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研究生: 吳慧璇
Sabrina
論文名稱: 可持續釋放抗菌物質之生物材料於抗生物膜之應用
Biomaterials with Sustained Release of Antimicrobial Agents for Antibiofilm Applications
指導教授: 李振綱
Cheng-Kang Lee
口試委員: 朱義旭
Yi-Hsu Ju
蔡伸隆
Shen-Long Tsai
楊佩芬
Pei-Fen Yang
王勝仕
Steven S.-S. Wang
劉懷勝
Hwai-Shen Liu
學位類別: 博士
Doctor
系所名稱: 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年: 2018
畢業學年度: 106
語文別: 英文
論文頁數: 82
中文關鍵詞: 抗菌膜抗菌過氧化氫銀納米粒子精油
外文關鍵詞: antibiofilm, antimicrobial, hydrogen peroxide, silver nanoparticles, essential oil
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  • 一般細菌及微生物會聚集附著於常與水接觸之材料表面形成生物膜,如生物膜形成於醫療裝置和材料表面常會造成傷口癒合困難及感染的併發症。本論文研究了幾種策略來防止生物膜形成或破壞已成熟之生物膜,生物膜的組成除了細菌外還有保護細菌之胞外多醣(EPS)基質,通常難以利用一般之抗生素將被保護之細菌殺死,而過氧化氫(H2O2)是一強氧化劑常也用於消毒滅菌,本研究在藻酸鈣水凝膠膜中摻入乙酰化澱粉(AS)和澱粉葡萄糖苷酶 及葡萄糖氧化酶(AMG-GOX),製備出可持續產生H2O2之凝膠膜,且證實其有抗微生物和生物膜之效果。納米銀顆粒(AgNPs)因其具有十分有效的抗微生物活性,近年來被廣泛研究及應用,藻酸鈣膜和磁性納米粒子(MNPs)表面可容易地接附上單寧酸,而可直接將溶液中之銀離子還原成AgNPs,具有AgNPs藻酸鈣膜和磁性粒子皆表現出優良之抗菌和抗生物膜活性。此外利用Tollen試劑可將AgNPs直接形成在包覆有羧甲基殼聚醣(CMCS)的MNP上, CMCS本身之殺菌的能力加上所形成之AgNP,可大幅增加此複合磁性粒子之抗微生物活性,利用磁場可使此磁粒更有效地嵌入生物膜而殺死其中之細菌。精油(EO)可有效殺死多種細菌和病毒,利用纖維素納米晶體(CNC)所形成的Pickering乳液可有效地將EO包覆起來而均勻分散於水中,EO微乳液可被均勻包埋於海藻酸鈣膜中,而持續釋放出EO達到抗菌和抗生物膜之效果。


    Generally, bacteria and microorganism aggregates and adheres to the surface of materials that are often in contact with water to form biofilm. For example, biofilm formation on medical devices and materials that often causes difficulty in wound healing and complications of infection. This dissertation studied several strategies to prevent biofilm formation or eradicate mature biofilm. The structure of biofilm that contains extracellular polysaccharide (EPS) as main matrix provides protection that makes it difficult to be disassembled by conventional antibiotic treatment. Reactive oxygen species hydrogen peroxide (H2O2) has been known as strong oxidizing agent used to kill bacteria. In this study, H2O2 producing system was developed by incorporating acetylated starch (AS) and dual enzyme amyloglucosidase-glucose oxidase (AMG-GOX) in calcium alginate hydrogel membrane. Sustained release of H2O2 from the presented AS-AMG-GOX loaded alginate hydrogel membrane proved to have antimicrobial and biofilm eradication effect. Silver nanoparticles (AgNPs) has been widely studied for its antimicrobial activity. In this study, AgNPs was grafted on two template, calcium alginate membrane and magnetic nanoparticles (MNPs). AgNPs green synthesis by tannic acid has been studied to be prepared in aqueous solution at room temperature. We prepared tannic acid-coated calcium alginate membrane to directly synthesize AgNPs on the template. AgNPs was effectively synthesized on tannic acid-coated membrane and exhibited antimicrobial and antibiofilm activity. AgNPs was grafted on carboxymethyl chitosan (CMCS) coated MNPs with Tollen’s reagent. CMCS itself has shown ability to kill bacteria and by grafting both CMCS and AgNPs on MNPs, its antimicrobial activity could be achieved. It also exhibited antibiofilm activity by killing cells embedded in biofilm more effectively. The advantage of MNPs@CMCS-AgNPs is retrievable using magnetic field. Essential oil (EO) has been studied to effectively kill several bacterial and viral pathogens due to the presence of different types of antimicrobial compounds in it. To provide protection against environment, EO was encapsulated. EO-in-water microemulsions could be obtained by one-step microencapsulation utilizing cellulose nanocrystals (CNC) based Pickering emulsion with composition of 20 mM NaCl and 0.1 wt% as the most stable and well-dispersed microemulsion. Furthermore, calcium alginate membrane was applied to provide more protection and control release. Essential oil in emulsion showed antimicrobial and antibiofilm activity.

    摘要 ii ABSTRACT iii ACKNOWLEDGEMENT iv TABLE OF CONTENTS v ABBREVIATIONS viii LIST OF FIGURE ix CHAPTER 1 1 INTRODUCTION 1 1.1 Background 1 1.2 Objectives 3 CHAPTER 2 6 LITERATURE REVIEW 6 2.1 Antimicrobial and antibiofilm agents 6 2.2 Antimicrobial and antibiofilm enzyme 8 2.3 Reactive Oxygen Species 10 2.3.1 Hydrogen peroxide-producing enzymes 11 2.3.2 Hydrogen peroxide detection 12 2.4 Amyloglucosidase 12 2.5 Encapsulation System 13 2.5.1 Calcium alginate encapsulation 13 2.5.2 Pickering emulsion-based encapsulation 14 2.6 Silver 16 2.6.1 Tannic acid-stabilized-silver nanoparticles 16 2.6.2 Silver-coated-magnetic nanoparticles 17 CHAPTER 3 19 MATERIALS AND METHODS 19 3.1 Materials 19 3.1.1 Bacteria 19 3.1.2 Enzyme 19 3.1.3 Chemical 19 3.1.4 Bacteria culture medium 20 3.1.5 Experiment reagent 20 3.1.6 Experiment instruments 21 3.2 Methods 22 3.2.1 Preparation of Acetylated Starch (AS) (Nata et al., 2014) 22 3.2.2 Calcium alginate membrane preparation 22 3.2.3 Calcium alginate membrane mechanical strength and chemical stability 22 3.2.4 Hydrogen peroxide detection assay 23 3.2.5 Antioxidant analysis 23 3.2.6 Bradford Assay 23 3.2.7 Fluorescence microscopy using LIVE/DEAD Baclight Bacterial Viability Kit 23 3.2.8 Attenuated Total Reflectance (ATR) spectra 24 3.2.9 Thermogravimetric analysis (TGA) 24 3.2.10 ICP optical emission spectrometer 24 3.3 Preparation and Characterization 24 3.3.1 AS-AMG-GOX Alginate membrane preparation 24 3.3.2 AS-AMG-GOX Alginate membrane stability 24 3.3.3 Hydrogen peroxide sustained production of AS-AMG-GOX Alginate membrane 25 3.3.4 Tannic acid-alginate membrane 25 3.3.5 Silver nanoparticles coating on TA-coated alginate membrane 25 3.3.6 Preparation of Carboxymethyl Chitosan (CMCS) and CMCS grafted magnetic nanoparticles (MNPs@CMCS) 25 3.3.7 In situ Deposition of Silver on MNPs and MNPs@CMCS (MNPs@Ag and MNPs@CMCS-Ag) 26 3.3.8 Preparation of essential oil emulsion 27 3.3.9 Encapsulation of essential oil emulsion in alginate membrane (EO-Alg) 27 3.3.10 EO-Alg membrane stability 27 3.3.11 Antibacterial assay 27 3.3.12 Biofilm disruption 28 CHAPTER 4 29 RESULTS AND DISCUSSION 29 4.1 Sustained release of reactive oxygen species from dual enzyme system 29 4.1.1 Characterization of AS-AMG-GOX Alginate membrane 29 4.1.2 Hydrogen peroxide release 30 4.1.3 Antimicrobial activity of AS-AMG-GOX membrane 31 4.1.4 Biofilm disruption by AS-AMG-GOX membrane 32 4.2 Sustained release of silver on tannic acid-coated alginate membrane 34 4.2.1 Alg, Alg/TA, Alg/TA/Ag, AlgTA and AlgTA/Ag membrane 34 4.2.2 Bacterial growth inhibition and killing effect 37 4.2.3 Biofilm disruption 38 4.3 Sustained release of silver on carboxymethyl chitosan(CMCS)-grafted magnetic nanoparticles 40 4.3.1 CMCS and CMCS grafted MNPs (MNPs@CMCS) 40 4.3.2 Silver deposited MNP@CMCS 41 4.3.3 Antimicrobial activity and biofilm disruption 43 4.4 Essential oil pickering emulsion in alginate membranes 48 4.4.1 Essential oil emulsion encapsulated in alginate membrane characterization 48 4.4.2 Antioxidant activity 49 4.4.3 Antibacterial activity 50 4.4.4 Biofilm disruption 51 CHAPTER 5 53 CONCLUSIONS 53 5.1 Sustained release of reactive oxygen species from dual enzyme system 53 5.2 Sustained release of silver on tannic acid-coated alginate membrane 53 5.3 Sustained release of silver on carboxymethyl chitosan(CMCS)-grafted magnetic nanoparticles 53 5.4 Essential oil pickering emulsion in alginate membranes 54 REFERENCES 55

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