臺灣博碩士論文加值系統

合成多功能性高分子材料的新概念，透過兩親性自組裝能有效生成單鏈高分子奈米粒子（SCNPs），因此在模擬蛋白質折疊結構方面取得重大的進展。在本論文中，我們開發一巨分子量兩親性接枝共聚合物，其在水溶液或有機溶劑中能進行自發性自組裝，形成直徑約為10 - 15 nm尺度均勻且穩定的SCNPs。SCNPs具有許多獨特的物理性質，包括特異的兩親特性、極低的黏度、良好的生物相容性及在血清存在下具有高度的結構穩定性，並可調節SCNPs的藥物負載量和粒徑。更重要的是，體外實驗證實裝載藥物的SCNPs能通過內吞作用被腫瘤細胞內化，進而逐步轉移到細胞核，表明這種獨特的SCNPs可以安全有效地應用於靶向藥物傳輸系統。

A new concept in synthesis of multifunctional polymeric materials has enabled significant progress in mimicking the folded structures of proteins by the efficient generation of single-chain polymer nanoparticles （SCNPs） via amphiphilic self-assembly. In this thesis, we developed an ultra-high-molecular-weight amphiphilic graft copolymer, which undergoes spontaneous self-assembly in aqueous solution or organic solvents to form uniform, stable SCNPs with a diameter approximately 10−15 nm. The resulting SCNPs possess a number of unique physical properties, including unique amphiphilic property, extremely low viscosity, good biocompatibility and high micellar stability in the presence of serum, as well as tunable drug-loading capacity and particle size. More importantly, in vitro experiments demonstrated that drug-loaded SCNPs were internalized by tumor cells through endocytosis, then subsequently progressively translocated to the nucleus, indicating that this unique SCNP system may serve as a safe and effective nanocarrier for targeted intracellular drug delivery.

摘要 I
Abstract II
致謝 III
目錄 IV
圖目錄 IX
表目錄 XVI
縮減表 XVII
第一章 緒論 1
1.1研究背景 1
1.2研究動機 5
第二章 文獻回顧 7
2.1點擊化學（Click Chemistry） 7
2.2縮水甘油基疊氮化聚合物（Glycidyl azide polymers，GAPs） 9
2.3聚乙二醇（Polyethylene glycol，PEG） 10
2.4高分子奈米載體藥物傳輸系統（Polymeric nanoparticles Drug delivery system） 12
2.5兩親接枝共聚高分子（Amphiphilic Graft Copolymer） 14
2.6單鏈高分子奈米粒子 （Single Chain Polymer Nanoparticles,SCNPs） 16
2.6.1 單鏈高分子粒子系統概述 16
2.6.2 單鏈高分子粒子之鑑定方法 19
2.6.3 單鏈高分子粒子未來展望 22
2.7阿黴素（Doxorubicin，DOX） 23
2.8葉酸對於腫瘤細胞的標靶特性 （Target Treatment Characteristics Of Folic Acid） 24
2.9文獻回顧總結 26
第三章 實驗材料與方法 27
3.1研究設計 27
3.2實驗材料 28
3.2.1實驗藥品 28
3.2.2實驗溶劑 32
3.2.3細胞實驗材料 34
3.3實驗儀器與設備參數 37
3.3.1旋轉塗佈機（Spin Coaters） 37
3.3.2桌上型酸鹼度計（pH Meter） 37
3.3.3酵素免疫分析儀（ELISA Reader） 38
3.3.4 CO2培養箱（CO2 incubators） 38
3.3.5冷凍離心機（Refrigerated Centrifuge） 39
3.3.6斜式旋轉濃縮機（Rotary Evaporation） 39
3.3.7流式細胞分選儀（Flow Cytometers） 39
3.3.8振盪混合器（Vortex Mixer） 40
3.3.9光致螢光光譜儀（Photoluminescence， PL） 40
3.3.10熱重分析儀（Thermogravimetric analysis，TGA） 42
3.3.11紫外線光譜儀（UV/VIis spectrophotometer ，UV/Vis） 42
3.3.12凝膠滲透層析儀（Gel permeation chromatography，GPC） 43
3.3.13螢光顯微鏡（Fluorescence microscope） 44
3.3.14原子力顯微鏡（Atomic Force Microscpoic，AFM） 44
3.3.15動態光散射儀（Dynamic Light Scattering，DLS） 45
3.3.16差示掃描量熱儀（Differential scanning calorimetry，DSC） 46
3.3.17高解析度場發射掃描式電子顯微鏡（Scanning Electron Microscope） 46
3.3.18傅里葉轉換紅外光譜（Fourier transform infrared spectroscopy） 47
3.3.19液態核磁共振光譜（Nuclear Magnetic Resonance Spectrometer，NMR） 48
3.3.20旋轉流變儀（Rehometer） 49
3.4實驗合成步驟 50
3.4.1合成Polyepichlorohydrin- Azide（PECH - N3） 50
3.4.2合成 Alkyne-functionalized polyethylene glycol（PEG - Alkyne） 51
3.4.3合成 Alkyne-functionalized folic acid （Propargyl Folate） 52
3.4.4合成 Polyepichlorohydrin - Polyethylene glycol（PECH - PEG） 、Folate-Polyepichlorohydrin-Polyethylene glycol （Folate-PECH-PEG） 54
3.5樣品製備 56
3.5.1微胞製備 56
3.5.2 DOX微胞（PECH - PEG / DOX）製備 56
3.5.3臨界聚集濃度（Critical Aggregate Concentration，CAC） 57
3.5.4 DOX藥物濃度檢量線（DOX Drug Concentration calibration curve） 57
3.5.5 DOX包覆能力（Drug Loading Capability） 58
3.5.6體外藥物釋放模擬 58
3.5.7微胞穩定性測試 59
3.6細胞生物性製備 60
3.6.1細胞培養基（Dulbecco's Modified Eagle Medium，DMEM） 60
3.6.2細胞解凍培養 60
3.6.3細胞繼代 60
3.6.4染色與數細胞 61
3.6.5細胞生物毒性測試 62
3.6.6螢光顯微鏡製備 62
3.6.7製備1X Annexin-binding buffer 63
3.6.8製備100 µg/mL Propidium iodide（PI） 63
3.6.9流式細胞儀（Flow cytometer） 63
第四章 結果與討論 65
4.1 材料鑑定 65
4.1.1 傅里葉轉換紅外光譜（Fourier - Transform Infrared Spectroscopy ,FTIR） 67
4.1.2 核磁共振氫譜1H NMR 69
4.1.3 凝膠滲透層析儀（Gel Permeation Chromatography,GPC） 74
4.1.4 熱重分析儀 ( Thermogravimetric analysis，TGA ) 76
4.1.5 熱差式分析儀 ( Differential scanning calorimetry，DSC ) 78
4.2 材料性質分析 82
4.2.1 高分子鏈段在有機溶劑下核磁共振氫譜1H NMR的運動 82
4.2.2 高分子鏈段在有機溶劑下的黏度（流變儀） 89
4.2.3 凝膠滲透層析儀GPC 94
4.2.4 THF有機溶劑下之高分子粒徑分析 96
4.2.5 PECH-PEG在非選擇性有機溶劑中的總結探討 98
4.3高分子於水溶液中性質分析 100
4.3.1高分子於重水中核磁共振氫譜結果 100
4.3.2高分子水溶液黏度性質（流變儀） 105
4.3.3水相凝膠滲透層析儀GPC 108
4.3.4 臨界聚集濃度Critical Aggregation Concentration 111
4.3.5 粒徑分析（DLS）、微胞形貌（AFM、SEM） 113
4.3.6 微胞穩定性分析 116
4.4藥物微胞分析 118
4.4.1藥物包覆率與粒徑分布 118
4.4.2掃描電子顯微鏡SEM、原子力顯微鏡AFM 122
4.4.3藥物微胞穩定性分析 125
4.4.4體外藥物釋放Drug release in vitro 127
4.5 葉酸靶向載體F-PECH-PEG材料鑑定 129
4.5.1 靶向載體F-PECH-PEG合成及結構鑑定 129
4.5.2 靶向載體F-PECH-PEG微胞性質測定 133
4.5.3 靶向藥物載體F-PECH-PEG/DOX性質測定 136
4.6 生物體外細胞實驗In Vitro 139
4.6.1 細胞毒性測試Cytotoxicity test 139
4.6.2 藥物毒殺實驗Cell Viability 141
4.6.3 流式細胞儀 （Flow cytometry） 143
4.6.4螢光顯微鏡CLSM 146
4.6.5流式細胞儀凋亡實驗 150
4.7 模擬腫瘤環境之生物體外細胞實驗In Vitro 154
4.7.1模擬腫瘤組織酸性環境細胞存活率實驗 154
4.7.2模擬腫瘤組織酸性環境下流式細胞儀實驗 156
4.7.3模擬腫瘤組織酸性環境下螢光顯微鏡結果 158
4.7.4模擬腫瘤組織酸性環境下流式細胞儀之細胞凋亡實驗 161
第五章 結論 163
第六章 未來展望 164
第七章 參考文獻 165
附件 176

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