摘 要

本研究探討離子對雙親分子HTMA-DS、雙十六碳鏈磷酸鹽(DHDP)及膽固醇形成陰陽離子液胞的行為。陰陽離子液胞是以機械分散法生成，並評估做為穩定劑之DHDP和膽固醇的添加對液胞穩定性與包覆效率的影響。在此研究中，利用液胞雙層膜包覆親油性藥物維他命E，以了解與藥物/化妝品傳輸載體系統之配方相關的重要參數。此研究是以高效液相層析儀偵測維他命E的濃度，並使用螢光偏極化法探討液胞雙層膜的排列特性。
液胞之物理穩定性是在室溫下由肉眼觀察、液胞大小及界面電位的隨時間觀測來決定，發現此陰陽離子液胞系統可穩定長達14個月，液胞的平均大小在55到125奈米之間，且具窄的粒徑分布，由界面電位值-40到-63 mV可確認液胞帶負電性。膽固醇與雙親分子之頭基及疏水性碳鏈的交互作用，顯然在陰陽離子液胞的穩定形成上扮演重要的角色。帶負電之DHDP分子的添加，可透過液胞間的電性排斥力提供障礙而避免液胞的聚集，以促進液胞的物理穩定性。電子穿透顯微鏡影像顯示液胞呈球狀且大小均勻。根據液胞之包覆效率的高低，依序為添加43 mol%、50 mol%及35 mol%膽固醇的液胞，隨著DHDP莫爾分率的增加，對維他命E的包覆效率則隨之下降。膽固醇和DHDP的加入明顯影響了液胞的雙層膜性質及包覆效率。毒性測試則顯示隨著液胞中DHDP莫爾分率的增加，似會使陰陽離子液胞系統較不具毒性。

ABSTRACT

The formation characteristics of catanionic vesicles formed by ion pair amphiphile HTMA-DS, dihexadecyl phosphate (DHDP), and cholesterol were investigated in this study. Catanionic vesicles were prepared with a mechanical dispersion approach. The addition of DHDP and cholesterol as stabilizing agents was applied in order to evaluate their effects on the vesicle stability and encapsulation efficiency. In the present study, vitamin E as a lipophilic drug was encapsulated within the vesicular bilayers to elucidate the key parameters involved in the formulation of the drug/cosmetic carrier system. The concentration of vitamin E was measured by HPLC with UV detector and a fluorescence polarization study was carried out to investigate the bilayer packing characteristic.
Physical stability of the catanionic vesicles determined by visual observation, vesicle size, and vesicle zeta potential was monitored with time at room temperature. This catanionic vesicle system was found to be stable for more than fourteen months. The mean sizes of vesicles ranged from 55 nm to 125 nm with narrow size distributions. Negatively charged characteristic of the vesicles was confirmed based on the zeta potential values that varied from -40 mV to -63 mV. The interactions of cholesterol with the headgroups and hydrophobic chains of amphiphiles apparently played important roles in the formation of stable catanionic vesicles. Negative charges of DHDP molecules provided a barrier to prevent aggregation of the vesicles and increased their physical stability with the electrostatic repulsive force between the vesicles. Transmission electron microscopy (TEM) images showed the spherical structure and homogenous size of the vesicles. The encapsulation efficiency values of the vesicles were found to be in the order with the added cholesterol concentration of 43 mol%, 50 mol%, and 35 mol%. It has been observed that with a higher molar fraction of DHDP, the vitamin E encapsulation efficiency became lower. The incorporation of cholesterol and DHDP significantly affected the bilayer properties of the catanionic vesicles as well as the encapsulation efficiency. With the cytotoxicity experiment, it was suggested that incorporation of more DHDP into the catanionic vesicles resulted in the less toxicity of the catanionic vesicle systems.

Anais, J. P., Razzouq, N., Carvalho, M., Fernandez, C., Astier, A., Paul, M., Astier, A., Fessi, H., and Lorino, A. M., “Development of alpha-Tocopherol Acetate Nanoparticles: Influence of Preparative Processes,” Drug Development and Industrial Pharmacy 35, 216-223, 2009.

Andersson, M., Hammarstrom, L., and Edwards, K., “Effect of bilayer phase transitions on vesicle structure and its influence on the kinetics of viologen reduction,” Journal of Physical Chemistry 99, 14531-14538, 1995.

Bhardwaj, U., and Burgess, D. J., “Physicochemical properties of extruded and non-extruded liposomes containing the hydrophobic drug dexamethasone,” International Journal of Pharmaceutics 388, 181-189, 2010.

Bhattacharya, S., De, S., and Subramanian, M., “Synthesis and vesicle formation from hybrid bolaphile/amphiphile ion-pairs. Evidence of membrane property modulation by molecular design,” Journal of Organic Chemistry 63, 7640-7651, 1998.

Bhattacharya, S., and Haldar, J., “Interactions between cholesterol and lipids in bilayer membranes. Role of lipid headgroup and hydrocarbon chain-backbone linkage,” Biochimica et Biophysica Acta 1467, 39-53, 2000.

Blanzat, M., Perez, E., Rico-Latters, R., Prome, D., Prome, J. C. and Lattes, A., “New catanionic glycolipids. 1. Synthesis, characterization, and biological activity of double-chain and gemini catanionic analogues of galactosylceramide (gal beta(1)cer),” Langmuir 15, 6163-6169, 1999.

Bonina, F., Montenegro, L., La Rosa, C., Gaspararri, F., and Leonardi, R., “Comparison of different separative techniques in the quantitative determination of active compound enclosed in liposomal systems,” International Journal of Cosmetic Science 16, 183-197, 1994.

Bretscher, M. S., and Munro, S., “Cholesterol and the Golgi-apparatus,” Science 261, 1280-1281, 1993.

Campbell, R. B., Balasubramanian, S. V., and Straubinger, R. M., “Phospholipid-cationic lipid interactions: influences on membrane and vesicle properties,” Biochimica et Biophysica Acta 1512, 27-39, 2001.

Carmona-Ribeiro, A. M., and Midmore, B. R., “Surface potential in charged synthetic amphiphile vesicles,” Journal of Physical Chemistry 96, 3542-3547, 1992.

Carrion, F. J., Maza, A. D., and Parra, J. L., “The influence of ionic strength and lipid bilayer charge on the stability of liposomes,” Journal of Colloid and Interface Science 164, 78-87, 1994.

Chien, C.-L., Yeh, S.-J., Yang, Y.-M., Chang, C.-H., and Maa, J.-R., “Formation and encapsulation of catanionic vesicles,” Journal of the Chinese Colloid and Interface Society 24, 31-45, 2002.

Chiu, S. W., Jakobsson, E., and Scott, H. L., “Combined Monte Carlo and molecular dynamics simulation of hydrated lipid-cholesterol lipid bilayers at low cholesterol concentration,” Biophysical journal 80, 1104-1114, 2001.

Chou, T.-H., and Chang, C.-H., “Thermodynamic characteristics of mixed DPPC/DHDP monolayers on water and phosphate buffer subphases,” Langmuir 16, 3385-3390, 2000

Chung, Y. C., Lee, H. J., and Park, J. Y., “Bilayer properties of the multiple-chain ion pair amphiphiles,” Bulletin of The Korean Chemical Society 19, 1249-1252, 1998.

Cuccovia, I. M., Feitosa, E., Chaimovich, H., Sepulveda, L., and Reed, W., “Size, electrophoretic mobility, and ion dissociation of vesicles prepared with synthetic amphiphiles,” Journal of Physical Chemistry 94, 3722-3725, 1990.

Degiorgio, V., and Piazza, R., “Electric birefringence of critical micellar solutions,” Physical Reviews Letters 55, 288-291, 1985.

Discher, B. M., Won, Y. Y., Ege, D. S., Lee, J. C. M., Bates, F. S., Discher, D. E., and Hammer, D. A., “Polymersomes: Tough vesicles made from diblock copolymers, “ Science 28, 1143-1146, 1999 .
Eastman, S. J., Siegel, C., Tousignant, J., Smith, A. E., Cheng, S. H., and Scheule R. K., “Biophysical characterization of cationic lipid:DNA complexes,” Biochimica et Biophysica Acta 1325, 41-62, 1997.

Feitosa, E., and Brown, W., “Fragment and vesicles structures in sonicated dispersions of dioctadecyldimethylammonium bromide,” Langmuir 13, 4810-4816, 1997.

Fukuda, H., Kawata, K., and Okuda, H., “Bilayer-forming ion-pair amphiphiles from single-chain surfactants,” Journal of the American Chemical Society 112, 1635-1637, 1990.

Gierula, M. P., Rόg, T., “Conformations, orientations and time scales characterising dimyristoylphosphatidylcholine bilayer membrane. Molecular dynamics simulation studies,” Acta Biochimica Polonica 44, 607-624, 1997.

Gregoriadis, G., and Davis, C., “Stability of liposomes in vivo and in vitro is promoted by their cholesterol content and the presence of blood-cells,“ Biochemical and biophysical Research Communications 89, 1287-1293, 1979.

Hauser, H., Gains, N., Eibl, H. J., Muller, M., and Wehrli, E., “Spontaneous vesiculation of aqueous lipid dispersions,” Biochemistry 25, 2126-2134, 1986.

Hirano, K., and Fukuda, H., “Polymerizable ion-paired amphiphiles,” Langmuir 7, 1045-1047, 1991.
Holmberg, K., “Handbook of applied surface and colloid chemistry,” John Wiley and Sons, England, 2002.

Huang, Z.-L., Hong., J.-Y., Chang, C.-H., and Yang, Y.-M., “Gelation of charged catanionic vesicles prepared by a semispontaneous process,” Langmuir 26, 2374-2382, 2010.

Kaler, E. W., Murthy, A. K., Rodriguez, B. E., and Zasadzinski, A. N., “Spontaneous vesicle formation in aqueous mixtures of single-tailed surfactants,” Science 245, 1371-1374, 1989.

Kirjavainen, M., Urtti, A., Jaaskelainen, I., Suhonen, T. M., Paronen, P., ValjakkaKoskela, R., Kiesvaara, J., and Monkkonen, J., “Interaction of liposomes with human skin in vitro - The influence of lipid composition and structure,” Biochimica et Biophysica Acta-Lipids and Lipid Metabolism 1304, 179-189, 1996.

Kuo, A.-T., personal communication.

Lasic, D. D., and Papahadjopoulos, D., “Liposomes and biopolymers in drug and gene delivery,” Solid State and Materials Science 1, 392-400, 1996.

Liu, N., and Park, H. J., “Chitosan-coated nanoliposome as vitamin E carrier,” Journal of Microencapsulation 26, 235-242, 2009.

Marques., E. F., Khan, A., Miguel, M. D., and Lindman, B., “Self-assembly in mixture of a cationic and an anionic surfactant-the sodium dodecyl-sulfate didodecyldimethylammonium bromide water-system,” Journal of Physical Chemistry 97, 4729-4736, 1993.

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

Marsh, D., and Smith, I. C. P., “Interacting spin label study of fluidizing and condensing effects of cholesterol on lechitin bilayers,” Biochimica et Biophysica Acta 298, 133-144, 1973.

McIntosh, T. J., Magid, A. D., and Simon, S. A., “Steric repulsion between phosphatidylcholine bilayers,” Biochemistry 26, 7325-7332, 1987.

McIntosh, T. J., Magid, A. D., and Simon, S. A., “Cholesterol modifies the short-range repulsive interactions between phosphatidylcholine membranes,” Biochemistry 28, 17-25, 1989.

McMullen, T. P. W., Lewis, R. N. A. H., and McElhaney, R. N., “Differential scanning calorimetric and fourier transform infrared spectroscopic studies of the effects of cholesterol on the thermotropic phase behavior and organization of a homologous series of linear saturated phosphatidylserine bilayer membranes,” Biophysical Journal 79, 2056-2065, 2000.

Moghimi, S. M., Symonds, P., Murray, J. C., Hunter, A.C., Debska, G., and Szewczyk, A. A., ”Two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy,” Molecular Therapy 11, 990-995,

Padamwar, M. N., and Pokharkar, V. B., “Development of vitamin loaded topical liposomal formulation using factorial design approach: drug deposition and stability,” International Journal of Pharmaceutics 320, 37-44, 2006.

Papahadjopoulos, D., Cowden, M., and Kimelberg, H., “Role of cholesterol in membranes effects on phospholipids-protein interactions, membrane permeability and enzymatic activity,” Biochimica et Biophysica Acta 330, 8-26, 1973.

Pavinatto, F. J., Pacholatti, C. P., Montanha, E. A., Caseli, L., Silva, H. S., Miranda, P. B., Viitala, T., and Oliveira Jr, O. N., “Cholesterol mediates chitosan activity on phospholipid monolayers and Langmuir-Blodgett films,” Langmuir 25, 10051-10061, 2009.

Regev, O., and Khan, A., ”Alkyl chain symmetry effects in mixed cationic-anionic surfactant systems,” Journal of Colloid and Interface Science 182, 95-109, 1996 .

Rόg, T., and Gierula, M. P., “Cholesterol effects on the phosphatidylcholine bilayer nonpolar region: A molecular simulation study,” Biophysical journal 81, 2190-2202, 2001 .

Rόg, T., Gierula, M. P., Vattulainen, I., and Karttunen, M. “Ordering effects of cholesterol and its analogues,” Biochimica et Biophysica Acta 1788, 97-121, 2009.

Segota, S., and Tezak, D. “Spontaneous formation of vesicles,” Advances in Colloid and Interface Science 121, 51-75, 2006.

Silva, B. F. B., Marques, E. F., Olsson, U., and Pons, R., “Headgroup effects on the unusual lamellar-lamellar coexistence and vesicle-to-micelle transition of salt-free catanionic amphiphiles,” Langmuir 26, 3058-3066, 2010.

Silvius., J. R., del Giudice, D., and Lafleur, M., “Cholesterol at different bilayer concentrations can promote or antagonize lateral segregation of phospholipids of differing acyl chain length,” Biochemistry 35, 15198-15208, 1996.

Simons, K., and Vaz, W. L. C., “Model systems, lipid rafts, and cell membranes,” Annual Review of Biophysics and Biomolecular Structure 33, 269-295, 2004.

Smaby, J. M., Momsen, M. M., Brockman, H. L., and Brown, R. E., “Phosphatidylcholine acyl unsaturation modulates the decrease in interfacial elasticity induced by cholesterol,” Biophysical Journal 73, 1492-1505, 1997 .

Soto-Arriaza, M. A., Sotomayor, C. P., Lissi, E. A., “Relationship between lipid peroxidation and rigidity in L-alpha-phosphatidylcholine-DPPC vesicles,” Journal of Colloid and Interface Science 323, 70-74, 2008.

Teixeira, C. V., Blanzat, M., Koetz, J., Rico-Lattes, I., and Brezesinski, G., “In plane miscibility and mixed bilayer microstructure in mixtures of catanionic glycolipids and zwitterionic phospholipids,” Biochimica et Biophysica Acta 1758, 1797-1808, 2006.

Tomasic, V., Popovic, S., Tusek, B. L., and Filipovic, V. N., “A novel catanionic surfactant: Hexadecyltrimethylammonium dodecyl sulfate,” Berichte der Bunsen-Gesellschaft-Physical Chemistry Chemical Physics 101 , 1942-1948, 1997 .

Tondre, C., and Caillet, C., “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.

Verma, D. D., Verma, S., Blume, G., and Fahr, A., “Particle size of liposomes influences dermal delivery of substances into skin,” International Journal of Pharmaceutics 258, 141-151, 2003.

Virden, J. W., and Berg, J. C., “NaCl-induced aggregation of dipalmitoylphosphatidylglycerol small unilamellar vesicles with varying amounts of incorporated cholesterol,” Langmuir 8, 1532-1537, 1992.

Vist, M. R., and Davis, J. H., “Phase-equilibria of cholesterol dipalmitoylphosphatidylcholine mixtures-H-2 Nuclear Magnetic-Resonance and Differential Scanning Calorimetry,” Biochemistry 29, 451-464, 1990.

Wang, X. Y., and Quinn, P. J., ”The distribution of alpha-tocopherol in mixed aqueous dispersions of phosphatidylcholine and phosphatidylethanolamine,” Biochimica et Biophysica Acta-Biomembranes 1509, 361-372, 2000.

Winterhalter, M., and Lasic, D. D., “Liposome stability and formation-experimental parameters and theories on the size distribution,” Chemistry and Physic of Lipids 64, 35-43, 1993.

Wlaschek, M., Tantcheva-Poor, I., Naderi, L., Ma, W. J., Schneider, A., Razi-Wolf, Z., Schuller, J., and Scharffetter-Kochanek, K., “Solar UV irradiation and dermal photoaging,” Journal of Photochemistry and photobiology B-Biology 63, 41-51, 2001.

Yeagle, P. L., “Phospholipid Headgroup Behavior in Biological Assemblies,” Account of Chemical Research 11, 321, 1978.

Yeagle, P. L., Albert, A. D., Boeszebattaglia, K., Young, J., and Frye, J., “Cholesterol dynamics in membranes,” Biophysical Journal 57, 413-424, 1990.

Yen, H.-T., personal communication.

Yokoyama, S., Inagaki, A., Imura, T., Ohkubo, T., Tsubaki, N., Sakai, H., and Abe, M., “Membrane properties of cationic liposomes composed of dipalmitoylphosphatidylcholine and dipalmityldimethylammonium bromide,” Colloids and Surfaces B: Biointerfaces 44, 204-210, 2005.

Yu, W.-Y., Yang, Y.-M., and Chang, C.-H., “Cosolvent effects on the spontaneous formation of vesicles from 1:1 anionic and cationic surfactant mixtures,” Langmuir 21, 6185-6193, 2005.