博碩士論文 105827005 詳細資訊



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姓名 蕭光紘(Kuang-Hung Hsiao)  查詢紙本館藏   畢業系所 生醫科學與工程學系
論文名稱 設計開發全氟碳複合奈米藥物載體對體表微生物多效抑菌功能之研究
(Development of Multifunctional Perfluorocarbon Drug Nano-Composites for Anti-Skin Microbes)
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摘要(中) 近十年，皮膚益生菌的研究層出不窮，其概念是源自於透過食用優格來促進腸胃道菌叢維持平衡並保持健康。透過過往的研究，表皮葡萄球菌 (Staphylococcus epidermidis/ S. epidermidis)藉由甘油的碳元刺激發酵反應，其發酵產物──短鏈脂肪酸可以有效地抑制痤瘡丙酸桿菌(Cutibacterium acnes/ C. acnes)生長。建立在此研究上，奈米科技可以提供一個同時乘載藥物，並提供益生菌碳元的新穎方法。近年來，有許多使用聚乙二醇(Polyethylene glycol, PEG)高分子或其衍生物來承載藥物的研究，而PEG高分子本身，係由環氧乙烷形成的聚合物，這樣的聚合物即是含有大量的碳元，亦可以提供表皮葡萄球菌產生短鏈脂肪酸。
本研究使用PEG衍生高分子作為藥物載體，包覆利福平(Rifampicin, RIF)及靛氰綠(Indocyanine green, ICG)形成奈米粒子，透過載體刺激益生菌產生活性，再釋放光治療及抗生素治療，三功效的治療方法來對抗嚴重的痤瘡病症。
在初步研究上，PEG之衍生物PEO-PPO-PEO高分子，與利福平、靛氰綠及全氟碳化物(Perfluorocarbon)透過超音波震盪形成雙層奈米粒子RIPNDs，其粒徑大小為240.7 ± 6.73 nm，表面電位為-20.9 ± 2.40 mV，而利福平與靛氰綠之包覆率為54.0 ± 10.5%及95.0 ± 4.84%。體外實驗針對痤瘡丙酸桿菌的胞殺效果上，在益生菌發酵液的環境及奈米粒子濃度高於20-μM ICG/3.8-μM RIF的條件下，並以雷射光(808 nm, 6 W/cm2)激發光治療效果，痤瘡丙酸桿菌得以根除。
而在進階的小鼠實驗中，被痤瘡丙酸桿菌感染的小鼠耳朵，透過發酵液及奈米粒子RIPNDs的光化治療後，相比於單用抗生素Rifampicin及單使用奈米粒子RIPNDs的光化治療，發炎程度下降了70.5%及23.1%，耳內抑菌程度增加97%及85%，其不只代表單用RIPNDs就可以有很好的治療效果，更衍生出發酵液在本研究的新穎性及重要性。
本研究奈米粒子RIPNDs不只可以有效抑制痤瘡丙酸桿菌，亦可以催化皮膚益生菌產生益生菌活性，這樣的治療方法我們稱之為光-化-益生抗菌治療，在未來臨床研究上擁有高度的潛力。
摘要(英) In past decade, researches of skin probiotics come out one and another. The concept is derived from the use of yogurt to promote the balance of the gastrointestinal flora and maintain body health. Previous studies show that Staphylococcus epidermidis (S. epidermidis) uses the carbon source from glycerol to stimulate the fermentation reaction. From fermentation products, short-chain fatty acids (SCFAs) can effectively inhibit Cutibacterium acnes (C. acnes, Propionibacterium acnes formerly) growth. Based on this research, nanotechnology can provide a novel way to simultaneously be a drug carrier and provide probiotic carbon source. In recent years, polyethylene glycol (PEG) polymers, which were formed from ethylene oxide, or their derivatives were studied to be drug vehicles. Such polymers contain a large amount of carbon source and be able to stimulate S. epidermidis generating SCFAs.
In this study, PEG-derived polymer was used as a drug carrier, encapsulating Rifampicin (RIF) and Indocyanine green (ICG) to form nanoparticles. Through the nanoparticles, the polymer shell stimulates the activity of probiotics, followed by phototherapy activation and antibiotics release. We anticipate this triple-functional treatment, named photo-chemo-biotic therapy, could combat severe acne symptoms.
In preliminary research, nanoparticle RIPNDs was fabricated by PEO-PPO-PEO polymer, rifampicin, indocyanine green and perfluorocarbon, forming a double-layer system through ultrasonic emulsification. The size and surface charge were 240.7 ± 6.73 nm and -20.9 ± 2.40 mV, respectively. And the encapsulation rates of rifampicin and indocyanine green were 54.0 ± 10.5% and 95.0 ± 4.84%, respectively. In vitro experiments aimed on the bactericidal effect of C. acnes, With the presence of probiotic fermentation product medium (FPM) and RIPNDs with concentration higher than 20-μM ICG/3.8-μM RIF under laser irradiation (808 nm, 6 W /cm2), C. acnes can be completely eradicated.
In the further mouse experiment, the infected mice ears by C. acnes were treated by photo-chemo-biotic therapy of RIPNDs. The degree of inflammation decreased 70.5% and 23.1% compared to rifampicin alone and nanoparticle RIPNDs with laser exposure (i.e. photo-chemo therapy), respectively. And the antibacterial effect increased 97% and 85%, respectively. This experiment not only shows RIPNDs gives a good therapeutic effect, but also point out the novelty and importance of probiotics effect.
To sum up the study, RIPNDs can not only effectively inhibit C. acnes growth, but also catalyze skin probiotics to generate probiotic activity. This photo-chemical-probiotic therapy are highly potential for use in the clinical anti-C. acnes treatment with reduced chemotoxicity
關鍵字(中) ★ 奈米粒子
★ 益生菌
★ 光治療
★ 全氟碳化物
★ 藥物輸送		 關鍵字(英) ★ Nanoparticle
★ Probiotics
★ Phototherapy
★ Perfluorocarbon
★ Drug delivery
論文目次 摘要 i
ABSTRACT ii
ACKNOWLEDGEMENT iv
TABLE OF CONTENTS vi
LIST OF FIGURES ix
LIST OF TABLES x
CHAPTER I INTRODUCTION 1
1.1 Overview of Polymer 1
1.2 Nanoparticles 1
1.3 Polymeric nanoparticles 2
1.3.1 Micelle 2
1.3.2 Nanocapsule 2
1.3.3 Polymersome 3
1.4 Application of Nanoparticles 3
1.5 Human microbiota 4
1.6 Probiotics 5
1.7Antibacterial activity of Short-Chain-Fatty-Acid 6
1.7.1 Disruption of electron transport chain 7
1.7.2 disruption of oxidative phosphorylation 7
CHAPTER II DEVELOPMENT OF RIFAMPICIN-INDOCYANINE GREEN-LOADED PERFLUOROCARBON NANODROPLETS FOR PHOTO-CHEMO-PROBIOTIC ANTIMICROBIAL THERAPY 8
2.1 Introduction 8
2.2 Materials and methods 11
2.2.1 Preparation and characterization of RIPNDs 11
2.2.2 Measurements of stability and release kinetics of entrapped molecules 14
2.2.3 Measurements of RIPND-induced hyperthermia effect and singlet oxygen generation 14
2.2.4 Microbial cultivation 15
2.2.5 Evaluation of effect of RIPNDs on microbial fermentation 15
2.2.6 Examination of effect of RIPND-mediated probiotic inhibition on C. acnes growth 16
2.2.7 In vitro antimicrobial efficacy of RIPNDs 16
2.2.8 Statistical analysis 17
2.3 Results and discussion 17
2.3.1 Characterization of RIPNDs 17
2.3.2 Thermal stability of RIPND-entrapped ICG and release rate of RIF 19
2.3.3 Effects of hyperthermia and singlet oxygen generation of RIPNDs 23
2.3.4 Effects of RIPNDs on microbial fermentation efficiency 27
2.3.5 Antimicrobial capability of RIPNDs to C. acnes 29
2.4 Conclusion 33
CHAPER III NOVEL RIFAMPICIN-INDOCYANINE GREEN-LOADED PERFLUOROCARBON NANODROPLETS PROVIDE EFFECTIVE IN VIVO PHOTO-CHEMO-PROBIOTIC ANTIMICROBILITY AGAINST PATHOGEN OF ACNE CUTIBACTERIUM ACNES 34
3.1 Introduction 34
3.2 Materials and methods 36
3.2.1. Fabrication and characterization of the RIPNDs 36
3.2.2 Microbial cultivation 37
3.2.3 Measurement of antimicrobial efficacy of the RIPNDs in vitro 37
3.2.4 Cell culture 38
3.2.5 In vitro cytotoxicity assay 39
3.2.6 Animal study 39
3.2.7 Evaluation of antimicrobial effect of the RIPNDs in vivo 40
3.2.8 Measurement of inflammatory response in vivo 41
3.2.9 Histological study 41
3.2.10 Statistical analysis 41
3.3 Results and discussion 41
3.3.1 Morphological and physicochemical analyses of the RIPNDs 41
3.3.2 Antibacterial effect of the RIPNDs in vitro 43
3.3.3 Cytotoxicity of the FPM and RIPNDs in vitro 45
3.3.4 Anti-inflammatory response of the RIPNDs in vivo 47
3.3.5 Antimicrobial effect of the RIPNDs in vivo 50
3.4 Conclusions 53
CHAPTER IV CONCLUSION 54
REFERENCES 55
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指導教授 黃俊銘 李宇翔(Chun-Ming Huang Yu-Hsiang Lee) 審核日期 2021-7-27
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