液胞 (vesicle) 為一種由界面活性劑自組裝形成之中空球殼結構，其球殼為雙層膜 (bilayer)。而微脂粒 (liposome) 是由天然界面活性劑–磷脂質 (phospholipid) 所製備之液胞，於藥物或DNA的輸送方面具有相當之應用潛力。然微脂粒的高昂造價，限制了其實務應用。離子對雙親分子 (ion pair amphiphile, IPA) 是由二種帶異性電荷之界面活性劑經等莫耳混合，並去除對離子後形成之複合物，其分子結構類似磷脂質，且製備成本低廉，可用於取代磷脂質來製備液胞，但IPA液胞仍有膠體懸浮液穩定性較低之問題。有研究顯示，在IPA液胞系統中加入膽固醇 (cholesterol, Chol) 去改變液胞雙層膜的排列結構，可提高液胞結構之穩定度，但膽固醇對於IPA液胞雙層膜之穩定機制仍有待進一步探討。另有文獻指出，IPA的兩個組成界面活性分子，對於IPA雙層膜的性質有不同的影響。本研究利用分子動態模擬 (molecular dynamics simulation)，從微觀上解析在含有膽固醇的IPA雙層膜中，IPA的不對稱碳鏈組合與膽固醇的添加，對於其雙層膜之結構特性的影響。我們選用兩種具相反對稱性的IPA：十六烷基三甲銨–十二烷基硫酸鹽 (hexadecyltrimethylammonium-dodecylsulfate, HTMA-DS) 和十二烷基三甲銨–十六烷基硫酸鹽 (dodecyltrimethyl-ammonium-hexadecylsulfate, DTMA-HS)，分別與膽固醇形成不同成份組成的凝膠態 (gel-state) 離子對雙親分子–膽固醇 (IPA-Chol) 雙層膜。隨著膽固醇濃度上升，兩種IPA-Chol雙層膜碳鏈的排列都越趨凌亂，且IPA間之距離增加，此現象和膽固醇對凝膠態磷脂質雙層膜之效應相符。兩種IPA-Chol雙層膜之水分子滲透係數 (permeability)，亦會隨著膽固醇的添加而降低，並於膽固醇莫耳分率 = 0.375時出現最小值。比較兩種由不同的IPA形成之IPA-Chol雙層膜，我們發現陰離子碳鏈較長的DTMA-HS-Chol雙層膜系統，相較於HTMA-DS-Chol，其碳鏈排列較不規則，膜機械強度較差，對水分子的滲透係數亦較高。上述差異是由於膽固醇和陰離子界面活性劑之間有較強親和力，加上IPA不對稱碳鏈組合的綜合影響。本研究提供了許多IPA-Chol雙層膜在分子層次上的資訊，有益於日後於不同的應用領域設計新型IPA。

Vesicle is a hollow spherical structure with bilayered shell(s) self-assembled from amphiphilic molecules in the aqueous phase. Liposomes, vesicles fabricated from phospholipids, have great potentials in various pharmaceutical fields such as drug or DNA deliveries. Yet, the high production costs of liposomes has hindered their practical applications. Ion pair amphiphile (IPA), a molecular complex composed of two oppositely charged surfactants, is structurally similar to a phospholipid and has been proposed as the cheaper lipid substitute. However, the IPA vesicles exhibit low colloidal stability in general, which limits their further application development. Reports have shown that the colloidal stability of IPA vesicles can be significantly enhanced via the addition of cholesterol, but the detailed stabilization mechanisms remain illusive. Furthermore, various studies have also suggested different roles for the cationic and anionic component within IPA membrane. In this work, we utilized molecular dynamics (MD) simulation to study how the cholesterol addition and the IPA chain arrangement affect the properties of IPA-Chol bilayer membrane. We focused on the gel-state IPA bilayers composed of one of the two IPAs with the reversed chain arrangements, hexadecyltrimethylammonium-dodecylsulfate (HTMA-DS) and dodecyltrimethylammonium-hexadecylsulfate (DTMA-HS). With increased cholesterol mole fraction (Xchol), the inter-complex spacing between IPAs increases while the alkyl chain ordering decreases, consistent with the cholesterol effects on the gel-state phospholipid bilayer. Upon the addition of cholesterol, the two asymmetric-IPA bilayers show different membrane characteristics, including alkyl chain ordering, atom mismatching, and mechanical moduli. The DTMA-HS-Chol systems, where the alkyl chain is longer for the anionic component, exhibit overall lower chain ordering and smaller mechanical strengths than the HTMA-DS-Chol systems. Due to the more disordered alkyl chain, DTMA-HS bilayer has higher water permeability than HTMA-DS system. Addition of cholesterol reduces the overall water permeability for both IPA systems with the minimum at Xchol = 0.375. The difference between two IPA bilayers is mainly originated from the interplay between the effects of IPA alkyl chain asymmetry and the biased interaction between cholesterol and the anionic surfactant. The presented results provide valuable molecular insights into the IPA-Chol mixtures and will be helpful for future IPA designs in various applications in the future.

Aiello, C.; Andreozzi, P.; Mesa, C. L.; Risuleo, G., “Biological activity of SDS-CTAB cat-anionic vesicles in cultured cells and assessment of their cytotoxicity ending in apoptosis,” Colloids Surf., B, 78, 2, 149-154, 2010.

Alwarawrah, M.; Dai, J.; Huang, J., “A molecular view of the cholesterol condensing effect in DOPC lipid bilayers,” J. Phys. Chem. B, 114, 22, 7516-7523, 2010.

Åman, K.; Lindahl, E.; Edholm, O.; Håkansson, P.; Westlund, P.-O., “Structure and dynamics of interfacial water in an Lα phase lipid bilayer from molecular dynamics simulations,” Biophys. J., 84, 1, 102-115, 2003.

Bemporad, D.; Essex, J. W.; and Luttmann, C., “Permeation of small molecules through a lipid bilayer:  a computer simulation study,” J. Phys. Chem. B, 108, 15, 4875-4884, 2004.

Berendsen, H. J. C.; Marrink, S. J., “Molecular dynamics of water transport through membranes: water from solvent to solute,” Pure Appl. Chem., 65, 12, 2513-2520, 1993.

Berkowitz, M. L., “Detailed molecular dynamics simulations of model biological membranes containing cholesterol,” Biochim. Biophys. Acta, 1788, 1, 86-96, 2009.

Bramer, T.; Dew, N.; Edsman, K., “Pharmaceutical applications for catanionic mixtures,” J. Pharm. Pharmacol., 59, 10, 1319-1334, 2007.

Chang, W. F., “The effects of additives on the physicochemical properties of catanionic vesicles formed by dihexdecyl-chained ion pair amphiphile,” Department of Chemical Engineering, National Cheng Kung University, Master Thesis, 2012.

Chen, J.; Long, P.; Li, H.; Hao, J., “Investigations into the bending constant and edge energy of bilayers of salt-free catanionic vesicles,” Langmuir, 28, 14, 5927-5933, 2012.

Chen, J.; Hao, J., “Molecular dynamics simulation of cetyltrimethylammonium bromide and sodium octyl sulfate mixtures: aggregate shape and local surfactant distribution,” Phys. Chem. Chem. Phys., 15, 5563-5571, 2013.

Clavero, E.; Rodriguez, J.; Laria, D., “Computer simulations of catanionic surfactants adsorbed at air/water interfaces. II. Full coverage,” J. Chem. Phys., 127, 124704, 2007.

Edholm, O., “Chapter 3 Time and length scales in lipid bilayer simulations,” Current Topics in Membranes, 60, 91-110, 2008.

Essmann, U.; Perera, L.; Berkowitz, M. L., “The origin of the hydration interaction of lipid bilayers from MD simulation of dipalmitoylphosphatidylcholine membranes in gel and liquid crystalline phases,” Langmuir, 11, 11, 4519-4531, 1995.

Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G., “A smooth particle mesh Ewald method,” J. Chem. Phys., 103, 8577-8593, 1995.

Feller, S. E.; MacKerell, A. D., Jr., “An improved empirical potential energy function for molecular simulations of phospholipids, J. Phys. Chem. B, 104, 31, 7510-7515, 2000.

Fukuda, H.; Kawata, K.; Okuda, H.; Regen, S. L., “Bilayer-forming ion pair amphiphiles from single-chain surfactants,” J. Am. Chem. Soc., 112, 4, 1635-1637, 1990.

Frenkel, D.; Smit, B., “Understanding molecular simulation (second edition),” Academic Press, 85, 2002.

Guàrdia, E.; Martí, J.; García-Tarrés, L.; Laria, D., “A molecular dynamics simulation study of hydrogen bonding in aqueous ionic solutions,” J. Mol. Liq., 117, 63-67, 2005.

Heimburg, T.; Wurz, U.; Marsh, D., “Binary phase diagram of hydrated dimyristoylglycerol-dimyristoylphosphatidylcholine mixtures,” Biophys. J., 63, 1369-1378, 1992.

Heller, H.; Schaefer, M.; Schulten, K., “Molecular dynamics simulation of a bilayer of 200 lipids in the gel and in the liquid crystal phase,” J. Phys. Chem., 97, 31, 8343-8360, 1993.

Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M., “LINCS: a linear constraint solver for molecular simulations,” J. Comput. Chem., 18, 1463-1472, 1997.

Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E., “GROMACS4: algorithms for highly efficient, load-balanced, and scalable molecular simulation,” J. Chem. Theory Comput., 4, 3, 435-447, 2008.

Hofsäß, C.; Lindahl, E.; Edholm, O., “Molecular dynamics simulations of phospholipid bilayers with cholesterol,” Biophys. J., 84, 4, 2192-2206, 2003.

Huang, J. B.; Zhao, G. X., “Formation and coexistence of the micelles and vesicles in mixed solution of cationic and anionic surfactant,” Colloid. Polym. Sci., 273, 156-164, 1995.

Huang, S. L., “Liposomes in ultrasonic drug and gene delivery,” Adv. Drug Deliv. Rev., 60, 10, 1167-1176, 2008.

Humphrey, W.; Dalke, A.; Schulten, K., “VMD - Visual Molecular Dynamics,” J. Molec. Graphics, 14, 33, 1996.

Hung, W.-C.; Lee, M.-T.; Chen, F.-Y.; Huang, H. W., “The condensing effect of cholesterol in lipid bilayers,” Biophys. J., 92, 3960-3967, 2007.

Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W., “Theory of self-assembly of hydrocarbon amphiphiles into micelles and bilayers,” J. Chem. Soc., Faraday Trans. 2, 72, 1525-1568, 1976.

Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L., “Comparison of simple potential functions for simulating liquid water,” J. Chem. Phys., 79, 926-935, 1983.

Kaler, E.; Murthy, A.; Rodriguez, B.; Zasadzinski, J., “Spontaneous vesicle formation in aqueous mixtures of single-tailed surfactants,” Science, 245, 4924, 1371-1374, 1989.

Khelashvili, G.; Pabst, G.; Harries, D., “Cholesterol orientation and tilt modulus in DMPC bilayers,” J. Phys. Chem. B, 114, 22, 7524-7534, 2010.

Khelashvili, G.; Kollmitzer, B.; Heftberger, P.; Pabst, G.; Harries, D., “Calculating the bending modulus for multicomponent lipid membranes in different thermodynamic phases,” J. Chem. Theory Comput., 9 ,9, 3866-3871, 2013.

Khelashvili, G.; Harries, D., “How cholesterol tilt modulates the mechanical properties of saturated and unsaturated lipid membranes,” J. Phys. Chem. B, 117, 8, 2411-2421, 2013.

Klauda, J. B.; Brooks, B. R.; MacKerell, A. D., Jr.; Venable, R. M.; Pastor, R. W., “An ab initio study on the torsional surface of alkanes and its effect on molecular simulations of alkanes and a DPPC bilayer,” J. Phys. Chem. B, 109, 5300-5311, 2005.

Krause, M. R.; Regen, S. L., “The structural role of cholesterol in cell membranes: from condensed bilayers to lipid rafts,” Acc. Chem. Res., 47, 3512-3521, 2014.

Kuo, A. T.; Chang, C. H.; Shinoda, W., “Molecular dynamics study of catanionic bilayers composed of ion pair amphiphile with double-tailed cationic surfactant,” Langmuir, 28, 21, 8156-8164, 2012.

Kuo, A. T.; Chang, C. H., “Cholesterol-induced condensing and disordering effects on a rigid catanionic bilayer: a molecular dynamics study,” Langmuir, 30, 1, 55-62, 2014.

Kuo, A. T.; Chang, C. H., “Elucidating the effects of cholesterol on the molecular packing of double-chained cationic lipid langmuir monolayers by infrared reflection-absorption spectroscopy,” J. Oleo Sci., 64, 4, 455-465, 2015.

Lasic, D. D., “Novel applications of liposomes,” Trends Biotechnol., 16, 7, 307-321, 1998.

Lee, W. H.; Tang, Y. L.; Chiu, T. C.; Yang, Y. M., “Synthesis of ion-pair amphiphiles and calorimetric study on the gel to liquid-crystalline phase transition behavior of their bilayers,” J. Chem. Eng. Data, 60, 4, 1119-1125, 2015.

Lewis, R. N.A.H.; McElhaney, R. N., “Membrane lipid phase transitions and phase organization studied by Fourier transform infrared spectroscopy,” Biochim. Biophys. Acta, 1828, 10, 2013.

Li, Y. Y., “Stability and encapsulation behavior of catanionic vesicles with addition of cholesterol,” Department of Chemical Engineering, National Cheng Kung University, Master Thesis, 2004.

Liu, Y. S.; Wen, C. F.; Yang, Y. M., “Development of ethosome-like catanionic vesicles for dermal drug delivery,” J. Taiwan Inst. Chem. Eng., 43, 6, 830-838, 2012.

Luzar, A., “Resolving the hydrogen bond dynamics conundrum,” J. Chem. Phys., 113, 10663-10675, 2000.

Marques, E. F.; Regev, O.; Khan, A.; Lindman, B., “Self-organization of double-chained and pseudodouble-chained surfactants: counterion and geometry effects,” Adv. Colloid Interface Sci., 100-102, 83-104, 2003.

Marrink, S. J.; Berendsen, H., “Simulation of water transport through a lipid membrane,” J. Phys. Chem., 98, 15, 4155-4168, 1994.

Marrink, S.-J.; Berkowitz, M.; Berendsen, H. J. C., “Molecular dynamics simulation of a membrane/water interface: the ordering of water and its relation to the hydration force,” Langmuir, 9, 3122-3131, 1993.

Martinez-Seara, H.; Róg, T.; Karttunen, M.; Vattulainen, I.; Reigada, R, “Cholesterol induces specific spatial and orientational order in cholesterol/phospholipid membranes,” PLoS One, 5, 6, e11162, 2010.

Martínez, L.; Andrade, R.; Birgin, E. G.; Martínez, J. M., “Packmol: a package for building initial configurations for molecular dynamics simulations,” J. Comput. Chem., 30, 13, 2157-2164, 2009.

Martyna, G. J., Klein, M. L., Tuckerman, M., “Nose−Hoover chains: the canonical ensemble via continuous dynamics,” J. Chem. Phys., 97, 2635-2643, 1992.

Martyna, G. J.; Tobias, D. J.; Klein, M. L., “Constant pressure molecular dynamics algorithms,” J. Chem. Phys., 101, 4177-4189, 1994.

McMullen, T. P. W.; Lewis, R. N.A.H.; McElhaney, R. N., “Cholesterol–phospholipid interactions, the liquid-ordered phase and lipid rafts in model and biological membranes,” Curr. Opin. Colloid Interface Sci., 8, 6, 459-468, 2004.

Mills, T. T.; Toombes, G. E. S.; Tristram-Nagle, S.; Smilgies, D-M.; Feigenson, G. W.; Nagle, J. F., “Order parameters and areas in fluid-phase oriented lipid membranes using wide angle X-ray scattering,” Biophys. J., 95, 2, 669-681, 2008.

Mills, T. T.; Huang, J.; Feigenson, G. W.; Nagle, J. F., “Effects of cholesterol and unsaturated DOPC lipid on chain packing of saturated gel-phase DPPC bilayers,” Gen. Physiol. Biophys., 28, 2, 126-139, 2009.

Miyoshi, T.; Lönnfors, M.; Slotte, J. P.; Kato, S., “A detailed analysis of partial molecular volumes in DPPC/cholesterol binary bilayers,” Biochim. Biophys. Acta, 1838, 12, 2014.

Olsen, B. N.; Schlesinger, P. H.; Baker, N. A., “Perturbations of membrane structure by cholesterol and cholesterol derivatives are determined by sterol orientation,” J. Am. Chem. Soc., 131, 13, 2009.

Parrinello, M.; Rahman, A., “Polymorphic transitions in single crystals: a new molecular dynamics method,” J. Appl. Phys., 52, 7182-7190, 1981.

Picas, L.; Rico, F.; Scheuring, S., “Direct measurement of the mechanical properties of lipid phases in supported bilayers,” Biophys J., 102, 1, L01-L03, 2012.

Pike, L. J., “Lipid rafts: bringing order to chaos,” J. Lipid Res., 44, 4, 655-667, 2003.

Robinson, A.; Richards, G.; Thomas, P.; Hann, M., “Behaviour of cholesterol and its effect on head group and chain conformations in lipid bilayers: a molecular dynamics study,” Biophys. J., 68, 164-170, 1995.

Rodgers, J. M.; Sørensen, J.; de Meyer, F. J.-M.; Schiøtt, B.; Smit, B., “Understanding the phase behavior of coarse-grained model lipid bilayers through computational calorimetry,” J. Phys. Chem. B, 116, 5, 1551-1569, 2012.

Róg, T.; Pasenkiewicz-Gierula, M., “Cholesterol effects on the phosphatidylcholine bilayer nonpolar region: a molecular simulation study,” Biophys. J., 81, 4, 2190-2202, 2001.

Róg, T.; Pasenkiewicz-Gierula, M., “Cholesterol effects on a mixed-chain phosphatidylcholine bilayer: a molecular dynamics simulation study,” Biochimie, 88, 5, 449-460, 2006.

Róg, T.; Pasenkiewicz-Gierulab, M.; Vattulainena, I.; Karttunene, M., “Ordering effects of cholesterol and its analogues,” Biochim. Biophys. Acta, 1788, 1, 97-121, 2009.

Saito, H.; Shinoda, W., “Cholesterol effect on water permeability through DPPC and PSM lipid bilayers: a molecular dynamics study,” J. Phys. Chem., 115, 51, 2011.

Schindler, H.; Seelig, J., “Deuterium order parameters in relation to thermodynamic properties of a phospholipid bilayer. Statistical mechanical interpretation,” Biochemistry, 14, 11, 2283-2287, 1975.

Segota, S.; Tezak, D., “Spontaneous formation of vesicles,” Adv. Colloid Interface Sci., 121, 1-3, 51-75, 2006.

Shieh, H.-S.; Hoard, L. G.; Nordman, C. E., “The structure of cholesterol,” Acta Crystallogr., Sect. B, 37, 1538-1543, 1981.

Shinoda, W.; Namiki, N.; Okazaki, S., “Molecular dynamics study of a lipid bilayer: convergence, structure, and long-time dynamics,” J. Chem. Phys., 106, 5731, 1997.

Shinoda, W.; Mikami, M.; Baba, T.; Hato, M., “Molecular dynamics study on the effect of chain branching on the physical properties of lipid bilayers: structural stability,” J. Phys. Chem. B, 107, 50, 14030-14035, 2003.

Shinoda, W.; Mikami, M.; Baba, T.; Hato M., “Molecular dynamics study on the effects of chain branching on the physical properties of lipid bilayers: 2. permeability,” J. Phys. Chem. B, 108, 26, 9346-9356, 2004.

Shinoda, W.; Shinoda, K.; Baba, T.; Mikami, M., “Molecular dynamics study of bipolar tetraether lipid membranes,” Biophys. J., 89, 5, 3195-3202, 2005.

Shiu, L. M., “The stability and encapsulation behavior of catanionic vesicles,” Department of Chemical Engineering, National Cheng Kung University, Master Thesis, 2002.

Simons, K.; Ikonen, E., “Functional rafts in cell membranes,” Nature, 387, 569-572, 1997.

Smondyrev, A. M.; Berkowitz, M. L., “Structure of dipalmitoyl-phosphatidylcholine/cholesterol bilayer at low and high cholesterol concentrations: molecular dynamics simulation,” Biophys. J., 77, 2075-2089, 1999.

Soussan, E.; Cassel, S.; Blanzat, M.; Rico-Lattes, I., “Drug delivery by soft matter: matrix and vesicular carriers,” Angew. Chem. Int. Ed., 48, 2, 274-288, 2009.

Stȩpniewski, M.; Bunker, A.; Pasenkiewicz-Gierula, M.; Karttunen, M.; Róg, T., “Effects of the lipid bilayer phase state on the water-membrane interface,” J. Phys. Chem. B, 114, 11784-11792, 2010.

Stockton, G. W.; Smith, I. C. P., “A deuterium nuclear magnetic resonance study of the condensing effect of cholesterol on egg phosphatidylcholine bilayer membranes. I. Perdeuterated fatty acid probes,” Chem. Phys. Lipids., 17, 251-263, 1976.

Sugii, T.; Takagi, S.; Matsumoto, T., “A molecular-dynamics study of lipid bilayers: effects of the hydrocarbon chain length on permeability,” J. Chem. Phys., 123, 184714, 2005.

Tondre, C.; Caillet, C., “Properties of the amphiphilic films in mixed cationic/anionic vesicles: a comprehensive view from a literature analysis,” Adv. Colloid Interface Sci., 93, 1-3, 115-134, 2001.

Tu, K; Tobias, D. J.; Blasie, J. K.; Klein, M. L., “Molecular dynamics investigation of the structure of a fully hydrated gel-phase dipalmitoylphosphatidylcholine bilayer,” Biophys. J., 70, 2, 595-608, 1996.

van der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.; Berendsen, H. J. C., “GROMACS: fast, flexible, and free,” J. Comput. Chem., 26, 16, 1701-1718, 2005.

Vermeer, L. S.; de Groot, B. L.; Réat, V.; Milon, A.; Czaplicki, J., “Acyl chain order parameter profiles in phospholipid bilayers: computation from molecular dynamics simulations and comparison with 2H NMR experiments,” Eur. Biophys. J., 36, 919-931, 2007.

Wu, C. J.; Kuo, A. T.; Lee, C. H.; Yang, Y. M.; Chang, C. H., “Fabrication of positively charged catanionic vesicles from ion pair amphiphile with double-chained cationic surfactant,” Colloid Polym. Sci., 292, 3, 589-597, 2014.