神經膠質母細胞瘤為腦瘤中最常見之惡性腫瘤，目前主流之治療方式為化學治療，但其治療效果並不顯著，一來由於腦中特化之血腦屏障結構，阻擋化療藥物進入腦腫瘤中進行治療；二來由於化療藥物無法進入血腦屏障的情況下，勢必需要增加藥物遞送的量，進而導致其他周圍組織器官之損害，產生不必要之副作用，因此如何提升血腦屏障之通透性並提升化療藥物累積量為現下藥物遞送的概念。近年來已有許多文獻證實使用聚焦式超音波配合負載藥物之微氣泡進行腦部藥物遞送之可行性，能夠同時達到提升血腦屏障之通透性、定點釋藥，並降低化療藥物之副作用等目的；然而，目前的影像工具僅能提供血腦屏障開啟位置的資訊，無法得知治療後藥物釋放至腦中的分布與濃度。超順磁性氧化鐵粒子(superparamagnetic iron oxide nanoparticle, SPIO)，為核磁共振之負向顯影劑，已被證實可藉由核磁共振造影觀測出血腦屏障開啟的區域。因此本研究的目的是將超順磁性氧化鐵粒子與化療藥物阿黴素(doxorubicin, DOX)鍵結在一起(SD複合物)，並負載於微氣泡上，簡稱為SD-MBs，希望藉由核磁共振造影動態監測SD-MBs被聚焦式超音波驅動釋藥後，超順磁性氧化鐵粒子在腦中分布的位置與濃度，由於顯影之處就表示阿黴素之存在，因而可以即時評估藥物遞送之訊息。此外，由於SD複合物具有趨磁性，我們也將評估是否能利用磁標靶強化藥物在腦中的累積量。
本研究先將帶有胺基的超順磁性氧化鐵粒子與阿黴素的羰基以共價鍵的形式鍵結在一起，再將鍵結好的複合物以疏水作用力吸附在微氣泡的磷脂質殼層，形成SD-MBs。之後最佳化乘載於微氣泡上的SD複合物，再對SD-MBs進行物理性質與穩定性量測分析，並評估其於超音波以及核磁共振影像下之顯影能力，以及是否能被聚焦式超音波驅動釋藥而對腦瘤細胞有毒殺的效果。另外，我們也將進行動物實驗，使用聚焦式超音波(1 MHz, 0.3 MPa, 5000 cycles, PRF = 1 Hz, duty cycle = 0.5 %, 施打4點, 照射時間 = 240 s/點)與無載藥之微氣泡配合靜脈注射SD複合物或SD-MBs開啟血腦屏障後，以核磁共振橫向遲緩率分布圖(R2-MAP)影像即時監測遞送至腦部的SD複合物，最後利用高效液相層析儀配合UV偵檢器定量腦中阿黴素的濃度。
結果顯示SD-MBs具有大量乘載SD複合物的能力、良好的穩定性、及超順磁性之特性，並且被聚焦式超音波擊破後能有效毒殺腦瘤細胞。而在活體評估藥物遞送情形的結果發現在有磁標靶的狀況下，可於目標區域增加藥物九倍的累積量(從0.25 μg提升到2.23 μg)；但亦發現若是提高聲壓造成腦組織出血的話，反而會因出血競爭而降低藥物遞送量；在核磁共振造影的部分，即時負向顯影的區域與血腦屏障開啟的位置相當一致，表示可由影像顯影程度而推回藥物濃度與分布位置。
本研究已經證實了SD-MBs可以和聚焦式超音波產生局部血腦屏障開啟並同時遞送SD複合物進腦組織供核磁共振造影偵測藥物分布位置。此外，SD複合物可藉由磁標靶而增加藥物累積量。未來工作將應用此藥物釋放平台至大鼠腦瘤模型，進一步在SD-MBs上修飾專一性標誌配位體，配合磁標靶，達到雙重標靶治療之目的。

第一章 緒論 1
1.1. 腦瘤 1
1.1.1. 神經膠質母細胞瘤(Glioblastoma multiforme, GBM) 2
1.1.2. 神經膠質母細胞瘤治療方法與瓶頸 3
1.1.3. 阿黴素(Doxorubicin, DOX) 4
1.2. 血腦屏障(Blood Brain Barrier, BBB) 5
1.2.1. 醫用超音波 7
1.2.2. 超音波與物質之作用 7
1.3. MRI/US 雙顯影試劑 9
1.3.1. 核磁共振顯影劑 9
1.3.2. 超順磁性氧化鐵粒子作為藥物載體 10
1.3.3. 超音波對比劑-微氣泡 11
1.3.4. 超順磁性氧化鐵粒子承載於微氣泡 12
1.4. 核磁共振顯影劑之藥物定量 15
1.5. 研究動機與目的 16
第二章 實驗材料與方法 18
2.1. 超順磁性氧化鐵粒子鍵結阿黴素(SPIO-DOX)之製備 18
2.2. 造影微氣泡(MBs)之製備 19
2.3. 超順磁性氧化鐵粒子鍵結阿黴素承載於微氣泡(SD-MBs)之製備 19
2.4. SD-MBs的物理性質分析 20
2.4.1. 粒徑分析 20
2.4.2. 光學性質量測 21
2.4.3. 磁性測量及磁吸力測試 21
2.4.4. SD鐵定量及SD-MBs之SPIO承載定量 21
2.4.5. SD及SD-MBs之DOX鍵結承載效率 22
2.4.6. 藥物滯留率分析 23
2.5. 仿體實驗 24
2.5.1. SD-MBs超音波聲學穩定性測試 24
2.5.2. 核磁共振對比影像之R2定量分析 25
2.6. 細胞實驗 27
2.6.1. 細胞培養 27
2.6.2. 藥物載體之毒性測試 27
2.7. 動物實驗架構 30
2.7.1. 高頻超音波影像系統 30
2.7.2. 商用高頻超音波Vevo®2100影像系統 31
2.7.3. 聚焦式超音波 32
2.8. 動物實驗流程 34
2.8.1 聚焦式超音波驅動微氣泡開啟血腦屏障進行藥物遞送之可行性流程 35
2.8.2 以MRI監測聚焦式超音波驅動微氣泡開啟血腦屏障之流程 36
2.8.3 動物犧牲取腦與組織切片 37
2.9. 動物實驗之腦內定量分析 38
2.9.1. 鼠腦MRI影像分析 38
2.9.2. 鼠腦DOX藥量分析 38
2.10. 統計分析 39
第三章 結果與討論 41
3. 概述 41
3.1. SD-MBs性質量測 41
3.1.1. SD-MBs光學性質 41
3.1.2. SD-MBs粒徑分布 43
3.1.3. SD-MBs磁性量測 44
3.1.4. SD鍵結效率 47
3.1.5. SPIO與DOX包覆於SD-MBs之承載量 48
3.1.6. SD-MBs藥物穩定性 52
3.1.7. SD-MBs聲學穩定性 53
3.1.8. MRI仿體實驗對比結果 54
3.2. 細胞實驗之毒性測試 56
3.3. 動物實驗 59
3.3.1. SD-MBs活體內存活時間 59
3.3.2. 血腦屏障開啟之參數選定 60
3.3.3. 活體內DOX藥量分析 62
3.3.4. 活體MRI影像對比定量分析 63
第四章 結論 68
第五章 未來展望 71
參考文獻 72

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