- 學位論文
奈米銀-甲殼素複合材料之創新合成技術研發及其應用
Novel Synthesis and Application of Silver Nanoparticles-Chitosan Composite Particles
摘要
奈米銀粒子已被廣泛應用於奈米科技、生物科技、醫療產業等領域之上。為了降低奈米銀對人體的潛在毒性，近年來已有多種合成方法被研發出來。其中，奈米銀粒子合成產品的穩定性及分佈性，是合成技術的重要參數。本實驗室研發出兩種合成系統，以製備不同尺寸的奈米銀-甲殼素複合微球粒子。於第一套合成系統中，我們研發出幫浦驅動針滴法，藉由單一合成步驟，即能將奈米銀粒子包埋於甲殼素基質之中。此方法能夠同步且同位地推動製備與穩定化的程序，而成功合成出奈米銀粒子-甲殼素複合微球體。運用幫浦驅動針滴法合成出的奈米銀粒子-甲殼素複合微球體之直徑經測量介於1.7~2.5 mm間，而包埋其中的奈米銀粒子直徑介於15±3.3 nm奈米間。此外，我們將製備出的複合微球藉由紫外光/可見光分光光度計、傅立葉轉換紅外線分光光度計、X光繞射儀、能量分散式分光光度計、掃描式電子顯微鏡、穿透式電子顯微鏡等儀器進行定性檢測，結果顯示奈米銀粒子均勻分布於甲殼素基質之中，而且微球體具有良好之多孔洞材質特性，能供多種領域應用所需。我們將此微球產品進行抗真菌試驗，結果顯示其對蛹蟲草真菌(Cordyceps militaris)具有顯著抑制功效，但是對牛樟芝真菌(Antrodia cinnamomea)則無作用。牛樟芝真菌是台灣重要的經濟作物，由於此微球產品具有抑制其它微生物之功效，或許日後可應用於牛樟芝的栽培上。於第二套合成系統中，我們研發出以聚甲基丙烯酸甲酯(Polymethyl methacrylate)製造的微流道晶片系統，而成功合成出更小的奈米銀粒子-甲殼素複合微球體，其直徑位於262~558 μm的微米等級。複合微球體的尺寸可藉由調整微流道設備的參數來控制，包括：甲殼素濃度、氫氧化鈉濃度、連續相流速、分散相流速等。我們將製備出的微球體運用紫外光/可見光分光光度計、傅立葉轉換紅外線分光光度計、能量分散式分光光度計、掃描式電子顯微鏡等儀器進行定性檢測，結果顯示奈米銀粒子均勻分布在甲殼素基質微球體粒子之中，且具有良好之多孔洞材質特性。此外，為了試驗此微球體對人類細胞是否具有毒性，我們使用NIH-3T3及MCF-7兩種細胞株進行MTT assay細胞毒性試驗，結果顯示即使微球體濃度高達1000 μg/mL，皆未出現可觀察到的細胞毒性。我們也以大腸桿菌（Escherichia coli）進行抗細菌試驗，結果顯示奈米銀-甲殼素微球具有與濃度呈正相關的抗菌能力。整體而言，我們成功研發出兩套能夠製備穩定且均勻的奈米銀粒子-甲殼素複合微球體的合成系統，且證明其合成產品具有良好的生物相容性、無毒性、抗細菌活性、抗真菌活性。我們的製備方法與合成產品可作為研發抗微生物試劑之參考，以期將來發展出在醫學及其他領域上之實質應用。
並列摘要
Silver nanoparticles (AgNPs) have been widely applied in various fields including nanotechnology, biotechnology, and medical industry. In order to reduce the AgNPs potential toxicity for human body, various synthesis strategies have been developed in recent years. The stability and distribution characteristics of the synthesized AgNPs are two crucial parameters in synthetic techniques. In our study, we established two synthetic systems to fabricate AgNPs-embedded chitosan particles with different size scales. In the first system, a novel approach for the one-step synthesis of AgNPs-embedded chitosan particles was proposed by using the pump-driven syringe method. The method was planned to simultaneously obtain and stabilize AgNPs in chitosan polymer matrix in situ, and the product was called silver nanoparticles-chitosan composite particles (Ag@chitosan). The diameters of the synthesized Ag@chitosan spheres ranged from 1.7 to 2.5 mm, and those of embedded AgNPs were measured to be 15±3.3 nm. Furthermore, we utilized various instruments to analyze the characteristics of the prepared Ag@chitosan spheres including the ultraviolet-visible absorbance spectrophotometer (UV-Vis), Fourier transform infrared spectrometer (FTIR), X-ray diffractometer (XRD), energy dispersive spectroscopy (EDS), scanning electron microscope (SEM), and transmission electron microscope (TEM). The results showed that the AgNPs were homogeneously distributed over the chitosan sphere, and these synthesized spheres possessed porous structure that could be utilized in multiple fields. Then, the antifungal effect of the Ag@chitosan spheres was evaluated, and it was found that they could inhibit the growth of Cordyceps militaris (Cm) but not Antrodia cinnamomea (Ac). Ac is an important cash crop with highly economic value in Taiwan, and our products might be an option to become a beneficial additive for cultivating Ac by inhibiting other fungi and bacteria. In the second system, we developed the microfluidic chip device made by Polymethyl methacrylate (PMMA) to synthesize the Ag@chitosan spheres with micrometer scale. The diameters of the Ag@chitosan spheres synthesized by the microfluidic method ranged from 262 to 558 μm, and their sizes could be controlled by altering the concentration of chitosan, concentration of NaOH, flow rate of continuous phase, and flow rate of dispersed phase. Moreover, we utilized various instruments to analyze the characteristics of the prepared Ag@chitosan spheres including the UV-Vis spectrophotometer, FTIR spectrometer, EDS, and SEM. The results showed that the AgNPs were also homogeneously distributed and the sphere structure was also porous as expected. In order to assess the possible toxicity of Ag@chitosan for human cells, we executed the MTT assay on both NIH-3T3 and MCF-7 cell lines. The results demonstrated that there was no observable toxicity even if the concentration of Ag@chitosan up to 1000μg/mL. Furthermore, the antibacterial effect of Ag@chitosan was evaluated by using Escherichia coli (E. Coli) broths, and the results showed that E. Coli growth could be inhibited by Ag@chitosan with dose-dependent relationship. Generally speaking, we have established two novel systems to synthesize homogeneous and stable AgNPs embedded in the chitosan particles successfully, and have confirmed the product to be highly biocompatible, non-toxic, antibacterial, and antifungal. Our synthetic systems and the synthesized products will provide a novel option of the antimicrobial research, and may be utilized in medicine and other fields in the future.