本研究利用幾丁聚醣(chitosan, CS )奈米粒子作為多酚化合物(polyphenol)-没食單寧(gallotannin, GT)之載體(carrier)，利用三聚磷酸鈉(tripolyphosphate , TPP)與幾丁聚醣之間電荷反應，以離子凝膠技術(ionic gelation)包埋(entrapped)或吸附(adsorbed)没食單寧以及使用多層乳化包覆技術(water-oil-water)包覆(encapsulated)没食單寧的方式製備出一系列没食單寧-幾丁聚醣奈米粒子。比較不同製備方法的奈米粒子：包埋、吸附與包覆之間的差異性。利用穿透式電子顯微鏡(Transmission electron microscopy , TEM)和動態光散射粒徑分析儀(Dynamic light scattering, DLS)分別測試奈米粒子的直徑大小、界面電位(Zeta potential)及形狀外貌…等；並針對影響奈米粒子的物化性質作探討。其奈米粒子的粒徑大小和承載沒食單寧的包覆率，皆會因不相同的製備過程而有不同地影響因素。測試没食單寧-幾丁聚醣奈米粒子對纖維母細胞(L929 mouse fibroblast)的活性測試以及對革蘭氏陽性菌(Gram-positive coccus)—金黃色葡萄球菌(Staphylococcus aureus)之抗菌活性，藉以量測出最小抑菌濃度(minimum inhibitory concentration, MIC)及最小殺菌濃度(minimum bactericidal concentration, MBC)。研究結果發現將沒食單寧包裹於幾丁聚醣奈米粒子中，顆粒大小圓潤、平均，其中更以W/O/W製備得之結果為最佳；在一般環境下，奈米粒子能夠保護核蕊物質--單寧，不會因受到外在環境而產生變化；而是在給予一個特定仿造生物體環境之條件下，例如：pH値、機械性質…等，造成奈米粒子的幾丁聚醣外殼會溶解破裂，使沒食單寧逐漸釋放出來。沒食單寧無細胞毒性但奈米粒子中的單寧濃度會對細胞生長造成影響，而其粒子對金黃色葡萄球菌具有抗菌活性。

This study presents the preparation and characteristic of chitosan (CS) nanoparticles as a carrier of polyphenol compound – gallotannin (GT). The preparation method of CS nanoparticles is using the electric charge interaction between sodium tripolyphosphate (TPP) and chitosan. Gallotannin is entrapped or absorbed by the ionic gelation technology with chitosan as well as using multi-layer emulsification encapsulation technology: water-oil-water encapsulation gallotannin to prepare a series of GT-CS nanoparticles. We also discuss the differences of these three nanoparticle preparation methods (entrapped, absorbed, encapsulated ).
  The nanoparticles’ diameter, zeta potential and the shape are measured by using transmission electron microscopy (TEM) and the dynamic light scattering (DLS). The influences on the physicochemical characterization of nanoparticles are also discussed. The cell’s activity with L929 mouse fibroblast and antibacterial activity with the Gram-positive coccus-Staphylococcus aureus of GT-CS nanoparticle were detected, and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) are measured. Those results show that gallotannin had encapsulated in chitosan nanoparticle, and the particle was round and size average, especially the W/O/W particles had the best effect. The nanoparticles could protect the core material-tannin under the normal environment. But in the specific condition such as creating the outer covering, chitosan nanoparticle could be dissolved and cause gallotannin to release gradually under the copy organisam environment, for example: pH, mechanical property, etc. Gallotannin is found non-toxicity but the tannin density in nanoparticle would influence on the cell growth. These particles had the antibacterial activity to Staphylococcus.

論文摘要................................. .I 
Abstract................................ III 
目錄.......................................V 
圖目錄...................................VII 
表目錄....................................IX

第一章前言 ....................................1 
第二章 文獻整理................................4 
 2.1 何謂奈米生醫材料..........................4 
  2.1.1 奈米材料之種類.........................5 
 2.2 奈米生醫材料之應用........................7 
 2.3 藥物控制釋放技術..........................8 
 2.4 幾丁質與幾丁聚醣..........................9 
  2.4.1 幾丁聚醣之應用.........................11 
  2.4.2 幾丁聚醣與其奈米粒子之溶劑系統.........11 
  2.4.3 以幾丁聚醣粒子為藥物載體...............12 
 2.5 幾丁聚醣奈米粒子之製備與應用..............12 
 2.6 多重乳化物之形成......................... 15 
 2.7 單寧及沒食單寧(Gallotannin)...............16 
第三章 實驗材料與方法..........................18 
 3.1 實驗藥品..................................18 
 3.2 實驗儀器..................................19 
 3.3 實驗架構..................................20 
 3.4 樣品製備..................................21 
  3.4.1 製備幾丁聚醣奈米粒子...................21 
  3.4.2 製備承載沒食單寧之幾丁聚醣奈米粒子.....21 
   3.4.2.1 離子凝膠法..........................21 
   3.4.2.2 W/O/W法.............................21 
 3.5 粒徑與Zeta potential分析..................22 
 3.6 穿透式電子顯微鏡分析......................22 
 3.7 FTIR分析..................................23 
 3.8 UV光譜儀測試分析..........................23 
  3.8.1 製作沒食單寧檢量線.....................23 
  3.8.2 包覆率檢測.............................23 
  3.8.3 不同pH值環境下奈米粒子之釋放率.........24 
 3.9 機械性質分析..............................24 
 3.10 抗菌試驗.................................24
  3.10.1 菌活性值之計算........................24 
  3.10.2 抗菌活性..............................25 
 3.11 纖維母細胞培養試驗.......................25 
  3.11.1 細胞活化..............................26 
  3.11.2 細胞繼代..............................26 
  3.11.3 培養細胞於樣品中......................26 
  3.11.4 細胞增生實驗 (MTT assay)..............27 
第四章 結果與討論..............................28 
 4.1 粒徑與Zeta potential分析..................28 
  4.1.1 幾丁聚醣奈米粒子.......................28 
  4.1.2 承載沒食單寧之幾丁聚醣奈米粒子.........32 
 4.2 穿透式電子顯微鏡分析(TEM) ................36 
 4.3 傅利葉紅外線光譜分析(FTIR) ...............53 
 4.4 UV光譜儀測試分析..........................54 
  4.4.1 包覆率.................................54 
  4.4.2 不同pH值環境下奈米粒子之釋放率.........58 
 4.5 機械性質分析..............................61 
 4.6 In vitro 抗菌活性測試.....................63 
 4.7 In vitro細胞活性試驗......................64 
  4.7.1 細胞型態之觀察.........................64 
  4.7.2 細胞增生試驗(MTT assay)................73 
第五章 結論....................................76 
第六章 參考文獻................................78 

圖 2-1 為幾丁質與幾丁聚醣結構圖...............10 
圖2-2 為幾丁聚醣與三聚磷酸鈉之間離子交鏈反應結構......14 
圖2-3 為單寧結構圖....................................17 
圖3-1 實驗架構圖............20 
圖4-1 幾丁聚醣濃度對包覆率之影響......................31 
圖4-2 轉速對奈米粒子粒徑之影響........................31 
圖4-3 為GT濃度對粒徑之影響............................35 
圖4-4 為油量對粒徑之影響..............................35 
圖4-5 為幾丁聚醣奈米粒子在TEM下型態...................39 
圖4-6 為幾丁聚醣奈米粒子在TEM下型態...................39 
圖4-7 為幾丁聚醣包埋沒食單寧奈米粒子在TEM下型態.......40 
圖4-8 為幾丁聚醣包埋沒食單寧奈米粒子在TEM下型態.......40 
圖4-9 為沒食單寧吸附於幾丁聚醣奈米粒子在TEM下型態.....41 
圖4-10 為沒食單寧吸附於幾丁聚醣奈米粒子在TEM下型態(水洗後) .....41 
圖4-11 為沒食單寧-幾丁聚醣奈米粒子在TEM下型態 (慢轉速) .....42 
圖4-12 為沒食單寧-幾丁聚醣奈米粒子在TEM下型態 (慢轉速) .....42 
圖4-13 為沒食單寧-幾丁聚醣奈米粒子在TEM下型態 (慢轉速) .....43 
圖4-14 為沒食單寧-幾丁聚醣奈米粒子在TEM下型態 (慢轉速) .....43 
圖4-15 為沒食單寧-幾丁聚醣奈米粒子在TEM下型態 (快轉速) .....44 
圖4-16 為沒食單寧-幾丁聚醣奈米粒子在TEM下型態 (快轉速) .....44 
圖4-17 為沒食單寧-幾丁聚醣奈米粒子在TEM下型態(適當轉速) .....45 
圖4-18 為沒食單寧-幾丁聚醣奈米粒子在TEM下型態(適當轉速) ....45 
圖4-19 為W/O/W奈米粒子在TEM下型態(多油量).........46 
圖4-20 為W/O/W奈米粒子在TEM下型態(多油量) ........46 
圖4-21 為W/O/W奈米粒子在TEM下型態(多油量) ........47 
圖4-22 為W/O/W奈米粒子在TEM下型態(多油量) ........47 
圖4-23 為W/O/W奈米粒子在TEM下型態(多油量) ........48 
圖4-24 為W/O/W奈米粒子在TEM下型態(適當油量) ......48 
圖4-25 為W/O/W奈米粒子在TEM下型態(適當油量) ......49 
圖4-26 為W/O/W奈米粒子在TEM下型態(適當油量) ......49 
圖4-27 為W/O/W奈米粒子在TEM下型態(適當油量) ......50 
圖4-28 為W/O/W奈米粒子在TEM下型態(適當油量) ......50 
圖4-29 為W/O/W奈米粒子在TEM下型態(適當油量) ......51 
圖4-30 為W/O/W奈米粒子在TEM下型態(適當油量) ......51 
圖4-31 為W/O/W奈米粒子在TEM下型態(適當油量) ......52 
圖4-32 為W/O/W奈米粒子在TEM下型態(適當油量) ......52 
圖4-33 為FTIR數據圖...............................54
圖4-34 沒食單寧之減量線...........................56
圖4-35 為沒食單寧濃度對包覆率之影響...............56
圖4-36 為沒食單寧濃度對乘載率之影響...............57
圖4-37 為油量對包覆率之影響.......................57
圖4-38 為油量對乘載率之影響.......................58
圖4-39 為GT-CS粒子於不同酸鹼環境下GT的釋放量......60
圖4-40 為W/O/W粒子於不同酸鹼環境下GT的釋放量......60
圖4-41 為CS的抗拉張力數據.........................62
圖4-42 為CS-TPP的抗拉張力數據.....................62
圖4-43 為24 小時對照組細胞生長情形................66
圖4-44 為48 小時對照組細胞生長情形................66
圖4-45 為72 小時對照組細胞生長情形................66
圖4-46 為24 小時GT=0.1mg/mL對細胞生長之影響.......67
圖4-47 為48 小時GT=0.1mg/mL對細胞生長之影響.......67
圖4-48 為72 小時GT=0.1mg/mL對細胞生長之影響.......67
圖4-49 為24 小時GT=0.25mg/mL對細胞生長之影響......68
圖4-50 為48 小時GT=0.25mg/mL對細胞生長之影響......68
圖4-51 為72 小時GT=0.25mg/mL對細胞生長之影響......68
圖4-52 為24 小時GT=0.5mg/mL對細胞生長之影響.......69
圖4-53 為48 小時GT=0.5mg/mL對細胞生長之影響.......69
圖4-54 為72 小時GT=0.5mg/mL對細胞生長之影響.......69
圖4-55 為24 小時GT=0.75mg/mL對細胞生長之影響......70
圖4-56 為48 小時GT=0.75mg/mL對細胞生長之影響......70
圖4-57 為72 小時GT=0.75mg/mL對細胞生長之影響......70
圖4-58 為24 小時GT=1.0mg/mL對細胞生長之影響.......71
圖4-59 為48 小時GT=1.0mg/mL對細胞生長之影響.......71
圖4-60 為72 小時GT=1.0mg/mL對細胞生長之影響.......71
圖4-61 為24 小時W/O/W奈米粒子對細胞生長之影響.....72
圖4-62 為48 小時W/O/W奈米粒子對細胞生長之影響.....72
圖4-63 為72 小時W/O/W奈米粒子對細胞生長之影響.....72
圖4-64 為不同濃度GT之細胞活性測試.................75
圖4-65 為W/O/W奈米粒子之細胞活性測試..............75

表4-1 為幾丁聚醣濃度對粒徑之影響..................30 
表4-2 為三聚磷酸鈉濃度對奈米粒子特性之影響........30 
表4-3 為GT-CS奈米粒子之DLS數據....................34 
表4-4 為不同製備方式之沒食單寧-幾丁聚醣奈米粒子於DLS下之數.......34 
表4-5 為幾丁聚醣外殼材之機械性質..................62 
表4-6 奈米粒子之抗菌活性..........................63

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