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研究生:莊景光
研究生(外文):Ching-Kuang Chuang
論文名稱:離子鍵結型奈米微粒製備與其對小腸上皮細胞滲透能力之探討
指導教授:宋信文
指導教授(外文):Hsing-Wen Sung
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學工程學系
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:2004
畢業學年度:92
語文別:中文
論文頁數:52
中文關鍵詞:奈米微粒幾丁聚醣聚麩氨酸小腸上皮細胞
外文關鍵詞:nanoparticlechitosanr-PGAtight junctionCaco-2 Cell
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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。
目 錄
內容 頁數
摘要 I
目錄 III
圖索引 VI
表索引 VIII

第一章 緒論
1.1 消化道 1
1.2 親水性分子在消化道的吸收途徑 3
1.3 Caco-2 cell monolayers 5
1.4 奈米微粒 5
1.5 離子鍵結型奈米微粒 6
1.6 幾丁質與幾丁聚醣 7
1.7 聚麩氨酸 8
1.8 研究動機與目的 9

第二章 製備小分子材料
2.1 研究目的 13
2.2 小分子幾丁聚醣的製備 13
2.2.1幾丁聚醣 13
2.2.2去聚合幾丁聚醣 13
2.3 小分子聚麩氨酸的製備 15
2.3.1發酵聚麩氨酸 15
2.3.2水解聚麩氨酸 17
2.4 膠體色層分析(GPC) 17
2.4.1幾丁聚醣之GPC分析 17
2.4.2聚麩氨酸之GPC分析 18
2.5 實驗結果與討論 18
2.5.1 幾丁聚醣膠體色層分析(GPC) 18
2.5.2 聚麩氨酸膠體色層分析(GPC) 19
2.6 結論 20

第三章 製備離子鍵結型奈米微粒
3.1 研究目的 21
3.2 離子鍵結型奈米微粒的製備 21
3.3 奈米微粒粒徑與表面電荷分析 22
3.4 穿透式電子顯微鏡 (TEM) 22
3.5 原子力顯微儀 (AFM) 23
3.6 實驗結果與討論 23
3.6.1巨觀分析 23
3.6.2粒徑分析 25
3.6.3表面電荷分析 29
3.6.4穿透式電子顯微鏡 (TEM) -- 31
3.6.5原子力顯微儀 (AFM) 32
3.7 結論 33

第四章 離子鍵結型奈米微粒對小腸上皮細胞滲透能力之探討
4.1 研究目的 34
4.2 Caco-2 cell monolayers的培養 34
4.3 Transepithelial electrical resistance (TEER ) 35
4.4 Confocal laser scanning microscopy (CLSM) 36
4.4.1製備FITC-labeled幾丁聚醣與奈米微粒 36
4.5 實驗結果與討論 --38
4.5.1 Transepithelial electrical resistance (TEER ) 38
4.5.2 Confocal laser scanning microscopy (CLSM) 42
4.6 結論 46
參考書目 47
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