Studies of magnetic chitosan/Fe3O4 micro/Nanoparticles for nattokinase delivery 劉佳瑜、
E-mail: [email protected]
ABSTRACT
In this study, chitosan/iron (II, III) oxide/ nattokinase micro/nano particles were produced by low-temperature spray-drying. The magnetic micro/nano particles were further study the transportation behavior between cells using an in vitro model of human oral squamous cell carcinoma (KOSC-3). In order to prepare micro/nano particles, three suspensions of 0.5、1、1.5％ (w/v) chitosan with iron oxide were first mixed with nattokinase and then spray-dried in a low temperature condition. The samples were analyzed by field emission scanning electron microscopy (FESEM). It was found that the size of all the particles were in the range of 767 nm to 2.1 μm. The cytotoxicity of micro/nano particles was determined by MTT assay. No observable toxicity was noted on KOSC-3 cells by incubation with chitosan/iron (II, III) oxide/ nattokinase micro/nano particles. In vitro studies performed on KOSC-3 cell showed a pronounced opening of the cell junctions obtained by transepithilial electrical resistance (TEER) assay. In the meantime, the medium in the outside of insert was taken and a QuantiProTM BCA assay kit was used to investigate the release and
transportation of nattokinase. The results indicated that the samples from multi-stage electromagnet collector had significantly improved transportation properties by the attraction of a magnet. However, the largest and smallest particles behave differently and that is worthy of further study.
Keywords : chitosan、Fe3O4、nattokinase、KOSC-3、drug release Table of Contents
封面內頁 簽名頁 授權書 iii 中文摘要 iv 英文摘要 v 誌謝 vi 目錄 vii 圖目錄 x 表目錄 xii 1. 緒論 1 2. 文獻回顧 2 2.1 藥物傳輸 簡介 2 2.1.1 藥物傳輸技術 2 2.1.2 藥物傳輸之途徑 3 2.1.3 奈米顆粒之藥物傳輸 3 2.1.4 藥物控制釋放 6 2.2 幾丁質與幾丁聚 醣簡介 7 2.2.1 幾丁質及其衍生物結構 7 2.2.2 幾丁聚醣藥物載體的特性 10 2.3 磁性材料 10 2.4 納豆激? 略 12 2.5 低溫噴霧 乾燥 15 3. 材料與方法 20 3.1 實驗架構 20 3.2 實驗材料 21 3.2.1 藥品 21 3.2.2 耗材 22 3.2.3 儀器設備 23 3.2.4 細胞株 24 3.2.5 培養基與試劑配製 25 3.3 幾丁聚醣/四氧化三鐵懸浮液之製備 29 3.4 低溫噴霧乾燥法製備磁性微奈米複合顆粒 29 3.5 產物 分析 32 3.5.1 場發射電子顯微鏡 (FESEM) 32 3.5.2 原子吸收光譜儀 (AAS) 32 3.5.3 傅立葉紅外線光譜儀 (FTIR) 32 3.5.4 酵素 活性測定 33 3.6 細胞分析 33 3.6.1 細胞活化 33 3.6.2 繼代培養 34 3.6.3 細胞毒性 (MTT) 試驗 34 3.6.4 細胞穿透電阻 (TEER) 試驗 35 3.6.5 釋放試驗 36 3.6.6 細胞存活率 (Trypan blue) 試驗 37 4. 結果與討論 38 4.1 磁性幾丁聚醣/四氧化三鐵/納豆激?
◎L奈米粒子製備 38 4.2 場發射電子顯微鏡 (FESEM) 觀察 40 4.3 原子吸收光譜儀 (AAS) 分析 48 4.4 傅立葉紅外線光譜儀 (FTIR) 分析 50 4.5 酵素活性測定 53 4.6 細胞培養 56 4.7 細胞毒性 (MTT) 試驗 58 4.8 細胞穿透電阻 (TEER) 試驗 61 4.9 釋放 試驗 64 4.10 細胞存活率 (Trypan blue) 試驗 67 5. 結論 70 參考文獻 72
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