利用玫瑰紅鍵結之甲殼素奈米銀粒子在發光二極體光照射下做為抗菌劑

Rose bengal-conjugated chitosan-Ag nanoparticles as antibacterial agents under LED light exposure

李佳蓉(Chia-Jung Li)

指導教授 ： 謝之真

國立臺灣大學/工學院/化學工程學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.10511

摘要

由於抗生素的濫用導致許多微生物對抗生素產生抗藥性已成為一個嚴重的問題，找出新的抗微生物療法實為當務之急。本研究中利用具光動力效應之光感物質玫瑰紅(Rose bengal)光感物質鍵結生物高分子─甲殼素之奈米銀粒子在可見光照射下做為抗菌劑。我們使用三個不同玫瑰紅接枝量的甲殼素做為奈米銀粒子製作過程中的保護劑。動態光散射分析顯平均粒徑為7.67 nm。
本研究利用大腸桿菌在添加玫瑰紅鍵結之甲殼素奈米銀粒子後在波長為540nm的發光二極體(LED)照射後培養並檢測菌液混濁度來觀察大腸桿菌的生長情況，以驗證玫瑰紅鍵結之甲殼素奈米銀粒子之抗菌效益。在發光二極體照射後使用singlet oxygen sensor green (SOSG) reagent偵測單態氧的產生。在發光二極體照射後玫瑰紅鍵結之甲殼素奈米銀粒子產生的單態氧比起相同濃度的玫瑰紅增強了約1.4倍，證實了金屬增強單態氧產生。實驗結果發現玫瑰紅鍵結之甲殼素奈米銀粒子的濃度增加以及較多玫瑰紅接枝量的之甲殼素奈米銀粒子，會導致更多單態氧產生。由於沒有發光二極體照射下的玫瑰紅鍵結之甲殼素奈米銀粒子之抗菌效益與奈米銀粒子大致相同，我們推斷單態氧的生長可提高玫瑰紅鍵結之甲殼素奈米銀粒子的抗菌能力。在26 mW/cm2 的發光二極體照射615秒(光劑量16 J/cm2)後，100 μg/ml的玫瑰紅鍵結之甲殼素奈米銀粒子能抑制大腸桿菌的生長到一定程度，而200 μg/ml的玫瑰紅鍵結之甲殼素奈米銀粒子可完全抑制抑制大腸桿菌的生長。這些結果顯示，在發光二極體照射下的玫瑰紅鍵結之甲殼素奈米銀粒子的抑菌效果來自於光動力之單態氧產生與奈米銀粒子之抑菌能力的結合。

關鍵字

奈米銀粒子 ； 甲殼素 ； 玫瑰紅 ； 光動力殺菌 ； 抗菌劑 ； 金屬增強單態氧產生

並列摘要

The evolving of new resistant strains of bacteria to current antibiotics has become a serious problem; therefore, there is a strong incentive to develop new anti-microbial strategies for reducing microbial contamination. In this study, a light-activated antimicrobial agent tagged with a biopolymer was used to incorporate with silver nanoparticles (Ag NPs) as a potential antimicrobial approach when exposed to visible light. First, chitosan (CS) was conjugated with rose bengal (RB), a well-known photosensitizer for photodynamic therapy (PDT). Then RB-CS was further used as the stabilizing agent in the synthesis of Ag NP via chemical reduction process. Three different amounts of RB grafted on chitosan (0.0895, 0.1937, 0.7118 μg RB/mg chitosan) were prepared for the fabrication of Ag NPs. Since the absorption peak of RB at 556 nm was much lower than the absorbance of Ag NPs, the UV-Vis absorption spectrum of RB-CS/Ag NPs didn’t reveal a significant peak at 556 nm. The result of dynamic light scattering analysis showed that RB-CS/Ag NPs had an average size of 7.67 nm.
The antibacterial acivity of the RB-CS/Ag NPs was tested against Gram-negative E. coli exposed with light emitting diode (LED) light irradiation. The effect of the RB-CS/Ag NPs on the growth of E. coli was investigated by monitoring culture turbidity. After LED light exposure, singlet oxygen sensor green (SOSG) reagent was employed to detect singlet oxygen generation. The amount of singlet oxygen generated from RB-CS/Ag NPs was ≈ 1.4 fold higher than the corresponding RB only produced. Metal enhanced singlet oxygen generation was observed. Our results revealed increasing concentration of RB-CS/Ag NPs led to more singlet oxygen generation, with a fixed concentration of RB-CS/Ag NP. The more RB was grafted, the more singlet oxygen was detected. Without LED irradiation, the antibacterial effect of RB-CS/Ag NPs and Ag NPs were about the same. It is speculated that the generation of singlet oxygen enhances the antibacterial activity of RB-CS/Ag NPs. Under 26 mW/cm2 LED light exposure for 615 sec (i.e., 16 J/cm2) , 100 μg/ml RB-CS/Ag NPs were capable of inactivating growth of E. coli to a certain extent. According to cell growth profiles, the lag phase of the treated bacteria exhibited a prolonged feature as compared to untreated bacteria. Growth was completely inhibited when bacteria were treated with 200 μg/ml RB-CS/Ag NPs. These results suggest under LED light irradiation RB-CS/Ag NPs can inhibit bacterial growth through the combined RB-associated singlet oxygen generation, Ag NP-induced inactivation and CS-triggered antimicrobial activity.

並列關鍵字

Silver nanoparticles ； chitosan ； rose bengal ； antimicrobial photodynamic therapy ； antibacterial agent ； metal enhanced singlet oxygen generation

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