臺灣博碩士論文加值系統

現今生醫材料中已發展出許多具抗菌能力的敷料，目的是對傷口處之細菌進行抑制以防止感染。例如含奈米銀的敷材雖能快速產生殺菌作用，但奈米銀顆粒也會隨擴散而消耗；本論文之研究方向為製備不具擴散能力的兩類抗菌材料並經CFU試驗檢測抗菌能力:第一類是製備帶胺基的幾丁聚醣與聚乙烯醇混合之電紡絲膜(PVA/CS)，其對大腸桿菌有優異的抗菌效果；透過與戊二醛及多巴胺進行表面修飾後可將酵素lysostaphin固定化於材料，其對金黃色葡萄球菌有優異的抗菌能力。第二類材料是利用水溶之抗菌胜肽ε-Polylysine與陰離子表面活性劑AOT結合，製備不溶水之複合物(AP)。以二甲基甲醯胺為溶劑溶解AP可與溶於二甲基甲醯胺之聚丙烯腈溶液混合，經電紡絲得具抗菌功能之非水溶的耐水性電紡絲膜(PAN/AP)。此材料以CFU法檢測抗菌效果發現，對大腸桿菌的抗菌能力可達與聚乙烯醇電紡絲膜接近的80 %的抗菌效果，而對金黃色葡萄球菌的抗菌能力則會比固定化酵素lysostaphin的75 %更好。此材料除了用CFU試驗檢測抗菌效果，也使用濁度試驗檢測確認金黃色葡萄球菌培養後濁度下降情形；接觸式殺菌試驗也展現材料對金黃色葡萄球菌優異的抗菌能力，顯示其對大腸桿菌以及金黃色葡萄球菌皆能作用。

Antimicrobial electrospun nanofibrous mats were prepared based on two kinds of biopolymers. Chitosan and hydrophobically modified chitosan (HMCS) are known for their antimicrobial activity, however, they can not be easily electrospun into nanofibers. Aqueous solution of polyvinylalcohol (PVA) was found to be easily electrospun into nanofibrous mat. By electrospinning chitosan (CS) mixed PVA solution (6 wt% PVA+ 1wt% CS) a nice nanofibrous mat (averaged fiber diameter ca 250 nm) could be obtained. The mat demonstrated very good antibacterial results against Escherichia coli. Epsilon-polylysine (PL), an antimicrobial agent has been approved to use in food industry, can easily form a water insoluble complex with anionic surfactant dioctyl sulfosuccinate (AOT). The AOT-PL (AP) complex is well dissolved in dimethylformamide and can be co-electrospun with polyacrylonitrile (PAN). The diameter of obtained electrospun fiber increases with the addition of AP from 800 nm for 33 wt% AP to 2.5 um for 67 wt% AP. The obtained AP mat is water durable. The antimicrobial activity test shows that bacteria could only be contact-killed by the AP mat. It has nearly 80 % antibacterial effect against Escherichia coli, and more than 75 % antibacterial activity against Staphylococcus aureus.

摘要 I
Abstract II
誌謝 III
縮寫表 IV
目錄 V
圖目錄 X
表目錄 XVIII
第一章 緒論 1
1.1 前言 1
1.2 研究內容簡介 2
第二章 文獻回顧 3
2.1 靜電紡絲 3
2.2 靜電紡絲幾丁聚醣 5
2.3 戊二醛 6
2.4 Lst酵素 7
2.5 疏水性修飾幾丁聚醣與胺基抗菌機制 7
2.6 靜電紡絲奈米網 8
2.7 靜電紡絲抗菌敷料 9
2.8 ε-Poly-lysine的抗菌應用 10
第三章 實驗材料與方法 12
3.1 實驗流程 12
3.2 實驗菌株、質體、藥品 13
3.3 實驗溶液 14
3.4 實驗設備 15
3.5 實驗方法:幾丁聚醣、疏水性幾丁聚醣、幾丁聚醣奈米網相關 17
3.5.1 聚乙烯醇與幾丁聚醣薄膜 17
3.5.2 聚乙烯醇與疏水修飾幾丁聚醣薄膜 17
3.5.3 戊二醛交聯與耐水性 17
3.5.4 產lysostaphin酵素 18
3.5.5 IMAC純化 18
3.5.6 測量蛋白質濃度、蛋白質電泳 19
3.5.7 測量蛋白質活性 20
3.5.8 PDA表面修飾及lysostaphin酵素固定化 20
3.5.9 醛基測量 21
3.5.10 聚乙烯醇與幾丁聚醣薄膜(奈米網) 21
3.6 材料性質鑑定: 幾丁聚醣、疏水性幾丁聚醣相關 22
3.6.1 傅立葉轉換紅外線光譜儀(FTIR/ATR) 22
3.6.2 熱重分析儀(TGA) 22
3.6.3 水接觸角(Water contact angle) 22
3.6.4 掃描式電子顯微鏡(SEM)觀察表面型態及厚度、元素分析 22
3.6.5 拉伸試驗 23
3.6.6 胺基測量 23
3.7 應用:抗菌評估 23
3.7.1 CFU(Colony Forming Unit)法 23
3.8 實驗方法: AOT-PL的製備與電紡絲含AOT-PL纖維相關 24
3.8.1 AOT-PL的製備、溶解度測試 24
3.8.2 PAN/AOT-PL靜電紡絲膜的製備 24
3.9 材料性質鑑定:電紡絲含AOT-PL纖維相關 24
3.9.1 表面積分析 24
3.10 應用:抗菌評估 25
3.10.1 CFU(Colony Forming Unit)法 25
3.10.2 濁度法 25
3.10.3 抑菌環寬 26
3.10.4 接觸殺菌 26
3.10.5 以生物膜的方式配合螢光顯微鏡測定抗菌效果 26
第四章 幾丁聚醣及疏水性幾丁聚醣電紡絲 27
4.1 聚乙烯醇與幾丁聚醣薄膜 27
4.2 聚乙烯醇與疏水修飾幾丁聚醣薄膜 30
4.3 戊二醛交聯與耐水性測試 31
4.4 聚乙烯醇與幾丁聚醣薄膜(奈米網) 33
4.5 傅立葉轉換紅外線光譜分析 35
4.6 熱重分析 36
4.7 水接觸角分析 39
4.8 膜厚與拉伸試驗 40
4.9 元素分析及胺基測量 41
4.10 薄膜抗菌評估 42
4.11 固定化酵素Lst抗菌評估 43
第五章 含AOT-PL電紡絲及其抗菌性 44
5.1 AOT-PL的溶解度測試 44
5.2 PAN與PAN/AOT-PL電紡絲 44
5.3 傅立葉轉換紅外線光譜分析 47
5.4 熱重分析 47
5.5 接觸角分析 49
5.6 膜厚與拉伸試驗 49
5.7 元素分析及胺基測量 50
5.8 表面積分析 51
5.9 PAN/AP抗菌評估 52
5.9.1 CFU法 52
5.9.2 濁度法 53
5.9.3 抑菌環寬與接觸殺菌法 55
5.9.4 以生物膜的方式配合螢光顯微鏡測定抗菌 56
第六章 結論 59
6.1 幾丁聚醣、疏水性幾丁聚醣關聯部分 59
6.2 含AOT-PL電紡絲及其抗菌性 60
第七章 參考文獻 61
第八章 附錄 66
8.1 聚乙烯醇與幾丁聚醣薄膜 66
8.1.1 不同電壓之6 wt% PVA/1 wt% CS(45%AA)薄膜SEM圖 66
8.1.2 不同距離之6 wt% PVA/1 wt% CS(45%AA)薄膜SEM圖 69
8.2 聚乙烯醇與疏水修飾幾丁聚醣薄膜 71
8.2.1 不同電壓之6 wt% PVA/0.75 wt% HMCS (45%AA)薄膜SEM圖 71
8.2.2 不同距離之6 wt% PVA/0.75 wt% HMCS (45%AA)薄膜SEM圖 74
8.3 聚乙烯醇與幾丁聚醣薄膜(奈米網) 76
8.3.1 不同流速之6 wt% PVA /1 wt% CS(70%AA)薄膜SEM圖 76
8.3.2 不同距離之6 wt% PVA /1 wt% CS(45%AA)薄膜SEM圖 76
8.3.3 不同距離之6 wt% PVA /1.5 wt% CS(70%AA)薄膜SEM圖 77
8.3.4 不同距離之8 wt% PVA/1 wt% CS (70%AA)薄膜SEM圖 78
8.3.5 不同距離之6 wt% PVA /1.5 wt% CS(45%AA)薄膜SEM圖 79
8.3.6 不同距離之6 wt% PVA /2 wt% CS(45%AA)薄膜SEM圖 80
8.3.7 不同距離之4 wt% PVA/1.5 wt% CS(70%AA)薄膜SEM圖 80
8.3.8 不同距離之4 wt% PVA/2 wt% CS(70%AA)薄膜SEM圖 81
8.3.9 不同距離之4 wt% PVA/2 wt% CS(45%AA)薄膜SEM圖 82
8.4 膜厚:幾丁聚醣、疏水性幾丁聚醣關聯部分 82
8.5 對大腸桿菌抗菌:幾丁聚醣、疏水性幾丁聚醣關聯部分 83
8.6 對金黃色葡萄球菌的抗菌:幾丁聚醣、疏水性幾丁聚醣關聯部分 84
8.7 膜厚:電紡絲含AOT-PL的耐水性纖維 85
8.8 對大腸桿菌抗菌:電紡絲含AOT-PL的耐水性纖維 85
8.9 對金黃色葡萄球菌的抗菌:電紡絲含AOT-PL的耐水性纖維 86

1. Jia, Y.-T., et al., Fabrication and characterization of poly (vinyl alcohol)/chitosan blend nanofibers produced by electrospinning method. Carbohydrate Polymers, 2007. 67(3): p. 403-409.
2. Jaworek, A. and A.T. Sobczyk, Electrospraying route to nanotechnology: An overview. Journal of Electrostatics, 2008. 66(3-4): p. 197-219.
3. Buruaga, L., et al., Electrospinning of waterborne polyurethanes. Journal of Applied Polymer Science, 2010. 115(2): p. 1176-1179.
4. Bhardwaj, N. and S.C. Kundu, Electrospinning: a fascinating fiber fabrication technique. Biotechnol Adv, 2010. 28(3): p. 325-47.
5. JAWOREK A., K.A., MAIN MODES OF ELECTROHYDRODYNAMIC SPRAYING OF LIQUIDS. 1998.
6. Ridolfi, D.M., et al., Electrospun poly(ethylene oxide)_chitosan nanofibers with cellulose nanocrystals as support for cell culture of 3T3 fibroblasts. 2017.
7. Fong, H.C., I. & Reneker, D.H., Beaded nanofibers formed during electrospinning. Polymer, 1998: p. 4585-4592.
8. Karakaş, H., ELECTROSPINNING OF NANOFIBERS AND THEIR APPLICATIONS. 2015.
9. Zhu, L., K. Liang, and Y. Ji, Prominent reinforcing effect of chitin nanocrystals on electrospun polydioxanone nanocomposite fiber mats. J Mech Behav Biomed Mater, 2015. 44: p. 35-42.
10. Chen, C.K. and S.C. Huang, Preparation of Reductant-Responsive N-Maleoyl-Functional Chitosan/Poly(vinyl alcohol) Nanofibers for Drug Delivery. Mol Pharm, 2016. 13(12): p. 4152-4167.
11. Huang, X.-J., D. Ge, and Z.-K. Xu, Preparation and characterization of stable chitosan nanofibrous membrane for lipase immobilization. European Polymer Journal, 2007. 43(9): p. 3710-3718.
12. Liu, Y., S. Wang, and W. Lan, Fabrication of antibacterial chitosan-PVA blended film using electrospray technique for food packaging applications. Int J Biol Macromol, 2018. 107(Pt A): p. 848-854.
13. Qin, X., et al., Characterization of poly (vinyl alcohol) nanofiber mats cross-linked with glutaraldehyde. Journal of Industrial Textiles, 2012. 43(1): p. 34-44.
14. Geng, X., O.H. Kwon, and J. Jang, Electrospinning of chitosan dissolved in concentrated acetic acid solution. Biomaterials, 2005. 26(27): p. 5427-32.
15. Wang, Y. and Y.-L. Hsieh, Crosslinking of polyvinyl alcohol (PVA) fibrous membranes with glutaraldehyde and PEG diacylchloride. Journal of Applied Polymer Science, 2010: p. NA-NA.
16. Cheng, P.-I., et al., High permselectivity of networked PVA/GA/CS-Ag+-membrane for dehydration of Isopropanol. Journal of Membrane Science, 2018. 564: p. 926-934.
17. Adriano, W.S., et al., STABILIZATION OF PENICILLIN G ACYLASE BY IMMOBILIZATION ON GLUTARALDEHYDE-ACTIVATED CHITOSAN. 2005.
18. Ma, L., Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering. Biomaterials, 2003. 24(26): p. 4833-4841.
19. Nithya, S., et al., Preparation, characterization and efficacy of lysostaphin-chitosan gel against Staphylococcus aureus. Int J Biol Macromol, 2018. 110: p. 157-166.
20. Chandra Ojha, S., et al., Purification and characterization of the antibacterial peptidase lysostaphin from Staphylococcus simulans: Adverse influence of Zn(2+) on bacteriolytic activity. Protein Expr Purif, 2018. 151: p. 106-112.
21. Wertheim, H.F.L., et al., The role of nasal carriage in Staphylococcus aureus infections. The Lancet Infectious Diseases, 2005. 5(12): p. 751-762.
22. Brown, A.F., et al., Staphylococcus aureus Colonization: Modulation of Host Immune Response and Impact on Human Vaccine Design. Front Immunol, 2014. 4: p. 507.
23. Le, K.Y., et al., Molecular determinants of staphylococcal biofilm dispersal and structuring. Front Cell Infect Microbiol, 2014. 4: p. 167.
24. Warfield, R., et al., Internally quenched peptides for the study of lysostaphin: An antimicrobial protease that kills Staphylococcus aureus. Org Biomol Chem, 2006. 4(19): p. 3626-38.
25. CHEN-GUANG LIU, et al., Linolenic Acid-Modified Chitosan for Formation of Self-Assembled Nanoparticles. J. Agric. Food Chem, 2005.
26. K. Mouryaa, V., N. N. Inamdara, and A. Tiwari, Carboxymethyl Chitosan And Its Applications. Advanced Materials Letters, 2010. 1(1): p. 11-33.
27. Zhu, A., et al., The aggregation behavior of O-carboxymethylchitosan in dilute aqueous solution. Colloids Surf B Biointerfaces, 2005. 43(3-4): p. 143-9.
28. Javvaji, V., et al., Reversible gelation of cells using self-assembling hydrophobically-modified biopolymers: towards self-assembly of tissue. Biomaterials Science, 2014. 2(7): p. 1016.
29. Vo, D.-T., C.G. Whiteley, and C.-K. Lee, Hydrophobically Modified Chitosan-Grafted Magnetic Nanoparticles for Bacteria Removal. Industrial & Engineering Chemistry Research, 2015. 54(38): p. 9270-9277.
30. Joerg C. Tiller*, C.-J.L., Kim Lewis†, and Alexander M. Klibanov, Designing surfaces that kill bacteria on contact. PNAS, 2001: p. 5981-5985.
31. Kong, M., et al., Antibacterial mechanism of chitosan microspheres in a solid dispersing system against E. coli. Colloids Surf B Biointerfaces, 2008. 65(2): p. 197-202.
32. Tan, H., et al., Quaternized chitosan as an antimicrobial agent: antimicrobial activity, mechanism of action and biomedical applications in orthopedics. Int J Mol Sci, 2013. 14(1): p. 1854-69.
33. Vo, D.T. and C.K. Lee, Cells capture and antimicrobial effect of hydrophobically modified chitosan coating on Escherichia coli. Carbohydr Polym, 2017. 164: p. 109-117.
34. Adina MATICA, G.M., Vasile OSTAFE, ANTIBACTERIAL PROPERTIES OF CHITIN AND CHITOSANS. 2017: p. pp. 39-54.
35. Thomas D. Brock, M.T.M., 微生物生物學. 1995.
36. I.M. Helandera, E.-L.N.-L., R. Ahvenainena, J. Rhoadesc, S. Roller, Chitosan disrupts the barrier properties of the outer membrane ofGram-negative bacteria. 2001: p. 235–244.
37. Ding, B., et al., Formation of novel 2D polymer nanowebs via electrospinning. Nanotechnology, 2006. 17(15): p. 3685-3691.
38. Bin Ding, M.W., Xianfeng Wang, Jianyong Yub, Gang Suna, Electrospun nanomaterials for ultrasensitive sensors. 2010.
39. Zhang, S., et al., Model derivation and validation for 2D polymeric nanonets: Origin, evolution, and regulation. Polymer, 2015. 74: p. 182-192.
40. Yu, H., et al., Fabrication of polylysine based antibacterial coating for catheters by facile electrostatic interaction. Chemical Engineering Journal, 2019. 360: p. 1030-1041.
41. Hong, K.H., Preparation and properties of electrospun poly(vinyl alcohol)/silver fiber web as wound dressings. Polymer Engineering & Science, 2007. 47(1): p. 43-49.
42. Agnė Matusevičiūtė, et al., Formation of PVA Nanofibres with Iodine
by Electrospinning. FIBRES & TEXTILES in Eastern Europe, 2012.
43. Ushimaru, K., Y. Hamano, and H. Katano, Antimicrobial Activity of epsilon-Poly-l-lysine after Forming a Water-Insoluble Complex with an Anionic Surfactant. Biomacromolecules, 2017. 18(4): p. 1387-1392.
44. Amariei, G., et al., Biocompatible antimicrobial electrospun nanofibers functionalized with epsilon-poly-l-lysine. Int J Pharm, 2018. 553(1-2): p. 141-148.
45. Liu, H., et al., The antimicrobial effects and synergistic antibacterial mechanism of the combination of ε-Polylysine and nisin against Bacillus subtilis. Food Control, 2015. 47: p. 444-450.
46. Cui, L., et al., Facilitation of α-polylysine in TGase-mediated crosslinking modification for gluten and its effect on properties of gluten films. Journal of Cereal Science, 2017. 73: p. 108-115.
47. Dai, X., et al., Antibacterial amphiphiles based on ε-polylysine: synthesis, mechanism of action, and cytotoxicity. RSC Advances, 2015. 5(85): p. 69325-69333.
48. 陳冠蓉, 基因重組融合酵素之表達生產以有效去除葡萄球菌生物膜. 2017.
49. Ball, V., Polydopamine films and particles with catalytic activity. Catalysis Today, 2018. 301: p. 196-203.
50. Yang, J.M., et al., Evaluation of membrane preparation method on the performance of alkaline polymer electrolyte: Comparison between poly(vinyl alcohol)/chitosan blended membrane and poly(vinyl alcohol)/chitosan electrospun nanofiber composite membranes. Electrochimica Acta, 2018. 266: p. 332-340.
51. Alavarse, A.C., et al., Tetracycline hydrochloride-loaded electrospun nanofibers mats based on PVA and chitosan for wound dressing. Mater Sci Eng C Mater Biol Appl, 2017. 77: p. 271-281.
52. Palla, M.S., et al., Isolation and molecular characterization of antifungal metabolite producing actinomycete from mangrove soil. Beni-Suef University Journal of Basic and Applied Sciences, 2018. 7(2): p. 250-256.
53. Jin, S.Y., et al., Effect of alkaline hydrolysis on cyclization reaction of PAN nanofibers. Materials & Design, 2017. 124: p. 69-77.
54. Li, X., et al., Fabrication of Polyacrylonitrile/polypyrrole (PAN/Ppy) composite nanofibres and nanospheres with core–shell structures by electrospinning. Materials Letters, 2008. 62(8-9): p. 1155-1158.