溫度敏感型三嵌段胺基酸水膠的合成與其在胜肽藥物口服劑型上的應用

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研究生：	鄭宇恩 Cheng, Yu-En
論文名稱：	溫度敏感型三嵌段胺基酸水膠的合成與其在胜肽藥物口服劑型上的應用 Synthesis and Application of Thermo-Sensitive Triblock Polypeptide Hydrogel for Oral Delivery of Peptides
指導教授：	朱一民 Chu, I-Ming
口試委員:	王潔 Wang, Jane 胡尚秀 Hu, Shang-Hsiu 魏毓宏 Wei, Yu-Hong 駱俊良 Lo, Chun-Liang
學位類別：	博士 Doctor
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：	英文
論文頁數：	68
中文關鍵詞：	水膠、降鈣素、溫度敏感型、口服傳輸
外文關鍵詞：	hydrogel, calcitonin, temperature responses, oral delivery
相關次數：	點閱：4 下載：0
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本研究旨在對新設計的胜肽藥物口服給藥材料進行研究探討。甲氧基化聚乙二醇-聚左旋丙胺酸 (methoxy-poly(ethylene glycol)-poly(L-alanine)，mPEG-PA) 為一已知溫度敏感型胺基酸水膠，能隨溫度產生不同的相變化特性。這種特性適合用來包覆胜肽或蛋白質藥。為增加對腸粘液的粘附能力，將甲氧基化聚乙二醇-聚左旋丙胺酸於末端接上帶電荷的離胺酸 (L-lysine) 形成新型胺基酸水膠。這種帶正電的材料將有助於與黏膜上帶負電的黏蛋白結合。該合成首先透過開環聚合，分別合成甲氧基化聚乙二醇-聚左旋丙胺酸與不同長度聚左旋賴氨酸 (poly(L-lysine)，PLL)。然後，甲氧基化聚乙二醇-聚左旋丙胺酸和聚左旋賴氨酸使用NHS酯反應合成三嵌段共聚物 (mPEG-PA-PLL)。
此研究調查了在不同pH值下降鈣素的釋放以及此三嵌段共聚物形成的水凝膠的生物相容性。細胞培養3天後，mPEG-PA-PLL4和mPEG-PA-PLL14的生物相容性分別約為79%和84%。於0.1N HCl溶液中，降鈣素在mPEG-PA-PLL4和mPEG-PA-PLL14前半小時的藥物釋放，具有爆發效應，其數值分別為14.5±4.8%和20.7±1.7%。而在pH 6.8的緩衝溶液中，mPEG-PA-PLL4和 mPEG-PA-PLL14在24小時內，從水凝膠中釋放的降鈣素則分別為65.9±18.5%和100.3±12.1%。這些研究數據顯示，此新設計的材料具有可成為口服胜肽藥物載體的良好潛力。

This work aims to conduct newly design material for oral delivery of peptide drugs. Methoxy poly(ethylene glycol)-poly(L-alanine) (mPEG-PA) is a thermo-sensitive hydrogel exhibiting a sol-to-gel phase transition property. This characteristic is appropriate for encapsulating peptide or protein drugs. To enhance the adhesion ability to intestinal mucus, a thermo-sensitive polymer, mPEG-PA, modified with charged amino acid lysine was developed. This positively charged material would help to bind the negative charged mucin on the mucus. The synthesis was conducted by individually synthesizing mPEG-PA and poly(L-lysine) (PLL) of different lengths via ring-opening polymerization. Then, mPEG-PA and PLL were combined using NHS ester reaction to synthesize the triblock copolymer (mPEG-PA-PLL).
This study investigated the release profile of calcitonin at different pH values and the biocompatibility of the synthesized triblock copolymer hydrogels. After 3 days of incubation, the biocompatibilities were approximately 79% and 84% for mPEG-PA-PLL4 and mPEG-PA-PLL14, respectively. In 0.1N HCl solution, the release of calcitonin had burst effect within the first half hours and were 14.5±4.8% and 20.7±1.7% for mPEG-PA-PLL4 and mPEG-PA-PLL14, respectively. In the pH 6.8 buffer solution, calcitonin released from the hydrogels with 24 hours were 65.9±18.5% and 100.3±12.1% for mPEG-PA-PLL4 and mPEG-PA-PLL14, respectively. The initial data showed the newly design material has good potential for oral delivery of peptide drugs.

摘要-------------------------------------------------------------iv
Abstract---------------------------------------------------------v
誌謝辭------------------------------------------------------------vi
Table of Contents------------------------------------------------vii
List of Figures--------------------------------------------------x
List of Tables---------------------------------------------------xii
CHAPTER 1 Introduction-------------------------------------------1
1.    Background-----------------------------------------------2
2.    Barriers to oral delivery--------------------------------5
2.1.    Biochemical barriers-----------------------------------6
2.1.1.    Enzymatic barriers-----------------------------------6
2.1.2.    pH gradient------------------------------------------7
2.2.    Physiological barriers---------------------------------7
2.2.1.    Intestinal mucosa barrier----------------------------7
2.3.    Cellular barriers--------------------------------------8
3.    Objectives-----------------------------------------------9
CHAPTER 2 State-of-The-Art: A Survey-----------------------------11
1.    General review-------------------------------------------12
2.    The strategies to oral peptide delivery------------------13
3.    Stimuli Sensitive/Responsive Hydrogels-------------------18
4.    Thermo-Sensitive Hydrogels-------------------------------19
5.    pH Sensitive Hydrogels-----------------------------------20
6.    Mucoadhesive Theories------------------------------------22
7.    Calcitonin-----------------------------------------------23
8.    The models of drug release-------------------------------24
CHAPTER 3 Experimental Methods-----------------------------------28
1.    Summary--------------------------------------------------29
2.    Flow chart-----------------------------------------------29
3.    Materials------------------------------------------------29
4.    End group modification of mPEG---------------------------31
5.    Synthesis of L-alanine N-carboxyanhydride (NCA-Ala) and Nepsilon-Carbobenzyloxy-L-lysine (Cbz-L-Lysine) carboxyanhydride (NCA-Lys(Z))----------------------------------------------------------31
6.    Synthesis of methoxy poly(ethylene glycol)-poly(D-alanine) (mPEG-PA) and poly(L-lysine(Z)) (PLL(Z))-------------------------32
7.    End group modification poly(L-lysine(Z)) (PLL(Z))--------33
8.    Synthesis of mPEG-PA-PLL triblock copolymer--------------33
9.    Characterization of mPEG-PA-PLL Copolymers and Polypeptide Hydrogels--------------------------------------------------------34
9.1.    Fourier-Transformed Infrared Spectroscopy (FT-IR)------34
9.2.    1H Nuclear Magnetic Resonance (1H-NMR) Spectroscopy----34
9.3.    MALDI-TOF Mass Spectroscopy----------------------------34
9.4.    Sol–Gel Phase Transition and Rheological properties----35
9.5.    Scanning Electron Microscopy (SEM)---------------------36
10.    In-vitro Degradation Testing of mPEG-PA-PLL-------------36
11.    Biocompatibility----------------------------------------37
12.    Methodological study of HPLC----------------------------37
13.    Encapsulation efficiency, loading content, and in-vitro drug-release studies--------------------------------------------------38
Chapter 4 Results and Discussions--------------------------------40
1.    The Synthesis and Characterization of mPEG-PA-PLL triblock copolymer--------------------------------------------------------41
2.    Sol–Gel Phase Transition and Rheological Properties------48
3.    SEM observation------------------------------------------50
4.    Biocompatibility Testing---------------------------------50
5.    In-vitro biodegradability--------------------------------52
6.    Encapsulation efficiency, loading content, and In-vitro drug-release studies--------------------------------------------------53
Chapter 5 Conclusions and future work----------------------------59
1.    Conclusions----------------------------------------------60
2.    Future Work----------------------------------------------61
References-------------------------------------------------------63
                                

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