Melchior, The preparation and characterization of liposomes containing X-ray contrast agents, Biochimica et biophysica acta, 756, 106, 1983.
[69] S. Benita, P. Poly, F. Puisieux, J. Delattre, Radiopaque liposomes: effect of formulation conditions on encapsulation efficiency, Journal of pharmaceutical sciences, 73, 1751-1755, 1984.
[70] K. Taylor, G. Taylor, I. Kellaway, J. Stevens, The stability of liposomes to nebulisation, International journal of pharmaceutics, 58, 57-61, 1990.
[71] G. Puglisi, M. Fresta, C. La Rosa, C. Ventura, A. Panico, G. Mazzone, Liposomes as a potential drug carrier for citicoline (CDP-choline) and the effect of formulation conditions on encapsulation efficiency, Die pharmazie, 47(3), 211-215, 1992.
[72] G. Betageri, Liposomal encapsulation and stability of dideoxyinosine triphosphate, Drug development and industrial pharmacy, 19, 531-539, 1993.
[73] S. Kulkarni, G. Betageri, M. Singh, Factors affecting microencapsulation of drugs in liposomes, Journal of microencapsulation, 12, 229-246, 1995.
[74] T. Nii, F. Ishii, Encapsulation efficiency of water-soluble and insoluble drugs in liposomes prepared by the microencapsulation vesicle method, International journal of pharmaceutics, 298, 198-205, 2005.
[75] S.A. Agnihotri, K.S. Soppimath, G.V. Betageri, Controlled release application of multilamellar vesicles: a novel drug delivery approach, Drug delivery, 17, 92-101, 2010.
[76] B. Maherani, E. Arab-Tehrany, A. Kheirolomoom, V. Reshetov, M.J. Stebe, M. Linder, Optimization and characterization of liposome formulation by mixture design, Analyst, 137, 773-786, 2012.
[77] D.G. Fatouros, K. Hatzidimitriou, S. Antimisiaris, Liposomes encapsulating prednisolone and prednisolone–cyclodextrin complexes: comparison of membrane integrity and drug release, European journal of pharmaceutical sciences, 13, 287-296, 2001.
[78] L. Paasonen, T. Laaksonen, C. Johans, M. Yliperttula, K. Kontturi, A. Urtti, Gold nanoparticles enable selective light-induced contents release from liposomes, Journal of controlled release, 122, 86-93, 2007.
[79] P.L. Biotechnology, Ion exchange chromatography: principles and methods, Pharmacia LKB biotechnology, 1991.
[80] A. Manosroi, P. Wongtrakul, J. Manosroi, H. Sakai, F. Sugawara, M. Yuasa, M. Abe, Characterization of vesicles prepared with various non-ionic surfactants mixed with cholesterol, Colloids and surfaces B: biointerfaces, 30, 129-138, 2003.
[81] J. Xi, R. Guo, X. Guo, Interactions of hemoglobin with lecithin liposomes, Colloid and polymer science, 284, 1139-1145, 2006.
[82] W.-Y. Yu, Y.-M. Yang, C.-H. Chang, Cosolvent effects on the spontaneous formation of vesicles from 1: 1 anionic and cationic surfactant mixtures, Langmuir, 21, 6185-6193, 2005.
[83] 劉晏如, 微脂粒配方與血液中蛋白質交互作用及其穩定性之研究, 國立清華大學化學工程學系碩士論文, 2002.
[84] J. Weinstein, S. Yoshikami, P. Henkart, R. Blumenthal, W. Hagins, Liposome-cell interaction: transfer and intracellular release of a trapped fluorescent marker, Science, 195, 489-492, 1977.
[85] 温智芳, 經皮藥物傳輸用類乙醇體陰陽離子液胞之研發, 國立成功大學化學工程學系碩士論文, 2013.
[86] 邱俊瑋, 未發表的研究成果, 國立成功大學化學工程學系, 2013.
[87] 劉育姍, 陰陽離子液胞包覆維他命 E 醋酸酯之行為探討, 國立成功大學化學工程學系碩士論文, 2011.
[88] Y.-M. Hao, K.a. Li, Entrapment and release difference resulting from hydrogen bonding interactions in niosome, International journal of pharmaceutics, 403, 245-253, 2011.
附錄
[1] A.M. Ponce, A. Wright, M.W. Dewhirst, D. Needham, Targeted bioavailability of drugs by triggered release from liposomes, Future Lipidology, 1 (2006) 25-34.
[2] T. Tagami, W.D. Foltz, M.J. Ernsting, C.M. Lee, I.F. Tannock, J.P. May, S.-D. Li, MRI monitoring of intratumoral drug delivery and prediction of the therapeutic effect with a multifunctional thermosensitive liposome, Biomaterials, 32 (2011) 6570-6578.
[3] J.H. Collier, P.B. Messersmith, Phospholipid strategies in biomineralization and biomaterials research, Annual Review of Materials Research, 31 (2001) 237-263.
[4] D. Needham, M.W. Dewhirst, The development and testing of a new temperature-sensitive drug delivery system for the treatment of solid tumors, Advanced drug delivery reviews, 53 (2001) 285-305.
[5] J.K. Mills, D. Needham, Lysolipid incorporation in dipalmitoylphosphatidylcholine bilayer membranes enhances the ion permeability and drug release rates at the membrane phase transition, Biochimica et Biophysica Acta (BBA)-Biomembranes, 1716 (2005) 77-96.
[6] M. Hossann, T. Wang, M. Wiggenhorn, R. Schmidt, A. Zengerle, G. Winter, H. Eibl, M. Peller, M. Reiser, R.D. Issels, Size of thermosensitive liposomes influences content release, Journal of Controlled Release, 147 (2010) 436-443.
[7] N.D. Winter, R.K. Murphy, T.V. O'Halloran, G.C. Schatz, Development and modeling of arsenic-trioxide-loaded thermosensitive liposomes for anticancer drug delivery, Journal of liposome research, 21 (2011) 106-115.
[8] X. Zhang, P.F. Luckham, A.D. Hughes, S. Thom, X.Y. Xu, Development of lysolipid-based thermosensitive liposomes for delivery of high molecular weight proteins, International journal of pharmaceutics, 421 (2011) 291-292.
[9] G.R. Anyarambhatla, D. Needham, Enhancement of the phase transition permeability of DPPC liposomes by incorporation of MPPC: a new temperature-sensitive liposome for use with mild hyperthermia, Journal of Liposome Research, 9 (1999) 491-506.
[10] D. Needham, G. Anyarambhatla, G. Kong, M.W. Dewhirst, A new temperature-sensitive liposome for use with mild hyperthermia: characterization and testing in a human tumor xenograft model, Cancer research, 60 (2000) 1197-1201.
[11] S.-Y. Jeong, S.L. Yi, S.-K. Lim, S.-J. Park, J. Jung, H.N. Woo, S.Y. Song, J.-S. Kim, J.S. Lee, J.S. Lee, Enhancement of radiotherapeutic effectiveness by temperature-sensitive liposomal 1-methylxanthine, International journal of pharmaceutics, 372 (2009) 132-139.
[12] 洪振益, 溫度效應對帶電陰陽離子液胞釋放行為的影響, 國立成功大學化學工程學系碩士論文, (2009).
[13] 李威漢, 陰陽離子界面活性劑的製備及其相轉移行為的熱卡分析, 國立成功大學化學工程學系碩士論文, (2010).
[14] 張維瀚, 添加劑對於陰陽離子雙層膜之相轉移行為的探討, 國立成功大學化學工程學系碩士論文, (2012) 1-126.
[15] L.H. Lindner, M.E. Eichhorn, H. Eibl, N. Teichert, M. Schmitt-Sody, R.D. Issels, M. Dellian, Novel temperature-sensitive liposomes with prolonged circulation time, Clinical Cancer Research, 10 (2004) 2168-2178.