溫度敏感型聚酯類水膠作為免疫抑制藥物傳遞系統之應用於同種異體皮膚移植

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研究生：	吳逸恩 Wu, I-En
論文名稱：	溫度敏感型聚酯類水膠作為免疫抑制藥物傳遞系統之應用於同種異體皮膚移植 Thermosensitive Polyester Hydrogel for Application of Immunosuppressive Drug Delivery System in Skin Allograft
指導教授：	朱一民 Chu, I-Ming 陳韻晶 Chen, Yun-Ching
口試委員:	黃振煌 Huang, Jen-Huang 姚少凌 Yao, Chao-Ling
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2022
畢業學年度：	110
語文別：	中文
論文頁數：	52
中文關鍵詞：	溫度敏感型、他克莫司、同種異體移植
外文關鍵詞：	thermosensitive, tacrolimus, allotransplantation
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他克莫司是一種的免疫抑制藥物，能夠用於抑制急性排斥反應，常用於同種異體移植後患者之治療，在血清中維持穩定的藥物濃度，會有比較良好的治療效果。然而，傳統的治療是利用口服或注射的途徑給藥，後者造成患者的不方便，前者會遇到藥物過量或患者在治療中依從性低下的問題。本研究選用溫度敏感型聚酯類水膠作為藥物載體，包覆疏水性免疫抑制藥物他克莫司，期望能藉由藥物控制釋放系統，達到緩慢且持續釋放的效果。材料的製備是以methoxy poly(ethylene glycol)作為起始劑，與D,L-lactide、caprolactone進行開環聚合反應，合成出mPEG-PLCL兩團聯共聚物，將這此種共聚物分別配製成水膠，比較不同組成水膠性質與包覆效果，以找出較適合包覆他克莫司之藥物載體。
其中，在本研究中我們嘗試不同的助溶劑以包覆他克莫司於mPEG-PLCL，最終發現以0.5 % 的PVP在藥物釋放以及包覆效果有最佳的效果。在體外藥物釋放以及動物實驗中，我們可以成功的讓藥物定點的穩定釋放30天也不會出現藥物突釋的現象。此外，從動物同種異體皮膚移植的實驗中，此劑型提高了移植的存活率。顯示出本研究的水膠配方具有傳送抑制免疫藥物，改善治療效果的潛力。

Tacrolimus (FK506) is a common immunosuppressive drug that is capable of suppressing acute rejection reactions, and is used to treat patients after allotransplantation. A stable and suitable serum concentration of tacrolimus is desirable for better therapeutic effects. However, daily drug administration via oral or injection routes is quite inconvenient and may encounter drug overdose or low patient compliance problems. In this research, our objective was to develop an extended delivery system using a thermosensitive hydrogel of poly ethylene glycol, D,L-lactide (L), and ϵ-caprolactone (CL) block copolymer, mPEG-PLCL, as a drug depot. The formulation of mPEG-PLCL and 0.5% PVP-dissolved tacrolimus was studied and the optimal formulation was obtained. The in vivo data showed that in situ gelling is achieved, a stable and sustained release of the drug within 30 days can be maintained, and the hydrogel was majorly degraded in that period. Moreover, improved allograft survival was achieved. Together, these data imply the potential of the current formulation for immunosuppressive treatments.

目錄
摘要    ………………………………………………………..................I
ABSTRACT    II
圖表目錄    4
第一章    文獻回顧    6
1.1    水膠介紹與種類    6
1.1.1    交聯方式    7
1.2    溫度敏感型聚酯類水膠 (THERMOSENSITIVE HYDROGELS)    9
1.2.1    相轉變行為(Phase transition behavior)    10
1.2.2    聚酯類水解機制(Hydrolysis)    11
1.3    藥物傳遞系統    12
1.3.1    藥物控制釋放方式    14
1.3.2    藥物釋放速率影響因子    15
1.4    移植排斥(TRANSPLANT REJECTION)    17
1.4.1    他克莫司 (Tacrolimus, FK506)    18
第二章    研究動機    20
第三章    材料與方法    22
3.1    MPEG-PLCL合成製備    22
3.2    化學結構鑑定與性質分析    23
3.2.1    氫原子核磁共振光譜儀 (1H-NMR)    23
3.2.2    凝膠滲透層析儀 (Gel permeation chromatography, GPC)    24
3.2.3    傅立葉轉換紅外線光譜儀 (Fourier transform infrared spectroscopy, FTIR)    …………………………………………………………………………..25
3.2.4    水膠相轉變行為測定(Sol-gel-sol phase transition)    25
3.2.5    水膠黏度分析    25
3.3    藥物傳輸系統於溫感性水膠之應用    26
3.3.1    Tacrolimus (FK506)濃度標準線建立    26
3.3.2    藥物配方與包覆率測試    26
3.3.3    體外降解實驗 (Degradation test in vitro)    27
3.3.4    體外藥物釋放實驗 (Drug release in vitro)    27
3.4    生物相容性測試    28
3.4.1    哺乳類動物細胞培養    28
3.4.2    生物毒性測試 (MTT assay& live/dead)    29
3.4.3    同種異體皮膚移植動物實驗    30
3.4.4    CD4 T cells, CD8 T cells, 以及 Regulatory T cells 之定量分析    31
第四章    實驗結果與討論    32
4.1    化學結構鑑定與性質分析    32
4.1.1    氫原子核磁共振光譜儀 (1H-NMR)    32
4.1.2    凝膠滲透層析儀 (Gel permeation chromatography, GPC)    33
4.1.3    傅立葉轉換紅外線光譜儀 (Fourier transform infrared spectroscopy, FTIR)    …………………………………………………………………………..35
4.1.4    水膠相轉變行為以及黏度測定(Sol-gel-sol phase transition)    35
4.2    藥物傳輸系統於溫感性水膠之應用    38
4.2.1    藥物配方與包覆率測試    38
4.2.2    體外降解實驗 (Degradation test in vitro)    39
4.2.3    體外藥物釋放實驗 (Drug release in vitro)    41
4.3    生物相容性與毒性測試測試 (MTT ASSAY)    43
4.4    同種異體皮膚移植結果分析    44
第五章    結論與未來工作    47
5.1    實驗結論    47
5.2    未來工作    47
第六章    參考資料    49

 

圖表目錄
圖 1. 水膠之廣泛應用性示意圖。    6
圖 2. 水膠之廣泛應用性示意圖。    7
圖 3. 常用於化學交聯常用之交聯劑。    8
圖 4. 高分子光交聯機制示意圖。    8
圖 5. 環境應答型水膠膨潤現象示意圖。    9
圖 6. 常見的溫度敏感型水膠之高分子化學結構。    10
圖 7. 成膠機制示意圖。    11
圖 8. 酯鍵之水解反應。    12
圖 9. 高分子材料降解類型示意圖。    12
圖 10. 不同給藥方式之藥物分布情形。    13
圖 11. 不同藥物釋放方式。    14
圖 12. PEG-PLGA-PEG(550-2810-550)水膠釋放(a)酮洛芬及(b)螺內酯的情形。    15
圖 13. PVP之化學結構。    16
圖 14. PVP作為藥物載體之多用途 (A)作為藥物分散劑; (B)在高分子水膠中給予賦形之示意圖。    16
圖 15. Tacrolimus化學結構。    18
圖 16. Tacrolimus作用機制。    19
圖 17. mPEG-PLCL合成(A)反應示意圖; (B)合成後之產物實體樣品圖。    23
圖18. MTT assay實驗組別示意圖。    30
圖 19. mPEG-PLCL(550-1405) LA/CL=80/20 1H-NMR圖譜。    33
圖 20. mPEG-PLCL之FT-IR圖譜。    35
圖 21. 隨溫度提高下之相變化 (A)不同濃度之mPEG-PLCL水膠溶膠-凝膠-溶膠相隨溫度轉變圖; (B) 25 wt% mPEG-PLCL水膠溶膠-凝膠-溶膠相隨溫度變化形態改變圖; (C) 25 wt% mPEG-PLCL水膠隨溫度變化之黏度測量。    37
圖 22. Tacrolimus藥物濃度檢量線。    39
圖 23. 25 wt% mPEG-PLCL體外降解之(A)重量損失率; (B)分子量變化率。    40
圖 24. Tacrolimus於25 wt% mPEG-PLCL體外藥物釋放測試 (A)藥物釋放量; (B)藥物隨天數之累積量。    42
圖 25. 以不同配方之水膠進行MTT assay生物毒性測試於 (A) Day1; (B) Day3。    44
圖 26. mPEG-PLCL 水膠包覆藥物定點注射延長了LEW大鼠的同種異體皮膚移植。(A) 移植物的存活率; (B) 皮膚通種異體移植物和組織周圍水膠巨觀與微觀圖。黑色箭頭表示同種皮膚的位置。白色和紅色箭頭表示 POD 30的水膠殘留。放大倍率為100倍，比例尺: 200 µm。(C) 外周血(CD4 T cells、CD8 T cells以及Treg cells) 定量分析。Naïve 和 Untreatment分別表示未受移植接受移植但未接收水膠注射給藥的對照組。    46

表 1. mPEG-PLCL氫原子訊號對照表。    33
表 2. mPEG-PLCL共聚物分子量表(g/mol)。    34
表 3. 以不同溶劑25 wt%之水膠並用於包覆FK506之包覆效率。    38
表 4. 血液中他克莫司脂濃度: 體內藥物來自於含有10 mg/mL 之他克莫司包覆於水膠之釋放。    45


                                

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