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研究生:黃連倉
研究生(外文):Lien-Tsang Huang
論文名稱:幾丁質仿肝素分子之化學改質
論文名稱(外文):chitin mimics chemical structure of heparin by chemical modification
指導教授:吳紹榮吳紹榮引用關係
指導教授(外文):Shao-Jung Wu
學位類別:碩士
校院名稱:明志科技大學
系所名稱:化工與材料工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:60
中文關鍵詞:肝素磺酸化羧甲基化幾丁聚醣硫醇水膠萄萄糖胺聚糖生物醫學緩衝液高分子生物體
外文關鍵詞:heparinSCM-chitosanthiolhydrogel
相關次數:
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肝素是一種含高度硫酸基的萄萄糖胺聚糖(GAG),在生物體內與多種結合蛋白連結形成蛋白多醣而呈現多種的生物活性。例如與許多凝血因子形成很強的複合物,從而抑制它們的活性,展現出優良的抗凝血活性。另外,肝素也可與生長因子及其受體特異性結合,進而調控細胞生長、遷移與發育及血管之增生與組織之修復。因此,肝素已經是一種具有高度醫療價質的生物醫學材料。本研究主要目標即是以幾丁質合成仿肝素結構之衍生物—磺酸化羧甲基化幾丁質(SCM-Chitin)與磺酸化羧甲基化幾丁聚醣(CM-Chitosan),並經由硫醇化反應使其可在室溫下因形成雙硫鍵而自成水膠(hydrogel)。如此合成之仿肝素結構的高分子水膠可與蛋白質藥物穩定結合並將其包埋於水膠中,故未來將可用於蛋白質藥物傳輸之載體。
論文第一部份的研究內容,是在探討經由合成步驟直接將幾丁質分子進行化學改質,使其成為帶有負電荷官能基之仿肝素結構多醣體。合成之產物SCM-Chitin是先經由羧甲基化反應得到羧甲基化幾丁質(CM-Chitin),再藉由磺酸化反應之後所得到的仿肝素之結構。另外,在合成過程中先將CM-Chitin進行脫乙醯基反應,得到羧甲基化幾丁聚醣(CM-Chitosan),而後進行磺酸化反應,最後得到SCM-Chitosan。產物之化學結構鑑定及羧甲基與磺酸基之取代度分別透過Fourier transform infrared spectroscopy(FTIR)、nuclear magnetic resonance(NMR)及Element Analysis(EA)來進行分析。
論文第二部份之研究,是將SCM-Chitosan藉由1-ethyl-3-(3-dimethylamin
opropyl)-carbodiimide(EDC)及N-hydroxysuccinimide(NHS)接上硫醇化合物。硫醇基氧化形成雙硫鍵可使SCM-Chitosan自成水膠,並可增進材料的黏膜附著性質,增進其未來應用於蛋白質藥物傳輸之潛力。首先活化SCM-Chitosan上羧基與半胱氨酸(cysteine)上胺基共價結合,而將硫醇接合於SCM-Chitosan高分子骨架上得到SCM-Chitosan-cysteine conjugate;另外,我們也嘗試先活化thioglycolic acid上羧基與SCM-Chitosan上胺基共價結合而得到SCM-Chitosan-thioglycolic acid conjugate。將硫醇化之產物與Ellman reagent結合後,在450nm波長下測定吸光度而得到產物之硫醇基含量。由硫醇基濃度的量測,測得每一克SCM-Chitosan-cysteine conjugate含有2425.22 硫醇基,而每一克的SCM-Chitosan-thioglycolic acid conjugate則僅含有78.22 硫醇基;接著把SCM-Chitosan接上硫醇後的產物(SCM-Chitosan-cysteine conjugate及SCM-Chitosan-thioglycolic acid conjugate)在37℃不同pH值環境下測定硫醇基濃度隨時間變化之關係以瞭解硫醇基氧化形成雙硫鍵的特性,並測量因形成雙硫鍵而形成的水膠在不同pH值中,其澎潤值的變化。由實驗結果顯示硫醇基在接近生理緩衝液的pH值中易形成雙硫鍵,且已形成雙硫鍵的產物在模擬胃腸液之不同pH值溶液中有不同的澎潤值,顯示合成之水膠的膨潤性質具有對pH值的敏感性。由本論文之研究結果顯示,經由硫醇化反應而得到之SCM-Chitosan水膠具備未來用於蛋白質藥物傳輸之潛力。
Heparin has been found to possess bioactivities such as anticoagulation and a large variety of cell-modulating activities. The aim of the study was to synthesize a heparin-like chitosan derivative with thermal responsive thiol groups, for the purpose of protein drug delivery in the future. The thiolated polymer should be capable of developing into hydrogels at 37℃ via the oxidation of thiol groups to disulfide bonds. The first step of this study, 6-O-carboxymethylchitin (6-O-CM-Chitin) was synthesized through a carboxymethylation reaction with chitin, then 6-O-CM-Chitin was directly sulfated, or deacetylated and sulfated using trimethylamine-sulfur trioxide to obtain sulfated 6-O-CM-Chitin (SCM-chitin) and sulfated 6-O-CM-chitosan (SCM-chitosan). The structure of the synthesized SCM-chitin and SCM-chitosan was characterized by FT-IR and 13C-NMR. The second step, we brought in the thiol groups to SCM-chitosan by covalently coupling of thioglycolic acid (TGA) or cysteine with SCM-chitosan, leading to the formation of two types of thiolated SCM-chitosan: SCM-chitosan-cysteine conjugates and SCM-chitosan-thioglycolic acid conjugates. Oxidation of the thiol groups on SCM-chitosan-TGA and SCM-chitosan-cystein to form disulfide bonds were determined by quantifying the amount of thiol groups on the modified SCM-chitosan with Ellman’s reagent before and after forming disulfide bonds. Moreover, thiolated SCM-chitosan displayed in situ-gelling features, due to the pH-dependent formation of inter- as well as intra-molecular disulfide bonds. The hydrogels prepared from the thiolated SCM-chitosans showed pH-responsive swelling properties in the simulated gastrointestinal (GI) tract medium. Therefore, according to the results of this study, the thiolated SCM-chitosans hydrogels might be used as potential protein drug delivery systems in the future.
目錄
中文摘要-------------------------------------------------------------Ι
英文摘要-------------------------------------------------------------Ⅲ
目錄----------------------------------------------------------------Ⅴ
表目錄--------------------------------------------------------------Ⅷ
圖目錄--------------------------------------------------------------Ⅸ
第一章 緒論-----------------------------------------------------------1
第二章 文獻回顧
2.1 幾丁質-----------------------------------------------------------3
2.2 幾丁聚醣---------------------------------------------------------4
2.3 幾丁質與幾丁聚醣在各方面之應用--------------------------------------5
2.3.1 生醫材料之應用--------------------------------------------------5
2.3.2 機能性食品之應用------------------------------------------------5
2.3.3 吸收、排泄體內重金屬之應用----------------------------------------6
2.3.4 廢水處理上之應用------------------------------------------------6
2.3.5 農業上的利用----------------------------------------------------7
2.3.6 紡織業之應用----------------------------------------------------7
2.4 肝素-------------------------------------------------------------7
2.4.1 肝素之簡介------------------------------------------------------7
2.4.2 肝素相關應用與研究----------------------------------------------11
2.5 幾丁質與幾丁聚醣的化學改質-----------------------------------------12
第三章 幾丁質(Chitin)改質近似於肝素(Haparin)
3.1 前言------------------------------------------------------------16
3.2 實驗原理--------------------------------------------------------16
3.2.1 羧甲基化幾丁質的製備--------------------------------------------16
3.2.2 去乙醯的原理---------------------------------------------------18
3.2.3 磺酸化羧甲基幾丁質的原理----------------------------------------19
3.2.4 磺酸化羧甲基幾丁聚醣的原理---------------------------------------20
3.2.5 實驗製程的合成步驟圖--------------------------------------------22
3.3 實驗藥品--------------------------------------------------------23
3.4 主要實驗儀器-----------------------------------------------------24
3.5 實驗方法--------------------------------------------------------25
3.5.1 水溶性羧甲基幾丁質(6-O-CM-Chitin)的製備-------------------------25
3.5.2 水溶性羧甲基幾丁聚醣(6-O-CM-Chitosan)的製備----------------------25
3.5.3 SCM-Chitin的製備----------------------------------------------26
3.5.4 SCM-Chitosan的製備--------------------------------------------26
3.5.5 傅立葉紅外線光譜儀(FTIR)測試方法-------------------------------27
3.5.6 核磁共振(NMR) 測試方法-----------------------------------------27
3.5.7 元素分析(EA)測試方法------------------------------------------28
3.5.8 分子量(gel-permeation chromatography, GPC)測試方法------------28
3.6 結果與討論
3.6.1 傅立葉紅外線光譜儀分析------------------------------------------29
3.6.2 核磁共振分析---------------------------------------------------32
3.6.3 元素分析------------------------------------------------------35
3.6.4 分子量測試分析-------------------------------------------------37
第四章 硫醇化SCM-chitosan
4.1 前言------------------------------------------------------------39
4.2 實驗藥品--------------------------------------------------------44
4.3 實驗儀器--------------------------------------------------------44
4.4 實驗合成流程-----------------------------------------------------44
4.5 實驗方法--------------------------------------------------------45
4.5.1 SCM-Chitosan-cysteine conjugate之製備-------------------------45
4.5.2 SCM-Chitosan-thioglycolic acid conjugate之製備----------------46
4.5.3 SCM-Chitosan-cysteine conjugate硫醇基含量之測量----------------46
4.5.4 SCM-Chitosan-thioglycolic acid conjugate硫醇基含量之測量-------47
4.5.5 雙硫鍵的形成測試-----------------------------------------------47
4.5.6 澎潤作用測試---------------------------------------------------48
4.5.6 氧化成膠時間評估-----------------------------------------------48
4.6 結果與討論-------------------------------------------------------48
4.6.1 硫醇含量分析---------------------------------------------------48
4.6.2 在37℃下硫醇基含量分析------------------------------------------49
4.6.3 澎潤作用分析---------------------------------------------------52
4.6.4 氧化成膠時間評估分析--------------------------------------------54
第五章 結論----------------------------------------------------------55

參考文獻-----------------------------------------------------------------------------------56

表目錄
表2-1 幾丁質與幾丁聚醣之生化特性----------------------------------------4
表2-2 幾丁質與幾丁聚醣應用在生醫材料------------------------------------5
表2-3 幾丁質與幾丁聚醣在機能性食品之應用---------------------------------6
表2-4 部分GAG-結合蛋白及其生物活性-------------------------------------10
表2-5 幾丁聚醣改質應用在組織工程上-------------------------------------14
表3-1 羧甲基化幾丁質的化學反應式---------------------------------------18
表3-2 (Me)3NSO3磺酸化反應機制----------------------------------------20
表3-3 高分子量的Chitin、CM-Chitin(H-form)、CM-Chitosan(H-form) 、SCM-
Chitin(Na-form)、SCM-Chitosan(Na-form)元素分析結果------------35
表3-4 低分子量的Chitin、CM-Chitin(H-form)、CM-Chitosan(H-form) 、SCM-
Chitin(Na-form)、SCM-Chitosan(Na-form)元素分析結果------------35
表3-5 高分子量的Mn,Mw分子量值----------------------------------------37
表3-6 低分子量的Mn,Mw分子量值----------------------------------------37
表4-1 1%SCM-Chitosan-cysteine conjugate在37℃不同pH值下硫醇含量變化----50
表4-2 3% SCM-Chitosan-TGA conjugate在37℃不同pH值下硫醇含量變化--------51

圖目錄
圖2-1 幾丁質的化學結構-------------------------------------------------3
圖2-2 幾丁聚醣的化學結構-----------------------------------------------4
圖2-3 肝素的化學結構--------------------------------------------------8
圖2-4 透明質酸--------------------------------------------------------8
圖2-5 硫酸軟骨素A型---------------------------------------------------8
圖2-6 硫酸軟骨素C型---------------------------------------------------9
圖2-7 硫酸角質素------------------------------------------------------9
圖2-8 硫酸真皮素------------------------------------------------------9
圖2-9 硫酸類肝素------------------------------------------------------9
圖3-1 去乙醯反應機制-------------------------------------------------19
圖3-2 SO3-pyridine反應機制-------------------------------------------20
圖3-3 6-O-CM-Chitin化學結構------------------------------------------20
圖3-4 SCM-Chitin化學結構---------------------------------------------20
圖3-5 6-O-CM-Chitosan化學結構----------------------------------------21
圖3-6 SCM-Chitosan化學結構-------------------------------------------21
圖3-7 合成步驟圖-----------------------------------------------------22
圖3-8 高分子量的Chitin、6-O-CM-Chitin、SCM-Chitin之FTIR光譜圖---------30
圖3-9 低分子量的Chitin、6-O-CM-Chitin、SCM-Chitin之FTIR光譜圖---------30圖3-10 高分子量6-O-CM-Chitin、6-O-CM-Chitosan、SCM-Chitosan之FTIR光譜圖--------------------------------------------------------------31
圖3-11 低分子量的6-O-CM-Chitin、6-O-CM-Chitosan、SCM-Chitosan之FTIR 光譜圖--------------------------------------------------------31
圖3-12 6-O-CM-CM-Chitin之13C NMR圖譜--------------------------------33
圖3-13 SCM-Chitin之13C NMR圖譜--------------------------------------33
圖3-14 6-O-CM-CM-Chitosan之13C NMR圖譜------------------------------34
圖3-15 SCM-Chitosan之13C NMR圖譜------------------------------------34
圖4-1 高分子硫醇化之結構----------------------------------------------40
圖4-2 雙硫鍵在thiomers與mucin間的形成機制-----------------------------40
圖4-3 原位交聯示意圖-------------------------------------------------41
圖4-4 SCM-Chitosan接cysteine----------------------------------------45
圖4-5 SCM-Chitosan接thioglycolic acid (TGA)-------------------------45
圖4-6 1%SCM-Chitosan-cysteine conjugate在37℃不同pH值下的硫醇含量變化------------------------------------------------------------51
圖4-7 3% SCM-Chitosan-TGA conjugate在37℃不同pH值下的硫醇含量變化------52
圖4-8 已形成雙硫鍵的SCM-Chitosan-cysteine conjugate在pH 1. 2及pH 7.4澎潤測試------------------------------------------------------53
圖4-9 已形成雙硫鍵的SCM-Chitosan-TGA conjugate在pH1.2及pH 7.4澎潤測試--54
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