功能性奈米材料應用於致病菌的標靶及抑制之研究

Abstract

細菌除了會污染食物之外還會造成許多感染性疾病。其中一種常見致病菌即能產生不同外毒素的金黃色葡萄球菌，金黃色葡萄球菌能造成包含燙傷樣皮膚綜合症、食物中毒、中毒性休克綜合徵和紅斑疹等疾病。每年有許多人死於金黃色葡萄球菌的感染，雖然抗生素可有效地治療細菌的感染，但是抗藥性菌株如甲氧西林耐藥性金黃色葡萄球菌的出現，已經使得細菌感染的治療變得更為困難，因此尋找新的治療方法及抗生素已是刻不容緩的課題。在天然物中，有許多具抗菌性的胜肽已被分離出來，研究中發現這些抗菌性的胜肽含有大量的精氨酸和色氨酸。研究中也指出帶正電的精氨酸可用來接近帶負電細菌，而疏水性的色氨酸可穿透菌膜。因此我們相信如果將抗菌胜肽修飾於奈米粒子上，基於多價作用的概念，抗菌活性應可藉此增加。所以，本論文的研究目標為合成金奈米粒子並將表面修飾上抗菌胜肽，研究此官能化金奈米粒子的抗生素活性。此外，在我們之前的研究中已發現一個五胜肽對金黃色葡萄球菌有極佳的標靶選擇能力。因此在這個研究中，我們首先利用一鍋反應修飾一段含有精氨酸、色氨酸以及五胜肽於金奈米粒子表面，實驗顯示合成出的功能化金奈米粒子對金黃色葡萄球菌以及甲氧西林耐藥性金黃色葡萄球菌具有標靶及抗菌的能力。令我們驚訝的是，此胜肽官能化金奈米粒子與腐生葡萄球菌，糞腸球菌，屎腸球菌，抗萬古黴素之糞腸球菌和抗萬古黴素耐藥性屎腸球菌間也擁有標靶辨識能力，這可能是因為疏水性的色氨酸和這些細菌表面的疏水性官能基所產生之作用力。此外，這些細菌的生長也受到我們合成的抗菌金奈米粒子的抑制。我們的實驗結果顯示被用來修飾在奈米粒子表面的胜肽分子本身並沒有抑制這些細菌生長的能力，因此我們推論具胜肽修飾的金奈米粒子抗菌能力主要是來自於金奈米粒子上的胜肽與標靶細菌間產生了多價作用力而提升了抗菌能力。巨噬細胞有藉由內化而吞噬粒徑小於十微米顆粒的趨勢，所以我們基於巨噬細胞的此特性可將抗菌金奈米粒子帶入甲氧西林耐藥性金黃色葡萄球菌感染的巨噬細胞中，藉此研究被細菌感染的巨噬細胞是否可利用此抗菌金奈米粒子的抗菌能力抑制細胞內細菌的生長。實驗結果顯示我們可以成功地利用我們的金奈米粒子有效地抑制巨噬細胞中甲氧西林耐藥性金黃色葡萄球菌的生長。在本論文的研究中，我們已成功合成出具抗菌活性的金奈米粒子，並且在體外試驗中及巨噬細胞中證明它的抗生素活性，此奈米粒子未來應有潛力用於醫療上的使用。

Pathogenic bacteria can contaminate food and cause infectious diseases. Staphylococcus aureus is one of the common pathogenic bacteria and can generate different exotoxins, which can lead to scalded skin syndrome, food poisoning, toxic shock syndrome, and erythematous rash. Many people die every year because of infections caused by S. aureus. Antibiotics can be used to effectively treat bacterial infections. However, the emerging of antibiotic-resistant strains such as methicillin-resistant S. aureus (MRSA) has caused some difficulties in treating bacterial infections. It is urgent to explore new therapeutics and antibiotics to deal with this situation. It has been discovered that naturally antibacterial peptides purified from natural products contain abundant arginines and tryptophans. Positively charged arginine and hydrophobic tryptophan can be used to target negatively charged bacteria and penetrate bacterial membrane, respectively. We believed that if a number of antibacterial peptides are functionalized on nanoparticles, antibiotic activity can be enhanced owing to multivalent interactions. Thus, the goal of this work was aimed to develop nanoparticle-based antibiotics based on the use of antibacterial peptide functionalized gold nanoparticles. Additionally, we previously have discovered that a penta-peptide has good targeting capacity toward S. aureus and MRSA. In this study, a peptide contained arginine, tryptophan and the penta-peptides was functionalized on the surface of gold nanoparticles through one-pot reactions. It was demonstrated that the generated gold nanoparticles possess targeting capacity and antibacterial activity toward S. aureus and MRSA. Unexpectedly, the antibacterial NPs also possess binding affinity toward S. saprophyticus, Enterococcus faecalis, Enterococcus faecium, vancomycin-resistant E. faecalis, and vancomycin-resistant E. faecium. It may be due to the interactions between hydrophobic tryptophan and the hydrophobic functionalities on the cell surface of these bacteria. Furthermore, the growth of these bacteria could be inhibited when incubating with the antibacterial gold nanoparticles. Nevertheless, the results showed that the free form peptides used to functionalize gold nanoparticles in this study do not have apparent antibacterial activity. The antibiotic activity of the generated gold nanoparticles is mainly derived from the multi-valent interactions of the peptide on the gold nanoparticles with target bacteria. Macrophages have the tendency to internalize particle (<10 m) through phagocytosis. Thus, it is possible to introduce the antibacterial gold nanoparticles to bacterium-infected macrophages and investigate the antibacterial effectiveness of the gold nanoparticles in macrophages. MRSA infected macrophages were used as the model samples. The feasibility of using the antibacterial peptide-functionalized gold nanoparticles to inhibit the cell growth of MRSA in macrophages was successfully demonstrated. In conclusion, functional gold nanoparticles that possess antibacterial activity have been successfully generated in this study. It has been demonstrated the feasibility of using the antibacterial gold nanoparticles as antibiotics in inhibition of the growth of bacteria in vitro and in bacterium-infected macrophages. It may have the potential to use the generated functional gold nanoparticles in medical uses.