Multi-functional Nanoparticles for biomedical applications｜國立清華大學博碩士論文庫

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研究生：	拉维 Raviraj
論文名稱：	Multi-functional Nanoparticles for biomedical applications
指導教授：	黃國柱 Hwang, Kuo-Chu
口試委員:	黃國柱宋信文江啟勳袁俊傑 Lin, Shyr-Yu
學位類別：	博士 Doctor
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	169
中文關鍵詞：	多功能纳米和生物医学应用
外文關鍵詞：	multi-functional nanoparticles and biomedical applications
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Multi-functional nanoparticles such as metal nanoparticles and core/shell metal-filled carbon nanoparticles acquire several important and unique properties such as ultra-high absorption co-efficient, photostability (for metal nanoparticles) and magnetic, fluorescent, biocompatibility and facile surface functionalization capability (for core/shell metal-filled carbon nanoparticles) and making them as novel nanoparticle vectors/cargoes for carrying DNA, siRNA, anti-cancer drugs and photosensitizers for several potential therapeutic technologies.
In brief, the thesis begins with the cellular uptake and gene transfection studies of lipid-Fe@CNPs in various cell lines including HeLa, U87MG and TRAMP-C1. Our results suggest that flexible binding between the DNA and the nanoparticle cargo is the key for high gene transfection efficiency and in contrast, tight binding results in very poor efficiencies. In vivo zebrafish gene transfection also showed that flexible binding has driven a better efficiency in the GFP expression, when compared to the tight binding between nanoparticles functionalized with tri methyl ammonium group (TMAEA) and naked DNA. In the case of mouse embryonic stem cells (MESCs), the lipid-Fe@CNPs has also exhibited a better GFP expression, in the presence of elongation factor (EF-1) promoter rather than cytomegalovirus (CMV) promoter. We further investigated the effects of surface functionality of Fe@CNPs on the cellular cytotoxicity and embryonic development in zebrafish. Both in vitro and in vivo zebrafish studies revealed that the Fe@CNPs with imidazolium, trimethyl ammonium and carboxylic acid functional groups induce most of the cellular events, such as, higher levels of ROS and apoptosis, lowering mitochondrial membrane potential, acidification of cells and blocking the cell cycle at S-phase, which all confers to the cellular death along with several abnormalities in zebrafish. Hence, bearing all these issues in mind, the Fe@CNPs with particular functionality could be used as a suitable candidate for potential therapeutic applications.
Among many nanoparticles, metal nanoparticles (M NPs) possess several unique properties such as tunable surface plasmon resonance (SPR), ultra-high absorption co-efficient and excellent photostability. Herein, we showed that singlet oxygen can be formed via direct photoexcitation of metal nanoparticles without any photosensitizers. Further, we also showed that morphology of metal nanoparticles / nanorods play a critical role in the sensitization of singlet oxygen. Overall, the results indicate that gold nanorods are potentially very promising dual functional nanomaterials with capabilities of simultaneously serving as near infrared (NIR) photodynamic therapy and photothermal therapy reagents for cancer treatments. In the last chapter, we have utilized lipid-coated Au NRs to serve as excellent photodynamic / photothermal reagents in the photo destruction of HeLa cells and also for the in vivo malignant tumors with B16F0 melanoma model. Under low laser fluencies, in the in vitro, we have detected reactive oxygen species (ROS), apoptosis, SOSG, and heat shock protein expression (HSP70) levels under photoexcitation at 550 and 940 nm wavelengths. In the in vivo, when we irradiated melanoma tumors with 780 nm, the tumor temperature has been raised until 46oC and whereas, 915 nm irradiation results in temperature of 43oC. The tumor growth curve shows appreciable growth delay when compared with all the other controls, including, chemotherapeutic drug doxorubicin. The experimental results clearly reveal that ROS mediated cell death is more dominant than the contribution from PTT.
Hence, our research provides a sufficient understanding of demonstrating multi-functional nanoparticles (metal and core/shell carbon nanoparticles) as excellent candidates for various biomedical applications.

金屬奈米顆粒以及包裹金屬的奈米碳球均為具多功能性奈米粒子其本身具有幾點重要和獨特的性能，如高吸收係數，光穩定性 (以金屬奈米粒子而言), 磁性，螢光，生物相容性和極簡單表層官能基化 (以包裹金屬的奈米碳球而言）, 同時在一些具有潛力的治療技術上，這些奈米粒子可作為去氧核醣核酸(ＤNA), 短干擾核醣核酸(Small interfering RNA, siRNA), 抗癌藥物以及光治療的新型生物載體
　　此論文則以研究利用脂質（lipid）修飾在具有包裹鐵的奈米碳球上，並以此奈米碳球為載體進行HeLa細胞, U87MG和TRAMP 的细胞吸收和基因轉殖研究為開端。我們的實驗结果闡訴，基因轉殖深深受到去氧核醣核酸(DNA) 與奈米粒子表面的官能基之間的作用力的影響，當其作用較強時，其基因轉殖能力較差；反言之，當作用力較具彈性時，其具有較好的轉植表現。在斑馬魚的實驗中，以綠色螢光蛋白(ＧＦP) 進行基因轉殖的表現的觀測，發現在表面修飾TMAEA的奈米碳球具有較差的螢光表現，而這表示去氧核醣核酸（ＤＮＡ）與TMAEA具有較強的作用力，讓GFP不易在斑馬魚中進行表達。而在小老鼠胚胎幹細胞(mESCs)的實驗中，Lipid-Fe@CNPs 在ＥＦ-1比巨細胞病毒（ＣＭＶ）具有較好的GFP的表徵。我們更進一步探討其奈米碳球表面的官能基對於斑馬魚的細胞毒性與胚胎生長上的影響。　在體內以及體外的斑馬魚研究中顯示，咪唑啉酮（imidazolium）三甲基氨（trimethyl ammonium）以及羧酸 (carboxylic acid group）的官能基團都會導致細胞死亡。因此，總結上述的內容，表面官能基化的包裹鐵金屬粒子的奈米碳球極在醫療上具有無限的潛力。
縱觀許多奈米粒子，金屬奈米粒子具有獨特的性質，如可調式的表面電漿共振(SPR)、高吸收係數以及極為優異的光穩定性…等等。然而，我們實驗證實金屬奈米粒子在沒有修飾任何光敏劑的情況下，照光產生單重態的氧。　我們也同時實驗證實不同形狀的金屬奈米粒子其晶面對於產生單重態的氧扮演著舉足輕重的角色。　總結，實驗的結果指出金奈米棒極有前途可能成為具有雙功能的奈米材料治療癌症，因為金奈米棒同時具有紅外光驅動的光動療以及光熱療的能力，可以成為抗癌試劑。　在最後一個章節，我們利用脂質包裹的金奈米棒當作一個極為優越的光動療/光熱療的試劑，在照光下摧毀HeLa細胞；並也同時進行生物體實驗，在B16F0黑色素惡性腫瘤下，在極低功率的光照射下，我們量測到具高氧化力的物種存在於生物體內及體外實驗中，並且發現加熱停止HSP70的運作，進而導致細胞死亡，而造成細胞死亡的原因為極為可能是光動療以及光熱療。
因此，我們研究提供對這些具功能性的金屬材料(如　金屬　以及　包裹金屬的奈米碳球)的認知，並提供實驗說明這一些材料如何在生化的運用上具有極佳的性質。

Table of Contents

Chapter 1 Flexible Binding is the Key for High Gene Transfection Efficiency: Lipid-Carbon Nanoparticles as Nano DNA Cargoes
1 Background and Introduction    1
1.1 Gene therapy    2
1.2 Intracellular barriers in DNA delivery    2
1.3 Non-viral delivery approaches    5
1.4 Discrepancies in tight binding between oligonucleotides and vectors    6
2 Experimental Section    7
2.1 Synthesis of Core/Shell Iron/Carbon Nanoparticles using Solid State Microwave Arcing    7
2.2 Surface functionalization of Fe@CNPs    8
2.3 Synthesis of lipid-folate conjugates    9
2.4 Preparation of lipid-Fe@CNPs    9
2.5 Surface modification of Fe@CNPs with N, N, N-trimethyl–N-2-methacryloxyethyl) ammonium chloride    9
2.6 Preparation of DNA-lipid-Fe@CNPs and DNA-Fe@CNPs-TMAEA complexes    10
2.7 Cell culture, materials and reagents    10
2.8 Transfection and cellular uptake assay    10
2.9 Cellular Uptake    11
2.10 Confocal Microscopy    11
2.11 Western blot analysis    11
2.12 Agarose gel electrophoresis    12
2.13 Annexin-V apoptosis assay    12
2.14 Reactive oxygen species (ROS) generation    12
2.15 Cytotoxicity assay    13
2.16 Incubation of lipid-Fe@CNPs conjugates under ATP depletion    13
2.17 Folic Acid Pre-treatment for blocking folate receptors    13
2.18 Microinjection of DNA-lipid-Fe@CNPs or DNA-Fe@CNPs-TMAEA into Zebra Fish Embryos    14
3 Results and Discussion    14
3.1 Surface functionalization of Fe@CNPs    14
3.2 Zeta-potential and Confocal microscopy    20
3.3 Cellular uptake and Gene transfection efficiency    22
3.4 Gel electrophoresis    27
3.5 Cytotoxicity Assays    29
3.6 In vivo cytotoxicity and gene expression in zebrafish    30
4 Conclusions    35
5 References    36
Chapter 2 Ultra-high Cellular Uptake Facilitates Efficient Gene Delivery Using Fluorescent Lipid-Fe@CNPs Conjugates labeled Embryonic Stem Cells
1 Background and Introduction    40
1.1 Stem Cells    40
1.2 Embryonic Stem Cells    41
1.3 Genetic Manipulation in ES cells    42
1.4 Embryonic stem cell labeling    45
2 Experimental Section    46
2.1 Synthesis of Core/Shell Iron/Carbon Nanoparticles using Solid State Microwave Arcing    46
2.2 Surface functionalization of Fe@CNPs    46
2.3 Characterization of Fe@CNPs    47
2.4 Preparation of lipid-Fe@CNPs nanoconjugates    48
2.5 Preparation of DNA-lipid-Fe@CNPs complexes    48
2.6 Cell culture, Materials and Reagents    48
2.7 Transfection and Cellular uptake assays    49
2.8 Cytotoxicity assays    50
3 Results and Discussion    50
4 Conclusions    58
5 References    59
Chapter 3 Effect of Surface Functionality of Carbon Nanoparticles on Cytotoxicity and Embryo Development in Zebrafish
1 Background and Introduction    62
2 Experimental Section    63
2.1 Synthesis of Core/Shell Iron/Carbon Nanoparticles using Solid State Microwave Arcing    63
2.2 Surface functionalization of Fe@CNPs    64
2.3 Cell culture, materials and reagents    64
2.4 Cytotoxicity assay    64
2.5 Annexin-V apoptosis assay    65
2.6 Reactive oxygen species (ROS) generation    65
2.7 Assessing the integrity of lysosomal membrane    66
2.8 Changes in the mitochondrial membrane potential    66
2.9 Analysis of Intracellular pH    66
2.10 Cell cycle analysis    67
2.11 Microinjection of different surface functionalized Fe@CNPs into Zebrafish Embryos    67
3 Results and Discussion    68
3.1 Characterization of Fe@CNPs    68
3.2 Cytotoxicity assay    71
3.3 ROS generation    73
3.4 Annexin-V apoptosis assay    74
3.5 Changes in the lysosomal membrane integrity    75
3.6 Changes in the mitochondrial membrane potential    76
3.7 Intracellular pH    77
3.8 Cell cycle analysis    78
3.9 Biocompatibility of different surface functionalized Fe@CNPs in zebrafish    ………………………………………………………………………………79
4 Conclusions    81
5 References    81
Chapter 4 Metal Nanoparticles Sensitize Formation of Singlet Oxygen
1 Background and Introduction    84
2 Experimental Section    86
2.1 Singlet oxygen phosphorescence measurements    86
2.2 Chemical trapping of singlet oxygen using Singlet Oxygen Sensor Green (SOSG)    86
2.3 Hydroperoxidation of cyclohexene    87
2.4 Generation of singlet oxygen by dark reaction    88

3 Results and Discussion    88
4 Conclusions    100
5 References    101
Chapter 5 Morphology Dependent Sensitization and Formation of Singlet Oxygen (1∆g) by Gold Nanorods and Silver Nanoparticles
1 Background and Introduction    105
2 Experimental Section    106
2.1 Singlet oxygen phosphorescence measurements    106
2.2 SOSG experiments    107
2.3 Hydroperoxidation of cyclohexene    107

2.4 Results and Discussion    108
3 Conclusions    123
4 References    124
Chapter 6 Gold Nanorods with triple function as intracellular fluorescent marker and Photothermal / Photodynamic Destruction of Malignant Tumors
1 Background and Introduction    129
1.1 Photodynamic therapy (PDT)    129
1.2 Modes of cell death    130
1.3 Gold nanomaterials for PDT-PTT applications    132
2 Experimental Section    134
2.1 Synthesis of lipid-coated Au NRs    134
2.2 Singlet oxygen phosphorescence measurements    134
2.3 Cell culture, materials and reagents    135
2.4 Cellular uptake studies using confocal laser scanning microscopy (CLSM) and flow cytometry    135
2.5 Cytotoxicity assays    136
2.6 ROS experiments    137
2.7 Annexin-V apoptosis assay    137
2.8 Singlet oxygen sensor green (SOSG) experiments    137
2.9 Assessing the mitochondrial membrane potential    138
2.10 Heat-shock protein expression analysis    138
2.11 In vivo fluorescence imaging    138
2.12 In vivo ROS and HSP 70 analysis    139
2.13 In vivo tumor growth study    139
2.14 Tissue sectioning and caspase 3 staining    140
2.15 In vivo thermal imaging    140
3 Results and Discussion    140
4 Conclusions    161
5 References    162


                                

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