臺灣博碩士論文加值系統

水膠是指具有3-D 網狀結構之高分子，且具有在水中膨潤但不溶解的性質。近幾年來水膠的研究重點著重於功能型水膠之設計，此種水膠會隨著外界環境變化而產生相對應的應答行為使得水膠產生形態上的改變。而藥物釋放所訴求的目的不外乎下列幾點︰增加藥效或延長藥物作用時間、降低藥物副作用、適時、適量將藥物帶到標的部位。水膠具有上述的功能，藉由其在不同的生理環境下產生收縮或膨潤的變化，進而達到控制釋放、延長藥效及藥物標的的作用，因此可將水膠應用在藥物釋放系統。
以幾丁聚醣做為藥物釋放之載體其適用範圍是以酸性環境為主。因此為了改變幾丁聚醣的酸鹼應答行為，我們利用化學方法做一修飾接上甲基(carboxymethyl group)，使其具有在酸性條件下收縮鹼性環境下膨潤的性質，依此計畫，可得到三種不同接枝位置的水溶性產物：N,O-carboxymethyl chitosan (NOCC)、N-carboxymethyl chitosan (NCC)、O-carboxymethyl chitosan (OCC)。經過實驗分析結果來看，經由FOURIER-TRANSFORM INFRARED SPECTROMETER (FT-IR)的分析可證明幾丁聚醣在經由三種不同化學方法後在其鏈上是有接上甲基(carboxymethyl group)，而在接枝的位置上經由NMR證明NOCC其胺基與第6個碳上的氧皆有受到化學修飾，且其接枝比分別為13.0 %及10.5 %。在體外抗酵素分解方面，幾丁聚醣分子鏈上經由化學修飾而導入carboxymethyl group，經由ninhydrin assay的分析，可加速被Lysozyme分解的速度。
在水膠性質測試方面，單獨使用NOCC以GP製成水膠，由於分子上的胺基在酸性下會質子化而帶正電，因此造成在測試其酸鹼可逆性質時，當NOCC水膠從酸性環境移入中性環境下帶正電的胺基會與帶負電的基形成吸引力，因而產生收縮現象。所以我們將胺基乙醯化成乙醯胺基以改善收縮現象，然而乙醯化後的水膠仍有兩個問題需要去改善，第一是在酸性環境下起始膨潤過高；第二是在酸性及鹼性環境下的膨潤差異度過低，因此分別以酸處理及加入alginate來加以改善。經實驗證明，經由加入alginate及乙醯化和醋酸處理後，NOCC/alginate GP交聯水膠是適用於intestine site-specific drug delivery system。
經由三種不同交聯結構所製備的NOCC/alginate水膠，GP是以胺基為交聯對象，因此NOCC分子會被交聯而形成網目結構，且alginate分子穿插於NOCC的網目結構中，因而形成semi-IPN (semi-interpenetrating polymer network)構造。Glutaraldehyde (GA)的交聯結構經由文獻及本實驗的FT-IR結果說明，GA是會與NOCC及alginate分子上的醇基(－OH)產生交聯反應。離子交聯方面，經由FT-IR證明解離帶負電的基會被鈣離子所交聯。
在藥物包覆及釋放方面， GP交聯水膠包覆率為100%；離子交聯水膠包覆率約為92%，而GA交聯水膠則因為交聯液為強酸環境(pH 0.46)，因此造成蛋白質結構denature，所以造成測不到其濃度。雖然GP交聯水膠發現交聯劑對蛋白質藥物有所影響，但由其釋放行為來看是有達到腸部標的釋放的目標，所以GP交聯水膠也可適用於其他非蛋白質藥物。另一方面，亦可使用不同的藥物包覆方式如吸附法，應可以改善GP對於蛋白質的影響。同樣地，離子交聯水膠亦有達到腸部標的釋放的目標，且其溫和的反應環境，因此此種水膠是一較理想的蛋白質藥物載體。

摘要-----------------------------------------------------------I
目錄----------------------------------------------------------IV
圖索引-------------------------------------------------------VII
表索引---------------------------------------------------------X
第一章 緒論
1-1 水膠 1
1-2 幾丁聚醣 1
1-3 研究動機與目的 3
第二章 水溶性幾丁聚醣衍生物的合成
2-1 研究目的 11
2-2 幾丁聚醣衍生物的製備 11
2-2-1 幾丁聚醣原料 11
2-2-2 N,O-carboxymethyl chitosan (NOCC)的製備 11
2-2-3 N,O-carboxymethyl chitosan (NCC)的製備 11
2-2-4 O-carboxymethyl chitosan (OCC)的製備 12
2-3 定性分析 15
2-3-1 傅立葉轉換紅外線光譜分析 15
2-3-2 核磁共振分析(NMR) 15
2-4 體外抗酵素分解 15
2-5 實驗結果與討論 18
2-5-1 NOCC之FT-IR分析 18
2-5-2 NCC之FT-IR分析 18
2-5-3 OCC之FT-IR分析 20
2-5-4 NOCC核磁共振分析(NMR) 21
2-5-4-1 定性分析 21
2-5-4-2 定量分析 21
2-5-5 體外抗酵素分解 24
2-6 結論 25
第三章 NOCC水膠製備
3-1 研究目的 26
3-2 NOCC水膠的製備 26
3-3 膨潤性質測試 27
3-4 酸鹼可逆性質 28
3-5 NOCC/alginate水膠之製備 29
3-6 實驗結果與討論 30
3-6-1 膨潤性質測試 30
3-6-2 酸鹼可逆性質 30
3-7 結論 39
第四章 不同交聯結構之水膠製備及蛋白質藥物包覆及釋放
4-1 研究目的 40
4-2 不同交聯結構之水膠製備 40
4-2-1 胺基交聯(GP交聯之水膠) 40
4-2-2 醛醇反應交聯(GA交聯之水膠) 40
4-2-3 離子交聯 41
4-3 不同交聯結構之水膠其酸鹼可逆性質測試 42
4-4 水膠交聯結構分析 43
4-5 蛋白質藥物包覆 44
4-5-1 GP交聯之水膠包覆蛋白質 44
4-5-2 GA交聯之水膠包覆蛋白質 44
4-5-3 離子交聯交聯之水膠包覆蛋白質 46
4-6 蛋白質定量(Bradford Method) 47
4-7 包覆率分析 47
4-8 不同交聯結構水膠之藥物釋放實驗 48
4-9 結果與討論 50
4-9-1 交聯結構分析 50
● GP交聯結構分析(胺基交聯) 50
● GA交聯結構分析(醛醇反應交聯) 50
● Calcium交聯結構分析(離子交聯) 54
4-9-2 不同交聯結構之水膠其酸鹼可逆性質測試 55
4-9-3 包覆率分析 56
4-9-4 不同交聯結構之水膠之藥物釋放行為 57
● GP交聯水膠 57
● Calcium交聯水膠 61
● in pH 7.4溶離液 63
● in pH 1.2溶離液 63
4-10 結論 64

1. Peppas, N.A., Hydrogels in Medicine and Pharmacy, Vol 1~III, CRC Press, Inc., Boca Raton, Florida, 1987.
2. Park, T.G. and Hoffman, A.S., “Estimation of temperature-dependent pore size in poly(N-isopropylacrylamide) hydrogel beads,” Biotechnology Progress. 10(1):82-6, 1994 .
3. Park, T.G. and Hoffman, A.S., “ Thermal cycling effects on the bioreactor performances of immobilized beta-galactosidase in temperature-sensitive hydrogel beads, ” Enzyme & Microbial Technology. 15(6):476-82, 1993.
4. Sershen, S.R., Westcott, S.L., Halas, N.J. and West, J.L. “ Temperature-sensitive polymer-nanoshell composites for photothermally modulated drug delivery,” Journal of Biomedical Materials Research. 51(3):293-8, 2000.
5. Vakkalanka, S.K., Brazel, C.S., and Peppas, N.A., “ Temperature- and pH-sensitive terpolymers for modulated delivery of streptokinase,” Journal of Biomaterials Science, Polymer Edition. 8(2):119-29, 1996.
6. Brannonpeppas, L. and Peppas, N.A., “Dynamic and Equilibrium Swelling Behavior of pH-Sensitive Hydrogels Containing 2-Hydroxyethyl Methacrylate, ” BIOMATERIALS. 11(9)： 635-644, 1990.
7. Kang, D.Y., TAO,PENG, Goosen, M.F.A., Min, J.M. and He, Y.Y., “ pH-sensitivity of hydrogels based on complex forming chitosan：polyether interpenetrating polymer network, ” Journal of Applied Polymer Science, 48：343-354,1993.
8. Don, L. and Hoffman, A., “ A novel approach for preparation of pH-sensitive hydrogels for enteric drug delivery, ” Journal of Controlled Release, 15：141-150, 1991.
9. Paavola, A., Yliruusi, J. and Rosenberg, P., “ Controlled release and dura mater permeability of lidocaine and ibuprofen from injectable poloxamer-based gels,” Journal of Controlled Release. 52(1-2)：169-78, 1998.
10. Na, K., Park, K.H., Kim, S.W. and Bae Y.H., “ Self-assembled hydrogel nanoparticles from curdlan derivatives: characterization, anti-cancer drug release and interaction with a hepatoma cell line (HepG2), ” Journal of Controlled Release. 69(2):225-36, 2000.
11. Hinds, K., Koh, J.J., Joss, L., Liu, F. and Baudys M., “ Kim SW. “ Synthesis and characterization of poly(ethylene glycol)-insulin conjugates, ” Bioconjugate Chemistry. 11(2):195-201, 2000.
12. Sato, S., Jeong, S.Y., Kim, S.W. and Mcrea, J.C., “ Glucose Stimulated Insulin Delivery Systems, ” Pure and Applied Chemistry. 56(10)：1323-1328, 1984.
13. Ishihara, K., Kobayashi, M. and Shinohara, I., “Insulin Permeation Through Amphiphilic Polymer Membranes Having 2-Hydroxyethyl Methacrylate Moiety, ” Polymer Journal. 16(8)：647-651, 1984.
14. Hirano, S. and Matsumura, T., “N-acyl derivatives of chitosan and their hydrolysis by chitonase,” Carbohydr. Res., 165, 120-122, 1987.
15. Brandenberg, G., Leibrock, L.G., Shuman, R., Malette, W.G. and Quigley, H., “Chitosan: a new topical hemostatic agent for diffuse capillary bleeding in brain tissue,” Neurosurgery., 15, 9-13, 1984.
16. Muzzarelli, R.A., Tanfani, F. and Emanuelli, M., “Sulfated N-carboxymethyl chitosan : Novel blood anticoagulants,” Carbohydr. Res., 126, 225-231, 1984.
17. Stanley, W.L., Watters, G.G., Kelly, S.H. and Olson, A.C., “Glucoamylase immobilized on chitin with glutaraldehyde,” Biotechnol. Bioeng., 20, 135-140, 1978.
18. Onsoyen, E. and Skaugrud, O., “Metal recovery using chitosan,” J. Chem. Tech. Biotechnol., 49, 395-404, 1990.
19. Muzzarelli, R.A.A., “Chitin and its derivatives: New trends of applied and research,” Carbohydr. Polym., 3, 52-57, 1993.
20. Ralston, G.B., Tracey, M.V. and Wrench, P.V., “The inhibition of fermentation in baker's yeast by chitin,” Biochim. Biophys. Acta., 93, 652-655, 1964.
21. Nishimura, S., Ikeuchi, Y. and Tokura, S., “The adsorption of bovine blood proteins onto the surface of O-carboxymethyl chitin,” Carbohydr. Res., 134, 305-312, 1984.
22. Inoue, K., Baba, Y. and 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.
23. Chandy, T. and Sharma, C.P., “Prostaglandin El-immobilized poly(vmyl alcohol)-blended chitosan membranes: blood compatibility and permeability properties,” J. Appli. Polym. Sci., 44, 2145-2156, 1992.
24. Kifime, K., Yamaguchi, Y. and Kishimoto, S., “Wound healing effect of chitin surgical dressing,” Trans. Soc. Biomat., XI, 216-220, 1988.
25. Pelletir, A., Lemire, L. and Sygnsch, J., “Chitin／chitosan transformation by thermo- chemical treatment,” Biotechnol. Bioeng., 36, 310-315, 1990.
26. Peluso, G., Petille, O., Ranieri, M., Santin, M.,Ambrosio, L., Calabro, D., Avallone, B. and Balsamo, G., “Chitosan-mediated stimulation of macrophage function,” Biomaterials, 15, 1215-1220, 1994.
27. Sakaguchi, T., Horikoshi, T. and Nakajima, A., “Adsorption of uraniumby chitin phosphate and chitosan phosphate,” Agric. Biol. Chem., 45, 2191-2195, 1981.
28. Hassan, E.E., Parish, R.C. and Gallo, J.M., “Optimized formulation of magnetic chitosan microspheres containing the anticancer agent, oxantrazole,” Pharm. Res., 9, 390-397, 1992.
29. Ohya, Y., Shiratami, M. and Ouchi, T., “Release behavior of 5-FU from chitosan gel nanospheres immobilizing 5-FU coated with polysaccharides,” J. Macro. Sci., 31, 629-642, 1994.
30. Ohya, Y., Takei, T. and Ouchi, T., “Thermo-sensitive release behavior of 5-FU from chitosan-gel microspheres coated with lipid layer,” J. Bioact. & Compat. Polym., 7, 242-256, 1992.
31. Nishioka, Y., Kyotani, S., Okamura, M., Miyazaki, M., Okazaki, K., Ohnishi, S., Yamamoto, Y., and Ito, K., “Release characteristics of cisplatin chitosan microspheres and effect of containing chitin,” Chem. Pharm. Bull., 38, 2871-2873, 1990.
32. Akbuga, J. and Bergisadi, N., “5-Fluorouracil-loaded chitosan microspheres: preparation and release characteristics,” J. Microencapsul., 13, 161-168, 1996.
33. Jameela, S.R., Misra, A. and Jayakrishnan, A., “Cross-linked chitosan microspheres as carriers for prolonged delivery of macromolecular drugs,” J. Biomat. Sci. Polym. Edn., 6, 621-632, 1994.
34. Jameela, S.R., Kumary, T.V., Lal, A.V. and Jayakrishnan, A., “Progesterone-loaded chitosan microspheres: a long acting biodegradable controlled delivery system,” J. Control. Rel., 52, 17-24, 1998.
35. Jameela, S.R. and Jayakrishnan, A., “Glutaraldehyde cross-linked chitosan microspheres as a long acting biodegradable drug delivery vehicle: studies on the in vitro release of mitoxantrone and in vivo degradation of microspheres in rat muscle,” Biomaterials, 16, 769-775, 1995.
36. Alexakis, T., Boadi, D.K., Quong, D., Groboillot, A., O'Neill, I., Poncelet, D. and Neufeld, R.J., “Microencapsulation of DNA within alginate microspheres and crosslinked chitosan membranes for in vivo application,” Appli. Biochem. Biotechnol., 50, 93-106, 1995.
37. Thanoo, B.C., Sunny, M.C. and Jayakrishnan, A., “Cross-linked chitosan microspheres: preparation and evaluation as a matrix for the controlled release of pharmaceuticals,” J. Pharm. Pharmacol., 44, 283-286, 1992.
38. Thacharodi, D. and Panduranga R.O., “Development and in vitro evaluation of chitosan-based transdermal drug delivery systems for the controlled delivery of propanolol hydrochloride,” Biomaterials, 16, 145-148, 1995.
39. Sezer, A.D. and Akbuga, J., “Release characteristics of chitosan treated alginate beads: I. Sustained release of a macromolecular drug from chitosan treated alginate beads,” J. Microencapsul., 16, 195-203, 1999.
40. Thacharodi, D. and Rao K.P., “Propranolol hydrochloride release behaviour of crosslinked chitosan membranes,” J. Chem. Technol. Biotechnol., 58, 177-181, 1993.
41. Groboillot, A.F., Champagne, C.P., Darling, G.D., Poncelet, D. and Neufeld, R.J., “Membrane formation by interfacial cross-linking of chitosan for microencapsulation of lactococcus lactis,” Biotechnol. Bioeng., 42, 1157-1163, 1993.
42. Freeman, A. and Dror, Y., “Immobilization of disguised yeast in chemically crosslinked chitosan beads,” Biotechnol. Bioeng., 44, 1083-1088, 1994.
43. Shinonaga, M.A., Kawamura, Y. and Yamane, T., “Immobilization of yeast cells with cross-linked chitosan beads,” J. Ferment. and Bioeng., 74, 90-94, 1992.
44. Chanda, J., Kuribayashi, R. and Abe, T., “Use of the glutaraldehyde-chitosan treated porcine pericardium as a pericardial substitute,” Biomaterials, 17, 1087-1091, 1996.
45. Thacharodi, D. and Rao, K.P., “Rate-controlling biopolymer membranes as transdermal delivery systems for nifedipine: development and in vitro evaluations,” Biomaterials, 17, 1307-1311, 1996.
46. Yao, K.D., Peng, T., Yin, Y.J. and Xu, M.X., “ Microcapsules/Microspheres related to chitosan,” J. Macromol. Sci. Rev. Marcomol. Chem. Phys., 35, 155-180, 1995.
47. Kim, J.H., Kim, J.Y., Lee, Y.M. and Kim, K.Y., “Controlled release of riboflavin and insuline through crosslinked poly(vinyl alcohol)/chitosan blend membrane,” J. Appli. Polym. Sci., 44, 1823-1828, 1992.
48. Patel, V.R. and Amiji, M.M., “ Preparation and Characterization of Freeze-Dried Chitosan-Poly(Ethylene Oxide) Hydrogels for Site-Specific Antibiotic Delivery in the Stomach,” Pharmaceutical Research. 13(4)：588-593, 1996.
49. Shu, X.Z., Zhu, K.J. and Song, Weihong, “ Novel pH-sensitive citrate cross-linked chitosan film for drug controlled release, ” International Journal of Pharmaceutics, 212(1)：19-28, 2001.
50. Jackson, D.S. and Princeton, N.J., “Chitosan-glycerol-water gel,” U.S. Patent, No. 4659700, 1-4, 1987.
51. 卓貴美, 圖解生理學, 五南圖書出版公司, 台北, 341, 2000.
52. Yeh, P.Y., Berenson-M.M., Samowitz,W.S., Kopeckova, P. and Kopecek, J., “ Site-Specific Drug-Delivery and Penetration Enhancement in the Gastrointestinal-Tract. ” Journal of Controlled Release, 36(1-2)：109-124, 1995.
53. Tarvainen, T., Nevalainen, T., Sundell, A., Svarfvar, B., Hyrsyla, J., Paronen, P. and Jarvinen, K., “Drug release from poly(acrylic acid) grafted poly(vinylidene fluoride) membrane bags in the gastrointestinal tract in the rat and dog, ” Journal of Controlled Release, 66(1)：19-26, 2000.
54. Ramkissoon-Ganorkar, C., Liu, F., Baudys, M. and Kim, S.W., “Modulating insulin-release profile from pH thermosensitive polymeric beads through polymer molecular weight,” Journal of Controlled Release, 59(3)：287-298,1999.
55. Nakamura, K., Maitani, Y., Lowman, A.M., Takayama, K., Peppas, N.A. and Nagai, T., “ Uptake and release of budesonide from mucoadhesive, pH-sensitive copolymers and their application to nasal delivery, ” Journal of Controlled Release, 61(3)：329-335, 1999.
56. Risbud MV, Hardikar AA, Bhat SV, Bhonde RR., “ pH-sensitive freeze-dried chitosan-polyvinyl pyrrolidone hydrogels as controlled release system for antibiotic delivery, ” Journal of Controlled Release, 68(1)：23-30, 2000.
57. Madsen, F. and Peppas, N.A., “ Complexation graft copolymer networks: swelling properties, calcium binding and proteolytic enzyme inhibition, ”Biomaterials, 20(18)：1701-1708, 1999.
58. Krogars, K., Heinamaki, J., Vesalahti, J., Marvola, M., Antikainen, O. and Yliruusi, J.,“Extrusion-spheronization of pH-sensitive polymeric matrix pellets for possible colonic drug delivery, ” International Journal of Pharmaceutics, 199(2)： 187-194, 2000.
59. Prasad, Y.V.R., Krishnaiah, Y.S.R. and Satyanarayana, S.,“In vitro evaluation of guar gum as a carrier for colon-specific drug delivery, ” Journal of Controlled Release, 51(2-3)： 281-287 ,1998.
60. Cappello, J., Crissman, J.W., Crissman, M., Ferrari, F.A., Textor, G., Wallis, O., Whitledge, J.R., Zhou, X., Burman, D., Aukerman, L. and Stedronsky, E.R.,“In-situ self-assembling protein polymer gel systems for administration, delivery, and release of drugs” Journal of Controlled Release, 53(1-3)：105-117, 1998.
61. Amiji, M., Tailor, R., Ly, M.K. and Goreham, J.,“Gelatin poly(ethylene oxide) semi-interpenetrating polymer network with pH-sensitive swelling and enzyme-degradable properties for oral drug delivery, ” Drug Development and Industrial Pharmacy, 23(6)：575-582, 1997.
62. Rodriguez, M., Vila-Jato, J.L. and Torres, D.,“Design of a new multiparticulate system for potential site-specific and controlled drug delivery to the colonic region, ” Journal of Controlled Release, 55(1)：67-77, 1998.
63. Carelli, V., Coltelli, S., Di Colo, G., Nannipieri, E. and Serafini, M.F.,“Silicone microspheres for pH-controlled gastrointestinal drug delivery,”International Journal of Pharmaceutics, 179(1)：73-83, 1999.
64. Khare, A.R. and Peppas, N.A.,“Release Behavior of Bioactive Agents from pH-Sensitive Hydrogels, ”Journal of Biomaterials Science-Polymer Edition, 4(3)：275-289, 1993.
65. Siegel, R.A. and Firestone, B.A.,“PH-Dependent Equilibrium Swelling Properties of Hydrophobic Poly-Electrolyte Copolymer Gels” Macromolecules, 21(11)：3254-3259, 1988.
66. Fujikawa, S., Yokota, T., Koga, K. and Kumada, S.I., “The continuous hydrolysis of geniposide to genpin using immobilized β-glucosidase on calcium alginate gel,” Biotechnol. Lett., 9, 697-702, 1987.
67. Kimura, Y., Okuda, H. and Archi, S., “Effects of geniposide isolated from Gardenia jasminoides on metabolic alterations in high sugar diet-fed rats,” Chem. Pharm. Bull., 30, 4444-4447, 1982.
68. Wang, C.J., Wang, S.W. and Lin, J.K., “Suppressive effect of geniposide on the hepatotoxicity and hepatic DNA binding of aflatoxin B1 in rats,” Caner. Lett., 60, 95-102, 1991.
69. Tseng, T.H., Chu, C.Y. and Wang, C.J., “Inhibition of penta-acetyl geniposide on AFB1-induced genotoxicity in C3H10T1/2 cells,” Cancer. Lett., 62, 233-242, 1992.
70. Miwa, T., Jap. J. Pharmacol., 2, 102-108, 1953.
71. Miwa, T., Jap. J. Pharmacol., 2, 139-143, 1953.
72. Miwa, T., Jap. J. Pharmacol., 3, 1-5, 1953.
73. Hayes, E.R. and Canada, W.,“ N,O-carboxymethyl chitosan and preparative method therefore, ”U.S. Patent, No. 4619995, 1986.
74. Muzzarelli, R.A.A., Ilari, P. and Petrarulo, M.,“Solubility and structure of N-carboxymethylchitosan,”International Journal of Biological Macromolecules, 16(4)：177-180, 1994.
75. Nudga, L.A., Plisko, E.A. and Danilov, S.N.,“O-alkylation of chitosan,”Journal of General Chemistry of The USSR, 2729-2732,1974.
76. Mi, F.L., Tan, Y.C., Liang, H.C., Huang, R.N. and Sung, H.W., “In vitro evaluation of a chitosan membrane cross-linked with genipin,”J. Biomater. Sci. Polymer Edn, 12(8)：835-850, 2001.
77. Sugimoto, M., Morimoto, M., Sashiwa, H., Saimoto, H. and Shigemasa, Y.,“Preparation and characterization of water-soluble chitin and chitosan derivatives, ”Carbohydrate Polymers, 36(1)：49-59, 1998.
78. Tokura, S., Nishi, N., Nishmura, S. and Somorin, O. “Lysozyme accessible fibers from chitin and its derivatives,”Sen-I Gakkaishi, 39, 507-511, 1983.
79. Kim Y.J., Yoon K.J., and Ko S.W.“Preparation and properties of alginate superabsorbent filament fibers crosslinked with glutaraldehyde,”Journal of Applied Polymer Science, 78 (10): 1797-1804, 2000.
80. John McMurry, Organic Chemistry, 4th ed, Brook/Cole Publishing Company, USA.
81. Sung, H.W., Huang, D.M., Chang, W.H., Huang, R.N. and Hsu, J.C., "Evaluation of Gelatin Hydrogel Crosslinked with Various Crosslinking Agents as Bioadhesives: In Vitro Study," J. Biomed. Mater. Res., 46, 520-530, 1999.
82. Bradford M.M., “A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Princple of Protein-Dye Binding,”Analytical Biochemistry, 72, 248-254, 1976.
83. Viness Pillay and Reza Fassihi, “In vitro release modulation from Crosslinked pellets for site-specific drug delivery to the gastrointestinal tract I. Comparison of pH-responsive drug release and associated kinetics,”Journal of Controlled Release, 59, 229-242, 1999.
84. Pepperman A.B. and Kuan J.W., “Controlled release formulations of alachlor based on calcium alginate,” Journal of Controlled Release, 34, 17-23, 1995.
85. Miyazaki S., Kubo W. and Attwood D., “Oral sustained delivery of theophylline using in-situ gelation of sodium alginate,” Journal of Controlled Release, 67, 275-280, 2000.