使用ε-聚賴氨酸（EPL）和抗生素聯合治療常見傷口病原體感染，以及使用EPL和絲素蛋白（SF）製備傷口修復光聚合水凝膠是本論文的兩個重點。EPL 是一種常用的抗菌肽，對哺乳動物具低毒性，可應用在於食品添加劑，以及作為細菌脂多醣的有效吸附劑。最近的研究也證實EPL具促進哺乳動物細胞附著的能力。
本論文首先探討 EPL 是否可以增強特定抗生素（即氨芐青黴素 (AMP)、慶大霉素 (GEN) 和四環素 (TCN)）針對四種重要傷口感染細菌的功效：綠膿桿菌、肺炎克雷伯菌、甲氧西林(MET)敏感金黃色葡萄球菌(MSSA)和甲氧西林耐藥葡萄球菌金黃色葡萄球菌(MRSA)，同時還研究了 EPL 提高甲氧西林對抗 MRSA 有效性的潛力。本論文採用棋盤法來評估 EPL 與每種抗生素在次最低抑制濃度下的組合效果。AMP-EPL、GEN-EPL 和 TCN-EPL 組合表現出針對綠膿桿菌、MSSA、肺炎克雷伯菌和MRSA部分協同作用或累加效應。此外，MET-EPL 還表現出針對 MRSA 的部分協同作用。通過活/死螢光染色和活細胞計數證實，在抗生素-EPL治療的情況下，觀察到細菌活力顯著下降。在這些組合中，GEN-EPL對MSSA表現出最有效的抗菌作用，在一小時內有效消除細菌。相反，AMP、MET與EPL的組合對抗MRSA以及TCN-EPL 對抗肺炎克雷伯菌的效果最差，需要十多個小時才能根除細菌生長。所有抗生素-EPL 處理均表現出對綠膿桿菌生物膜形成的抑制活性，並增強體外預先形成的生物膜破壞。在豬皮膚模型上也觀察到了類似的生物膜形成抑制作用。重要的是，當用 Balb/3t3 成纖維細胞進行測試時，在任何抗生素-EPL治療中均未檢測到顯著的細胞毒性。這些發現凸顯了EPL作為抗生素輔助療法的潛力，可增強抗生素對抗細菌傷口病原體的有效性，同時還能控制生物膜生成，而不損害哺乳動物細胞活力。
良好的傷口癒合水凝膠必須具有有效的抗菌特性，並能為細胞粘附和增殖創造有利的環境。儘管SF具有傷口癒合特性，但它缺乏這些基本品質。這項研究的第二個目的是利用SF和EPL的特性來製造可光聚合水凝膠用於傷口修復。利用三種不同濃度的甲基丙烯酸縮水甘油酯(GMA)對 SF進行修飾，產生SF-GMA(L)、SF-GMA(M)和SF-GMA(H)衍生物。此外，也將EPL修飾GMA (EPL-GMA)。隨後，將 SF-GMA 與 EPL-GMA 混合，形成可光交聯的 SF-GMA-EPL 水凝膠。含有20% EPL-GMA的水凝膠SF-GMA(L)-EPL、SF-GMA(M)-EPL和SF-GMA(H)-EP在所有製備的水凝膠樣品中表現出最高的抗菌活性和哺乳動物細胞粘附能力。此些水凝膠的羥基自由基(·OH)清除效率範圍在69%至74%之間，在去除細菌脂多醣方面的效率則約為60%。水凝膠的吸水能力與其內部孔隙的大小相關。這些水凝膠降解緩慢，其降解產物具有細胞相容性。此外，水凝膠表現出彈性特性，儲能模量（G'）範圍為300至600 Pa。總結而言，本研究開發的水凝膠表現出出色的生物和物理特性，使其非常適合作為促進傷口癒合用敷料。

The development of combination therapy against common wound pathogens by using Epsilon-ply-L-lysine (EPL) and antibiotics, and the fabrication of wound repair photopolymerizable hydrogel by using EPL and silk fibroin (SF) are the two focuses of this dissertation. EPL is an often-used antimicrobial peptide known for its low mammalian toxicity. Its primary application lies in the realm of food additives, and it serves as an effective adsorbent for bacterial lipopolysaccharides (LPS). Notably, recent studies have unveiled an additional intriguing property of EPL, showcasing its capability for promoting mammalian cell attachment.
In the initial phase of this study, I examined whether EPL enhances the efficacy of specific antibiotics, namely ampicillin (AMP), gentamicin (GEN), and tetracycline (TCN), against four significant bacterial wound pathogens: Pseudomonas aeruginosa PAO1, Klebsiella pneumoniae CG43, methicillin-sensitive Staphylococcus aureus (MSSA) ATCC 25923, and methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591. Additionally, the potential of EPL to improve the effectiveness of methicillin (MET) against MRSA was investigated. The checkerboard approach was employed to assess the combinatorial effects of EPL with each antibiotic at sub-minimal inhibitory concentrations. AMP-EPL, GEN-EPL, and TCN-EPL combinations exhibited activity against P. aeruginosa, K. pneumoniae, MSSA, and MRSA, with outcomes ranging from synergy to partial synergy or additive effects. Additionally, MET-EPL demonstrated partial synergy against MRSA. A noteworthy decrease in bacterial viability was observed in the presence of antibiotic-EPL treatments, as confirmed through live/dead fluorescence staining and viable cell counts. Among these combinations, GEN-EPL exhibited the most potent antimicrobial effect against MSSA, effectively eliminating the bacteria within one hour. Conversely, the combinations of AMP and MET with EPL against MRSA, as well as TCN-EPL against K. pneumoniae, were identified as the least potent, requiring over 10 hours to eradicate bacterial growth. All antibiotic-EPL treatments exhibited inhibitory activity against P. aeruginosa biofilm formation and enhancement of preformed biofilm disruption in vitro. A similar inhibition of biofilm formation on a porcine skin model was also observed. Importantly, no significant cytotoxicity was detected in any of the antibiotic-EPL treatments when tested with Balb/3t3 fibroblasts. These findings highlight the potential of EPL to enhance the effectiveness of antibiotics against bacterial wound pathogens while also promoting biofilm control without compromising mammalian cell viability.
To achieve an optimal wound-healing hydrogel, it is imperative to possess effective antibacterial properties and create a conducive environment for cell adhesion and proliferation. Despite Bombyx mori SF exhibiting inherent wound-healing properties, it lacks these essential qualities. The second aim of this study is to fabricate photo-polymerizable hydrogel by utilizing the properties of SF and EPL for wound repair. Three different concentrations of glycidyl methacrylate (GMA) were utilized to modify SF, resulting in SF-GMA(L), SF-GMA(M), and SF-GMA(H) derivatives. Additionally, a methacrylated form of EPL, i.e., (EPL-GMA) was synthesized. Subsequently, SF-GMA was blended with EPL-GMA to create photo-crosslinkable SF-GMA-EPL hydrogels. The hydrogels SF-GMA(L)-EPL, SF-GMA(M)-EPL, and SF-GMA(H)-EPL, containing 20% EPL-GMA, exhibited the highest levels of antimicrobial activity and mammalian cell adhesion ability among all the fabricated hydrogel samples. The hydrogels were evaluated for their •OH radical scavenging efficiency, which was found to range between 69% and 74%. Additionally, these hydrogels displayed a 60% efficiency in the removal of bacterial lipopolysaccharides. The water absorption capacity of the hydrogels aligned with the size of their internal pores. These hydrogels demonstrated a gradual degradation pattern, and the degradation products were found to be cytocompatible. Furthermore, the hydrogels exhibited elastomeric properties, with a storage modulus (G') ranging from 300 to 600 Pa. In conclusion, the hydrogels developed in this study showcase outstanding biological and physical characteristics, making them well-suited to facilitate wound healing.

Acknowledgments i
摘要 ii
Abstract iv
Abbreviation vi
Table of contents viii
List of figures xi
List of tables xv
Chapter 1. Introduction and review of literature 1
1. 1 Antimicrobial peptide 2
1. 1. 1 Epsilon-poly-L-lysine 3
1. 1. 1. 1 Mechanism of antibacterial action of EPL 4
1. 1. 1. 2 Biomedical applications of EPL 5
1. 2 Antibiotic resistance 8
1. 3 Other Serious Side effects of antibiotic use 9
1. 4 Combination therapy to treat bacterial infection 10
1. 4. 1 Combination of AP and antibiotics 10
1. 4. 1. 1 Antibacterial mechanism of AP and antibiotic combination 11
1. 4. 1. 2 EPL in combination study 13
1. 5 Silk 15
1. 5. 1 B. mori SF 16
1. 5. 1. 1 Wound healing and SF in wound healing 17
1. 6 Photopolymerizable hydrogel 19
1. 6. 1 SF-GMA-based photopolymerizable hydrogel 20
1. 6. 2 EPL-GMA-based photopolymerizable hydrogel 20
Chapter 2. Combination effect of epsilon-poly-L-lysine and antibiotics against common bacterial pathogens 22
2. 1 Introduction 23
2. 2 Materials and Methods 24
2. 2. 1 Bacterial strain and culture condition 24
2. 2. 2 Chemicals and antimicrobial agents 25
2. 2. 3 EPL spotting assay 25
2. 2. 4 Determination of minimum inhibitory concentration 25
2. 2. 5 Checkerboard assay and fractional inhibitory concentration 25
2. 2. 6 Fluorescence spectroscopy and microscopy analysis 26
2. 2. 7 Time-kill assay 26
2. 2. 8 In vitro anti-biofilm assay 27
2. 2. 9 Ex vivo anti-biofilm assay 27
2. 2. 9 Cytotoxicity 27
2. 3 Results and Discussion 28
2. 3. 1 Bacterial susceptibility towards EPL and antibiotics 28
2. 3. 2 Combinatorial effects of EPL and commonly used antibiotics on bacterial growth 30
2. 3. 3 Antibiotic-EPL combination on total bacterial count and cell viability 33
2. 3. 4 Effect of treatment time on antibiotic-EPL antibacterial efficiency 35
2. 3. 5 Effect of antibiotic-EPL on P. aeruginosa biofilm under in vitro condition 37
2. 3. 6 Effect of antibiotic-EPL on P. aeruginosa biofilm under ex vitro condition 39
2. 3. 7 Antibiotic-EPL cytocompatibility study on Balb/3t3 cell 40
2. 4 Conclusion 42
Chapter 3. Fabrication and in vitro evaluation of photo crosslinkable Silk fibroin –Epsilon-poly-L-lysine hydrogel for wound repair 43
3. 1 Introduction 44
3. 2 Materials and Methods 46
3. 2. 1 Extraction of SF fibers from B. mori cocoon 46
3. 2. 2. Preparation and storage of SF solution 46
3. 2. 3 Synthesis of SF-GMA and EPL-GMA graft polymers 46
3. 2. 4 Evaluation of GMA grafting on SF and EPL by FTIR and NMR 47
3. 2. 5. Fabrication of SF-GMA-EPL hydrogel by photopolymerization 48
3. 2. 6 Antibacterial properties of SF-GMA-EPL hydrogel 48
3. 2. 7 Cell attachment and proliferation on SF-GMA-EPL hydrogel 49
3. 2. 8 Hydroxyl radicle scavenging activity of SF-GMA-EPL hydrogel 50
3. 2. 9 Determination of bacterial LPS removal by SF-GMA-EPL hydrogel 50
3. 2. 10 Degradation of SF-GMA-EPL hydrogel and cytotoxicity of degradation product 50
3. 2. 11 Scanning electron microscopy analysis of SF-GMA-EPL hydrogel 51
3. 2. 12 Swelling ratio of SF-GMA-EPL hydrogel 51
3. 2. 13 Rheological analysis of SF-GMA-EPL hydrogel 52
3. 3 Results and discussion 52
3. 3. 1 Extraction of SF fibers and preparation of SF solution 52
3. 3. 2 Preparation of SF-GMA and EPL-GMA graft polymers and SF-GMA-EPL hydrogel 53
3. 3. 3. Evaluation of SF-GMA and EPL-GMA polymers 54
3. 3. 4 Antibacterial efficiencies of SF-GMA-EPL hydrogel 57
3. 3. 5 Bacterial growth inhibition dynamics of SF-GMA-EPL hydrogel 60
3. 3. 6 Cell attachment and proliferation on SF-GMA-EPL hydrogel 61
3. 3. 7 LPS adsorption and OH radicle scavenging ability of SF-GMA-EPL hydrogel 65
3. 3. 8 Degradation ratio of SF-GMA-EPL hydrogel 66
3. 3. 9 Physical characterization of SF-GMA-EPL hydrogel 68
3. 4 Conclusion 71
Chapter 4. Future perspectives 73
References 75
Publication list 92

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