臺灣博碩士論文加值系統

本研究致力於藥物傳輸系統的開發。利用油酸修飾幾丁聚醣 (oleoyl chitosan, OCS)包覆奈米磁性粒子(Fe3O4)和抗癌藥阿黴素(doxorubicin, DOX)。合成原理以三聚磷酸鹽(sodium tripolyphosphate, TPP) 作為OCS的離子交聯劑，讓DOX和Fe3O4侷限於高分子基材中，製備成奈米載體。載體表面OCS會再以化學鍵結方式進行葉酸 (Folic acid, FA)再修飾，使藥物載體對癌細胞更具專一性。
結果顯示奈米載體粒徑大小約為200~250 nm，為圓球型，最高達到96%的藥物包覆效率(encapsulation efficiency, EE)及40%藥物承載量(loading efficiency, LE)，且經由體外藥物釋放證實載體具有pH敏感性。載體在經過葉酸修飾後，並不影響EE與LE值。
在體外細胞毒性測試上，50% FA-DOX-MOCNP有良好抑制癌細胞的作用，其DOX濃度為17.25 μg/ml能讓癌細胞達到半數致死率(IC50)，分別為DOX-MOCNP的13%和DOX的15%。
在螢光顯微鏡觀察中，影像能反映出磁導作用，證實藥物載體標靶治療的可能性。載體被癌細胞吞噬後，Fe3O4會存在細胞質中，DOX則進入細胞核內，因此可推測細胞核內大量的DOX能讓癌細胞死亡。

We prepared magnetic oleoylchitosan nanoparticles (MOCNP) containing an anticancer drug (doxorubicin; DOX) based on ionic gelation process of OCS and sodium tripolyphosphate (TPP) in order to develop an efficient drug delivery system for cancer treatment.
MOCNP were 200~250 nm in size and with a spherical shape. The drug encapsulation efficiency was up to 90% with drug loading efficiency at 40%. In vitro release studies showed pH-dependent with rapid release at pH 5.5, whereas at pH 7.4 there was a sustained release after a burst release. After modification with folic acid (FA), EE and LE were not significantly different.
The anti-tumor effects of these drug-containing particles were examined using human glioblastoma cells (U87) in vitro and evaluated via MTT assay. MOCNP showed no cytotoxicity, but nanoparticles containing the anticancer drug showed higher antitumor effect when compared to free DOX.
The fluorescence imaging results indicated nanoparticles could be uptaken by U87 cells. These results suggested that nanoparticles may be a promising carrier for DOX delivery in cancer therapy. In addition, MOCNP may provide a therapeutic benefit by delivering drugs efficiently to magnetically targeted tumor tissues.

目錄
長庚大學博碩士論文著作授權書 III
致謝 IV
縮寫表 V
中文摘要 VI
Abstrate VII
目錄 VIII
圖目錄 XIII
表目錄 XVI
第一章 緒論 1
1.1 前言 1
1.2 研究動機與目的 2
第二章 文獻回顧 4
2.1 藥物傳輸系統的發展 4
2.1.1 標靶治療 5
2.1.2 控制釋放 7
2.1.3 藥物釋放動力學模式 14
2.2 核殼奈米粒子 20
2.2.1 奈米載體的製備方式 20
2.3 生物相容性材料 22
2.3.1 幾丁聚醣 23
2.3.2 疏水性幾丁聚醣 24
2.3.3 超順磁四氧化三鐵 25
2.4 阿黴素 (doxorubicin, DOX) 29
第三章 實驗器材與方法 31
3.1 實驗藥品 31
3.2 實驗設備 33
3.3 實驗架構 35
3.4 實驗方法 36
3.4.1 磁性奈米粒子製備- Fe3O4 36
3.4.2 幾丁聚醣接枝油酸 (oleolychitosan, OCS) 36
3.4.3 葉酸改質油酸修飾幾丁聚醣 37
3.4.4 奈米載體合成 38
3.4.5 螢光奈米載體合成 40
3.4.6 材料毒性測定 41
3.4.7 奈米載體於細胞内的作用 42
3.4.8 奈米載體之物化性分析 44
3.4.8.1 OPA 分析 44
3.4.8.2 FA 定量分析 46
3.4.8.3 藥物包覆率測定 47
3.4.8.4 體外藥物釋放測定 48
3.4.8.5 動態光散射分析 (DLS) 49
3.4.8.6 介面電位分析 (Zeta potential) 49
3.4.8.7 傅力葉轉換紅外線光譜儀分析 (FTIR) 49
3.4.8.8 穿透式電子顯微鏡分析 (TEM) 49
3.4.8.9 原子力顯微鏡分析 (AFM) 50
3.4.8.10 掃描式電子顯微鏡分析 (SEM)/能量散佈光譜儀 (EDS) 50
3.4.8.11 熱重量分析 (TGA) 51
3.4.8.12 X光繞射儀分析 (XRD) 51
3.4.8.13 超導量子干涉磁化儀分析 (SQUID) 51
3.4.8.14 感應偶合電漿放射光譜儀(ICP-OES) 52
3.4.8.15 細胞存活率分析 (MTT assay) 52
3.4.9 體內血液毒性測試 53
第四章 結果與討論 54
4.1 製備奈米載體之參數探討 54
4.1.1 OCS:TPP質量比對載體之影響 55
4.1.2 Fe3O4濃度對載體之影響 63
4.1.3 OCS濃度對載體之影響 70
4.1.4 油酸取代比率對載體之影響 76
4.1.4.1 OPA定量油酸取代比率 76
4.1.4.2 以不同油酸取代比率的OCS包覆藥物之結果探討 77
4.2 藥物載體之最佳化條件 82
4.3 磁性奈米載體之葉酸修飾 86
4.3.1 光譜定量葉酸接枝率 86
4.3.2 葉酸接枝率對載體之影響 87
4.4 最佳化磁性奈米載體之物性分析 95
4.4.1 載體形態之穿透式電子顯微鏡分析(TEM) 95
4.4.2 原子力顯微鏡分析 (AFM) 97
4.4.3 傅力葉轉換紅外線光譜儀分析 (FTIR) 99
4.4.4 磁性載體之XRD分析 101
4.4.5 磁性載體之SQUID分析 103
4.4.6 能量散佈光譜分析(EDS) 105
4.4.7 熱重量分析(TGA) 107
4.4.8 感應偶合電漿放射光譜儀(ICP-OES) 111
4.5 磁性奈米載體之體外細胞實驗 112
4.5.1 細胞毒殺測試 112
4.5.2 癌細胞吞噬情形 117
4.5.3 葉酸對癌細胞吞噬作用的影響 118
4.6 磁性奈米載體之體內血液毒性測試 121
第五章 結論 122
參考文獻 124

圖目錄
圖 2-1 EPR效應之示意圖 7
圖 2-2 葉酸修飾後載體其主動標靶和藥物傳遞之示意圖 7
圖 2-3 傳統藥物傳遞和控制釋放的釋放模式比較圖 9
圖 2-4 控制系統釋放機制示意圖 13
圖 2-6 奈米載體型式示意圖 20
圖 2-7 帶正電高分子和三聚磷酸鹽(TPP)離子交聯示意圖 22
圖 2-8 幾丁聚醣化學結構 23
圖 2-9 油酸改質幾丁聚醣的化學結構 24
圖 2-10 鐵磁性物質之磁滯曲線示意圖 25
圖 2-11 順磁性物質的磁滯曲線示意圖 26
圖 2-12 保護劑對飽和磁化率的影響 27
圖 2-13 阿黴素和道諾黴素的化學結構 29
圖 2-14 嵌入作用使DNA長鏈扭曲變形示意圖 30
圖 3-1 實驗流程示意圖 40
圖 3-2 標示FITC之化學合成示意圖 41
圖 3-3 細胞培養之磁場引導示意圖 43
圖 3-4 OPA檢測機制示意圖 44
圖 3-5 OPA的濃度校正曲線 45
圖 3-6 FA的濃度校正曲線 46
圖 3-7 DOX的濃度校正曲線 47
圖 4-1 探討OCS和TPP質量比對粒徑和表面電位之影響 57
圖 4-2 探討OCS和TPP質量比對EE和LE之影響 58
圖 4-3 探討OCS和TPP質量比對體外藥物釋放的影響 62
圖 4-4 探討Fe3O4濃度對粒徑和表面電位之影響 65
圖 4-5 探討Fe3O4濃度對EE和LE之影響 67
圖 4-6 探討Fe3O4濃度對體外藥物釋放的影響 69
圖 4-7 探討OCS濃度對粒徑和表面電位之影響 71
圖 4-8 探討OCS濃度對EE和LE之影響 73
圖 4-9 探討OCS濃度對體外藥物釋放的影響 74
圖 4-10 探討油酸取代比率對EE之影響 78
圖 4-11 探討油酸取代比率對體外藥物釋放的影響 80
圖 4-12 探討醋酸濃度和DOX濃度對粒徑大小和表面電位之影響 82
圖 4-13 探討醋酸濃度和DOX濃度對EE和LE之影響 83
圖 4-14 探討DOX濃度對EE和LE之影響 84
圖 4-15 粒徑分佈圖 89
圖 4-16 探討FA接枝率對體外藥物釋放的影響 91
圖 4-17 探討FA接枝率對體外藥物釋放的影響 93
圖 4-18 穿透式電子顯微鏡分析圖 96
圖 4-19 MOCNP之AFM 3D示意圖 98
圖 4-20 MOCNP之AFM 2D示意圖 98
圖 4-21 FTIR分析圖 100
圖 4-22 XRD分析圖 102
圖 4-23 SQUID分析圖 104
圖 4-24 EDS分析圖 106
圖 4-25 TGA分析圖 108
圖 4-26 TGA分析之微分圖 110
圖 4-27 載體於不同反應時間之細胞毒性分析 114
圖 4-28 藥物載體於不同反應時間之細胞毒性分析 114
圖 4-29 藥物載體於不同DOX濃度之細胞毒性分析 115
圖 4-30 50% FA-MOCNP對U87作用之螢光顯微鏡圖 116
圖 4-31 藥物載體對U87作用之螢光顯微鏡圖 116
圖 4-32 藥物載體於癌細胞內之吞噬情形 118
圖 4-33 葉酸對細胞吞噬的影響 120

表目錄
表 2-1 Peppas動力學方程式其n值所代表之釋放機制 19
表 3-1 製程之各參數條件 39
表 3-2 各實驗動物血液檢驗之標準值 53
表 4-1 MOCNP 於不同OCS和TPP質量比的製備條件下其粒徑和表面電位58
表4-2 DOX-MOCNP 於不同OCS和TPP質量比的製備條件下其粒徑、表面電 位、EE和LE 59
表 4-3 DOX-MOCNP 於不同OCS和TPP質量比條件下之釋放動力學模式 63
表 4-4 MOCNP 於不同Fe3O4濃度的製備條件下其粒徑和表面電位 66
表 4-5 DOX-MOCNP 於不同Fe3O4濃度的製備條件下其粒徑、表面電位、EE和LE 66
表 4-6 DOX-MOCNP 於不同Fe3O4濃度條件下之釋放動力學模式 70
表 4-7 MOCNP 於不同OCS濃度的製備條件下其粒徑和表面電位 72
表 4-8 DOX-MOCNP於不同OCS濃度的製備條件下其粒徑、表面電位、EE和LE 72
表 4-9 DOX-MOCNP 於不同OCS濃度條件下之釋放動力學模式 75
表 4-10 OCS於不同油酸添加量的製備條件下其胺基量變化量和取代程度 77
表 4-11 MOCNP於不同油酸取代比率的製備條件下其粒徑和表面電位 79
表 4-12 DOX-MOCNP 於油酸取代比率的製備條件下其粒徑、表面電位、EE和LE 79
表 4-13 DOX-MOCNP 於不同油酸取代比率下之釋放動力學模式 81
表 4-14 文獻中奈米粒子對DOX之EE和LE 85
表 4-15 FA改質OCS之接枝率 86
表 4-16 不同FA接枝率載體之粒徑和表面電位 88
表 4-17 不同FA接枝率藥物載體的粒徑、表面電位、EE和LE 88
表 4-18 不同FA接枝率藥物載體之釋放動力學模式 92
表 4-19 不同FA接枝率藥物載體之釋放動力學模式 94
表 4-20 XRD分析之晶體粒徑 102
表 4-21 SQUID分析之飽和磁化強度和殘留磁化強度 105
表 4-22 EDS分析之元素含量比 106
表 4-23 TGA分析之重量損失百分比 109
表 4-24 磁性奈米載體之性質 111
表 4-25 各藥物載體之IC50 115
表 4-26 血液毒性測試結果 121

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