玻尿酸奈米銀粒子之合成及其抗菌與抑制黑色素瘤細胞之功效

The Synthesis, Antibacterial Activities and the Inhibition of Human Malignant Melanoma Cells of Hyaluronic Acid Silver Nanoparticles

呂傑堯(Chieh-Yao Lu)

指導教授 ： 施養佳 ； 鄧金培 ； 張培均

亞洲大學/健康學院/生物科技學系碩士班/碩士(2011年)

摘要

近幾年來，金屬奈米粒子的合成是熱門的研究方向，而常被合成之金屬奈米粒子包括: 奈米金、奈米銀及奈米鐵粒子等。特別是銀離子，因具有良好的導電性、化學穩定性、催化和抗菌活性，所以常被製成銀奈米粒子，用在抗菌、除臭、抗發炎及促進傷口癒合等方面。而製備奈米粒子的方法很多，其中化學還原法是最常被使用的方法。化學還原法製備良好奈米粒子最重要的三項條件是:保護劑、還原劑與金屬鹽類的濃度。本研究所使用的保護劑為玻尿酸(HA)，還原劑為硼氫化鈉(NaBH4)，金屬鹽類為硝酸銀(AgNO3)。玻尿酸為脊椎動物普遍存在的生物性高分子。因為玻尿酸在結構上富含OH基團及帶大量負電，所以具強效的保濕性並且可以降低活性氧化物(ROS)對細胞造成傷害。玻尿酸之性質隨著分子量的不同也有所改變，因此普遍應用於生物醫學、藥物遞送系統及化妝保養品方面。本研究之主要目的為合成玻尿酸奈米銀粒子及探討其生物功能性之應用潛力。本研究分為三個部分進行: (1)合成玻尿酸奈米銀粒子，並探討還原劑濃度、玻尿酸濃度及硝酸銀濃度等最佳合成條件，亦比較其物理性質包含:穩定性、粒徑及光學特性。(2)評估玻尿酸奈米銀粒子之抗菌活性，探討玻尿酸奈米銀粒子是否具有抑制大腸桿菌與表皮金黃色葡萄球菌之能力。 (3)評估玻尿酸奈米銀粒子是否具抑制人類黑色素瘤細胞之功能。第一部分之研究結果顯示，可以利用還原劑硼氫化鈉調控玻尿酸奈米銀粒子的粒徑大小。而還原劑濃度最佳條件為0.002M時，可合成粒徑大小約為15nm且吸光值為398nm之玻尿酸奈米銀粒子。若將還原劑濃度調高為0.01M時候，粒徑大小約為21nm且吸光值為404nm之玻尿酸奈米銀粒子。在第二部分的結果顯示，當AgNO3濃度為0.26mg/ml、HA濃度為0.05 mg/ml 及NaBH4為0.002M時，玻尿酸奈米銀粒子為抑制大腸桿菌及表皮金黃色葡萄球菌之最佳合成條件，其最大抑菌圈可達14mm。更進一步的抑菌實驗結果顯示，當玻尿酸奈米銀粒子之AgNO3濃度稀釋為45µg/ml時，已經能完全抑制表皮金黃色葡萄球菌及大腸桿菌之生長。第三部分之研究目的為評估玻尿酸奈米銀粒子抑制黑色素瘤細胞之功效。本實驗以固定AgNO3及NaBH4之合成濃度，但改變不同之玻尿酸的濃度進行合成，再以AgNO3濃度(0.26mg/ml)為標準，將實驗樣本分別稀釋成5種不同濃度並與黑色素瘤細胞進行24、48及72小時培養。實驗結果顯示，當玻尿酸奈米銀粒子之AgNO3濃度稀釋為8 µg/ml時，其抑制率可達到90%。隨著培養時間越長抑制率也跟著提高，在72小時幾乎已經完全抑制黑色素瘤細胞之生長。綜合以上實驗結果證明，玻尿酸奈米銀粒子的確具有抑制腐敗菌與黑色素瘤細胞生長之功效。未來還可繼續將此玻尿酸奈米銀粒子應用於其他皮膚病原菌或不同的腫瘤細胞進行更深入之研究。由於玻尿酸奈米銀粒子之抗菌效果比單純的銀離子抑菌效果更好，所以若將玻尿酸奈米銀粒子應用於開發具抗菌能力之生醫材料上，未來應具有高度商品化之潛力及產業競爭力。

關鍵字

玻尿酸 ； 奈米銀粒子 ； 抗菌活性 ； 黑色素瘤細胞

並列摘要

In recent years, the synthesis of metal nanoparticles is a popular research field. The metals are often used including: gold-nano, silver-nano, iron-nano particles. In particular silver ions, due to they have good electrical conductivity, chemical stability, catalytic and antibacterial activity, so they are often made of silver nanoparticles. They can be used in anti-bacterial, deodorizing, anti-inflammatory and promote wound healing and so on. There are many different methods for the preparation of nanoparticles, in which chemical reduction method is the most commonly used method. The most important three conditions for the preparation of nanoparticles by chemical reduction method include: protective agent, reducing agent and metal salt concentrations. Protective agent used in this study was hyaluronic acid (HA); reducing agent was sodium borohydride (NaBH4) and metal salts of silver nitrate (AgNO3). Hyaluronic acid is common biopolymers in vertebrate. Because the structure of HA is rich in OH group and it carries a large number of negative charged. The moisture effect of HA is very strong and it can reduce the activity of oxide (ROS) of cell damage. The characteristics of HA depends on the different molecular weight, so HA is widely used in biomedical, drug delivery and cosmetic products. The main purpose of this study is to synthesize HA-Ag nanoparticles and to study their biological effects and potential applications. This study is divided into three parts: (1) To synthesize HA-Ag nanoparticles and to investigate the concentration of reducing agent, hyaluronic acid and silver nitrate concentration to find out the best synthesis conditions. The physical properties of HA-Ag nanoparticles included: stability, particle size and optical characteristics were also been analyzed. (2) To evaluate the antimicrobial activity of HA-Ag nanoparticles to E. coli and Staphylococcus epidermidi. (3) To evaluate the inhibition effects of HA-Ag nanoparticles to human malignant melanoma cells. The first part of the study showed that reducing agent (NaBH4) was able to control the size of HA-Ag nanoparticles. Its optimum concentration of NaBH4 was 0.002M, which produced the 15nm size, 398nm of absorption of HA-Ag nanoparticles. If the concentration of reducing agent increased to 0.01M, and the HA-Ag nanoparticles sized up to 21nm and the absorbance value shifted to 404nm. In the second part of experiments, 0.26mg/ml of AgNO3, 0.05 mg/ml of HA and 0.002M of NaBH4 were the best condition for the synthesis of HA-Ag nanoparticles to inhibit E. coli and S. epidermidi, and the largest inhibition diameter was 14mm. The results of the inhibition effects of HA-Ag nanoparticles by the second method indicated that both pathogens were totally inhibited by the nanoparticles which concentration was diluted to 45 μg/ml of AgNO3. The third part of this experiment was to evaluate the inhibition effect of HA-Ag nanoparticles to human malignant melanoma cells. The concentration of AgNO3 and NaBH4 were fixed and prepared with two different HA concentrations to produce HA-Ag nanoparticles as stock. The stock solution was then diluted into 5 different concentrations to treat cells for 24, 48 and 72 hours. The results indicated that the dilution of 8 μg/ml of AgNO3, the inhibition rate of human malignant melanoma cells reached 90%. The inhibition rate increased along with the raise of cultivation time, and the melanoma cells almost died after 72 hours. In summary, according to the above results, they were provide the strong evidences for the inhibition effects of HA-Ag nanoparticles to pathogens and human malignant melanoma cells. In the future, HA-Ag nanoparticles can be used to treat different pathogens or tumor cells for further investigation. Due to HA-Ag nanoparticles have better inhibition ability then Ag+, they can be applied in the development of anit-bacterial biomaterial products. In the future, HA-Ag nanoparticles must have the high potential commercialization and the strong industrial competitiveness.

並列關鍵字

Hyaluronic acid ； Silver nanoparticles ； Antibacterial Activities ； Human Malignant Melanoma Cells

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