臺灣博碩士論文加值系統

本研究主要在探索奈米中孔洞二氧化矽材料於生物影像及具生物辨識功能的藥物輸送系統上之應用。我們利用離子交換的方法將擁有正價介面電荷的中孔洞二氧化矽奈米顆粒與具負價電荷之三價釓金屬錯合物(Gd-DTPA)結合在一起，形成一具有高弛緩率(relaxivity)的核磁共振顯影劑。相較於現今臨床上使用的顯影劑，此顯影劑上的順磁中心(釓金屬錯合物)可緩慢的由動物體內排出。此特性可應用於核磁共振顯影上長時間的血管造影。另一方面，於細胞影像及細胞治療上的應用，我們也成功的合成出兩種具螢光及順磁性質的多功能性中孔洞奈米矽材。這多功能的奈米中孔洞材二氧化矽同樣具有高的弛緩率，好的光穩定性並能高效率的標定細胞。最重要的是此奈米材料對細胞具有很低的毒性。利用光學顯微鏡及核磁共振顯影技術均可清楚的觀察到以奈米顆粒標定過的細胞於動物體內的位置。
於本研究的最後一部份，我們成功的發展出具辨識功能奈米輸送系統。將單源抗體(monoclonal antibody; Herceptin)嫁接於中孔洞二氧化矽奈米粒子的表面並有效的辨識Her2/neu蛋白過度表現的乳癌細胞。我們發現抗體於奈米粒子上的分布密度愈高表現出對特定細胞的選擇性愈佳。並且利用再修飾上聚乙二醇分子(polyethylene glycol)，可有效的降低具低密度抗體的中孔洞二氧化矽奈米粒子對非Her2/neu蛋白過度表現細胞的非專一性黏附。最後我們利用共軛焦螢光顯微鏡及穿隧式電子顯微鏡證明此奈米顆粒主要利用受體介導的內吞作用機制(receptor-mediated endocytosis)進入細胞中。

In this research, we explored the bio-applications of mesoporous silica nanomaterials for bio-imaging and targeted drug delivery. Mesoporous silica nanoparticles (MSNs) with positive surface charge incorporated with Gd(DTPA) by electrostatic force show the high r1 and r2 relaxivity, which are much higher than those of free Gd(DTPA). These composite nanomaterials show the slower excretion rate from animal body and could be a potential blood-pool MRI contrast agent for angiography. For cellular imaging and effective cell therapy, we also successfully synthesized two types of multifunctional MSNs nanoprobes with fluorescence and paramagnetism. Both nanomaterials show high sensitivity to MRI, good photo-stability, high cell labeling efficiency and low cytotoxicity. The nanoprobes labeled cells could be clearly visualized by MRI and optical modalities.
In the last part, green fluorescence MSNs were modified with monoclonal antibody, Herceptin, to target the Her2/neu over-expressing breast cancer cell. MSNs with highest density of Herceptin on the outer surface show the highest selectivity to the targeted cell and the further modification with polyethylene glycol (PEG) can prevent from the non-specific targeting for the MSNs with low density of Herceptin. We also used the confocal microscope and TEM to demonstrate the Herceptin functionalized MSNs could be engulfed by Her2/neu positive breast cancer cells through the receptor-mediated endocytosis.

摘要………………………………………………………………... I
Abstract…………………………………………………………... II
Table of Contents……………………………………………….. III
List of Tables…………………………………………………….. VI
List of Figures…………………………………………………… VII
List of Schemes…………………………………………………. XVII
Chapter 1 Introduction………………………………………….. 1
1.1 Nano-materials on Bio-imaging………………………………………….. 1
1.2 Nanomedicine……………………………………………………………. 8
1.3 Porous Silica on Biomedicine……………………………………………. 17
Chapter 2 Mesoporous Silica Nanomaterials Incorporated
with [Gd(DTPA)H2O]2- for Biomedical and
Molecular Imaging Applications…………………. 23
2.1 Introduction…………………………………………………………….. 23
2.1.1 Biomedical and Molecular Imaging……………………………….. 23
2.1.2 A Brief Introduction of Magnetic Resonance Imaging (MRI)…….. 26
2.1.3 Relaxivity of Gd3+ complexes: A Simple Introduction of Theory…. 30
2.2 [Gd(DTPA)H2O]2- Incorporated with Mesoporous Silica Nanoparticles
Through Ion Exchange to be a High Relaxivity Blood-Pool MR
Contrast Agent………………………………………………………….. 33
2.2.1 Experiments………………………………………………………… 33
2.2.1.1 Synthesis of Mesoporous Silica Nanoparticles Incorporated
with [Gd(DTPA)H2O]2-………………………………………. 33
2.2.1.2 Physical and Chemical Characterizations……………………. 35
2.2.1.3 Characterization on Magnetic Resonance Properties………… 37
2.2.2 Results and Discussion 39
2.2.2.1 Physical and Chemical Properties of MPG500N and MPG500P. 39
IV
2.2.2.2 Magnetic Resonance Properties of MPG500N-Gd and
MPG500P-Gd………………………………………………….. 45
2.2.2.3 Solvent Effect on the Release of [Gd(DTPA)H2O]2- from
MPG500P-Gd………………………………………………….. 55
2.2.2.4 Results of Intravenous Injection into Mice…………………... 57
2.2.3 Summary…………………………………………………………… 60
2.3 High Contrast Paramagnetic Fluorescent Mesoporous Silica
Nanomaterials as Multifunctional Cell-Imaging Probes
(Gd-Dye@MSN and Gd-Dye@MSN-R)................................................. 61
2.3.1 Experiments………………………………………………………… 61
2.3.1.1 Synthesis of Gd-Dye@MSN and Gd-Dye@MSN-R………… 61
2.3.1.2 Materials and Magnetic Resonance Characterization………... 62
2.3.1.3 Cell Cultures and Assays……………………………………... 64
2.3.2 Results and Discussion……………………………………………... 69
2.3.2.1 Physical and Chemical Properties of Gd-Dye@MSN and
Gd-Dye@MSN-R…………...................................................... 69
2.3.2.2 Relaxivity and Phantom Images……………………………… 73
2.3.2.3 Gd-Dye@MSN-R for 3T3-L1 Cell Labeling………………… 78
2.3.3 Conclusion………………………………………………………….. 86
Chapter 3 Green Fluorescence Meosporous Silica
Nanoparticles Functionalized with Herceptin
Targeting Her2/neu Over-expressing Breast
Cancer Cells………………………………………... 87
3.1 Introduction…………………………………………………………….. 87
3.2 Experiments…………………………………………………………….. 92
3.2.1 Synthesis of Green Fluorescence Mercapto (Outer surface)-
Mesoporous Silica Nanoparticles (GFMO@MSN) Functionalized
with Herceptin or Polyethylene Glycol (PEG5000) ………………… 92
3.2.2 Cell Culture, Assays and Materials Characterization………………. 96
3.3 Results and Discussions………………………………………………... 101
3.3.1 Physical and Chemical Properties………………………………….. 101
3.3.2 Modification of Polyethylene Glycol (PEG) Prevents MSN from
Non-specific Binding to Cells……………………………………… 109
3.3.3 Effect of HP5k-GFMO@MSN with Different GFMO@MSN /
Herceptin Ratios on Cell Recognition……………………………… 116
V
3.3.4 Effect of Further Modification with PEG5k………………………… 124
3.3.5 Competition Reaction and TEM : The Evidence of HP5k-
GFMO@MSN-1 Engulfed by BT474 through Receptor- mediated
Endocytosis………………………………………………………… 129
3.4 Conclusion……………………………………………………………… 138
Chapter 4 Conclusion and Perspective 139
Reference 143

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