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研究生(外文):Hsin-Yi Wang
論文名稱(外文):Development of functionalized nanoparticles with pH-responsive dextran-based shells for doxorubicin delivery and controlled release
指導教授(外文):Wen-Hsuan Chiang
口試委員(外文):Shang-Shiu HuChih-Kang Peng
外文關鍵詞:imidazolenanoparticlepH responsiveDextran
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本研究為了開發具備pH應答功能的奈米微粒作為化療藥物doxorubicin (DOX)的傳輸載體,我們將硬脂酸(stearic acid)及組胺酸衍生物N-acetyl-histidine經酯化反應共價鍵結於親水性葡聚醣(dextran)鏈段上得到雙性dextran-stearic acid-histidine (Dex-SA-His)高分子。雙性Dex-SA-His與疏水poly(lactic-co-glycolic acid)鏈段及DOX分子於水溶液經由自組裝排列可形成載藥奈米複合微粒,其疏水核心與親水殼層分別由PLGA及Dex-SA-His鏈段所構成。動態光散射及穿透式電子顯微鏡量測結果分別說明載藥複合微粒之水力直徑約96 nm且呈現球形結構。憑藉富含dextran的親水殼層及紮實緻密的PLGA核心,載藥複合微粒在含有fetal bovine serum的磷酸鹽緩衝溶液中及大量稀釋作用下皆能保持良好分散的穩定微粒結構。隨著溶液pH值由7.4降低至5.0時,由於載藥複合微粒的殼層之histidine單元內imidazole官能基大幅質子化導致殼層中的正電荷大量增加,此舉促使殼層內的滲透壓增加而使水分子流入造成殼層膨潤結構漸趨鬆散,進而加速DOX分子向外釋放。體外細胞毒性實驗結果顯示,與不具有pH應答性的載藥奈米微粒相比,具有pH應答功能的載藥複合微粒能明顯毒殺人類乳癌細胞MCF-7 cells且具有較低的IC50值,其細胞毒殺能力近似游離DOX分子。上述實驗結果初步證明本計畫所開發具有pH值應答功能的奈米複合微粒可應用於DOX分子的傳輸及控制釋放。
In this study, we grafted hydrophilic dextran (Dextran) polymer with Stearic acid and N-Acetyl-Histidine to make it a pH-responsive amphiphilic polymer (Dex- SA-His ), the Dex-SA-His segments are covered on the surface of the hydrophobic PLGA core through the process of hydrophobic force to develop high biocompatibility, degradability and pH responsive drug delivery carrier. The results of the dynamic light scattering and transmission electron microscope show that the nanoparticles have a spherical structure, and the particle size is about 100 nm. In the acidic environment, the surface of the particles is partially positive charge due to the protonation of the imidazole functional group on N-Acetyl-Histidine, which promotes the external aqueous solution to penetrate into the particles easily. Zeta potential is used to analyze the surface charge of the particles under different pH environments. The surface of the particles shows a positive charge in an acidic environment, and the electrostatic repulsion force generated between the positive and positive charges loosens the structure results in swelling phenomenon. This carrier can encapsulate doxorubicin (DOX) by hydrophobic force, and DLE can reach more than 60%. Use dynamic light scattering instrument to measure the particle size change under different pH environments, it can be confirmed that the volume change rate of the drug carriers in an acid environment increases 48%. The drug release experiments show that when environmental pH reduced from 7.4 to 5.0, the dextran shell with pH responsive releases more drugs due to the loose structure, which is beneficial to increase the therapeutic effect. The protection of the polymer shell does indeed prevent the drug leaking in the physiological environment. The above experimental results show that the nanoparticles with pH response functioned are developed by this research indicate excellent potential in the drug delivery system of DOX.
摘要 i
Abstract ii
目錄 iii
表目錄 v
圖目錄 vi
一、研究動機 1
二、文獻回顧 2
2-1 腫瘤微環境 ( Tumor microenvironment ) 2
2-2 高滲透長滯留效應 (Enhanced permeability and retention (EPR) effect) 4
2-3 葡聚醣 ( Dextran ) 5
2-4 聚乳酸乙醇酸 ( poly(lactic-co-glycolic acid), PLGA ) 7
2-5 組胺酸 ( Histidine ) 8
2-6 富含葡聚醣之pH應答型高分子微胞 10
2-7 化療藥物 DOX介紹 12
三、實驗方法與分析 13
3-1 實驗儀器與設備 13
3-2 實驗藥品 14
3-3 Dex-SA與Dex-SA-His的合成及性質分析 15
3-3-1 DMSO 除水 15
3-3-2 Dex-SA合成 15
3-3-3 Dex-SA-His 合成 17
3-4 Dex-SA-His的化學組成鑑定 18
3-4-1 傅立葉轉紅外線光譜儀分析 18
3-5 載體製備與性質探討 19
3-5-1 奈米複合微粒載體製備 19
3-5-2 載藥複合微粒製備 20
3-5-3 動態光散射之奈米粒子結構分析 ( Dynamic Light Scattering, DLS) 21
3-5-4 Zeta potential分析 22
3-5-5 穿透式電子顯微鏡 (TEM) 23
3-6 載藥複合微粒之分析 24
3-6-1 藥物包覆含量之測定 24
3-6-2 製備DOX之檢量線 24
3-6-3 體外藥物釋放試驗 25
3-6-4 Free DOX、複合微粒與載藥複合微粒之體外細胞毒性試驗 27
四、結果與討論 30
4-1 Dex-SA與Dex-SA-His之基本性質檢測分析 30
4-1-1 FT-IR鑑定高分子官能基 30
4-1-2 利用1H-NMR測定高分子組成測定 31
4-2 複合微粒之性質及結構探討 32
4-2-1 複合微粒之粒徑及型態分析 32
4-2-2 複合微粒之pH應答結構變化 37
4-3 載藥奈米複合微粒的特性分析 40
4-3-1 載藥奈米複合微粒之pH應答結構變化 40
4-3-2 載藥複合微粒之結構穩定性分析 43
4-3-3 體外藥物釋放實驗 45
4-3-4 體外細胞毒性實驗 47
五、結論 49
六、參考文獻 50
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