臺灣博碩士論文加值系統

本研究主要是以熱感應性的Poly(N-isopropylacrylamide-co-N-methylol acrylamide (poly(NIPAAm-NMA))高分子材料及魚鱗胜肽做為材料，並使用特殊的紡絲技術將此二種材料製備成電紡纖維，在此研究中，我們改變紡絲條件以及進料的成分比率，探討不同的實驗變因對於電紡纖維形態結構的影響，此外，而為了使電紡纖維的結構穩定，因而在此研究中，利用熱烘的方式將熱感應性的poly(NIPAAm-NMA)高分子材料交聯固定，此外並分別使用戊二醛與1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) 對電紡纖維中的胜肽結構進行交聯固定，而後將具交聯結構的電紡纖維進行耐水性實驗，藉由耐水性實驗來評估戊二醛與EDC穩定胜肽結構的效率。接著我們以電紡纖維做為載體，分別以直接紡絲以及含浸載負藥物的方式將藥物載入電紡纖維中，並觀察不同的載藥方式對於藥物釋放速率的影響。

In this study, the thermo-sensitive Poly(N-isopropylacrylamide-co-N-methylol acrylamide (poly(NIPAAm-NMA)) and fish peptide were used as materials to manufacture the electrospun fibers by the method of electrospinning. The effects of various variables on the morphology of electrospun fibers were investigated. In order to stabilize the morphology of the electrospun fibers, the poly(NIPAAm) segments were crosslinked by heating process, and the glutaraldehyde and1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were used to crosslinked the peptide segments respectively. Moreover, the electrospun fibers were used as drug carriers to load with the model drug, caffeine. The caffeine was loaded into the electrospun fibers by the method of swelling process and electrospinning process respectively. The effect of loading methods on the caffeine release rate was investigated.

目錄

中文摘要………………………………………………………………… Ι
英文摘要………………………………………………………………… Ⅱ
目錄……………………………………………………………………… Ⅲ
圖目錄…………………………………………………………………… Ⅵ
表目錄…………………………………………………………………… Ⅷ
一、緒論…………………………………………………………………… 1
1.1 前言……………………………………………………………… 1
1.2 研究動機與目的………………………………………………… 1
二、文獻回顧……………………………………………………………… 2
2.1 熱感應高分子 聚(氮-異丙基丙烯醯胺) ( polyN-isopropylacrylamide ) 介紹 2
2.2 電紡絲原理……………………………………………………… 3
2.3 影響電紡絲之因素……………………………………………… 3
(1) 製程條件…………………………………………………… 3
(2) 溶液特性…………………………………………………… 3
2.4 蛋白質交聯方法………………………………………………… 5
2.4.1 戊二醛………………………………………………………… 8
2.4.2 EDC交聯反應機制……………………………………………… 11
三、實驗…………………………………………………………………… 12
3.1 實驗藥品………………………………………………………… 12
3.2 實驗儀器………………………………………………………… 15
3.3 實驗方法………………………………………………………… 17
3.3.1 實驗架構……………………………………………………… 17
3.3.2 poly(NIPPAm-co-NMA)合成方法……………………………… 18
3.3.3電紡纖維之製備………………………………………………… 18
3.3.4 熱烘交聯處理………………………………………………… 20
3.3.5 戊二醛交聯處理……………………………………………… 20
3.3.6 EDC胜肽交聯反應……………………………………………… 22
3.3.7 電紡纖維耐水試驗…………………………………………… 22
3.3.8 Bradford protein–binding assay蛋白質定量…………………… 22
3.3.9 SDS 聚丙烯醯胺膠電泳法分析……………………………… 23
3.3.9-1 材料…………………………………………………………… 23
3.3.9-2 檢測方法……………………………………………………… 23
3.3.10 控制釋放……………………………………………………… 25
四、結果討論……………………………………………………………… 27
4.1 電紡纖維的型態結構…………………………………………… 27
4.1.1 胜肽含量對電紡纖維外觀型態的影響……………………… 27
4.1.2 改變溶劑的比例對電紡纖維外觀型態的影響……………… 29
4.1.3 固含量與溶劑含量不同對電紡纖維的影響………………… 33
4.2 SDS 聚丙烯醯胺膠電泳法分析（SDS-PAGE）…………………… 35
4.3 熱烘交聯處理…………………………………………………… 37
4.3.1 熱烘交聯反應時間對電紡纖維外觀型態的影響…………… 37
4.4 電紡纖維之耐水性……………………………………………… 46
4.4 以戊二醛進行胜肽交聯………………………………………… 49
4.4.1-1 戊二醛交聯反應時間對電紡纖維的影響…………………… 49
4.4.1-2 戊二醛交聯對耐水性的影響………………………………… 49
4.4.2-1 戊二醛蒸氣交聯反應方式對電紡纖維形態的影響………… 56
4.4.2-2 戊二醛濃度對於電紡纖維形態的影響……………………… 56
4.5 以EDC進行胜肽交聯…………………………………………… 59
4.6 電紡纖維中胜肽成分交聯效率檢測…………………………… 64
4.7 控制釋放………………………………………………………… 68
4.7.1 咖啡因在不同纖維比例、不同溫度及pH值釋放試驗………… 68
4.7.2 咖啡因在不同溫度下載入纖維……………………………… 82
五、結論…………………………………………………………………… 88
參考文獻…………………………………………………………… 90









圖目錄

圖一、電紡設備架設圖…………………………………………………… 4
圖二、蛋白質交聯反應…………………………………………………… 6
圖三、戊二醛作為初期的胜肽交聯……………………………………… 7
圖四、戊二醛在酸或鹼性的水溶液中本身聚合反應…………………… 9
圖五、戊二醛與蛋白質形成共價交聯可能的反應路徑………………… 10
圖六、EDC 在蛋白質交聯下的反應…………………………………… 11
圖七、實驗流程架構圖…………………………………………………… 17
圖八、戊二醛交聯處理架設圖…………………………………………… 21
圖九、SDS 聚丙烯醯胺膠進行胜肽電泳分析…………………………… 25
圖十、胜肽含量對電紡纖維外觀型態的影響…………………………… 28
圖十一、紡絲溶液分層的情形…………………………………………… 30
圖十二、電紡過程中針頭有塞住的情況………………………………… 30
圖十三、從SEM觀察發現纖維中有許多Bead(珠子)的型態…………… 32
圖十四、從SEM觀察發現纖維型態以不規則環狀四散………………… 32
圖十五、固含量與溶劑含量不同對電紡纖維的影響…………………… 34
圖十六、SDS 聚丙烯醯胺膠電泳法分析………………………………… 36
圖十七、熱烘交聯處理不同時間對奈米纖維的影響-1………………… 38
圖十八、熱烘交聯處理不同時間對奈米纖維的影響-2………………… 39
圖十九、熱烘交聯處理不同時間對奈米纖維的影響-3………………… 40
圖二十、熱烘交聯處理不同時間對奈米纖維的影響-4………………… 41
圖二十一、熱烘交聯處理不同時間對奈米纖維的影響-5……………… 42
圖二十二、熱烘交聯處理不同時間對奈米纖維的影響-6……………… 43
圖二十三、熱烘交聯處理不同時間對奈米纖維的影響-7……………… 44
圖二十四熱烘交聯處理不同時間對奈米纖維的影響-8………………… 45
圖二十五、電紡纖維之耐水性-1………………………………………… 47
圖二十六、電紡纖維之耐水性-2………………………………………… 48
圖二十七、戊二醛交聯處理不同時間對奈米纖維的影響-1…………… 50
圖二十八、戊二醛交聯處理不同時間對奈米纖維的影響-2…………… 51
圖二十九、戊二醛交聯處理不同時間對奈米纖維的影響-3…………… 52
圖三十、戊二醛交聯處理不同時間後浸泡1小時對奈米纖維的影響-1…… 53
圖三十一、戊二醛交聯處理不同時間後浸泡1小時對奈米纖維的影響-2… 54
圖三十二、戊二醛交聯處理不同時間後浸泡1小時對奈米纖維的影響-3… 55
圖三十三、奈米纖維在戊二醛交聯處理擺放位置的影響……………… 57
圖三十四、戊二醛濃度在交聯處理的影響……………………………… 58
圖三十五、電紡纖維中EDC含量佔胜肽含量的影響…………………… 60
圖三十六、電紡纖維中EDC含量佔胜肽含量的耐水試驗-1…………… 61
圖三十七、電紡纖維中EDC含量佔胜肽含量的耐水試驗-2…………… 62
圖三十八、電紡纖維的耐水試驗及蛋白質染色………………………… 63
圖三十九、電紡纖維中胜肽成分交聯效率檢測-1……………………… 66
圖四十、電紡纖維中胜肽成分交聯效率檢測-2………………………… 66
圖四十一、咖啡因在不同纖維比例、不同pH值釋放試驗-1…………… 71
圖四十二、咖啡因在不同纖維比例、不同溫度釋放試驗-1……………… 72
圖四十三、咖啡因在不同纖維比例、不同溫度及pH值釋放試驗-1…… 73
圖四十四、咖啡因在不同纖維比例、不同pH值釋放試驗-2…………… 77
圖四十五、咖啡因在不同纖維比例、不同溫度釋放試驗-2…………… 78
圖四十六、咖啡因在不同纖維比例、不同溫度及pH值釋放試驗-2…… 79
圖四十七、咖啡因在不同纖維比例、不同pH值釋放試驗-3…………… 80
圖四十八、咖啡因在不同纖維比例、不同溫度釋放試驗-3…………… 81
圖四十九、含浸載負藥物方式的載藥實驗……………………………… 85
圖五十、含浸載負藥物方式的載藥實驗………………………………… 85
圖五十一、載藥方式不同在不同溫度比較累計釋放率-1……………… 87
圖五十一、載藥方式不同在不同溫度比較累計釋放率-2……………… 88
圖五十一、載藥方式不同在不同溫度比較累計釋放率-3……………… 89




表目錄
表一、紡絲溶液之配製組成……………………………………………… 19
表二、以胜肽所配製之標準溶液………………………………………… 22
表三、胜肽含量對於電紡纖維外觀型態的影響………………………… 27
表四、電紡纖維中胜肽成分交聯效率檢測……………………………… 65
表五、咖啡因在不同纖維比例、不同溫度及pH值釋放試驗…………… 70
表六、咖啡因在不同纖維比例、不同溫度及pH值釋放試驗…………… 71
表七、咖啡因在不同纖維比例、不同溫度及pH值釋放試驗…………… 75
表八、咖啡因在不同纖維比例、不同溫度及pH值釋放試驗…………… 76
表九、含浸載負藥物的方式將藥物載入電紡纖維……………………… 83
表十、含浸載負藥物的方式進行藥物釋放試驗………………………… 85

1.Fu-Yin Hsu, Yi-Sheng Hung, Hau-Min Liou, Chia-Hao Shen, “Electrospun hyaluronate–collagen nanofibrous matrix and the effects of varying the concentration of hyaluronate on the characteristics of foreskin fibroblast cells”, Acta Biomaterialia Vol.6, 2140-2147, 2010
2.Jayarama Reddy Venugopal, Yanzhong Zhang, Seeram Ramakrishna, “In Vitro Culture of Human Dermal Fibroblasts on Electrospun Polycaprolactone Collagen Nanofibrous Membrane”, Artificial Organs, Vol.30(6), 440-446
3.In-Sung Yeo, Ju-Eun Oh, Lim Jeong, Taek Seung Lee, Seung Jin Lee, Won Ho Park, “Byung-Moo Min Collagen-Based Biomimetic Nanofibrous Scaffolds: Preparation and Characterization of Collagen/Silk Fibroin Bicomponent Nanofibrous Structures”, Biomacromolecules, Vol.9 (4), 1106-1116, 2008
4.Yuanyuan Duan, Zhongyi Wang, Wei Yan, Shaohai Wang, Shaofeng Zhang, Jun Jia, “Preparation of collagen-coated electrospun nanofibers by remote plasma treatment and their biological properties”, Journal of Biomaterials Science, Vol.18(9), 1153-1164, 2007
5.Ramasamy Sripriya, Muthusamy Senthil Kumar, Praveen Kumar Sehgal, “Improved collagen bilayer dressing for the controlled release of drugs, Journal of Biomedical Materials Research Part B: Applied Biomaterials”, Vol.70(2), 389-396, 2004
6.Wan Meng, Se-Yong Kim, Jiang Yuan, Jung Chul Kim,Oh Hyeong Kwon, Naoki Kawazoe, Guoping Chen,Yoshihiro Ito, Inn-Kyu Kang, “Electrospun PHBV/collagen composite nanofibrous scaffolds for tissue engineering”, Journal of Biomaterials Science, Polymer Edition, Vol.18(1), 81-94, 2007
7.Falguni Patia, Basudam Adhikarib, “Santanu Dharaa,Isolation and characterization of fish scale collagen of higher thermal stability”, Bioresource Technology, Vol.101(10), 3737-3742, 2010
8.Catarina Pinto Reis, Ronald J. Neufeld, António J. Ribeiro, Francisco Veiga, “Nanoencapsulation II. Biomedical applications and current status of peptide and protein nanoparticulate delivery systems, Nanomedicine: Nanotechnology, Biology and Medicine”, Vol.2(2), 53-65, 2006
9.R.A. Thakur, C.A. Florek, J. Kohn, B.B. Michniak, “Electrospun nanofibrous polymeric scaffold with targeted drug release profiles for potential application as wound dressing”, International Journal of Pharmaceutics, Vol.364, 87-93, 2008
10.Wang Y, Zhang CL, Zhang Q, Li P. “Composite electrospun nanomembranes of fish scale collagen peptides/chito-oligosaccharides: antibacterial properties and potential for wound dressing”, International Journal of Nanomedicine, Vol.6, 667-676, 2011
11.Zeng-xiao Cai, Xiu-mei Mo, Kui-hua Zhang, Lin-peng Fan, An-lin Yin, Chuang-long He, Hong-sheng Wang, “Fabrication of Chitosan/Silk Fibroin Composite Nanofibers for Wound-dressing Applications”, Int. J. Mol. Sci., Vol.11, 3529-3539, 2010
12.Xin Liu, Tong Lin, Jian Fang, Gang Yao, Hongqiong Zhao, Michael Dodson, Xungai Wang, “In vivo wound healing and antibacterial performances of electrospun nanofibre membranes”, Journal of Biomedical Materials Research Part A, Vol.94(2), 499-508, 2010
13.E.J. Chong, T.T. Phan, I.J. Lim, Y.Z. Zhang, B.H. Bay, S. Ramakrishna, C.T. Lim, “Evaluation of electrospun PCL/gelatin nanofibrous scaffold for wound healing and layered dermal reconstitution”, Acta Biomaterialia, Vol.3(3), 321-330, 2007
14.Xin Liu, Tong Lin, Yuan Gao, Zhiguang Xu, Chen Huang, Gang Yao, Linlin Jiang, Yanwei Tang, Xungai Wang, “Antimicrobial electrospun nanofibers of cellulose acetate and polyester urethane composite for wound dressing”, Journal of Biomedical Materials Research Part B: Applied Biomaterials, Vol.100(6), 1556-1565, 2012
15.Jiantao Lin, Caihong Li, Yi Zhao, Jianchan Hu, Li-Ming Zhang, Co-electrospun “Nanofibrous Membranes of Collagen and Zein for Wound Healing”, ACS Appl. Mater. Interfaces, Vol.4 (2), 1050-1057, 2012
16.Kyong Su Rhoa, Lim Jeongb, Gene Leea, Byoung-Moo Seod, Yoon Jeong Parka, Seong-Doo Honge,Sangho Roha, Jae Jin Choa, Won Ho Parkb, Byung-Moo Min, “Electrospinning of collagen nanofibers: Effects on the behavior of normal human keratinocytes and early-stage wound healing”, Biomaterials, Vol.27(8), 1452-1461, 2006
17.Christine Cochrane, Mark G. Rippon, Alan Rogers, Rob Walmsley, Derek Knottenbelt, Phil Bowler, “Application of an in vitro model to evaluate bioadhesion of fibroblasts and epithelial cells to two different dressings”, Biomaterials, Vol.20(13), 1237-1244, 1999
18.邱文英, “溫度感應型磁性乳膠顆粒之製備與研究”, 台灣大學, 博士論文 (2004)
19.林建中, “應用高分子化學”, 高立圖書出版, 1982
20.何信法, “以電紡法製造電紡纖維及其應用”, 台南科技大學, 碩士論文 (2007)
21.Reza Lalani, Lingyun Liu, “Electrospun Zwitterionic Poly(Sulfobetaine Methacrylate) for Nonadherent, Superabsorbent, and Antimicrobial Wound Dressing Applications”, Biomacromolecules, Vol.13(6), 1853-1863, 2012
22.Milena Ignatova, Nevena Manolova, Nadya Markova, Iliya Rashkov, “Electrospun Non-Woven Nanofibrous Hybrid Mats Based on Chitosan and PLA for Wound Dressing Applications”, Macromol. Biosci, Vol.9, 102-111, 2009
23.Ko Eun Park, Hyun Ki Kang, Seung Jin Lee, Byung-Moo Min, Won Ho Park, “Biomimetic Nanofibrous Scaffolds:  Preparation and Characterization of PGA/Chitin Blend Nanofibers”, Biomacromolecules, Vol.7(2), 635-643, 2006
24.Hyung Kil Noh, Sung Won Lee, Jin-Man Kim, Ju-Eun Oh, Kyung-Hwa Kim, Chong-Pyoung Chung, Soon-Chul Choi, Won Ho Park, Byung-Moo Min, “Electrospinning of chitin nanofibers: Degradation behavior and cellular response to normal human keratinocytes and fibroblasts”, H.K. Noh et al. / Biomaterials, Vol.27, 3934-3944, 2006
25.Shaoping Zhong, Wee Eong Teo, Xiao Zhu, Roger W. Beuerman, Seeram Ramakrishna, Lin Yue Lanry Yung, “An aligned nanofibrous collagen scaffold by electrospinning and its effects on in vitrofibroblast culture”, Journal of Biomedical Materials Research Part A, Vol.79(3), 456-463, 2006
26.Jolanta Janiszewska, Joanna Swieton, Andrzej W. Lipkowsk, Zofia Urbanczyk-Lipkowska, “Low molecular mass peptide dendrimers that express antimicrobial properties”, Bioorganic & Medicinal Chemistry Letters, Vol.13(21), 3711-3713, 2003
27.Greg T. H., “Bioconjugate techniques Bioconjugate Techniques (Second Edition) ”, Academic press, 2008
28.Nicholas Light, Allen J. Bailey, “Collagen cross-links: location of pyri~inolin~ in type I collagen”, FEBS, Vol.182(2), 503-508, 1985
29.Andrea Heinz , Christoph K.H. Ruttkies, Günther Jahreis, Christoph U. Schräder , Kanin Wichapong Wolfgang Sippl , Fred W. Keeley , Reinhard H.H. Neubert , Christian E.H. “Schmelzer, In vitro cross-linking of elastin peptides and molecular characterization of the resultant biomaterials”, Biochimica et Biophysica Acta, Vol.1830, 2994-3004, 2013
30.Paul Bornstein, “Covalent cross-links in collagen: a personal account of their discovery”, Matrix Biology, Vol.22, 385-391, 2003
31.Samuel B. Adeloju, Abdulazeez T. Lawal, “Fabrication of a bilayer potentiometric phosphate biosensor by cross-link immobilization with bovine serum albumin and glutaraldehyde”, Analytica Chimica Acta, Vol.691, 89-94, 2011
32.Myung Chul Chang, Junzo Tanaka, “FT-IR study for hydroxyapatite/collagen nanocomposite cross-linked by glutaraldehyde”, Biomaterials, Vol.23, 4811-4818, 2002
33.Katsumi Mera, Mime Nagai, Jonathan W.C. Brock, Yukio Fujiwara, Toshinori Murata, Toru Maruyama, John W. Baynes, Masaki Otagiri, Ryoji Nagai, “Glutaraldehyde is an effective cross-linker for production of antibodies against advanced glycation end-products”, Journal of Immunological Methods, Vol.334, 82-90, 2008
34.Netty Zegers, Koen Gerritse, Carla Deen, Wim Boersma and Eric Claassen, “An improved conjugation method for controlled covalent coupling of synthetic peptides to proteins using glutaraldehyde in a dialysis method”, Journal of Immunological Methods, Vol.130, 195-200, 1990
35.Dhan B. Khadka, Michael C. Cross, Donald T. Haynie, “A Synthetic Poly peptide Electrospun Biomaterial”, ACS Appl. Mater. Interfaces, Vol.3, 2994-3001, 2011
36.Thanavel Rajangam, Hyun-Jong Paik, Seong Soo A. An, “Fabricating Fibrinogen Microfibers with Aligned Nanostructure, as Biodegradable Threads for Tissue Engineering”, Bull. Korean Chem. Soc, Vol.33(6), 2075-2078, 2012
37.Jui-Yang Lai, Ya-Ting Li, “Functional Assessment of Cross-Linked Porous Gelatin Hydrogels for Bioengineered Cell Sheet Carriers”, Biomacromolecules, Vol.11, 1387-1397, 2010
38.Dhan B. Khadka, MS, Donald T. Haynie, PhD, “Protein- and peptide-based electrospun nanofibers in medical biomaterials, Nanomedicine: Nanotechnology”, Biology, and Medicine, Vol.8, 1242-1262, 2012
39.Tsaffrir Zor, Zvi Selinger, “Linearization of the Bradford Protein Assay Increases Its Sensitivity: Theoretical and Experimental Studies”, Analytical Biochemistry, Vol.236, 302-308, 1996
40.John P. Hachmann, Joseph W. Amshey, “Models of protein modification in Tris–glycine and neutral pH Bis–Tris gels during electrophoresis: Effect of gel pH”, Analytical Biochemistry, Vol.342, 237-245, 2005