小腸上皮細胞(epithelial cell)扮演著隔開人體與外界環境的角色，主要是由細胞內的酵素與連接細胞之間的蛋白質錯合物構成屏障的功能。而由於細胞膜主要是由脂雙層所構成，因此親脂性分子可以直接穿過細胞膜(transcellular pathway)通過上皮細胞；反之，親水性分子無法直接穿過疏水性的細胞膜，需要經由細胞與細胞之間的空隙(paracellular pathway)通過上皮細胞。位於上皮細胞之間的蛋白質錯合物tight junction為paracellular pathway的主要屏障，其功用為選擇性地讓一些親水性分子進出上皮細胞。正因為小腸上皮細胞之間tight junction的阻礙，造成以往經由口服投藥的親水性蛋白質藥物在小腸無法有效地吸收，進而影響藥物治療疾病的功效。
本研究為了解決親水性藥物在小腸不良吸收的問題，利用可逆性地調控tight junction開合的方法提升小腸吸收親水性藥物的能力。根據文獻記載幾丁聚醣能夠暫時性地打開tight junction，是一種有效而且安全的paracellualr permeability enhancers (PPEs)，將幾丁聚醣與親水性藥物合併服用，可以增加親水性藥物在小腸的吸收，進而提升藥物的生體可用率。因此本研究的目的是希望以幾丁聚醣為基材製備藥物載體，借由幾丁聚醣具有打開小腸上皮細胞之間的tight junction的特性，將親水性藥物藉由藥物載體完整地帶過小腸上皮細胞。
在製備藥物載體時，為了提升親水性藥物的包覆率與維持藥物的活性，我們採用了ionic gelation方法製備奈米微粒，製備流程為混合帶正電荷的幾丁聚醣與帶負電荷的聚麩氨酸水相高分子溶液，可以在瞬間得到經由離子鍵結的奈米微粒。這種製備方法的優點在於製備過程不需要提高溫度也不需使用有機溶劑，整個過程都在溫和的水相環境中進行，有利於維持藥物的活性。再加上將藥物載體製備成奈米微粒，能夠增加藥物傳遞效率、改善藥物釋放模式和提高藥物標的性能等優點。
本實驗分為兩大部分:第一部分為探討不同分子量與濃度的製備條件，以找出形成奈米微粒的最佳條件，接著探討製備出來的奈米微粒各項性質，包括粒徑分析、表面電荷分析與外觀型態的觀察等;第二部分則是以Caco-2 cell monolayers做為體外model，評估此種奈米微粒對於小腸上皮細胞的滲透能力。
第一部分的實驗結果顯示，以分子量160,000和14,000的聚麩氨酸與分子量5,000的幾丁聚醣在不同濃度下製備奈米微粒，透過粒徑儀的分析，我們可以定義出奈米微粒形成的範圍，之後藉由粒徑儀、TEM和AFM的分析，可進一步地證明我們製備出來的微粒為大小均一的奈米小球。最後依據奈米微粒表面電荷的分析，我們可以篩選出來我們所期望表面為帶正電荷幾丁聚醣的奈米微粒。
在第二部份的實驗裡，transepithelial electrical resistance (TEER) 的實驗結果發現，表面帶正電荷幾丁聚醣的奈米微粒能夠有效地打開並可逆性地調控tight junction，同時表面帶正電荷幾丁聚醣的奈米微粒比同濃度幾丁聚醣溶液有較好的調控細胞與細胞之間tight junction能力。而由confocal laser scanning microscopy (CLSM) 的實驗結果，顯示製備出來的奈米微粒明顯地藉由paracellular pathway穿透Caco-2 cell monolayers，此結果提供了奈米微粒打開tight junction的直接證據。綜合以上的實驗結果，顯示此種親水性奈米級藥物載體可以有效地增加親水性藥物在小腸的吸收，藉此提升藥物的生體可用率。

1. 姚富洲, “生命科學,” 合記出版公司,台北, 2002.
2. 卓貴美, “圖解生理學,” 五南圖書出版公司,台北, 339-362 ,2000.
3. 朱家瑜, “人體組織學,” 藝軒出版公司, 台北, ,2000.
4. Ward, P.D., Tippin, T.K., Thakker, D.R., “Enhancing paracellular permeability by modulating epithelial tight junctions,” Pharmaceutical Science & Technology Today, 3, 346-358, 2000.
5. Diamond, J.M., “The epithelial junction: bridge, gate, and fence,” Physiologist, 20, 10-8, 1977.
6. Ballard, S.T., Hunter, J.H., Taylor, A.E., “Regulation of tight-junction permeability during nutrient absorption across the intestinal epithelium,” Annu. rev. nutr, 15, 35-55, 1995.
7. Hidalgo, I.J., Raub, T.J., Borchardt, R.T., “Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability,” Gastroenterology, 96, 736-49, 1989.
8. Wilson, G., “Transport and permeability properties of human Caco-2 cells: an in vitro model of the intestinal epithelial cell barrier,” J. control. release., 11, 25-40, 1990.
9. Delie, F. and Rubas, W., “A human colonic cell line sharing similarities with enterocytes as a model to examine oral absorption: advantages and limitations of the Caco-2 model,” Crit. rev. ther. drug carr. syst., 14, 221-286, 1997.
10. Liu, D.Z., LeCluyse, E.L., Thakker, D.R., “Dodecylphosphocholine- mediated enhancement of paracellular permeability and cytotoxicity in Caco-2 cell monolayers,” J. pharm. sci, 88, 1161-1168, 1999.
11. Liu, D.Z., Morris-Natschke, S.L., Kucera, L.S., Ishaq, K.S., Thakker, D.R., “Structure-activity relationships for enhancement of paracellular permeability by 2-alkoxy-3-alkylamidopropyl- phosphocholines across Caco-2 cell monolayers”, J. pharm. sci, 88, 1169-1174, 1999.
12. Artursson, P., Palm, K., Luthman, K., “Caco-2 monolayers in experimental and theoretical predictions of drug transport,” Adv. drug deliv. rev., 46, 27-43, 2001.
13. 李昂, “奈米顆粒在藥物輸遞的應用,” 化工資訊, 10, 44-55, 2001.
14. Soppimath, K.S., Aminabhavi, T.M., Kulkarni, A.R., Rudzinski, W.E., “Biodegradable polymeric nanoparticles as drug delivery devices,” J. control. release., 70, 1-20, 2001.
15. Hu, Y., Jiang, X., Ding, Y., Ge, H., Yuan, Y., Yang, C., “Synthesis and characterization of chitosan-poly(acrylic acid) nanoparticles,” Biomaterials, 23, 3193-3201, 2002.
16. Hans, M.L. and Lowman, A.M., “Biodegradable nanoparticles for drug delivery and targeting,” Current Opinion in Solid and Materials Science, 6, 319-327, 2002.
17. Pan, Y,. Li, Y.J., Zhao, H.Y., Zheng, J.M., Xu, H., Wei, G., Hao J.S. Cui, F.D., “Bioadhesive polysaccharide in protein delivery system: chitosan nanoparticles improve the intestinal absorption of insulin in vivo,” Int. j. pharm., 249, 139-147, 2002.
18. Hirano, S. and Matsumura, T., “N-acyl derivatives of chitosan and their hydrolysis by chitonase,” Carbohydr. res, 165, 120-122, 1987.
19. Brandenberg, G., Leibrock, L.G., Shuman, R., Malette, W.G., Quigley, H., “Chitosan: a new topical hemostatic agent for diffuse capillary bleeding in brain tissue,” Neurosurgery, 15, 9-13, 1984.
20. Muzzarelli, R.A., Tanfani, F., Emanuelli, M., “Sulfated N-carboxymethyl chitosan : Novel blood anticoagulants,” Carbohydr. res, 126, 225-231, 1984.
21. Stanley, W.L., Watters, G.G., Kelly, S.H., Olson, A.C., “Glucoamylase immobilized on chitin with glutaraldehyde,” Biotechnol. bioeng., 20, 135-140, 1978.
22. Onsoyen, E. and Skaugrud, O., “Metal recovery using chitosan,” J. chem. technol. biotechnol., 49, 395-404, 1990.
23. Muzzarelli, R.A.A., “Chitin and its derivatives: New trends of applied and research,” Carbohydr. res, 3, 52-57, 1993.
24. Ralston, G.B., Tracey, M.V., Wrench, P.V., “The inhibition of fermentation in baker’s yeast by chitin,” Biochimca et Biophysica Acta, 93, 652-665, 1964.
25. Nishimura, S., Ikeuchi, Y., Tokura, S., “The adsorption of bovine blood proteins onto the surface of O-carboxymethyl chitin,” Carbohydr. res, 134, 305-312, 1984.
26. Inoue, K., Baba, Y., Yoshizuka, K., “Selectivity series in the adsorption of mental ions on a resin prepared by crosslinking copper(Ⅱ)-complexed chitosan,” Chem. lett., 1281-1284, 1988.
27. Chandy, T. and Sharma, C.P., “Prostaglandin E1-immobilized poly(vmyl alcohol)-blended chitosan membranes: blood compatibility and permeability properties,” Journal of Application Polymer Science, 44, 2145-2156, 1992.
28. Kifime, K., Yamaguchi, Y., Kishimoto, S., “Wound healing effect of chitin surgical dressing,” Trans. Soc. Biomat., XI, 216-220, 1988.
29. Pelletir, A., Lemire, L., Sygnsch, J., “Chitin / chitosan transformation by thermo- chemical treatment,” Biotechnol. bioeng., 36, 310-315, 1990.
30. Peluso, G., Petille, O., Ranieri, M., Samtin, M., Ambrosio, L., Calabro, D., Avallone, B., Balsamo, G., “Chitosan-mediated stimulation of macrophage function,” Biomaterials, 15, 1215-1220, 1994.
31. Sakaguchi, T., Horikoshi, T., Nakajima, A., “Adsorption of uraniumby chitin phosphate and chitosan phosphate,” Agric. biol. chem., 45, 2191-2195, 1981.
32. Peniston, Q.P. and Johnson, E.L., “Process for depolymerization of chitosan,” U.S. Patent, No. 3922260, 1-5, 1975.
33. MacLaughlin, F.C., Mumper, R.J., Wang, J., Tagliaferri, J.M., Gill I., Hinchcliffe, M., Rolland, A.P., “Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid deliver,” J. control. release., 56, 259-272, 1998.
34. Gross, A., Bacterial γ-poly(glutamic acid). In: Kaplan, D.L. (Ed.) Biopolymers from Renewable Resources, Springer-Verlag, New York, 195-219, 1998.
35. Richard, A. and Margaritis, A., “Poly(glutamic acid) for biomedical applications,” Critical Reviews in Biotechnology, 21, 219-32, 2001.
36. Yoon, S.H., Do, J.H., Lee, S.Y., Chang, H.N., “Production of poly-γ-glutamic acid by fed-batch culture of Bacillus licheniformis,” Biotechnol. lett., 22, 585-588, 2000.
37. Thorne, C.B., Housewright, R.D., Leonard, C.G., “Production of glutamyl polypeptide by Bacillus Subtilis,” U.S. Patent, NO. 2895882, 1-4, 1959.
38. Goto, A. and Kunioka, M., “Biosynthesis and Hydrolysis of Poly(γ-glutamic acid) from Bacillus subtilis IFO3335,” Bioscience Biotechnology Biochemistry, 56, 1031-1035, 1992.
39. Kotze, A.F., Luessen, H.L., de Boer, A.G., Verhoef, J.C., Junginger, H.E., “Chitosan for enhanced intestinal permeability: prospects for derivatives soluble in neutral and basic environments,” European Journal of Pharmaceutical Sciences, 7, 145-151, 1999.
40. Thanou, M., Verhoef, J.C., Junginger, H.E., “Chitosan and its derivatives as intestinal absorption enhancers,” Adv. drug deliv. rev., 50, Suppl 1, S91-101, 2001.
41. Kotze, A.F., Thanou, M.M., Luebetaen, H.L., de Boer, A.G., Verhoef, J.C., Junginger, H.E., “Enhancement of paracellular drug transport with highly quaternized N-trimethyl chitosan chloride in neutral environments: in vitro evaluation in intestinal epithelial cells (Caco-2),” J. pharm. sci., 88, 253-257, 1999.
42. Thanou, M., Verhoef, J.C., Junginger, H.E., “Oral drug absorption enhancement by chitosan and its derivatives,” Adv. drug deliv. rev., 52, 117-126, 2001.
43. Kotze, A.F., Thanou, M.M., Luessen, H.L., de Boer, B.G., Verhoef, J.C., Junginger, H.E., “Effect of the degree of quaternization of N-trimethyl chitosan chloride on the permeability of intestinal epithelial cells (Caco-2),” European Journal of Pharmaceutics & Biopharmaceutics, 47, 269-274, 1999.
44. Kotze, A.F., Luessen, H.L., de Leeuw, B.J., de Boer, B.G., Verhoef, J.C., Junginger, H.E., “N-trimethyl chitosan chloride as a potential absorption enhancer across mucosal surfaces: in vitro evaluation in intestinal epithelial cells (Caco-2),” Pharm. res., 14, 1197-1202, 1997.
45. 陳家全, “生物電子顯微鏡學,” 行政院國家科學委員會精密儀器發展中心”, 新竹, 1997.
46. Yamashita, S., Konishi, K., Yamazaki, Y., Taki, Y., Sakane, T., Sezaki, H., Furuyama, Y., “New and better protocols for a short-term Caco-2 cell culture system,” J. pharm. sci., 91, 669-679, 2002.
47. Borchard, G., Lueßen, H.L., de Boer, Albertus, G., Verhoef, J., C., Lehr, C.M., “The potential of mucoadhesive polymers in enhancing intestinal peptide drug absorption. III: Effects of chitosan-glutamate and carbomer on epithelial tight junctions in vitro,” J. control. release., 39, 131-138, 1996.
48. Zengshuan, M. and Lim, L.Y., “Uptake of chitosan and associated insulin in Caco-2 cell monolayers: a comparison between chitosan molecules and chitosan nanoparticles,” Pharm. res., 20, 1812-1819, 2003.