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標題: | 利用微乳液系統合成奈米空心球之生化應用 Protein Encapsulated Hollow Silica Nanospheres Templated by Reverse Microemulsion |
作者: | Jen-Hsuan Chang 張仁瑄 |
指導教授: | 牟中原(Chung-Yuan Mou) |
關鍵字: | 軟模板製備法,反微乳液系統,奈米二氧化矽球,小角X 光散射儀,超氧化物歧化酶,活性氧化物,海拉細胞, hollow silica nanospheres,reverse microemulsion,SAXS,BimodelSchulzSpheres model,superoxide dismutase,reactive oxygen species,HeLa cells, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 奈米/微米尺度的空心材料,近幾年受到廣泛的研究及開發,其主要原因是該材料可用於物質分離、藥物輸送以及催化反應等相關應用。本實驗中所利用的奈米空心球製備法,屬於軟模板製備法,使用環己烷作為系統油相、不帶電的 CA-520 為界面活性劑、水,三者混和後於溶液中形成油包水的反微乳液系統。TEOS (tetraethylorthosilicate)、有機矽烷 (amino-propyltrimethoxy silane, APTMS) 為矽源,氨水作為催化劑,經由水解、縮合反應,於軟模板中形成奈米二氧化矽球,藉由簡單的去離子水洗滌奈米矽球而得到均勻的奈米空心球。然而,奈米空心二氧化矽球的形成機制在過往的文獻中並無完整且深入的探討,因此本實驗主要著重於其空心二氧化矽球的形成機制以及該材料在生化領域上的應用。
於本研究第一部分,我們使用同步輻射所提供的小角 X 光散射儀,探討奈米球於反微胞系統中形成機制。從實驗結果光譜圖中及擬合之數據結果推論奈米球之形成機制為成核-成長的機制。透過元素分析儀以及固態核磁共振儀了解奈米球經過水洗滌後形成空心球,水洗滌前後的結構以及元素成分,進一步推論奈米球水洗滌前後,形成奈米空心球的反應機制。了解到水洗滌過程中,實心球內的矽聚合物被水洗滌出,而形成奈米二氧化矽空心球。 本研究的第二部分,嘗試使用反微胞系統合成之奈米空心球成功包覆超氧化物歧化酶 (SOD@HSNs)。為了證實 SOD 確實包覆於空心球中,我們將 SOD 鍵結螢光分子 (SOD-RITC),利用鹽酸溶液水洗吸附於空心球的 SOD-RITC 以及 SOD-RITC@HSNs,並且透過螢光分析儀分析 SOD-RITC 水洗後的螢光強度,亦算出 SOD 的包覆效率,約為 1.25%。不僅如此,透過負染分析,於電子顯微鏡下觀察到 SOD 被包覆於空心球中。接著,活性鑑定結果實驗顯示,SOD 包覆於空心球後,與未包覆的 SOD 比較,剩餘約 25% 之活性。雖然包覆 SOD 過程中會損失部分活性,但更重要的是,透過活性分析,證明奈米二氧化矽殼確實可以用來保護 SOD,免於胰蛋白酶及尿素分子的降解。最後,我們想了解 SOD@HSNs 於細胞中是否能協助細胞清除細胞中的活性氧, 而回升細胞生存率。因此將 SOD@HSNs 進行細胞實驗,而選用的細胞為海拉細胞。透過共軛交顯微鏡與流式細胞儀分析,觀察到海拉細胞在短時間內可以吞噬大量 SOD@HSNs。在細胞的應用端,細胞毒性鑑定實驗顯示海拉細胞對於 SOD@HSNs 無明顯細胞毒性反應,亦不影響細胞增殖。 最後,我們對於 SOD@HSNs 於細胞中是否可以清除活性氧化物相當感興趣。於實驗中餵食海拉細胞 100 μg/mL 的 SOD@HSNs,再將海拉細胞浸泡於 500 毫莫耳的巴拉刈,24 小時候進行細胞毒性分析,發現 SOD@HSNs 保護約 12% 的 海拉細胞免於活性氧的破壞而凋亡。 本研究成功利用微乳液系統合成奈米矽球,並且在合成過程中包覆 SOD 酵素,並且成功送入海拉細胞,保護海拉細胞免於活性氧的凋亡。在過去的文獻中,使用奈米矽球包覆酵素送入細胞進行蛋白治療的研究很少見,未來期待將此系統應用於包覆兩種以上生物分子或酵素,進行連續性反應。 Traditionally hollow silica nanospheres (HSNs) are prepared through coating the targeting materials over the templates, then the templates are removed, and the HSNs are obtained. However, the synthetic procedures were complicated and difficult to reduce the particle size to 100 nm. Uniform hollow silica nanospheres have synthesized by water-in-oil reverse microemulsion (w/o) method. However, the formation machenism has not been studied. In the first part, we studied the formation mechanism of HSNs with time-resolved small-angle X-ray scattering (SAXS) from National Synchrotron Radiation Research Center (NSRRC). The BimodelSchulzSpheres model was used to fit our SAXS data, and the nucleation-growth mechanism was proposed for the formation of the hollow nanoparticles. Moreover, elemental analysis and solid state nuclear magnetic resonance were used to identify the major substance which was washed out during the formation of HSNs. The encapsulation of enzymes in biocompatible silica nanoparticles has been studied extensively for many applications. In the second part, superoxide dismutase (SOD) was encapsulated into HSNs (SOD@HSNs) by water-in-oil microemulsion system. In order to quantify the loading efficiency of SOD@HSNs, SOD was conjugated with RITC and the amount of entrapped SOD-RITC was determined by the fluorescence intensity via a calibration curve. Comparison of desorption and leakage behavior of SOD-RITC adsorbed on HSNs and SOD-RITC@HSNs and negative stain analysis were utilized to prove that SOD was indeed inside HSNs. Then, the enzymatic activity of SOD@HSNs was determined to be about 25% of native SOD. Furthermore, we demonstrated that the silica shell could protect SOD from denaturants, such as trypsin and urea. Because SOD is an antioxidant, we would test the SOD@HSN as an antioxidant to rescue cells from reactive oxygen species (ROS) stress. The flow cytometry and confocal microscopy analysis showed that SOD@FITC-HSNs internalized HeLa cells with high efficiency.The SOD@FITC-HSNs were highly biocompatible by cell viability and long-term cellular proliferation examinations. Finally, the cellular antioxidant activity of SOD@HSNs was demonstrated. Delivery of 100 μg/mL SOD@HSN into HeLa cells could protect ~12% HeLa cells from death after challenged with 500 mM paraquat, a ROS producer. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17426 |
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