臺灣博碩士論文加值系統

鐵鉑合金奈米粒子在2000年首次被發表以來，由於它高密度的磁紀錄特性而被當作硬碟的碟片材料。在可以控制合成的粒子直徑大小，超順磁的效果，同時的也在細胞核的傳送載體、核磁共振顯影和電腦斷層掃描顯影之顯影劑上有著高度的潛力。本實驗以穿透式電子顯微鏡、磁性特性，以及X光晶格繞射了解其物理特性，且顯示在不同合成方法下可控制其直徑大小從3~4 奈米, 5~6奈米 以及 12~14奈米，同時在核磁共振照影以及電腦斷層掃描照影下，也都具有加強顯影的效果。細胞實驗中也顯示其高度的生物相容性。此外，C3H/HEN品系的老鼠用來測試鐵鉑奈米顆粒的器官分布，在12小時的時間後12奈米直徑的鐵鉑奈米顆粒主要會集中於脾臟(241.5μg/g)以及肺臟(120.4μg/g)並隨時間漸漸降低。 6奈米直徑的鐵鉑奈米顆粒主要集中於肺臟(247.9μg/g)以及脾臟(204.9μg/g)並其組織中濃度在48小時達最大值。而3奈米直徑的鐵鉑奈米顆粒主要集中於脾臟(146.6μg/g)、肺臟(96.5μg/g)以及肝臟(41.9μg/g)，亦在48小時達最大值。值得注意的是12奈米直徑的鐵鉑奈米顆粒在48小時仍可有102.2μg/g之血中濃度。相較下較小粒徑之奈米粒子在12小時前即達到最高血中濃度。另外，三種尺寸之奈米粒子在腦中之濃度皆在24小時達最高；12奈米與3奈米最高，6奈米較低。
在X射線電腦斷層顯影（CT）上，在等鐵離子濃度下12nm粒子有最高之顯影對比，次為程度接近之3nm粒子，6nm粒子最差。但三者在100微莫耳濃度即有可偵測之CT對比值，訊號分別為緩衝液之1.39、1.43、及1.13倍。在磁振顯影方面12nm粒子之顯影效果最佳，在1毫莫耳濃度以上即有可偵測對比。3及6nm粒子顯影能力相近，需25毫莫耳以上之濃度。
為評估鐵鉑奈米顆粒在癌症治療的專一標定治療潛力，我們在鐵鉑奈米顆粒表面接上抗Her2的抗體，並且利用具有Her2高度表現的小鼠膀胱癌細胞及其核酸干擾降表現之細胞株為研究模型。在細胞實驗中，核磁共振T2訊號上顯示接上抗Her2抗體的鐵鉑奈米顆粒確能選擇性的標的具有高度表現Her2抗原的細胞。且12奈米粒子較3奈米之對比性更佳。
另外，在動物活體實驗中，將鐵鉑奈米顆粒從尾靜脈打入具膀胱癌的老鼠，結果在核磁共振T2的訊號上，3~4奈米和12~14奈米的鐵鉑奈米顆粒分別成功的改變訊號強度9%以及29%。最後，我們成功的將入細胞核生太接上鐵鉑奈米粒，並證明其入核效果，顯示鐵鉑奈米顆粒具攜帶基因訊息入核進行基因治療之可行性。總之，鐵鉑奈米顆粒在作為核磁共振和電腦斷層掃描之多模式加強顯影劑以及攜帶藥物至特定細胞及其胞器上有著很高的發展潛力。

The FePt alloy nanoparticle (FePt-np) was first introduced in 2000. It was originally designed for the high-density magnetic recordings due to its superparamagnetic property, stability, high electron density and controllable small size. This research project explore the potential for using FePt-np as novel cellular and sub-cellular nuclear targeted vehicles with dual contrast property in combined X-ray computed tomography (CT scan) and magnetic resonance imaging (MRI).
The physical properties of the nanoparticles such as TEM, magnetic property, and XRD were characterized. The nanoparticles presented a tunable sizes from 3~4 nm, 5~6nm and 12~14nm in diameter. The in vitro biocompatibility evaluation revealed satisfactory results in both cytotoxicity and hemolysis tests. The biological distribution of the as synthesized particles in C3H/HEN mice reavealed that 12nmFePt-nps mainly accumulated in the spleen (241.5μg/g) and lung (120.4μg/g) in 12 hr then gradually decreased toward normal level. The 6nm particles mainly accumulated in the lung (247.9μg/g) and spleen (204.9μg/g) and reached plateau at 48hr. Tissue accumulation of 3nm FePt-nps also reached plateau at 48hr in the spleen (146.6μg/g), lung (96.5μg/g) and liver (41.9μg/g). The 12nm particles presented longer circulation time that a high blood concentration of 102.2μg/g could still be detected after 48hr. On the other hand, 3 and 6nm particles reached highest blood concentration before 12hr. All three types of nanoparticles reached highest concentration in the brain at 24hr with highest concentration for 12 and 3nm particles then 6nm particles.
We then measured the image contract effect of the particles in CT and MRI. At equal iron concentration, 12 nanometer particles presented the highest image contrast in CT followed by 3nm and 6nm particles. All three nanoparticles show detectable image contrast at above 100�嵱 (1.39, 1.43. and 1.13 folds to the phosphate buffer control). The 12nm particles presented the best T2* MRI contrast property with a detection limit at 1mM. 3 and 6nm particles required more than 25mM of iron concentration for detectable image contrast. Furthermore, in order to improve the specificity of FePt in targeting cancer diagnostics and therapy, the anti-Her2 monoclonal antibody was conjugated onto the nanoparticles. The MBT-2 cells with high endogenous Her2 expression and the control knock down cell line (MBT-KD) were applied as the model system. to the targeting nanoparticles demonstrated specific taggeting to MBT cells under T2 pulse sequence. The 12nm particles outperform the 3nm size particles.
In the tumor bearing C3H/HEN mice, we further demonstrated that significant inversed MRI contrast (9% for 3nm particles and 29% for the 12nm particles) of the tumor lesion in the T2 sequence could be achieved after tail vein injection of the anti-Her2 tagged 3nm and 12nm nanoparticles. Finally, the nuclear –targeting for improved gene delivery was investigated for combined cellular targeting, trans-membrane transport and nuclear trafficking. The NLS-FePt-nps were successfully synthesized. Their sub-cellular delivery, The NLS-peptide tagged nanoparticles show preferred uptake by HeLa cell nuclei over the non-NLS-labeled nanoparticles. In conclusion, FePt-nps show a promising potental for multi-modal molecular imaging in both MRI and CT and as a therapeutic vector for target specific organelles in specific cells.

中文摘要 4
Abstract 6
I. INTRODUCTION 8
1. Nanotechnology 8
1.1. The general characteristics of nanoparticles 9
1.1.1. Surface modification 9
1.1.2. Magnetic property 10
1.1.3. Catalytic property 11
1.1.4. Optical property 12
1.2. FePt- nanoparticles 12
2. Cancer 13
2.1 Cancer Epidemiology 13
2.2 Cancer Diagnosis 14
2.3 Molecular imaging of diagnostic medicine 15
2.3.1. The computed tomography 15
2.3.2. The magnetic resonance imaging (MRI) 16
2.4 Nanoparticles for molecular imaging probe 17
2.4.1. Nanoparticle for imaging probe of X-ray computed tomography 18
2.4.2. Nanoparticle for magnetic resonance imaging 19
2.5 The cancer therapy 20
2.5.1. Gene therapy for cancer 20
2.5.2. Challenge of Targeted nuclear delivery 21
2.5.3. Nanoparticles for nuclear targeting 21
2.5.4. The Ligand-directed targeting of nanoparticles 22
2.5.5. The kinetic properties of nanoparticles 23
2.6 The Development of a noninvasive molecular diagnostic contrast agent by MRI and CT and a nuclear transport vector 23
II. Materials and Methods 26
FePt-nps preparation and characterization 26
Cell lines culture 28
Animal model 31
The in vitro biocompatibility evaluation 31
Tumor targeting 35
III. Results 37
1. Anti-Her2 antibody tagged nanoparticles as MR imaging contrast agent 37
1.1. Physical property characterization of the nanoparticles 37
1.2. Cytotoxicity and hemolysis analysis 38
1.3. Cellular uptake and biodistribution of FePt-nps 39
1.4. The in vitro targeting assay by MR imaging 42
1.5. The in vivo targeting and tracing by MR imaging. 42
1.6. Nuclear targeting of NLS tagged FePt-nps 44
IV. Discussion 45
Size dependent property of FePt-nanoparticles 46
Biodistribution of FePt-nps 48
Magnetic Resonance Imaging of Biological Targets 49
Nuclear targeting of FePt-nps 51
Conclusion 53
V. Figures 54
VI. References 68
VII. Appendix 74
VIII. About the author 81

張仕欣，王崇人, ”金屬奈米粒子的吸收光譜” ,化學, 1998, 56, 209-222
吳亞娜，謝達斌,”超順磁性奈米粒子在腫瘤表面分子診斷及作為治療用途之評估及應用” 國立成功大學分子醫學研究所碩士論文,民國94年

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