臺灣博碩士論文加值系統

痤瘡發炎主要由痤瘡丙酸桿菌所引起，痤瘡丙酸桿菌可以月桂酸殺滅，但
月桂酸屬難溶於水之脂肪酸，需添加二甲基亞碸增加月桂酸溶解度。本研究以
兩性高分子形成微胞並包覆月桂酸之劑型，可減少二甲基亞碸(DMSO)的毒性且
提高月桂酸抗菌效果。本研究中設計三種不同分子量的PCL-PEG-PCL，並分別
利用氫核磁共振光譜、紅外線光譜、熱示差掃瞄卡量計及凝膠透層析儀分析合
成高分子之化學結構，並用1,6-二苯基-1,3,5-己三烯做為螢光探針觀察其臨界微
胞濃度。將PCL-PEG-PCL 以薄膜水合法製備微胞，以粒徑分析儀、表面電荷分
析及穿透式顯微鏡觀察，微胞未包覆月桂酸前的粒徑介於49.8-198.1nm，而當包
覆月桂酸後，微胞粒徑範圍下降至24.7-89.3 nm，並且觀察到含有不規則型態之
粒子，其表面電荷也因月桂酸的羧酸基去質子化由-3.0 mV 至-9.7 mV 下降為-4.2
mV 至-18.4 mV。在以2-萘基溴甲基酮與微胞中月桂酸反應後，利用高效液相層
析儀測定月桂酸包覆量；以PC50E40C50 的聚己內酯-聚乙二醇-聚己內酯可搭載
323.75 μg/mL 月桂酸，隨PCL 鏈長加長，藥物包覆率隨之增加。在抑菌及殺菌
測試中，比較微胞包覆月桂酸(微胞劑型)及月桂酸溶於5% DMSO 的劑型效力，
以最小抑菌濃度(MIC)及最小殺菌濃度(MBC)做評估；在5% DMSO 中，月桂酸
的MIC 與MBC 分別為20 ug/mL 和80 μg/mL，而微胞劑型MIC 與MBC 分別介
於10-20 μg/mL 與40-80 μg/mL，以微胞劑型只需較少月桂酸量便可達同樣抑菌
和殺菌功效。以不同劑型對小鼠纖維母細胞和人類角質細胞做生物相容性評
估，結果可見微胞劑型的生物相容性明顯優於溶劑劑型。因此利用PCL-PEG-PCL
包覆月桂酸可避免添加有機溶劑助溶的麻煩，並且仍可達到更佳的效果，未來
可進一步觀察在痤瘡性發炎的動物體內，預期能同樣有效殺菌且降低痤瘡性的
發炎。

Acne vulgaris is known as infection of pathogenic bacteria, Propionibacterium
acnes (P. acnes). Lauric acid (LA) was found to kill P. acnes effectively. However,
LA is a sparingly soluble fatty acid in water in which dimethly sulfoxide (DMSO) is
used to increase solubility. In this study, amphiphilic micelles encapsulating LA was
developed to reduce DMSO-induced toxicity and improve bactericidal effect of LA.
Poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-PEG-PCL)
triblock copolymer was sythysized by ring-opening polymerization. Different
molecular weights of PCL-PEG-PCL were synthesized and chararerized by 1H
nuclear magnegtic resonance, Fourier transform infared spectroscopy, differential
scanning calorimeter and gel permeation chromatography, respectively. The critical
micelle concentration(CMC) of the micelle solution was determined by using
fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene(DPH). The results of particle size
analyzer and transmission electron microscopy show that the averge particles size of
placebo micelles and LA-encapsulated micelles were 49.8-198.1 nm and 24.7-89.3
nm, respectively. Upon encapsulation of LA, zeta potential decreased from -3.0 ~
-9.7mV to -4.2 ~ -18.4 mV, because of depronated carboxyl group in LA. A
High-performance liquid chromatography was used to quantitate LA encapsulated in
the micelles, after encapsulated LA was derived with naphthacyl ester. As the results,
the payload of PC50E40C50 for LA was 323.75 μg/mL which was the highest among
various PCL-PEG-PCL. In addition, as the length of PCL increased, the drug loading
contents decreased. The effect of LA-encapsulated micelles(micelle formulation)
were compared with LA dissolved in 5% DMSO(free LA formulation). For free LA
formulation, as free lauric acid dissolved in 5% DMSO, the minimum inhibitory
concentration(MIC) and minimum bactericidal concentration(MBC) were 20 μg/mL
and 80μg/mL, respectively. In contrasty, MIC and MBC of micelle formulation were
only 10-20 μg/mL and 40-80 μg/mL, respectively. Therefore, the micelle formulation
requires slightly lower LA to reach the same antibactirials effecacy of free LA
formulation. The cytotoxicities of free LA formulation and micelle formulation in
fibroblast and keratinocyte were conducted. It is found that micelle formulation
I I I
posses better biocompatibility over free LA. In summary, this study demonstrated that
PCL-PEG-PCL was a potential drug carrier to encapsulate LA. It worthshile to
further examine the efficacy to against the infection of P.acnes in animal study.

目錄
摘要...............................................................................................................................I
Abstract ....................................................................................................................... II
致謝.............................................................................................................................IV
目錄..............................................................................................................................V
圖索引......................................................................................................................VIII
表索引..........................................................................................................................X
中英文及縮寫對照表.................................................................................................XI
第一章緒論................................................................................................................. 1
1.1 研究背景........................................................................................................ 1
1.2 研究動機與目的............................................................................................ 1
第二章文獻回顧......................................................................................................... 3
2.1 痤瘡................................................................................................................. 3
2.1.1 痤瘡丙酸桿菌簡介............................................................................. 3
2.1.2 痤瘡致病機制..................................................................................... 3
2.1.3 痤瘡治療............................................................................................. 5
2.2 脂肪酸............................................................................................................. 8
2.2.1 脂肪酸抑制微生物.............................................................................. 8
2.2.2 脂肪酸抗菌機制................................................................................ 10
2.2.3 脂肪酸衍生與分析............................................................................ 12
2.3 奈米藥物載體與殺菌藥劑遞送.................................................................. 14
2.3.1 微胞之結構........................................................................................ 14
2.3.2 生物相容性良好之高分子材料........................................................ 14
2.3.3 微胞的製備........................................................................................ 15
2.3.4 殺菌藥物載體.................................................................................... 17
第三章材料與實驗方法........................................................................................... 20
3.1 研究方法與架構.......................................................................................... 20
3.2 實驗用儀器.................................................................................................. 22
3.3 實驗用藥品.................................................................................................. 24
3.4 藥物載體聚己內酯-聚乙二醇-聚己內酯合成與鑑定............................... 27
3.4.1 聚己內酯-聚乙二醇-聚己內酯合成................................................ 27
3.4.2 聚己內酯-聚乙二醇-聚己內酯鑑定................................................ 29
3.5 微胞製備與鑑定.......................................................................................... 31
3.5.1 製備不含月桂酸微胞....................................................................... 31
3.5.2 製備搭載月桂酸的微胞................................................................... 31
3.5.3 微胞性質鑑定................................................................................... 33
3.6 月桂酸衍生及定量分析.............................................................................. 34
3.6.1 衍生反應........................................................................................... 34
3.6.2 製作月桂酸衍生物檢量線............................................................... 35
3.6.3 定量微胞溶液中月桂酸量............................................................... 35
3.6.4 月桂酸衍生物定量分析................................................................... 36
3.7 微胞載體對於痤瘡丙酸桿菌的抑菌效能.................................................. 37
3.7.1 實驗藥品配置................................................................................... 37
3.7.2 菌瓶開封及菌株活化....................................................................... 38
3.7.3 痤瘡丙酸桿菌保存........................................................................... 38
3.7.4 最小抑菌濃度(minimum inhibitory concentration)......................... 38
3.7.5 最小殺菌濃度(minimum bactericidal concentration) ...................... 41
3.8 微胞溶液細胞毒性測試.............................................................................. 43
3.8.1 實驗藥品配置................................................................................... 43
3.8.2 小鼠纖維母細胞培養(L929)............................................................ 44
3.8.3 人類角質細胞培養(CCD1106KERtr).............................................. 46
3.8.4 細胞毒性測試................................................................................... 48
3.9 統計分析...................................................................................................... 48
第四章結果與討論................................................................................................... 49
4.1 聚己內酯-聚乙二醇-聚己內酯性質鑑定................................................... 49
4.1.1 聚己內酯-聚乙二醇-聚己內酯的傅立葉轉換紅外線光譜分析.... 49
4.1.2 聚己內酯-聚乙二醇-聚己內酯的氫核磁共振光譜分析................ 51
4.1.3 聚己內酯-聚乙二醇-聚己內酯的凝膠滲透層析儀分析................ 56
4.1.4 聚己內酯-聚乙二醇-聚己內酯的臨界微胞濃度測定.................... 56
4.1.5 聚己內酯-聚乙二醇-聚己內酯的熱示差掃瞄卡量計分析............ 59
4. 2 聚己內酯-聚乙二醇-聚己內酯包覆月桂酸之微胞性質分析.................. 62
4.2.1 微胞粒徑大小及分佈....................................................................... 64
4.2.2 微胞表面電荷分析........................................................................... 65
4.2.3 微胞表面型態觀察........................................................................... 66
4.3 月桂酸定量分析.......................................................................................... 71
4.3.1 脂肪酸衍生相對位置....................................................................... 71
4.3.2 月桂酸衍生物檢量線....................................................................... 72
4.3.3 月桂酸濃度與載藥率....................................................................... 74
4.4 微胞劑型對痤瘡丙酸桿菌生長的影響...................................................... 76
4.4.1 最小抑菌濃度測試........................................................................... 76
4.4.2 最小殺菌濃度測試........................................................................... 80
4.5 細胞毒性測試.............................................................................................. 85
4.5.1 月桂酸對於鼠纖維母細胞之影響................................................... 85
4.5.2 月桂酸對於人類角質細胞之影響................................................... 90
第五章結論及未來展望........................................................................................... 95
參考文獻..................................................................................................................... 97
附件........................................................................................................................... 104
附件一最小抑菌濃度與最小殺菌濃度圖解................................................. 104
附件二凝膠滲透液相層析儀分析結果......................................................... 105
附件三臨界微胞濃度實驗之原始數據......................................................... 108
附件四臨界微胞濃度計算............................................................................. 110
附件五粒徑分析原始數據............................................................................. 112
附件六細菌之生長曲線................................................................................. 114
附件七奈米粒子裝載抗菌藥物的遞送方式................................................. 115

圖 2-1 痤瘡形成的三個階段....................................................................................... 4
圖 2-2 不同程度的痤瘡病症....................................................................................... 6
圖 2-3 自由脂肪酸在菌體內的作用機制................................................................. 12
圖 2-4 微胞結構示意圖............................................................................................. 14
圖 2-5 製備微胞流程................................................................................................. 16
圖 3-1 實驗架構......................................................................................................... 21
圖 3-2 聚己內酯-聚乙二醇-聚己內酯合成反應式.................................................. 28
圖 3-3 聚己內酯-聚乙二醇-聚己內酯合成裝置...................................................... 28
圖 3-4 微胞製備實驗流程......................................................................................... 32
圖 3-5 月桂酸衍生反應式......................................................................................... 35
圖 3-6 分光光度計測量月桂酸衍生後的光吸收特徵值......................................... 36
圖 3-7 測試最小抑菌濃度實驗流程......................................................................... 40
圖 3-8 測試最小殺菌濃度實驗流程......................................................................... 42
圖 3-9 鼠纖維母細胞之細胞型態............................................................................. 45
圖 3-10 人類角質細胞之細胞型態........................................................................... 47
圖 3-11 人類角質細胞之生長曲線圖....................................................................... 47
圖4-1 三種分子量的聚己內酯-聚乙二醇-聚己內酯之FT-IR 分析...................... 51
圖 4-2 PCL20-PEG40-PCL20 之1H-NMR 分析............................................................ 53
圖 4-3 PCL50-PEG40-PCL50 之1H-NMR 分析............................................................ 54
圖 4-4 PCL100-PEG40-PCL100 之1H-NMR 分析......................................................... 55
圖 4-5 PC20E40C20 的臨界微胞濃度測定................................................................... 57
圖 4-6 PC50E40C50 的臨界微胞濃度測定................................................................... 58
圖 4-7 PC100E40C100 的臨界微胞濃度測定................................................................. 58
圖 4-8 PC20E40C20 之DSC 分析結果圖...................................................................... 60
圖 4-9 PC50E40C50 之DSC 分析結果.......................................................................... 60
圖 4-10 PC100E40C100 之DSC 分析結果..................................................................... 61
圖 4-11 聚己內酯-聚乙二醇-聚己內酯微胞包覆月桂酸示意圖............................. 62
圖 4-12 加入熱水水合後的MC20 ............................................................................. 63
圖 4-13 經各轉速離心後的微胞溶液(MC20) ........................................................... 63
圖 4-14 以薄膜水合法製備完成的微胞水溶液....................................................... 63
圖 4-15 微胞直徑與表面電荷................................................................................... 65
圖 4-16MC20 微胞之型態.......................................................................................... 67
圖 4-17MC20LA 微胞之型態..................................................................................... 67
圖 4-18MC50 微胞之型態.......................................................................................... 68
圖 4-19MC50LA 微胞之型態..................................................................................... 68
圖 4-20MC100 微胞之型態......................................................................................... 69
圖 4-21MC100LA 微胞之型態................................................................................... 69
圖 4-22 以純月桂酸製備微胞.................................................................................... 70
圖 4-23 利用高效能液相層析儀分析月桂酸與油酸經衍生後產物....................... 71
圖 4-24 各濃度月桂酸衍生化後之HPLC 分析圖譜............................................... 72
圖 4-25 各濃度月桂酸衍生化後之檢量線............................................................... 73
圖 4-26 FREE LA 在液態培養之抑菌效果................................................................ 77
圖 4-27MC20LA 在液態培養之抑菌效果................................................................. 78
圖 4-28MC50LA 在液態培養之抑菌效果................................................................. 78
圖 4-29MC100LA 在液態培養之抑菌效果............................................................... 79
圖 4-30 FREE LA 之殺菌結果..................................................................................... 81
圖 4-31 微胞劑型之殺菌效果................................................................................... 81
圖 4-32 純月桂酸製備與控制組(PBS)之菌落生長情形......................................... 82
圖 4-33 FREE LA 之菌落生長觀察............................................................................. 82
圖 4-34MC20LA 之菌落生長觀察............................................................................. 83
圖 4-35MC50LA 之菌落生長觀察............................................................................. 83
圖 4-36MC100LA 之菌落生長觀察........................................................................... 84
圖 4-37 不同濃度月桂酸對纖維母細胞之影響....................................................... 86
圖 4-38 各分子量微胞與纖維母細胞共培養後之細胞存活率結果....................... 86
圖 4-39 不同濃度月桂酸與鼠纖維母細胞共培養24 小時後細胞型態................. 87
圖 4-40 微胞劑型與鼠纖維母細胞共培養24 小時後細胞型態............................. 88
圖 4-41 棕櫚酸鹽對細胞產生脂肪毒性................................................................... 89
圖 4-42 不同濃度月桂酸對人類角質細胞之影響................................................... 90
圖 4-43 各分子量微胞與人類角質細胞共培養後之細胞存活率結果................... 91
圖 4-44 PBS 與人類角質細胞共培養24 小時後細胞型態..................................... 91
圖 4-45 不同濃度月桂酸與人類角質細胞共培養24 小時後細胞型態................. 92
圖 4-46 微胞劑型與人類角質細胞共培養24 小時後細胞型態............................. 93

表 2-1 藥物副作用與新型藥劑傳遞系統................................................................... 7
表 2-2 脂肪酸抗微生物之研究................................................................................... 9
表 2-3 以高分子搭載抗菌劑之研究......................................................................... 19
表3-1 儀器設備目錄................................................................................................. 22
表 3-2 藥品目錄......................................................................................................... 24
表 3-3 聚己內酯-聚乙二醇-聚己內酯合成進藥量.................................................. 27
表 3-4 DSC 升降溫循環條件..................................................................................... 29
表 3-5 微胞製備比例及縮寫..................................................................................... 32
表4-1 聚己內酯-聚乙二醇-聚己內酯在傅立葉轉換紅外線光譜儀的特徵峰....... 50
表 4-2 聚己內酯-聚乙二醇-聚己內酯之分子量與臨界微胞濃度.......................... 56
表 4-3 聚己內酯-聚乙二醇-聚己內酯的熱示差掃描卡量計分析結果................... 61
表 4-4 微胞粒徑與分佈............................................................................................. 64
表 4-5 各濃度月桂酸衍生化後之積分面積............................................................. 73
表 4-6 月桂酸濃度及載藥率..................................................................................... 75
表 4-7 最小抑菌濃度測試結果................................................................................. 77
表 4-8 純月桂酸與PCL-PEG-PCL 包覆月桂酸微胞之最小殺菌濃度.................. 80
表 4-9 鼠纖維母細胞與不同月桂酸劑型培養後的細胞存活率............................. 89
表 4-10 人類角質細胞與不同月桂酸劑型培養後的細胞存活率........................... 94

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