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請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77769
完整後設資料紀錄
DC 欄位值語言
dc.contributor.advisor陳慧文
dc.contributor.authorYou-Ting Chenen
dc.contributor.author陳宥廷zh_TW
dc.date.accessioned2021-07-11T14:34:30Z-
dc.date.available2020-08-08
dc.date.copyright2018-08-08
dc.date.issued2018
dc.date.submitted2018-07-24
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77769-
dc.description.abstract流感病毒每年在全球造成許多發病、重症及死亡,是全球相當關心的公共衛生議題,傳統的治療策略為使用拮抗病毒蛋白質的藥物,然而帶有抗藥性突變的流感病毒毒株的出現,成為臨床治療上一大挑戰。Diphyllin與Bafilomycin是強效的Vacuolar ATPase阻斷劑,先前被證實對流感病毒具有抑制效果,然而,它們極差的水溶性和可能的副作用限制了它們的臨床應用。此研究的核心目標即是設計新穎的抗流感病毒治療策略,利用奈米顆粒來裝載與遞送抗病毒藥物,以提高藥物的安全性與效性。本研究中,我們分別製備了裝載Diphyllin和Bafilomycin的奈米顆粒,以動態光散射儀與穿透性電子顯微鏡觀察奈米顆粒的型態、粒徑與表面電位,並以高效液相色譜分析藥物在奈米顆粒中的包覆率。實驗結果顯示,我們所製備的奈米顆粒能有效地遞送進入Fcwf-4、ARPE-19、MH-S等多種細胞內部,並緩慢的釋放。在MDCK及MH-S細胞中顯示,以奈米顆粒包覆遞送之藥物比未包覆之藥物具有較低的細胞毒性,與顯著較高的抗流感病毒活性。在活體的安全性研究中,分別由靜脈內及鼻腔內給予小鼠Diphyllin奈米顆粒,觀測其體重變化及血液生化參數,發現小鼠對於Diphyllin奈米顆粒有良好的耐受性,並在流感病毒攻毒之後,有給予Diphyllin奈米顆粒之組別與未給藥組相比,有較少的體重減輕。另一方面,我們也製備了外覆紅血球膜的奈米顆粒,透過紅血球膜與流感病毒的交互作用,此奈米顆粒能夠專一地被流感病毒感染後的細胞攝入,有機會發展為標靶治療藥物之平台。總結來說,本研究顯示以奈米載體遞送抗病毒藥物為一新穎、有潛力之治療策略,能夠對病毒傳染病之藥物發展帶來更多希望。zh_TW
dc.description.abstractInfluenza virus infections are a major public health concern and cause significant morbidity and mortality worldwide. Conventional treatments against the disease are designed to target viral proteins. Nevertheless, the emergence of new influenza viral strains carrying drug-resistant mutations that can outpace the development of pathogen-targeting antivirals presents a major clinical challenge. Diphyllin and bafilomycin are potent vacuolar ATPase inhibitors, and previously known to have anti-influenza virus activity. However, their poor water solubility and potential off-target effect may limit the clinical application. The central objective of this project is to generate a novel anti-influenza therapeutic strategy, integrating nanoparticle technology to enhance host-targeting antiviral delivery towards improved drug safety and efficacy. In this study, we successfully constructed diphyllin-loaded nanoparticles and bafilomycin-loaded nanoparticles. The shape, size, and zeta potential of these nanoparticles were measured by dynamic light scattering and transmission electron microscopy, and the drug encapsulation was analyzed by high-performance liquid chromatography. The nanoparticles can be efficiently delivered intracellularly to multiple cell lines including Fcwf-4, ARPE-19, and MH-S cells. Moreover, the drug-loaded nanoparticles have a sustained drug release profile. The nanoformulated drugs exhibited lower cytotoxicity as compared to the free drug formulation in MDCK and MH-S cells. Furthermore, drug-loaded nanoparticles demonstrated prominent broad-spectrum anti-influenza activity in vitro. In an in vivo safety study that evaluated body weight and blood chemistry parameters following intravenous or intranasal administration, diphyllin nanoparticles were well-tolerated in mice. Furthermore, after infection, the mice treated with diphyllin-loaded nanoparticles had less body weight loss as compared to the control group. In addition, to further improve targeting delivery of nanoparticles to virus-infected cells, fluorescent nanoparticles were coated with the red blood cell membrane to validate the viral infection-specific targeting of nanoparticles. Collectively, this work highlights nanoformulated vacuolar ATPase inhibitors as potential host-targeted treatments against influenza.en
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Previous issue date: 2018
en
dc.description.tableofcontentsContents
中文摘要 I
Abstract III
List of figures IX
List of tables XI
Chapter 1 Introduction 1
1.1 Background and epidemiology of influenza virus 1
1.2 Influenza virus replication 2
1.3 Treatment of influenza virus infection 5
1.4 Vacuolar ATPase (V-ATPase) and its inhibitors 6
1.5 Application of diphyllin 8
1.6 Application of bafilomycin 8
1.7 Nanoparticulate drug delivery systems 10
1.8 The objective of this study 11
Chapter 2 Materials and methods 13
2.1 Cells and viruses 13
2.2 Preparation of diphyllin-loaded nanoparticles 14
2.3 Preparation of bafilomycin-loaded nanoparticles 15
2.4 Preparation of DiD-load nanoparticles 15
2.5 Preparation of RBC membrane-coated DiD-loaded nanoparticles 16
2.6 Transmission electron microscopy (TEM) and dynamic light scattering (DLS) 17
2.7 High-performance liquid chromatography (HPLC) 18
2.8 Drug release studies 18
2.9 Cellular uptake studies 19
2.10 Cytotoxicity evaluation of free drugs and drug-loaded nanoparticles in vitro 19
2.11 Assessment of the half inhibitory concentration (IC50) in vitro 20
2.12 Assessment of antiviral activity 20
2.13 Virus titration 21
2.13.1 Hemagglutination assay (HA assay) 21
2.13.2 50% of Tissue Culture Infective Dose (TCID50) 21
2.13.3 Plaque assay 22
2.13.4 Plasmid construction and quantitative PCR (qPCR) of influenza virus 23
2.14 In vivo tolerance of diphyllin nanoparticles 25
2.15 Establishment of animal models of influenza virus challenge 25
2.16 Evaluation of antiviral efficacy of diphylliny nanoparticles in vivo 26
2.17 Targeted delivery to virus-infected cells by RBC membrane-coated nanoparticles 26
2.18 Ethics statement 27
2.19 Statistical analyses 28
Chapter 3 Results 29
3.1 Characterization of diphyllin-loaded nanoparticles 29
3.2 Characterization of bafilomycin-loaded nanoparticles 29
3.3 Characterization of DiD-load nanoparticles 30
3.4 Characterization of RBC membrane-coated Did-loaded nanoparticles 31
3.5 Release kinetics of drug-loaded nanoparticles 31
3.6 Assessment of cellular uptake of nanoparticles 32
3.7 CC50 of free drugs and drug-loaded nanoparticles 32
3.8 IC50 of free drug and drug-loaded nanoparticles 33
3.9 Antiviral activity of diphyllin-loaded nanoparticles 34
3.10 Antiviral activity of bafilomycin-loaded nanoparticles 34
3.11 Toxicology of diphyllin nanoparticles in vivo 35
3.12 Establishment of 50% mice lethal dose of influenza virus (H1N1) and determination of viral titer in the lung, trachea, and BAL 36
3.13 Antiviral efficacy of diphylliny nanoparticles in vivo 36
3.14 Targeted delivery to virus-infected cells by RBC membrane coated-nanoparticles 37
Chapter 4 Discussion 38
Chapter 5 Figures and tables 45
Chapter 6 References 72
dc.language.isoen
dc.subjectBafilomycinzh_TW
dc.subject奈米顆粒zh_TW
dc.subjectDiphyllinzh_TW
dc.subjectVacuolar ATPase 阻斷劑zh_TW
dc.subject流感病毒zh_TW
dc.subjectInfluenza virusen
dc.subjectVacuolar ATPase inhibitoren
dc.subjectDiphyllinen
dc.subjectBafilomycinen
dc.subjectNanoparticlesen
dc.title以裝載Vacuolar ATPase 阻斷劑之奈米顆粒用於抗流
感病毒感染之效果
zh_TW
dc.titleAntiviral Efficacy of the Nanoparticulate Vacuolar
ATPase Blockers against Influenza Virus Infection
en
dc.typeThesis
dc.date.schoolyear106-2
dc.description.degree碩士
dc.contributor.coadvisor胡哲銘
dc.contributor.oralexamcommittee陳怡寧,徐維莉,王金和
dc.subject.keyword流感病毒,Vacuolar ATPase 阻斷劑,Diphyllin,Bafilomycin,奈米顆粒,zh_TW
dc.subject.keywordInfluenza virus,Vacuolar ATPase inhibitor,Diphyllin,Bafilomycin,Nanoparticles,en
dc.relation.page80
dc.identifier.doi10.6342/NTU201801911
dc.rights.note有償授權
dc.date.accepted2018-07-25
dc.contributor.author-college獸醫專業學院zh_TW
dc.contributor.author-dept獸醫學研究所zh_TW
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