臺灣博碩士論文加值系統

過去幾年來，癌症已經成為世界上最具破壞性的疾病之一。而到目前為止，癌症的治療大多是靠手術切除、化療、或是放射性療法。然而這些治療方式的成功率不高，而且也有不少副作用。為了增加癌症治療的效率，我們希望能發展一種多功能性的高分子微胞，同時具備藥物傳輸、基因治療與影像輔助的功能。這種高分子微胞由疏水性的硬脂酸內核與帶正電的聚乙烯亞胺外殼組成，而在聚乙烯亞胺外會接枝聚乙二醇。疏水性內核用來包覆超順磁氧化鐵奈米粒子，它是一種磁振造影的對比劑，具有很高的T2弛緩率及敏感度。而在親水性外殼的部份，聚乙烯亞胺將當作基因傳輸的載體，而聚乙二醇則是為了增加微胞在血液中循環的時間。

在此研究中，我們以TNBS assay、核磁共振光譜與穿透式電子顯微鏡證實成功製備出聚乙二醇-聚乙烯亞胺-硬脂酸微胞。利用DNA retardation assay則顯示出此微胞具有良好的DNA吸附能力。而在包覆超順磁氧化鐵奈米粒子的部份，實驗中以甲苯為溶劑，將氧化鐵溶液加到微胞溶液中，然後在4℃以5瓦的功率用超音波探針進行包覆。由粒徑分析儀測量後，得到直徑200奈米(PDI = 0.2)的微胞，另外，藉由穿透式電子顯微鏡則可以看到微胞內部包覆著許多氧化鐵粒子。經過原子吸收光譜的分析，則可得知此方法有60%的包覆率。將本樣品進行磁振造影的掃描分析後則發現，自行製備的氧化鐵微胞相較已上市的Resovist®有更高的T2弛緩率。此外，在活體實驗中，經過尾靜脈注射後的小鼠，其肝、腎、攝護腺的T2加權影像都有明顯的加深。由這些實驗結果可以說明，此微胞可以作為一種有效的磁振造影導引之藥物與基因傳輸系統。

Over the past several years, cancer has become one of the most devastating diseases worldwide. Nowadays, cancer treatment is much dependent on surgery, chemotherapy and radiotherapy. However, these methods were less successful and had major side effects. In order to improve the efficiency of cancer treatment, we focus on the development of multifunctional polymeric micelles for drug delivery, gene therapy and diagnostic imaging application. The developed polymeric micelles were composed of a hydrophobic stearic acid (SA) core and a positively charged polyethylenimine (PEI) outer shell which was modified by polyethylene glycol (PEG). The hydrophobic core served as a reservoir for superparamagnetic iron oxide nanoparticles (SPIONs) which is a magnetic resonance imaging (MRI) contrast agent with remarkably high T2 relaxivity and sensitivity. Of the hydrophilic shell, the cationic polymer PEI was used for non-viral transfection and PEG was used for prolonging blood circulation time.

In this study, we have successfully developed PEG-PEI-SA micelles confirmed by TNBS assay, nuclear magnetic resonance (NMR), transmission electron microscopy (TEM). And the DNA retardation assay demonstrated that PEG-PEI-SA micelles showed good DNA binding efficiency. The encapsulation was performed by addition of SPIONs in toluene to PEG-PEI-SA micelles solution, followed by sonication at 5 W for 1 min at 4℃. Dynamic light scattering (DLS) measurement revealed ~200nm particles (PDI = 0.2) were formed, while TEM analysis demonstrated that clusters of SPIONs were encapsulated in the hydrophobic core. And the encapsulation efficiency was about 60% which was analyzed by atomic absorption spectrometry (AAS). MRI scanning of the samples revealed that the SPIONs-loaded micelles had higher T2 relaxivity than Resovist®. In addition, in vivo study showed that the liver, kidney and prostate turned dark on T2 weighted images after tail vein injection. These results indicated that the SPIONs-loaded micelles can serve as an efficient image-guided drug and gene delivery system.

致謝 I
摘要 II
Abstract III
目錄 V
圖目錄 VII
表目錄IX
第一章 序論 1
1.1 奈米粒子與醫學 1
1.2 癌症 2
1.3 癌細胞及其環境 3
1.4 奈米粒子與癌症治療 6
1.5 微胞(micelles)的組成與特性 7
1.6 基因療法(Gene therapy) 9
1.7 磁振造影(Magnetic Resonance Imaging; MRI) 12
1.8 研究動機與目的 15
第二章 實驗材料與方法 17
2.1 實驗藥品 17
2.2 實驗儀器 18
2.3 合成聚乙烯亞胺-硬脂酸微胞(PEI-SA micelles) 20
2.4 在聚乙烯亞胺-硬脂酸微胞表面修飾聚乙二醇(PEG-PEI-SA micelles) 21
2.5 粒徑分析(Z-Average)與表面電位(zeta potential)分析 23
2.6 核磁共振光譜(1H NMR)分析 23
2.7 質體DNA之大量分離 23
2.8 DNA吸附能力分析(DNA Retardation Assay) 26
2.9 包覆超順磁氧化鐵奈米粒子(SPIONs) 27
2.10 比較微胞在PEG修飾與SPIONs包覆前後對細胞增生的影響 29
2.11 細胞吞噬能力分析 32
2.12 MRI弛緩率測量 34
2.13 動物實驗 34
第三章 結果與討論 36
3.1 合成聚乙烯亞胺-硬脂酸微胞(PEI-SA micelle) 36
3.2 在微胞表面修飾聚乙二醇 37
3.3 聚乙二醇-聚乙烯亞胺-硬脂酸微胞的粒徑與表面電位 40
3.4 核磁共振光譜(1H NMR)分析 41
3.5 DNA吸附能力分析 44
3.6 包覆超順磁氧化鐵奈米粒子 45
3.7 穿透式電子顯微鏡分析 52
3.8 比較微胞在PEG修飾與SPIONs包覆前後對細胞增生的影響 54
3.9 細胞吞噬能力分析 59
3.10 MRI弛緩率測量 64
3.11 動物實驗 66
第四章 結論 70
第五章 參考文獻 71

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