林峰輝Lin, Feng-Huei臺灣大學:醫學工程學研究所吳錫芩Wu, Hsi-ChinHsi-ChinWu2010-06-022018-06-292010-06-022018-06-292009U0001-1407200915261100http://ntur.lib.ntu.edu.tw//handle/246246/184656對於遺傳性或基因缺陷、變異所造成的疾病，基因治療提供一種嶄新且具有潛力的治療方式。這當中，基因載體的選擇對於基因物質（例如：核酸）是否能成功的轉染至活細胞內佔有舉足輕重的地位。本研究是利用溼式化學法合成氫氧基磷灰石，在過程中於不同時間點（中程、後段）添加鐵源前驅物以提供鐵離子及改變鐵/ 鈣比例變化。結果發現在形成氫氧基磷灰石的過程中，可製備出磁性氫氧基磷灰石奈米顆粒，並帶有大小不同的磁性。同時，研究中也與磁性天然骨礦物進行分析比較。性氫氧基磷灰石的物理及化學性質分別以：X光繞射儀針對晶體結構及晶胞參數進行分析；掃瞄式電子顯微鏡、原子力顯微鏡、（高解析）穿透式電子顯微鏡針對型態及晶格排列進行分析；化學組成成份利用感應耦合電漿進行分析；各官能基以傅立葉變換紅外線光譜來進行分析；超導量子干涉元件則是用來進行磁性量測。所製備出的其中一種磁性氫氧基磷灰石奈米顆粒（中程）是以鐵離子置換氫氧基磷灰石中的鈣離子而得，且仍然能維持原結構完整性並未發生損毀的現象。磁性氫氧基磷灰石（中程）的尺寸約20至50奈米大小且呈球體形狀。另一種製備出的磁性氫氧基磷灰石（後段）與磁性自然骨礦物奈米顆粒則是分別以磁鐵礦異質生長分別於氫氧基磷灰石和自然骨礦物晶粒上。觀察磁性氫氧基磷灰石（後段）與磁性自然骨礦物時，有小於10奈米大小的球體顆粒緊密環繞於呈棒/ 針狀的氫氧基磷灰石表面和自然骨礦物晶粒周圍。研究中也發現，這些磁性氫氧基磷灰石皆具有超順磁性且磁性隨著鐵/ 鈣比例的增加而增加。此外，以乳酸脫氫酶分析磁性氫氧基磷灰石具有良好的生物相容性。性氫氧基磷灰石奈米顆粒相較於單純磁鐵礦奈米顆粒而言具有與質體核酸的高度結合性，同時具有保護質體核酸的效果來抵抗核酸酵素的分解。當施予一外加磁場於磁性氫氧基磷灰石與磁性自然骨礦物奈米顆粒時，可有效增加轉染神經膠細胞源神經營養因子基因於大鼠骨髓間質幹細胞，且無細胞毒性的表現。研究中亦發現，即使僅攜帶少量的質體核酸於磁性奈米顆粒上，經過轉染的大鼠骨髓間質幹細胞所分泌的神經膠細胞源神經營養因子表現量仍能有效達到治療效果的劑量水平。研究藉由鐵源前驅物添加於反應過程中，合成出鐵置換及異質磊晶形式之氫氧基磷灰石為基質的磁性奈米顆粒，這些奈米顆粒具有適當的物化和生物性質，在生物醫學領域研究上相當具有發展空間應。本研究中，磁性氫氧基磷灰石奈米顆粒已被證明具有與質體核酸結合及避免酵素分解的保護能力。利用磁性氫氧基磷灰石奈米顆粒轉染大鼠骨髓間質幹細胞時給予外加磁場，能顯著增加神經膠細胞源神經營養因子的表現量。載負低劑量的質體核酸時，神經膠細胞源神經營養因子仍能持續被經過轉染的大鼠骨髓間質幹細胞所分泌，持續達兩週且達到治療所需程度。因此，我們認為磁性氫氧基磷灰石奈米顆粒極具有發展潛力作為非病毒載體應用於基因治療上。Gene therapy has great potential to revolutionize the treatment of human diseases in both genetic and acquired origin. Of the was that, the successful transfer of genetic materials (such as nucleic acid) into living cells is the most important issue depending on the development of the gene delivery carrier. In the study, nanoparticles (NPs) of synthetic hydroxyapatite (Hap) were rendered magnetic by treatment with iron ions using a wet-chemical process. During Hap precipitation processes, two kinds of magnetized Hap (mHap) NPs (mHap-Inter and mHap-Post) were fabricated by iron precursor addition at different time points (inter- and post-) in various ratios of Fe:Ca (XFe/ Ca) and were used to compare with magnetic natural bone mineral (mNBM). he physical and chemical properties of two types of mHap NPs were, respectively, evaluated the crystal structure and cell parameters by X-ray diffraction; morphology and lattice arrangement by scanning electron microscopy, atomic force microscopy and (high-resolution) transmission electron microscopy; composition analysis by inductively coupled plasma; functional groups by Fourier transform infrared spectroscopy; magnetization by superconducting quantum interference device. The mHap-Inter NPs, which iron substituted for calcium in Hap, was remained unaltered lattice constants without collapse phenomenon. The size distribution of mHap-Inter was around 20 to 50 nm with sphere shape. On the other hand, the mHap-Post and mNBM NPs were the result of the hetero-epitaxial growth of magnetite on the Hap and NBM crystallites. The magnetic NPs with sphere shape less than 10 nm in diameters were tightly surrounded on Hap or NBM crystallites which were revealed randomly in rod and needle-like shape. The magnetization of all groups of mHap NPs increased with the increasing of XFe/ Ca and possessed superparamagnetic property. Moreover, all the mHap NPs were examined with good biocompatibility using the analysis of lactate dehydrogenase assay.he mHap NPs displayed a high binding affinity with plasmid DNA (pDNA) and appeared to exert a protective effect on the bound plasmid against enzymatic degradation compared to magnetite NPs. The mHap-Inter, mHap-Post and mNBM NPs showed substantial increases in the glial cell line-derived neurotrophic factor (GDNF) gene transfection for rat marrow-derived mesenchymal stem cells (MSCs) under an applied magnetic field, and were shown to be non-cytotoxic. The amount of GDNF recovered in the medium approached therapeutic levels despite the small amount of plasmid delivered by the mHap NPs. he hydroxyapatite-based magnetic NPs were synthesized in two different types: iron-substitution and hetero-epitaxial growth during precipitation processes by iron addition. The mHap-Inter and mHap-Post NPs possessed the appropriate physico-chemical and biological properties with great promising to be applied in the biomedical applications. The mHap NPs were demonstrated to possess binding affinity with pDNA and protected the plasmid against enzymatic digestion. The mHap-Inter, mHap-Post and mNBM NPs significantly increased GDNF expression in rat MSCs transfection when magnetic field was applied (P<0.05). GDNF could be maintained for as long as two weeks and the accumulated amount achieved therapeutic levels even for low pDNA loading. According to the above facts, we believe that the mHap NPs have great potential as a novel non-viral vector for gene therapy.CONTENTS要 IXBSTRACT XIBBREVIATION XIVHAPTER 1 INTRODUCTION 1.1. Basis of Nanotechnology in Medicine 1.2. Gene Therapy 5.2.1. Germline Gene Therapy 7.2.2. Somatic Gene Therapy 7.3. Motivation 11.4. The Objective of This Study 11HAPTER 2 LITERATURE REVIEW 12.1. Gene Delivery System 12.1.1. Viral Vector 13.1.2. Non-Viral Vector 19.2. Non-Viral Inorganic Nanoparticles Delivery Vector 25.2.1. Calcium Phosphate 26.2.2. Iron Oxide 28.2.3. Magnetofection 29.3. Basic Concepts of Magnetism 30HAPTER 3 THEORETICAL BASIS 37.1. Hydroxyapatite (Hap) 37.1.1. Preparation of Hydroxyapatite 37.1.2. Apatite Compound and Metal Ions Substitution 38.2. The Delivery Pathway of Cellular Uptake 41.2.1. Endocytosis 41.2.2. Magnetofection 45.3. The Benefits and Strategies for magnetic Hap as Gene Delivery Carrier 47HAPTER 4 MATERIALS AND METHODS 48.1. Preparation of Synthetic Hydroxyapatite (Hap) Nanoparticles (NPs), and Magnetic Hydroxyapatite (mHap) and Natural Bone Mineral (NBM) Nanoparticles 48.2. Characteristics of Hydroxyapatite and Magnetic Hydroxyapatite 49.2.1. X-ray Diffraction (XRD) 49.2.2. Inductively Coupled Plasma Mass Spectrometer (ICPMS)/ Inductively Coupled Plasma Optical Emission Spectrometry (ICPOES) 50.2.3. Fourier Transform Infrared Spectroscopy (FTIR) 50.2.4. Scanning Electron Microscopy (SEM) 50.2.5. Atomic Force Microscopy (AFM) 51.2.6. Transmission Electron Microscopy (TEM)/ High Resolution Transmission Electron Microscopy (HRTEM) 51.2.7. Dynamic Light Scattering (DLS) 51.2.8. Superconducting Quantum Interference Device (SQUID) 52.3. In vitro Biocompatibility Test of Magnetic Hydroxyapatite Nanoparticles 52.3.1. Cell Culture 52.3.2. Lactate Dehydrogenase (LDH) Assay 53.4. Preparation of Plasmid DNA-Nanoparticles (pDNA-NPs) Transfection Complexes 54.5. Characteristics of Plasmid DNA and Nanoparticles Transfection Complexes 55.5.1. Gel Electrophoresis 55.5.2. Zeta-Potential 56.6. In vitro Biocompatibility Test of Plasmid DNA-Nanoparticles Transfection Complexes 57.6.1. Cell Culture 57.6.2. Lactate Dehydrogenase Assay 58.7. Transfection 58.7.1. Transfection with Magnetic Nanoparticles 58.7.2. Transfection with a Lipid Transfection Reagent 59.8. Evaluation of Transfection Efficiency and Transfection Complexes Association with and Internalization into Cells 60.8.1. Fluorescence Microscopy 60.8.2. Enzyme-linked Immunosorbent Assay (ELISA) 60.8.3. Transmission Electron Microscopy (TEM) 61.9. Statistical Analysis 62HAPTER 5 RESULTS 63.1. Characteristics of Hydroxyapatite (Hap) and Magnetic Hydroxyapatite (mHap) 63.1.1. Magnetic hydroxyapatite crystallites by inter-adding iron precursor (mHap-Inter) 63.1.2. Magnetic hydroxyapatite crystallites by post-adding iron precursor (mHap-Post) 79.2. Characteristics of Plasmid DNA and Nanoparticles (pDNA-NPs) Transfection Complexes 96.2.1. Binding Capacity by Gel Electrophoresis 96.2.2. Surface Charge by Zeta-Potential 100.2.3. In vitro Biocompatibility Test by Lactate Dehydrogenase Assay (LDH) 102.3. Evaluation of Transfection Efficiency and Transfection Complexes Association with and Internalization into Cells 105.3.1. Green Fluorescent Protein (GFP) Experssion by Fluorescence Microscope 105.3.2. Glial Cell Line–derived Neurotrophic Factor (GDNF) Expression by Enzyme-linked Immunosorbent Assay (ELISA) 107.3.3. Cellular Uptake by Transmission Electron Microscope (TEM) 110HAPTER 6 DISCUSSION 112HAPTER 7 CONCLUSION 121EFERENCE 122URRICULUM VITAE 128application/pdf8551771 bytesapplication/pdfen-US氫氧基磷灰石磁性生醫陶瓷基因治療非病毒載體hydroxyapatitemagneticbioceramicsgene therapynon-viral delivery vector[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/184656/1/ntu-98-D94548010-1.pdf