本研究是以定比例與構成之微脂體為基材，配合不同結構之四級銨鹽化幾丁聚醣聚合物(分別為N-(2-羥基)丙基-3-三甲基氯化銨幾丁聚醣(HTCC)與O-(2-羥基)丙基-3-三甲基氯化銨幾丁聚醣(O-HTCC))，經吸附作用合成出一新型藥物釋放載體，其物化性質測試包括：四級銨鹽化程度(DQ)，傅立葉紅外光光譜儀(FTIR)，穿透式電子顯微鏡(TEM)、電性及光散射儀等測試。在物化測試中，結果顯示其四級銨鹽化程度皆在五成以上，以及吸附的明確性與明顯的正電性質。
並進行抗菌性，細胞毒性與穿透性之分析。抗菌實驗，將四級銨鹽化幾丁聚醣聚合物溶於純水與微脂體 ,針對臨床上常感染的金黃色葡萄球菌(Staphylococcus aureus) 所做測試。結果顯示，微脂體包覆N-(2-羥基)丙基-3-三甲基氯化銨幾丁聚醣(HTCC)與N-(2-羥基)丙基-3-三甲基氯化銨幾丁聚醣(HTCC)溶液對金黃色葡萄球菌的最小殺菌濃度(MBC)分別為917±29μg/ml和1017±29μg/ml，最小抑菌濃度（MIC）分別為162±3μg/ml和196±6μg/ml。O-(2-羥基)丙基-3-三甲基氯化銨幾丁聚醣(O-HTCC)與微脂體包覆O-(2-羥基)丙基-3-三甲基氯化銨幾丁聚醣 (O-HTCC)，對金黃色葡萄球菌的最小殺菌濃度(MBC)分別為59.2±1.7μg/ml和54.2 ±1.7μg/ml，而最小抑菌濃度（MIC）分別為18±1μg/ml和15±1μg/ml ,顯示微脂體包覆四級銨鹽化幾丁聚醣聚合物之的最小殺菌濃度與最小抑菌濃度皆優於單純四級銨鹽化幾丁聚醣聚合物溶液，代表抗菌性最佳。細胞毒性研究證明利用微脂體做為四級銨鹽化幾丁聚醣聚合物之包覆或塗層載體，在鼠類纖維母細胞(L-929 fibroblast)與黑色素細胞(B16-F10 melanoma)上，進行MTT實驗與細胞形態分析，證明不會造成細胞毒殺作用。在細胞與體外穿透實驗中，細胞上使用共軛焦雷射掃描式顯微鏡(CLSM)測試微脂體包覆四級銨鹽化幾丁聚醣聚合物與四級銨鹽化幾丁聚醣聚合物塗層微脂體，對於無塗層微脂體之細胞穿透效果，實驗結果證明 , 無論是微脂體包覆四級銨鹽化幾丁聚醣聚合物四級銨鹽化幾丁聚醣聚合物塗層微脂體，對於無塗層微脂體之穿透效果皆較高(P<0.05)。體外穿透實驗使用三周齡SD品系雄鼠之背部皮膚，以Franz Cell進行測試，實驗結果證明鞠酸置於幾丁聚醣聚合物塗層微脂體，對於鞠酸置於無塗層微脂體或單純鞠酸之穿透效果皆較高。(P<0.05)美白實驗部分，評估鞠酸(Kojic acid)於四級銨鹽化幾丁聚醣聚合物塗層微脂體與無塗層微脂體中，對於鼠類黑色素細胞 (B16-F10)之美白現象。實驗結果證明，高濃度鞠酸下，四級銨鹽化幾丁聚醣聚合物之包覆載體不僅對鼠類纖維母細胞(L-929)穿透與融合速度皆快，在黑色素細胞中，穿透與融合速度皆快，並對黑色素有較佳抑制效果(P<0.05)。

In this study, derivatives of chitosan, N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC) and O-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (O-HTCC) were coated onto the liposomes made of cholesterol and 1, 2-palmitoyl-sn-glycero-3-phosphatidylcholine (DPPC). These coated liposomes were loaded with kojic acid for skin whitening. The appearance of liposome was examined using transmission electron microscope (TEM), and the coating of HTCC or O-HTCC to the liposome was confirmed by infrared spectroscopy. The zeta potential of liposome was measured. The antimicrobial activity of liposome against Staphylococcus aureus (ATCC 6538P) was evaluated based on the MIC and MBC values. The results showed that the antimicrobial activity of HTCC or O-HTCC loading liposome was higher than that of pure HTCC or O-HTCC. By labeling with Dil, the fusion of liposome with the cell membrane of L929 fibroblast and B16-F10 melanoma was improved by the coating of HTCC or O-HTCC and loading of chitosan derivatives. Based on the results of Franz cell experiment, the penetration of kojic acid (KA) through skin was improved by using HTCC-coating liposomes. The cytotoxicity of O-HTCC and O-HTCC-loading liposome was determined using L929 fibroblasts in vitro via MTT test and morphology of L-929 cells. The results showed that cytotoxicity was not observed when concentration of O-HTCC was below 100μg/ml. Furthermore, the cell proliferation of L929 was not affected by HTCC-coating liposomes, while that of B16-F10 was reduced slightly with the increase of the concentration of HTCC-loading liposome. The degree of skin whitening was determined based on the melanin content in B-16-F10 cells. The results showed that the level of melanin synthesis was lower when KA was delivered using HTCC-coating liposome instead of traditional liposome.

1.Shimogaki, H., Tanaka, Y., Tamai, H., Masuda, M., “In vitro and in vivo evaluation of ellagic acid on melanogenesis inhibition”, International Journal of Cosmetic Science, Vol. 22, pp. 291-303 (2000)
2.Sheu, S. C., “The newest practical physiological anatomy”, Yung-Da, Taipei, Taiwan (2002)
3.Elias, P.M., “Epidermal lipids, barrier function, and desquamation”, Journal of Investigative Dermatology, Vol. 80, pp. 44S-49S (1983)
4.Byorksten, J. A., “A common molecular basis for the aging syndrome”, Journal of the American Geriatrics Society, Vol. 6, pp. 740 (1958)
5.Hunt, G., Kyne, S., Ito, S., Wakamatsu, K., Todd, C. and Thody, A., “Eumelanin and phaeomelanin contents of human epidermis and cultured melanocytes”, Pigment Cell Res., Vol. 8(4) pp. 202-8.1995.
6.Cooksey, C. J., Garratt, P. J., Land, E. J., Pavel, S., Ramsden, C. A., Riley, P. A., Smit, N. P., “Evidence of the indirect formation of the catecholic intermediate substrate responsible for the autoactivation kinetics of tyrosinase”, J. Biol. Chem., Vol. 272 (42) pp. 26226-35, (1997)
7.Prota, G., “The role of peroxidase in melanogenesis revisited”, Pigment Cell Res., Suppl 2, pp. 25-31 (1992)
8.Ozeki, H., Ito, S., Wakamatsu, K., Thody, A. , “Spectrophotometer characterization of eumelanin and pheomelanin in hair”, Pigment Cell Res., Vol. 9(5), pp. 265-70 (1996)
9.Prota, G., “Melanins and Melanogenesis”, Cosmetic and Tolletries Magazine, 111: 43-51 (1996)
10.Keith, C. B., “Lawrence M., Melanins: Hair dyes for the future”, Cosmetics & Toiletries Magazine, 109: 59-64 (1994)
11.Mayer, A. M., “Polyphenol oxidases in plants-Recent progress”, Phytochem., Vol. 26, 11-20 (1987)
12.Whitaker, J. R., “Polyphenol oxidase”, In Food Enzymes, Structure and Mechanism, Chapman & Hall, New York, pp. 271-307 (1995)
13.Quevedo, W. C., Jr, Szabo, G., Virks, J., Sinesi, S. J., “Melanocyte populations in UV-irradiated human skin”, J. Invest Dermatol. , Vol. 45(4), pp. 295-8 (1965)
14.Romero, C., Aberdam, E., Larnier, C., Ortonne, J. P., “Retinoic acid as modulator of UVB-induced melanocyte differentiation”, Involvement of the melanogenic enzymes expression, J. Cell Sci., Vol. 107 ( Pt 4):1095-103 (1994)
15.Gonzalez, S., Moran, M., Kochevar, I. E., “Chronic photodamage in skin of mast cell-deficient mice”, Photochem. Photobiol., Vol. 70(2), pp. 248-53 (1999)
16.Lowell, A., “Golgsmith: Biochemistry and Physiology of the skin”, Oxford University Press, UK (1983)
17.Fuller, B. B., Viskochil, D. H., “The role of RNA and protein synthesis in mediating the action of MSH on mouse melanoma cells”, Life Sci., Vol. 24(26), pp. 2405-15 (1979)
18.Jimenez, M ., Kameyama, K., Maloy, W. L., Tomita, Y., Hearing, V. J., “Mammalian tyrosinase: biosynthesis, processing, and modulation by melanocyte-stimulating hormone”, Proc. Natl. Acad. Sci., USA, Vol. 85(11), pp. 3830-4 (1988)
19.Wong, G., Pawelek, J., “Melanocyte-stimulating hormone promotes activation of pre-existing tyrosinase molecules in Cloudman S91 melanoma cells”, Nature, 19 255(5510), pp. 644-6 (1975)
20.“Solar and ultraviolet radiation”, Lyon, 1992. IARC monographs on the evaluation of carcinogenic risks to humans (2006)
21.Linda, L. H., Pharm, D., “Use of hydroquinone as a bleaching cream”, Ann. Pharmacotherapy, Vol. 27, pp. 592-593 (1993)
22.Paul, D. M.; Cotton, D. W. K.,” The Molecular Biology of Skin” , Blackwell Scientific Publications Oseny Mead, Oxford OX2 0EL (1976)
23.Erol, D. D., Calis, U., Yulug, N., “Synthesis and antimicrobial activities of some dithiocarbamate derivatives of kojic acid”, Boll. Chim. Farm., Vol. 134, pp. 620-623 (1995)
24.Milot, C., McBrien, J., Allen, S., Guibal, E., “Influence of physico-chemical and structural characteristics of chitosan flakes on molybdate removal”, J. Appl. Polym. Sci., Vol. 68, pp. 571-580 (1998)
25.Shahidi, F., Arachchi, J. K.V., Jeon, Y. J. ,”Food applications of chitin and chitosans”, Trends in Food Science & Technology Vol. 10, pp. 37-51 (1999)
26.Lepri, L., Desideri, P. G., Coas, V., “Separation and identification of water-soluble food dyes by ion-exchange and soap thin-layer chromatography”, J. Chromatogr., Vol. 161 ,pp. 279-86 (1978)
27.Turkov, J., Brod, C., Stamberg, J., Proteolytic, “dry biopolymeric composition for treatment of wounds, and method of using same”, U.S. Patent, No. 4613502: 1-6 (1986)
28.Sashiwa, H., Shigemasa, Y., Roy, R., “Chemical modification of chitosan. Part 9: Reaction of N-carboxyethylchitosan methyl ester with diamines of acetal ending PAMAM dendrimers”, Carbohydrate Polymers., Vol. 47, pp. 201-208 (2002)
29.Ranter, B. D., Johnston, A. B., Lenk, T. J., “Biomaterial surfaces”, Journal of Biomedical Materials Research, Vol. 21, pp. 59-89 (1987)
30.Tanigawa, T., Tanaka, Y., Sashiwa, H., Saimoto, H., Shigemasa, Y., “Various biological effects of chitin derivatives”, In Advances in chitin and chitosan, Elsevier Applied Science, London and New York, 206-215 (1992)
31.Tan, S. C., Khor, E., Tan, T. K., Wong, S. M., “The degree of deacetylation of chitosan: advocation the first derivative UV-spectrophotometry method of determination” , Talanta, Vol.45, pp.713-719 (1998)
32.Papineau, A. M., Hoover, D. G., Knorr, D., Farkas, D. F., “Antimicrobial effect of water-soluble chitosans with high hydrostatic pressure”, Food Biotechnology, Vol. 5, pp. 45-57 (1991)
33.Cevc, G., Blume, G., “Hydrocortisone and dexamethasone in very deformable drug carriers have increased biological potency, prolonged effect, and reduced therapeutic dosage”, Biochim. Biophys. Acta., Vol. 1663, pp. 61–73 (2004)
34.Cevc G., Blume G., “Lipid vesicles penetrate into intact skin owing to the transdermal osmotic gradients and hydration force”, Biochim. Biophys. Acta., Vol. 1104(1), pp.226–32 (1992)
35.Maria, Teresa, De, Magistris, “Mucosal delivery of vaccine antigens and its advantages in pediatrics”, Advanced Drug Delivery Reviews, Vol. 58(1), pp. 52-67 (2006)
36.Mezei, M., Gulasekharam, V., “Liposomes – a selective drug delivery system for the topical route of administration. I. Lotion dosage form”, Life Sci., Vol. 26, pp. 1473-1477 (1980)
37.Kato, A., Ishibashi, Y., Miyake, Y., “Effect of egg yolk lecithin on transdermal delivery of bunazosin hydrochloride”, J. Pharm. Pharmacol., Vol. 39, pp. 399–400 (1987)
38.Mishra, D., Garg, M., Dubey, V., Jain, S,, Jain, N. K., “Elastic liposomes mediated transdermal delivery of an anti-hypertensive agent: propranolol hydrochloride”, J. Pharm. Sci., Vol. 96(1), pp. 145-55, (2007)
39.Nishihata, T., Kotera, K., Nakano, Y., Yamazaki, M., “Rat percutaneous transport of diclofenac and influence of hydrogenated soya phospholipids”, Chem. Pharm. Bull., Vol. 35, pp. 3807-3812 (1987)
40.Priborsky, J., Muhlbachiba, E., “Evaluation of in vitro percutaneous absorption across human skin and in animal model”, J. Pharm. Pharmacol. Vol. 43, pp. 468-472 (1990)
41.Song, Y. K., Kim, C. K., “Topical delivery of low-molecular-weight heparin with surface-charged flexible liposomes”, Biomaterials, Vol. 27(2), pp. 271-80 (2006)
42.Barry, B. W., “Novel mechanisms and devices to enable successful transdermal drug delivery”, Eur. J. Pharm. Sci., Vol. 14, pp. 101–114 (2001)
43.El, Maghraby, G. M., Williams, A. C., Barry, B. W., “Skin delivery of oestradiol from deformable and traditional liposomes: mechanistic studies”, J. Pharm. Pharmacol., Vol. 51, pp. 1123–1134 (1999)
44.El, Maghraby, G. M., Williams, A. C., Barry, B. W., “Oestradiol skin delivery from ultradeformable liposomes: refinement of surfactant concentration”, Int. J. Pharm., Vol. 196 , pp. 63–74 (2000a)
45.Kirjavainen, M., Urtti, A., Jaaskelainen, I., Suhonen, T. M., Paronen, P., Koskela, R. V., Kiesvaara, J., Monkkonen, J., “Interaction of liposomes with human skin in vitro – the influence of lipid composition and structure”, Biochim. Biophys. Acta., Vol. 1304 , pp. 179–189 (1996)
46.Kirjavainen, M., Urtti, A., Koskela, R. V., Kiesvaara, J., Monkkonen, J., “Liposome-skin interactions and their effects on the skin permeation of drugs”, Eur. J. Pharm. Sci., Vol. 7 ,pp. 279–286 (1999a)
47.Cevc, G., Blume, G., :Lipid vesicles penetrate into intact skin owing to the transdermal osmotic gradients and hydration force”, Biochim. Biophys. Acta., Vol. 1104, pp. 226–232 (1992)
48.Yokomizo, Y., Sagitani, H., “Effects of phospholipids on the percutaneous penetration of indomethacin through the dorsal skin of the guinea pig, in vitro”, J. Control. Release, Vol. 38, pp. 267–274 (1996a)
49.Zellmer, S., Pfeil, W., Lasch, J., “Interaction of phosphatidylcholine liposomes with the human stratum corneum”, Biochim. Biophys. Acta., Vol. 1237, pp. 176–182 (1995)
50.Torchilin, V. P., “Recent advances with liposomes as pharmaceutical carriers”, Nat. Rev. Drug Discov., Vol. 4(2), pp. 145-60 (2005)
51.El, Maghraby, G. M., Williams, A. C., Barry, B. W., “Can drug-bearing liposomes penetrate intact skin?” J. P. P., Vol. 58, pp. 415–429 (2006)
52.Foldvari, M., Gesztes, A., Mezei, M., “Dermal drug delivery by liposome encapsulation: clinical and electron microscopic studies”, J. Microencaps., Vol. 7, pp. 479–489 (1990)
53.Lim, S. H., Hudson, S. M., “Synthesis and antimicrobial activity of a water-soluble chitosan derivative with fiber-reactive group”, Science direct, 313-319 (2004)
54.Kim, G. E., Lee, J. H., Jung, S. H., Seo, E. S., Jin, S. D., Kim, G. J., Cha, J., Kim, E. J., Park, K. D., Kim, D., “Enzymatic synthesis and characterization of hydroquinone galactoside using Kluyveromyces lactis lactase”, J. Agric. Food Chem., Vol. 8;58(17), pp. 9492-7 (2010)
55.Mady, M. M., Darwish, M. M., Khalil, S., Khalil, W. M., “Biophysical studies on chitosan-coated liposomes”, Eur. Biophys. J., Vol. 38 ,pp. 1127-1133 (2009)
56.Kita, K., Dittrich, C., “Drug delivery vehicles with improved encapsulation efficiency: taking advantage of specific drug-carrier interactions”, Drug Deliv., Vol. 8 , pp. 329-342 (2011)
57.Bolean, M., Sim&atilde;o, A. M., Favarin, B. Z., Mill&aacute;n, J. L., Ciancaglini, P., “The effect of cholesterol on the reconstitution of alkaline phosphatase into liposomes”, Biophys. Chem., Vol. 152 , pp. 74-79 (2010)
58.Du, W. L., Niu, S. S., Xu, Y. L., Xu, Z. R., Fan, C. L., “Antibacterial activity of chitosan tripolyphosphate nanoparticles loaded with various metal ions”, Carbohydrate Polymers., Vol. 75, pp. 385–389 (2009)
59.Coelho, M. A., Berumen, L., Avrameas, S., “Properties of protein polymers as substratum for cell growth in vitro”, J. Cell Physiol., Vol. 83, pp. 379, (1974)
60.Uchida, Y., Izume, M., Ohtakara, A., “Chitin and chitosan”, Elsevier Applied Science, London and New York (1988)
61.Huang, L. L. H, Sung, H. W., Tsai, C. C., Huang, D. M., “Biocompatibility study of a biological tissue fixed with a naturally occuring crosslinking reagent”, J. Biomed. Mater. Res., Vol. 42, pp. 568-576 (1998)
62.Warner, R. R., Myers, M. C., Taylor, D. A., “Electron probe analysis of human skin: determination of the water concentration profile”, J. Invest. Dermatol., Vol. 90, pp. 218–224 (1988)
63.Rojanasakul, Y., Wang, L. Y., Bhat, M., Glover, D. D., Malanga, C. J., Ma, J. K., “The transport barrier of epithelia: a comparative study on membrane permeability and charge selectivity in the rabbit”, Pharm. Res., Vol. 9 , pp. 1029-1034 (1992)
64.Piemi, M. P., Korner, D., Benita, S., Marty, J. P., “Positively and negatively charged submicron emulsions for enhanced topical delivery of antifungal drugs”, J. Control. Rel., Vol. 58, pp. 177-187 (1999)
65.Kirjavainen, M., Urtti A., Jaaskelainen, I., Suhonen, T. M., Paronen, P., Valjakka-Koskela, R., Kiesvaara, J., Monkkonen, J., “Interaction of liposomes with human skin in vitro—the influence of lipid compositionan d structure”, Biochim. Biophys. Acta., Vol. 1304, pp. 179–89 (1996)
66.Hofland, H. E. , Bouwstra, J. A., Bodde, H. E., Spies, F., Junginger, H. E., “Interactions between liposomes and human stratum corneum in vitro: freeze fracture electron microscopical visualization and small angle X-ray scattering studies”, Br. J. Dermatol., Vol. 132 ,pp. 853–866 (1995)
67.Yoo, J., Shanmugam, S., Song, C. K., Kim, D. D., Choi, H. G., Yong, C. S., Woo, J. S., Yoo, B. K., “Skin penetration and retention of L-ascorbic acid 2-phosphate using multilamellar vesicles”, Arch. Pharm. Res., Vol. 31, No 12, pp. 1652-1658 (2008)
68.Li, X. H., Guo, L., Sun, Y., Zhou, J., Gu, Y. L., Li, Y. U., “Baicalein inhibits melanogenesis through activation of the ERK signaling pathway”, Int. J. Mol. Med., Vol. 25(6), pp. 923-7 (2010)