本研究利用由微藻萃取之脂肪酸混合物—藻油，以均質－擠壓製程製備藥物傳輸載體，並分析乳化液的載體粒徑分布，探討每一操作階段的粒徑、PDI值變化及材料利用率。研究中利用兩種不同組成之微藻油製備藥物傳輸載體，經由均質－擠壓製程後所形成的載體粒徑皆落在100～200 nm之間，界面電位呈現負值，其負電來源為脂肪酸解離。載體穩定天數最高可大於250天，物理穩定性佳。單獨以均質方式製備之載體乳化液的粒徑呈多峰分布，穩定性僅一天。接續進行擠壓製程後，載體粒徑分布逐漸趨向單一分布，平均載體粒徑降低，分散液穩定性獲得提升。結果說明擠壓製程具有調整載體平均粒徑的效果，經由2000 nm、1000 nm、600 nm孔徑的擠壓膜分別可得到平均粒徑190 nm、150 nm、130 nm的載體。最後以總有機含碳量分析計算經製備程序後的材料利用率約77%，原因為部分材料滯留在擠壓步驟的PC膜上。在溶血實驗及細胞毒性實驗中，所有組成的載體低於約0.9 mM時皆可視為無毒。以上結果顯示藻油載體有生物相容性佳的優勢，且均質－擠壓製程製備的載體有高穩定性、製備流程簡便等優點，具有應用的前景。

In this study, fatty acid mixtures cultivated from microalgae were utilized to fabricate drug delivery carriers by a homogenization-extrusion approach. Both of homogenization and extrusion are mechanical operations, and have potential for large-scale preparation. A fabrication approach for microalgal oil carriers was then explored. Physical properties, such as initial size, zeta potential and stability of the carrier, were analyzed by dynamic light scattering technique. Total organic carbon analysis was carried out to evaluate the material utilization after fabrication. Furthermore, hemolysis assay and cell viability assay were performed to evaluate the cytotoxicity of the carrier dispersions to human cells.
The carriers fabricated by the homogenization-extrusion approach had size between 100 to 200 nm with a negative charge, while the negatively charged character could be explained by the partial dissociation of the fatty acid molecules. The carriers were stable up to 371 days and possessed high physical stability. The utilization is about 77%. It is because that the extrusion membrane has higher infinity with oil than with water, and thus some aggregates will remain on the membranes in the extrusion operation. The homogenization-extrusion approach could be apply on a 600-mL scale, and physical properties of the carriers are similar to that obtained on a 60-mL scale. In hemolysis and cell viability tests, the carrier dispersions could be considered as non-toxic at a carrier concentration lower than approximately 1.7 mM. Carriers fabricated by the homogenization-extrusion approach have the advantages of high stability, good biocompatibility and simple fabrication.

Aboagla, E. M. E., and Maeda, T., “Arbutin's suppression of cryodamage in goat sperm and its mechanism of cryoprotection,” Theriogenology, 76, 538-546, 2011.
Aditya, N. P., Hamilton, I. E., and Norton, I. T., “Amorphous nano-curcumin stabilized oil in water emulsion: physico chemical characterization,” Food Chemistry, 224, 191-200, 2017.
Ali, M. H., Moghaddam, B., Kirby, D. J., Mohammed, A. R. and Perrie, Y., “The role of lipid geometry in designing liposomes for the solubilisation of poorly water soluble drugs,” International Journal of Pharmaceutics, 453, 225-232, 2013.
Aramaki, Y., Matsuno, R., Nitta, F., Arima, H., and Tsuchiya, S., “Negatively charged liposomes inhibit tyrosine phosphorylation of 41-kDa protein in murine macrophages stimulated with LPS,” Biochemical and Biophysical Research Communications, 220, 1-6, 1996.
Aramaki, Y., Takano, S., and Tsuchiya, S., “Induction of apoptosis in macrophages by cationic liposomes,” FEBS Letters, 460, 472-476, 1999.
Asai, Y. and Watanabe, S., “Interaction of a-tocopherol acetate with phosphatidylcholine and their formation of small dispersed particles,” Chemical and Pharmaceutical Bulletin, 46, 1785-1789, 1998.
Bhardwaj, U. and Burgess, D. J., “Physicochemical properties of extruded and non-extruded liposomes containing the hydrophobic drug dexamethadone,” International Journal of Pharmaceutics, 388, 181-189, 2010.
Blandamer, M. J., Briggs, B., Cullis, P. M., Rawlings, B. J. and Engberts, J., “Vesicle-cholesterol interactions: Effects of added cholesterol on gel-to-liquid crystal transitions in a phospholipid membrane and five dialkyl-based vesicles as monitored using DSC,” Physical Chemistry Chemical Physics, 5, 5309-5312, 2003.
Bouarab, L., Maherani, B., Kheirolomoom, A., Hadan, M., Aliakbarian, B., Linder, M. and Arab-Tehrany, E., “Influence of lecithin-lipid composition on physico-chemical properties of nanoliposomes loaded with a hydrophobic molecule.” Colloids and Surfaces B: Biointerfaces, 115, 197-204, 2014.
Bui, T. T., Suga, K., and Umakoshi, H., “Roles of sterol derivatives in regulating the properties of phospholipid bilayer systems,” Langmuir, 32, 6176-6184, 2016.
Chen, C. and Tripp, C. P., "An infrared spectroscopic based method to measure membrane permeance in liposomes," Biochimica et Biophysica Acta, 1778, 2266-2272, 2008.
Choosakoonkriang, S., Wieyhoff, C. M., Anchordoquy, T. J., Koe, G. S., Smith, J. G. and Middaugh, R. C., “Infrared spectroscopic characterization of the interaction of cationic lipids with plasmid DNA,” The Journal of Biological Chemistry, 276, 8037-8043, 2001.
Cistola, D. P., Hamilton, J. A., Jackson, D. and Small, D. M., “Ionization and phase behavior of fatty acids in water: app lication of the Gibbs phase rule,” Biochemistry, 27, 1881-1888, 1988.
Crosasso, P., Ceruti, M., Brusa, P., Arpicco, S., Dosio, F. and Cattel, L., “Preparation, characterization and properties of sterically stabilized paclitaxel-containing liposomes,” Journal of Controlled Release, 63, 19-30, 2000.
Doskocz, J., Dałek, P., Foryś, A., Trzebicka, B., Przybyło, M., Mesarec, L., Iglič, A., and Langner, M., “The effect of lipid phase on liposome stability upon exposure to the mechanical stress,” Biochimica et Biophysica Acta (BBA) - Biomembranes, 1862, 183361, 2020.
Dagastine, R. R., Manica, R., Carnie, S. L., Chan, D. Y. C., Stevens, G. W. and Grieser, F., “Dynamic forces between two deformable oil droplets in water,” Journal of Dairy Science, 313, 210-213, 2006.
Douliez, J. P., Houssou, B. H., Fameau, A. L. and Navailles, L., “Self-assembly of bilayer vesicles made of saturated long chain fatty acid," Langmuir, 32, 401−410, 2016.
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-Biomembranes, 1325, 41-62, 1997.
Eloy, J. O., de Souza, M. C., Petrilli, R., Barcellos, J. P. A., Lee, R. J., and Marchetti, J. M., “Liposomes as carriers of hydrophilic small molecule drugs: strategies to enhance encapsulation and delivery,” Colloids and Surfaces B: Biointerfaces, 123, 345-363, 2014.
Frisken, B. J., Asman, C. and Patty, P. J., “Studies of vesicle extrusion,” Langmuir, 16, 928-933, 2000.
Feitosa, E., Jansson, J. and Lindman, B., “The effect of chain length on the melting temperature and size of dialkyldimethylammonium bromide vesicles,” Chemistry and Physics of Lipids, 142, 128-132, 2006.
Fournier, I., Barwicz, J., Auger, M., and Tancrede, P., “The chain conformational order of ergosterol- or cholesterol-containing DPPC bilayers as modulated by amphoteric in B: a FTIR study,” Chemistry and Physics of Lipids 151, 41-50, 2008.
Gebicki, J. M. and Hicks, M., “Ufasomes are stable particles surrounded by unsaturated fatty acid membranes,” Nature, 243, 232-234, 1973.
Gonnet, M., Lethuaut, L., and Boury, F., “New trends in encapsulation of liposoluble vitamins, ” Journal of Controlled Release, 146, 276-290, 2010.
Gruszecki, W. I., and Strzałka, K., “Carotenoids as modulators of lipid membrane physical properties,” Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 1740, 108-115, 2005.
Grudzinski, W., Nierzwicki, L., Welc, R., Reszczynska, E., Luchowski, R., Czub, J., and Gruszecki, W. I., “Localization and orientation of xanthophylls in a lipid bilayer,” Scientific Reports, 7, 9619, 2017.
Håkansson, A., “Emulsion Formation by Homogenization Current Understanding and Future Perspectives,” Annual Review of Food Science and Technology, 10, 239-258, 2019.
Håkansson, A., and Rayner, M., “General Principles of Nanoemulsion Formation by High-energy Mechanical,” Nanoemulsions: Formulation, Applications, and Characterization, 103-139, 2018.
Hope, M. J., Nayar, R., Mayer, L. D. and Cullis, P. R., “Reduction of Liposome Size and Preparation of Unilamellar Vesicles by Extrusion Techniques,” Liposome Technology, 1, 123-139, 1993.
Henson, M. A., Bongers, P., Maindarkar, S. N. and Raikar, N. B., “Incorporating emulsion drop coalescence into population balance equation models of high pressure homogenization,” Colloids and Surfaces A: Physicochemical and Engineering Aspects, 396, 63-73, 2012.
Hara, M., Yuan, H., Yang, Q., Hoshino, T., Yokoyama., Mikake, J., “Stabilization of liposomal membranes by thermozeaxanthins: carotenoid-glucoside esters,” Biochimica et Biophysica Acta, 1461, 147-154, 1999.
Herrington, K. L., Kaler, E. W., Miller, D. D., Zasadzinski, J. A. and Chiruvolu, S., “Phase-behavior of aqueous mixtures of dodecyltrimethylammonium bromide (DTAB) and sodium dodecyl-sulfate (SDS),” Journal of Physical Chemistry, 97, 13792-13802, 1993.
Hincha, D. K., “Effects of alpha-tocopherol (vitamin E) on the stability and lipid dynamics of model membranes mimicking the lipid composition of plant chloroplast membranes,” FEBS Letters, 582, 3687-3692, 2008.
Holmberg, K., “Handbook of applied surface and colloid chemistry,” 2002.
Hong, S. S., Kim, S. H. and Lim, S. J., “Effects of triglycerides on the hydrophobic drug loading capacity of saturated phosphatidylcholine-based liposomes,” International Journal of Pharmaceutics, 483, 142-150, 2015.
Huang, H., Belwal, T., Liu, S., Duan, Z., and Luo, Z., “Novel multi-phase nano-emulsion preparation for co-loading hydrophilic arbutin and hydrophobic coumaric acid using hydrocolloids,” Food Hydrocolloids, 93, 92-101, 2019.
Innocente, N., Biasutti, M., Venir, E., Spaziani, M. and Marchesini, G., “Effect of high-pressure homogenization on droplet size distributions and rheological properties of model oil-in-water emulsions,” Journal of Dairy Science, 92, 1864-1875, 2009.
Immordino, M. L., Brusa, P., Arpicco, S., Stella, B., Dosio, F. and Cattel, L., “Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes containing docetaxel,” Journal of Controlled Release, 91, 417-429, 2003.
Inoue, T., Yanagihara, S. I., Misono, Y. and Suzuki, M., "Effect of fatty acids on phase behavior of hydrated dipalmitoylphosphatidylcholine bilayer: saturated versus unsaturated fatty acids," Chemistry and Physics of Lipids, 109, 117-133, 2001.
Israelachvili, J. N., Mitchell, D. J. and Ninham, B. W., “Theory of self-assembly of hydrocarbon amphiphiles into micelles and bilayers,” Journal of the Chemical Society-Faraday Transactions II, 72, 1525-1568, 1976.
Jayme, M. L., Dunstan, D. E. and Gee, M. L., “Zeta potentials of gum arabic stabilised oil in water emulsions,” Food Hydrocolloids, 13, 459-465, 1999.
Kawakami, K., Nishihara, Y. and Hirano, K., “Liposome/emulsion transition induced by -tocopheryl acetate” Langmuir, 15, 7454-7460, 1999.
Kafrawy, O., Zerouga, M., Stillwell, W. and Jenski, L. J., “Docosahexaenoic acid in phosphatidylcholine mediates cytotoxicity more effectively than other -3 and -6 fatty acids,” Cancer Letters, 132, 23-29, 1998.
Kaler, E. W., Murthy, A. K., Rodriguez, B. E. and Zasadzinski, J. A. N., “Spontaneous vesicle formation in aqueous mixtures of single-tailed surfactants,” Science, 245, 1371-1374, 1989.
Keough, K. M. W. and Davis, P. J., “Gel to liquid-crystalline phase-transitions in water dispersions of saturated mixed-acid phosphatidylcholines,” Biochemistry, 18, 1453-1459, 1979.
Kodati, V. R., El-Jastimi, R., and Lafleur, M., “Contribution of the intermolecular coupling and librotorsional mobility in the methylene stretching modes in the infrared spectra of acyl chains,” J. Phys. Chem., 98, 12191-12197, 1994.
Kranenburg, M. and Smit, B., “Phase behavior of model lipid bilayers,” Journal of Physical Chemistry B, 109, 6553-6563, 2005.
Kuo, J.-H., Jan, M.-S., Chang, C.-H., Chiu, H.-W., and Li, C.-T., “Cytotoxicity characterization of catanionic vesicles in RAW 264.7 murine macrophage-like cells,” Colloids and Surfaces B: Biointerfaces 41, 189-196, 2005.
Kuo, J.-H., Chang, C.-H., Lin, Y.-L. and Wu, C.-J., “Flow cytometric characterization of interactions between U-937 human macrophages and positively charged catanionic vesicles,” Colloids and Surfaces B: Biointerfaces, 64, 307-313, 2008.
Lakowicz, J. R. (Ed.)., “Principles of fluorescence spectroscopy,” Springer Science & Business Media, 2013.
Lasic, D. D., “Liposomes: from physics to applications,” Elsevier Amsterdam,” New York, 265-318, 1993.
Lasic, D. D., “Liposomes in gene delivery,” CRC Press, New York, 67-112, 1997.
Lasic, D. D. and Papahadjopoulos, D., “Liposomes and biopolymers in drug and gene delivery,” Current Opinion in Solid State & Materials Science, 1, 392-400, 1996.
Lee, W. H., Tang, Y. L., Chiu, T. C. and Yang, Y. M., “Synthesis of ion-pair amphiphiles and calorimetric study on the gel to liquid-crystalline phase transition behavior of their bilayers,” Journal of Chemical and Engineering Data, 60, 1119-1125, 2015.
Lentz, B. R., “Membrane “fluidity” as detected by diphenylhexatriene probes,” Chemistry and Physics of Lipids, 50, 171-190, 1989.
Lewis, R. N. A. H. and McElhaney, R. N., “The structure and organization of phospholipid bilayers as revealed by infrared spectroscopy,” Chemistry and Physics of Lipids 96, 9-21, 1998.
Liu, N. and Park, H.J., “Chitosan-coated nanoliposome as vitamin E carrier,” J. Microencapsulation, 26, 235–242, 2009.
Lloyd, J. B. F., “Synchronized excitation of fluorescence emission spectra,” Nature Physical Science, 231, 64, 1971.
Lopes, S., Neves, C., Eaton, P. and Gameiro, P., “Cardiolipin, a key component to mimic the E. coli bacterial membrane in model system membranes,” Biophysical Journal, 100, 626-626, 2011.
Manosroi, A., Wongtrakul, P., Manosroi, J., Sakai, H., Sugawara, F., Yuasa, M. and Abe, M., “Characterization of vesicles prepared with various non-ionic surfactants mixed with cholesterol,” Colloids and Surfaces B: Biointerfaces, 30, 129-138, 2003.
Marsh, D., “CRC handbook of lipid bilayers,” 1990.
Massey, J. B., “Interfacial properties of phosphatidylcholine bilayers containing vitamin E derivatives,” Chemistry and Physics of Lipids, 109, 157-174, 2001.
Matos, A. P., “Essential fatty acid from microalgae,” Inform, 27, 22-26, 2016.
Merino-Montero, S., Montero, M. T. and Hernandez-Borrell, J., “Effects of lactose permease of Escherichia coli on the anisotropy and electrostatic surface potential of liposomes,” Biophysical Chemistry, 119, 101-105, 2006
Morigaki, K. and Peter, Walde, P., “Fatty acid vesicles,” Current Opinion in Colloid & Interface Science, 12, 75-80, 2007.
Michel, N., Fabiano, A. S., Polidori, A., Jack, R. and Pucci, B., “Determination of phase transition temperatures of lipids by light scattering,” Chemistry and Physics of Lipids, 139, 11-19, 2006.
Murai, M., Aramaki, Y., and Tsuchiya, S., “Identification of the serum factor required for liposome-primed activation of mouse peritoneal-macrophages-modified alpha (2)-macroglobulin enhances Fc-gamma receptor-mediated phagocytosis of opsonized sheep red-blood-cells,” Immunology, 86, 64-70, 1995.
McMullen, T. P. W., Ruthven, N. A. H., Ronald L. and McElhaney, 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, 80, 2056-2065, 2000.
Namania, T., Ishikawa, T., Morigaki, K. and Walde, P., “Vesicles from docosahexaenoic acid,” Colloids and Surfaces B: Biointerfaces, 54, 118-123, 2007.
New, R. R. C., “Liposomes: a practical approach,” Oxford University Press, New York, 1-32, 1990.
Oliver, A. E., Hincha, D. K., Crowe, L. M., and Crowe, J. H., “Interactions of arbutin with dry and hydrated bilayers,” Biochimica et Biophysica Acta -Biomembranes, 1370, 87-97, 1998.
Patty, P. J. and Frisken, B. J., “The pressure-dependence of the size of extruded vesicles,” Biophys Journal, 85, 996-1004, 2013.
Reis, O., Winter, R. and Zerda, T. W., “The effect of high external pressure on DPPC-cholesterol multilamellar vesicles: a pressure-tuning Fourier transform infrared spectroscopy study,” Biochimica et Biophysica Acta, 1279, 5-16, 1996.
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.
Ruozi, B., Belletti, D., Tombesi, A., Tosi, G., Bondioli, L., Forni, F. and Vandelli, M. A., “AFM, ESEM, TEM, and CLSM in liposomal characterization: a comparative study,” International Journal of Nanomedicine, 6, 557-563, 2011.
Shen J., Wang Y., Fan P., Jiang L., Zhuang W., Han Y., and Zhang H., “Self-assembled vesicles formed by C18 unsaturated fatty scids and sodiumdodecyl sulfate as a drug delivery system,” Colloids and Surfaces A, 568, 66-754, 2019.
Segota, S. and Tezak, D., “Spontaneous formation of vesicles,” Advances in Colloid and Interface Science, 121, 51-75, 2006.
Shi, X. M., and Chen, F., “Stability of lutein under various storage conditions,” Food/Nahrung, 41, 38-41,1997.
Shrewry, Peter., “HEALTHGRAIN methods: analysis of bioactive components in small grain cereals,” Academic Press, 2016.
Stillwell, W. and Issall, S. R., “Review: Docosahexaenoic acid: membrane properties of a unique fatty acid,” Chemistry and Physics of Lipids, 126, 1–27, 2003.
Sujak, A., Gabrielska, J., Grudziński, W., Borc, R., Mazurek, P., and Gruszecki, W. I., “Lutein and zeaxanthin as protectors of lipid membranes against oxidative damage: the structural aspects,” Archives of Biochemistry and Biophysics, 371, 301-307, 1999.
Sujak, A., Mazurek, P., and Gruszecki, W. I., “Xanthophyll pigments lutein and zeaxanthin in lipid multibilayers formed with dimyristoylphosphatidylcholine,” Journal of Photochemistry and Photobiology B: Biology, 68, 39-44, 2002.
Tan, C., Xia, S., Xue, J., Xie, J., Feng, B., and Zhang, X., “Liposomes as vehicles for lutein: preparation, stability, liposomal membrane dynamics, and structure,” Journal of Agricultural and Food Chemistry, 61, 8175-8184, 2013.
Teo, Y. Y., Misran, M., Low, K. H., “Effect of PEGylated lipid and lecinol S-10 on physico-chemical properties and encapsulation efficiency of palmitoleate-palmitoleic acid vesicles,” Journal of Liposome Research, 24, 241-248, 2014.
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.
Vorauer-Uhl, K. and Wagner, A. J., “Liposome Technology for Industrial Purposes,” Journal of drug delivery, 2011, 1-9, 2011.
Vist, M. R. and Davis, J. H., “Phase-equilibria of cholesterol dipalmitoylphosphatidylcholine mixtures - deuterium nuclear magnetic-resonance and differential scanning calorimetry,” Biochemistry, 29, 451-464, 1990.
Wu, Y., Manna, S., Petrochenko, P., Koo, B., Chen, L., Xu, X., Choi, S., Kozak, D. and Zheng, J., “Coexistence of oil droplets and lipid vesicles in propofol drug products,” International Journal of Pharmaceutics, 577, 118998, 2020.
Wang, X. and Quinn, P. J., “The distribution of K-tocopherol in mixed aqueous dispersions ofphosphatidylcholine and phosphatidylethanolamine,” Biochimica et Biophysica Acta, 1509, 361-372, 2000.
Ward, O. P. and Singh, A., “Omega-3/6 fatty acids: Alternative sources of production,” Process Biochemistry, 40, 3627-3652, 2005.
Watry, M. R., Tarbuck, T. L. and Richmond, G. I., “Vibrational sum-frequency studies of a series of phospholipid monolayers and the associated water structure at the vapor/water interface,” Journal of Physical Chemistry B, 107, 512-518, 2003.
Wolfangel, P. and Muller, K., “Chain order in lipid bilayers: FTIR and solid state NMR studies on bilayer membranes from 1,2-Dimyristoyl-sn-glycero-3-phosphoglucose,” Journal of Physical Chemistry, 107, 9918-9928, 2003.
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 and Polymer Science, 292, 589-597, 2014.
Yang, C. H., Chang, N. F., Chen, Y. S., Lee, S. M., Lin, P. J., and Lin, C. C. “Comparative study on the photostability of arbutin and deoxy arbutin: Sensitivity to ultraviolet radiation and enhanced photostability by the water-soluble sunscreen, benzophenone-4,” Bioscience, Biotechnology, and Biochemistry, 77, 1127-1130, 2013.
Yatcilla, M. T., Herrington, K. L., Brasher, L. L., Kaler, E. W., Chiruvolu, S. and Zasadzinski, J. A., “Phase behavior of aqueous mixtures of cetyltrimethylammonium bromide (CTAB) and sodium octyl sulfate (SOS),” Journal of Physical Chemistry, 100, 5874-5879, 1996.
余欣盈, “以藻油製備包覆葉黃素之液胞的可行性,” 國立成功大學化學工程學系碩士論文, 2017.
林妤臻, “藻油液胞的藥物包覆特性,” 國立成功大學化學工程學系碩士論文, 2019.
黃曉貞, “以醱酵策略提升本土破囊壺菌DJ3之DHA生產效能,” 國立成功大學化學工程學系碩士論文, 2015.
楊政穎, “包覆維他命E醋酸脂之藻油載體的製備及特性分析,” 國立成功大學化學工程學系碩士論文, 2018.