Antifungal effects, via apoptotic features, and its synergism of silver nanoparticles : 은나노의 세포자살 기작을 통한 항균작용과 시너지 효과

다국어 초록 (Multilingual Abstract)

Silver nanoparticles are well known to have antimicrobial effects, but the mechanism(s) have not been revealed clearly. In this study, it was found that silver nanoparticles exerted its antifungal effect via apoptosis and synergistic effects. Candida albicans exposed to silver nanoparticles showed the increased reactive oxygen species (ROS) production, measured by DHR-123 staining and then we investigated that silver nanoparticles induced the accumulation of hydroxyl radicals on C. albicans as an oxidative stress response to the drug. There are many of studies have suggested that the accumulation of ROS induces and regulates the induction of apoptosis. Furthermore, hydroxyl radicas, which is one of the more active oxygen among the reactive oxygen species (ROS) formed, are considered to be important thing for cell death. Therefore, we assumed that hydroxyl radicals are related to apoptosis and the effect of thiourea as hydroxyl radicals scavenger was investigated. In addition, the reduction of mitochondrial membrane potential through flow cytometry analysis after staining these with DiOC6 or JC-1, and the release of cytochrome c from mitochondria were also assured. The apoptotic effects of silver nanoparticles were detected by fluorescence microscope using Annexin V-FITC, TUNEL, DAPI and FITC-VAD-FMK staining further confirmed diagnostic markers of yeast apoptosis including phosphatidylserine externalization, DNA and nuclear fragmentation, and activation of metacaspase. The results indicated that silver nanoparticles possess antifungal effects with another mechanism promoting such as apoptosis and we thought hydroxyl radicals generated by silver nanoparticles plays a significant role in mitochondrial dysfunctional apoptosis. Since then, the effects of a combination of silver nanoparticles with conventional antibiotics against various pathogenic bacteria were investigated. We showed the anti-microbial and anti-biofilm activities of silver nanoparticles alone or in combination with conventional antibiotics against pathogenic bacteria. Additionally, we conducted relevant experiments including the measuring of hydroxyl radical formation and permeability to investigate the important causative for synergy effects.

목차 (Table of Contents)

• CONTENTS
• INTRODUCTION 1
• MATERIALS AND METHODS 3
• Preparation of silver nanoparticles (nano-Ag) 3
• In Vitro antifungal activity 5
• In Vitro antibacterial activity 5
• Intracellular reactive oxygen species (ROS) accumulation 5
• Measurement of mitochondrial membrane potential (켱m) 6
• Cytochrome c release 7
• Annexin V ? propidium iodide (PI) double staining 8
• Measurement of DNA damages 8
• Measurement of metacaspase activation 9
• Combination assay 9
• Determination of biofilm formation by tissue culture plate method (TCP) 10
• Hydroxyl radical formation assay 11
• Antibacterial activity with detergents and ATPase inhibitors 11
• Chapter I. Silver nanoparticles induce apoptotic cell death in Candida albicans through the increase of hydroxyl radicals
• RESULTS 12
• In vitro antifungal activity 12
• Intracellular reactive oxygen species (ROS) accumulation 13
• Measurement of mitochondrial membrane potential (켱m) 17
• Cytochrome c release 19
• Annexin V ? propidium iodide (PI) double staining 21
• Measurement of DNA damages 23
• Measurement of metacaspase activation 24
• DISCUSSION 26
• Chapter II. Synergistic effect with silver nanoparticles and its involved mechanisms
• RESULTS 30
• In vitro antibacterial activity 30
• Combination assayn 32
• Determination of biofilm formation by tissue culture plate method (TCP) 34
• Hydroxyl radical formation assay 36
• Antibacterial activity with detergents and ATPase inhibitors 38
• DISCUSSION 41
• REFERENCE 44
• ABSTRACT 53