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研究生： 方磊
Lei Fang
論文名稱： 微脂粒膜內外間的pH gradient對親水性藥物5-fluorouracil包覆效率以及被包覆的疏水性藥物camptothecin釋放率的影響
The effect of pH gradient between inside and outside of liposomes on the encapsulation efficiency of 5-fluorouracil, a hydrophilic drug, and the releasing ratio of the encapsulated camptothecin, a hydrophobic drug
指導教授： 朱一民
I-Ming Chu
吳文桂
Wen-Guey Wu
口試委員:
學位類別： 碩士
Master
系所名稱： 生命科學暨醫學院 - 生命科學系
Department of Life Sciences
論文出版年： 2004
畢業學年度： 92
語文別： 中文
論文頁數： 50
中文關鍵詞： pH梯度 、硫酸銨 、微脂粒 、5-氟脲嘧啶 、喜樹鹼 、雙十六碳鏈磷酸鹽 、包覆效率 、藥物釋放率 、硫酸銨梯度
外文關鍵詞： pH gradient, ammonium sulfate, liposome, 5-fluorouracil, camptothecin, dicetyl phosphate, encapsulation efficiency, drug releasing ratio, 5-FU, CPT, DCP, dihexadecyl phosphate, DHP, ammonium sulfate gradient
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    • 根據文獻記載，5-FU的包覆效率無論包覆脂質的不同都不會超過6%。我們嘗試以微脂粒以及pH梯度的方式對5-FU進行包覆，以減低其副作用與細胞毒性。實驗結果發現5-FU無法以pH梯度的方式來有效提升微脂粒對它的包覆效率。因此我們想到可以從改善微脂粒膜的性質或是尋找與5-FU形成錯合物的物質等兩個方向再來著手。此外我們推測造成這種結果的原因可能跟5-FU本身的兩個2級胺結構有關。2級胺的pKa會比位於雜環中兩個環交界處的3級胺更遠離7，因此將更容易帶電而無法以pH梯度的方式送入微脂粒中。
    pH梯度藉著微脂粒內外離子濃度的不平衡，可以把親水性藥物送進微脂粒內。那反過來說，我們想知道到被包覆的疏水性藥CPT在釋放時採用pH梯度的方式是否會減低藥物釋放率。我們以25 mM的HSPC
    ，4 mM的DCP，以及1.1 mM的CPT為最佳的配方。結果發現以250 mM硫酸銨溶液水合形成的實驗組其藥物釋放率為35%，而沒有梯度也就是僅以沖提液150 mM氯化鈉溶液/25mM HEPES溶液來水合的對照組其藥物釋放率僅為10%。我們推測原因跟pKa與滲透壓兩者皆無關。而本身帶負電，常添加來製備帶負電微脂粒的DCP可能在該溶液環境下，使得CPT被膜盛載的能力降低。這仍須進一步的證實。

    目錄 1 簡介---p1 1.1 磷脂質，膽固醇，DCP與微脂粒---p1 1.2 膜的通透與pH梯度 (ΔpH)---p7 1.3 親水性藥物5-FU與疏水性藥物CPT---p13 2 實驗目的---p19 3 實驗藥品，器材與步驟與檢測儀器的操作步驟---p20 3.1 實驗藥品或材料---p20 3.2 實驗器材或設備---p21 3.3 實驗步驟與方法---p23 3.3.1 5-FU的包覆---p23 3.3.2 被包覆CPT的釋放---p24 3.4 檢測儀器的操作步驟與方法---p25 3.4.1 雷射粒徑儀LPA (5-FU與CPT部份的粒徑)---p25 3.4.2 HPLC (5-FU部份藥物的包覆效率)---p26 3.4.3 螢光儀 (CPT部份的藥物釋放率)---p26 4 結果與討論---p29 4.1 微脂粒對5-FU的包覆---p29 4.1.1 微脂粒對5-FU的包覆效率---p29 4.1.2 5-FU包覆效率適當取樣範圍的確定---p31 4.1.3 微脂粒對5-FU包覆的探討---p31 4.2 被微脂粒包覆的CPT的藥物釋放---p35 4.2.1 脂質，DCP與CPT組成比例的決定---p35 4.2.2 水合液的選擇與藥物釋放---p35 4.2.3 pH梯度對CPT微脂粒藥物釋放率影響的進一步確定--- p36 4.2.4 被微脂粒包覆的CPT其藥物釋放的探討---p36 5 總結---p41 6 參考文獻---p42 附錄 雷射粒徑儀 (Laser particle analyzer) 原理與操作步驟的簡 介---p46 圖目錄 圖1.1 醯烴長鏈的命名方式---p1 圖1.2 磷脂質的頭基及其命名方式---p2 圖1.3 (磷)脂質自組合成聚集體的型態---p2 圖1.4 單層微脂粒的立體切面圖---p2 圖1.5 膠體態與液晶態的磷脂質的排列方式---p4 圖1.6 不同溫度下磷脂質焓 (enthalphy) 的變化---p4 圖1.7 脂雙層的膜中磷脂質與膽固醇的排列方式---p6 圖1.8 不同濃度膽固醇組成的脂雙層其熱力學性質的變化---p6 圖1.9 DCP的結構式---p6 圖1.10 各種物質對磷脂質脂雙層膜的通透與否---p8 圖1.11 粒線體與葉綠體內部構造的比較---p9 圖1.12 粒線體與葉綠體其電子傳遞鏈的示意圖---p9 圖1.13 弱鹼性藥doxorubicin以硫酸銨溶液製造的pH梯度來載送--- p11 圖1.14 弱酸性藥nalidixic acid以醋酸鈣溶液造成的 pH梯度來載送的示意圖---p12 圖1.15 5-FU的結構式---p14 圖1.16 5-FU在體內所造成相關的新代謝機制示意圖---p14 圖1.17 CPT的發現與研究進程---p16 圖1.18 拓樸異構酶I被CPT抑制的示意圖---p16 圖1.19 CPT內酯型與羧酸型的化學結構---p18 圖1.20 兩種應用在臨床的CPT衍生物---p18 圖3.1真空旋轉濃縮機裝置的示意圖---p21 圖3.2 5-FU部份的流程圖---p27 圖3.3 CPT部份的流程圖---p27 圖3.4 (A) 5-FU濃度與HPLC的UV偵檢器對其所測得的積分面積間的對 應關係 (B) CPT濃度與螢光儀對其所測得的積分面積間的對應 關係---p28 圖4.1 不同微脂粒磷脂質PC的組成與pH梯度的有無對5-FU包覆效率的 影響---p30 圖4.2 包覆有5-FU的微脂粒通過充填有Sephadex G-50的管柱分離未 包覆藥，其UV積分面積對流出管數 (每管1.5 ml) 之間的關 係---p32 圖4.3 運用pH梯度 (pHout = 7.5，pHin = 4.0) 對以EPC所形成的微 脂粒包覆各種藥物的程度與穩定性---p34 圖4.4 Codeine的結構式---p34 圖4.5 常見1級，2級與3級胺基的pKb---p34 圖4.6 以pH梯度包覆CPT與沒有pH梯度包覆CPT的微脂粒兩者進行藥物 釋放，隨時間微脂粒粒徑與藥物釋放率的變化---p37 圖4.7 藥物釋放前後同管數序號的沖提流出液中CPT的含量比---p38 圖4.8 隨著藥物釋放時間具有完整內酯環的CPT濃度的變化---p39 圖A-1 與y軸夾的散射角è以及與z軸夾的散射角f示意圖。入射光朝 著y軸的方向而電場則朝著z軸的方向---p47 圖A-2 入射光照到一群障礙物並散射到受照者的觀測。由其電場強 度集合以及d角可得總體散射強度---p47 圖A-3 光學分析光譜的示意圖---p47

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