請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47398
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 周涵怡(Han-Yi Elizabeth Chou) | |
dc.contributor.author | Chia-Ying Lee | en |
dc.contributor.author | 李佳盈 | zh_TW |
dc.date.accessioned | 2021-06-15T05:58:04Z | - |
dc.date.available | 2010-09-09 | |
dc.date.copyright | 2010-09-09 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-17 | |
dc.identifier.citation | 1. Ofek, P., Fischer, W., Calderon, M., Haag, R. & Satchi-Fainaro, R. In vivo delivery of small interfering RNA to tumors and their vasculature by novel dendritic nanocarriers. FASEB J (2010).
2. Kim, S.H., Jeong, J.H., Kim, T.I., Kim, S.W. & Bull, D.A. VEGF siRNA delivery system using arginine-grafted bioreducible poly(disulfide amine). Mol Pharm 6, 718-726 (2009). 3. Shen, Y. Advances in the development of siRNA-based therapeutics for cancer. IDrugs 11, 572-578 (2008). 4. Devi, G.R. siRNA-based approaches in cancer therapy. Cancer Gene Ther 13, 819-829 (2006). 5. Aagaard, L. & Rossi, J.J. RNAi therapeutics: principles, prospects and challenges. Adv Drug Deliv Rev 59, 75-86 (2007). 6. Abdelrahim, M., Safe, S., Baker, C. & Abudayyeh, A. RNAi and cancer: Implications and applications. J RNAi Gene Silencing 2, 136-145 (2006). 7. Brink, P.R., Robinson, R.B., Rosen, M.R. & Cohen, I.S. In vivo cellular delivery of siRNA. IDrugs 13, 383-387 (2010). 8. Shi, M.L., Zhao, Z.H., Wang, Y. & Chen, H.P. [In vivo delivery of siRNA]. Yi Chuan 31, 683-688 (2009). 9. Wang, J., Lu, Z., Wientjes, M.G. & Au, J.L. Delivery of siRNA Therapeutics: Barriers and Carriers. AAPS J (2010). 10. Manjunath, N. & Dykxhoorn, D.M. Advances in synthetic siRNA delivery. Discov Med 9, 418-430 (2010). 11. Tiemann, K. & Rossi, J.J. RNAi-based therapeutics-current status, challenges and prospects. EMBO Mol Med 1, 142-151 (2009). 12. Barton, G.M. & Medzhitov, R. Retroviral delivery of small interfering RNA into primary cells. Proc Natl Acad Sci U S A 99, 14943-14945 (2002). 13. Xia, H., Mao, Q., Paulson, H.L. & Davidson, B.L. siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol 20, 1006-1010 (2002). 14. Shen, C., Buck, A.K., Liu, X., Winkler, M. & Reske, S.N. Gene silencing by adenovirus-delivered siRNA. FEBS Lett 539, 111-114 (2003). 15. Aagaard, L., et al. A facile lentiviral vector system for expression of doxycycline-inducible shRNAs: knockdown of the pre-miRNA processing enzyme Drosha. Mol Ther 15, 938-945 (2007). 16. Wang, Y., Gao, S., Ye, W.H., Yoon, H.S. & Yang, Y.Y. Co-delivery of drugs and DNA from cationic core-shell nanoparticles self-assembled from a biodegradable copolymer. Nat Mater 5, 791-796 (2006). 17. Luo, D., Han, E., Belcheva, N. & Saltzman, W.M. A self-assembled, modular DNA delivery system mediated by silica nanoparticles. J Control Release 95, 333-341 (2004). 18. Passineau, M.J., Zourelias, L., Machen, L., Edwards, P.C. & Benza, R.L. Ultrasound-assisted non-viral gene transfer to the salivary glands. Gene Ther (2010). 19. Ueno, Y., Hirashima, N., Inoh, Y., Furuno, T. & Nakanishi, M. Characterization of biosurfactant-containing liposomes and their efficiency for gene transfection. Biol Pharm Bull 30, 169-172 (2007). 20. Yu, G., et al. In vitro non-viral lipofectamine delivery of the gene for glial cell line-derived neurotrophic factor to human umbilical cord blood CD34+ cells. Brain Res 1325, 147-154 (2010). 21. Tempone, A.G., Mortara, R.A., de Andrade, H.F., Jr. & Reimao, J.Q. Therapeutic evaluation of free and liposome-loaded furazolidone in experimental visceral leishmaniasis. Int J Antimicrob Agents (2010). 22. Landen, C.N., Jr., et al. Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery. Cancer Res 65, 6910-6918 (2005). 23. Sorensen, D.R., Leirdal, M. & Sioud, M. Gene silencing by systemic delivery of synthetic siRNAs in adult mice. J Mol Biol 327, 761-766 (2003). 24. Filion, M.C. & Phillips, N.C. Toxicity and immunomodulatory activity of liposomal vectors formulated with cationic lipids toward immune effector cells. Biochim Biophys Acta 1329, 345-356 (1997). 25. Wu, S.Y. & McMillan, N.A. Lipidic systems for in vivo siRNA delivery. AAPS J 11, 639-652 (2009). 26. Kneuer, C., et al. The influence of physicochemical parameters on the efficacy of non-viral DNA transfection complexes: a comparative study. J Nanosci Nanotechnol 6, 2776-2782 (2006). 27. Semple, S.C., et al. Efficient encapsulation of antisense oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novel small multilamellar vesicle structures. Biochim Biophys Acta 1510, 152-166 (2001). 28. Dincer, S., Turk, M. & Piskin, E. Intelligent polymers as nonviral vectors. Gene Ther 12 Suppl 1, S139-145 (2005). 29. Kulkarni, R.P., Wu, D.D., Davis, M.E. & Fraser, S.E. Quantitating intracellular transport of polyplexes by spatio-temporal image correlation spectroscopy. Proc Natl Acad Sci U S A 102, 7523-7528 (2005). 30. Li, H., Wang, J., Zhou, T., Zhang, Y. & Zhang, Z. An investigation of the effects of nanosize delivery system for antisense oligonucleotide on esophageal squamous cancer cells. Cancer Biol Ther 7(2008). 31. Pavan, G.M., et al. PAMAM Dendrimers for siRNA Delivery: Computational and Experimental Insights. Chemistry (2010). 32. Akita, H., et al. Multi-layered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process. Biomaterials 30, 2940-2949 (2009). 33. Zhu, C., et al. Co-delivery of siRNA and paclitaxel into cancer cells by biodegradable cationic micelles based on PDMAEMA-PCL-PDMAEMA triblock copolymers. Biomaterials 31, 2408-2416 (2010). 34. Xiong, X.B., Uludag, H. & Lavasanifar, A. Biodegradable amphiphilic poly(ethylene oxide)-block-polyesters with grafted polyamines as supramolecular nanocarriers for efficient siRNA delivery. Biomaterials 30, 242-253 (2009). 35. Lim, S.Y., Kim, J.H., Lee, J.S. & Park, C.B. Gold nanoparticle enlargement coupled with fluorescence quenching for highly sensitive detection of analytes. Langmuir 25, 13302-13305 (2009). 36. Obonyo, O., Fisher, E., Edwards, M. & Douroumis, D. Quantum dots synthesis and biological applications as imaging and drug delivery systems. Crit Rev Biotechnol (2010). 37. Gao, X., Cui, Y., Levenson, R.M., Chung, L.W. & Nie, S. In vivo cancer targeting and imaging with semiconductor quantum dots. Nat Biotechnol 22, 969-976 (2004). 38. Farkas, J., et al. Effects of silver and gold nanoparticles on rainbow trout (Oncorhynchus mykiss) hepatocytes. Aquat Toxicol 96, 44-52 (2010). 39. Stelzer, R. & Hutz, R.J. Gold nanoparticles enter rat ovarian granulosa cells and subcellular organelles, and alter in-vitro estrogen accumulation. J Reprod Dev 55, 685-690 (2009). 40. King-Heiden, T.C., et al. Quantum dot nanotoxicity assessment using the zebrafish embryo. Environ Sci Technol 43, 1605-1611 (2009). 41. Lin, C.H., et al. The chemical fate of the Cd/Se/Te-based quantum dot 705 in the biological system: toxicity implications. Nanotechnology 20, 215101 (2009). 42. Jacobsen, N.R., et al. Lung inflammation and genotoxicity following pulmonary exposure to nanoparticles in ApoE-/- mice. Part Fibre Toxicol 6, 2 (2009). 43. Talelli, M., et al. Superparamagnetic iron oxide nanoparticles encapsulated in biodegradable thermosensitive polymeric micelles: toward a targeted nanomedicine suitable for image-guided drug delivery. Langmuir 25, 2060-2067 (2009). 44. Taylor, E.N. & Webster, T.J. The use of superparamagnetic nanoparticles for prosthetic biofilm prevention. Int J Nanomedicine 4, 145-152 (2009). 45. Vittorio, O., Raffa, V. & Cuschieri, A. Influence of purity and surface oxidation on cytotoxicity of multiwalled carbon nanotubes with human neuroblastoma cells. Nanomedicine (2009). 46. Nygaard, U.C., et al. Single-walled and multi-walled carbon nanotubes promote allergic immune responses in mice. Toxicol Sci 109, 113-123 (2009). 47. Ding, L., et al. Molecular characterization of the cytotoxic mechanism of multiwall carbon nanotubes and nano-onions on human skin fibroblast. Nano Lett 5, 2448-2464 (2005). 48. Kahru, A. & Dubourguier, H.C. From ecotoxicology to nanoecotoxicology. Toxicology (2009). 49. Fu, C.C., et al. Characterization and application of single fluorescent nanodiamonds as cellular biomarkers. Proc Natl Acad Sci U S A 104, 727-732 (2007). 50. Yu, S.J., Kang, M.W., Chang, H.C., Chen, K.M. & Yu, Y.C. Bright fluorescent nanodiamonds: no photobleaching and low cytotoxicity. J Am Chem Soc 127, 17604-17605 (2005). 51. Chang, Y.R., et al. Mass production and dynamic imaging of fluorescent nanodiamonds. Nat Nanotechnol 3, 284-288 (2008). 52. Faklaris, O., et al. Detection of single photoluminescent diamond nanoparticles in cells and study of the internalization pathway. Small 4, 2236-2239 (2008). 53. Neugart, F., et al. Dynamics of diamond nanoparticles in solution and cells. Nano Lett 7, 3588-3591 (2007). 54. Schrand, A.M., et al. Are diamond nanoparticles cytotoxic? J Phys Chem B 111, 2-7 (2007). 55. Santin, M., et al. In vitro host response assessment of biomaterials for cardiovascular stent manufacture. J Mater Sci Mater Med 15, 473-477 (2004). 56. Higuchi, Y., Kawakami, S. & Hashida, M. Strategies for in vivo delivery of siRNAs: recent progress. BioDrugs 24, 195-205 (2010). 57. Wang, J.J., Zheng, Y., Yang, F., Zhao, P. & Li, H.F. Survivin small interfering RNA transfected with a microbubble and ultrasound exposure inducing apoptosis in ovarian carcinoma cells. Int J Gynecol Cancer 20, 500-506 (2010). 58. Akinaga, H. Magnetic-field-sensing materials composed of metal-semiconductor hybrid nanostructures. J Nanosci Nanotechnol 5, 250-254 (2005). 59. Mercer, J. & Helenius, A. Virus entry by macropinocytosis. Nat Cell Biol 11, 510-520 (2009). 60. Roth, M.G. Clathrin-mediated endocytosis before fluorescent proteins. Nat Rev Mol Cell Biol 7, 63-68 (2006). 61. Carver, L.A. & Schnitzer, J.E. Caveolae: mining little caves for new cancer targets. Nat Rev Cancer 3, 571-581 (2003). 62. Schutze, S., Tchikov, V. & Schneider-Brachert, W. Regulation of TNFR1 and CD95 signalling by receptor compartmentalization. Nat Rev Mol Cell Biol 9, 655-662 (2008). 63. Sigismund, S., et al. Clathrin-independent endocytosis of ubiquitinated cargos. Proc Natl Acad Sci U S A 102, 2760-2765 (2005). 64. Cinar, H. & Barnes, E.M., Jr. Clathrin-independent endocytosis of GABA(A) receptors in HEK 293 cells. Biochemistry 40, 14030-14036 (2001). 65. Damke, H., Baba, T., van der Bliek, A.M. & Schmid, S.L. Clathrin-independent pinocytosis is induced in cells overexpressing a temperature-sensitive mutant of dynamin. J Cell Biol 131, 69-80 (1995). 66. Sandgren, K.J., et al. A differential role for macropinocytosis in mediating entry of the two forms of vaccinia virus into dendritic cells. PLoS Pathog 6, e1000866 (2010). 67. Perry, J.W. & Wobus, C.E. Endocytosis of murine norovirus 1 into murine macrophages is dependent on dynamin II and cholesterol. J Virol 84, 6163-6176 (2010). 68. Kalin, S., et al. Macropinocytotic uptake and infection of human epithelial cells with species B2 adenovirus type 35. J Virol 84, 5336-5350 (2010). 69. Nakase, I., et al. Cell-surface accumulation of flock house virus-derived peptide leads to efficient internalization via macropinocytosis. Mol Ther 17, 1868-1876 (2009). 70. Lu, J.J., Langer, R. & Chen, J. A novel mechanism is involved in cationic lipid-mediated functional siRNA delivery. Mol Pharm 6, 763-771 (2009). 71. Kerr, M.C. & Teasdale, R.D. Defining macropinocytosis. Traffic 10, 364-371 (2009). 72. Bartneck, M., et al. Rapid uptake of gold nanorods by primary human blood phagocytes and immunomodulatory effects of surface chemistry. ACS Nano 4, 3073-3086 (2010). 73. Albertazzi, L., Serresi, M., Albanese, A. & Beltram, F. Dendrimer internalization and intracellular trafficking in living cells. Mol Pharm 7, 680-688 (2010). 74. Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408 (2001). 75. Inoue, K., et al. Effects of multi-walled carbon nanotubes on a murine allergic airway inflammation model. Toxicol Appl Pharmacol 237, 306-316 (2009). 76. Kulkarni, M., Greiser, U., O'Brien, T. & Pandit, A. Liposomal gene delivery mediated by tissue-engineered scaffolds. Trends Biotechnol 28, 28-36 (2010). 77. Abrams, M.T., et al. Evaluation of efficacy, biodistribution, and inflammation for a potent siRNA nanoparticle: effect of dexamethasone co-treatment. Mol Ther 18, 171-180 (2010). 78. Fischer, D., Li, Y., Ahlemeyer, B., Krieglstein, J. & Kissel, T. In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis. Biomaterials 24, 1121-1131 (2003). 79. Peng, L., Liu, M., Xue, Y.N., Huang, S.W. & Zhuo, R.X. Transfection and intracellular trafficking characteristics for poly(amidoamine)s with pendant primary amine in the delivery of plasmid DNA to bone marrow stromal cells. Biomaterials 30, 5825-5833 (2009). 80. Agarwal, A., Unfer, R. & Mallapragada, S.K. Investigation of in vitro biocompatibility of novel pentablock copolymers for gene delivery. J Biomed Mater Res A 81, 24-39 (2007). 81. Mok, H., Lee, S.H., Park, J.W. & Park, T.G. Multimeric small interfering ribonucleic acid for highly efficient sequence-specific gene silencing. Nat Mater 9, 272-278. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47398 | - |
dc.description.abstract | 發展安全且高效能的生物載體在癌症以及許多疾病的治療上面扮演至關重要的角色。以基因標靶治療為例,也就是利用設計特定基因序列的遺傳序列送入病人體內,藉由調整該基因的表現來治療相關的遺傳或後天疾病,這種治療方式已廣泛應用在許多癌症的臨床治療上面。其中短片段雙股核醣核酸干擾小體(siRNA)係藉由設計與特定基因相關的短片段雙股核醣核酸序列送入體內,抑制該基因的表現量來治療與該基因相關的疾病的方式,已被視為十分具有潛力也廣泛應用在臨床治療的重要研究領域。目前雖然有許多備受矚目的載體,但其本身的結構以及組成均可能造成對生物體的毒性,不僅如此,這些多功能的載體在生物體內長期累積可能造成免疫以及其方面的傷害,因此能夠發展新一代結合影像診斷及標靶治療於一身,最重要的是具有活體相容性的多功能奈米載體,更是生醫材料領域研究的當務之急!
螢光奈米鑽石是經由高速粒子束撞擊,再以熱能結合創造出晶格缺陷,這個真空的晶格空缺使螢光奈米鑽石在約560 nm波長的光激發之下,可以有效且穩定地放出約700 nm波長的大紅色永恆的螢光。而且,螢光奈米鑽石具有高光穩定性、完全無光漂白、且沒有blinking的獨特性質,使螢光觀察得以長期且穩定的執行。 螢光奈米鑽石不具生物毒性,而且其表面特性很容易處理成含氧的官能基,再進一步修飾得以與核酸分子或是蛋白質作結合。 本篇研究將以口腔癌細胞為基礎,系統性的研究螢光奈米鑽石與標的細胞及活體之間的生物交互作用,並找出適合的生物分子裝載條件,期望能達到最終發展口腔癌治療的依據。我們以短片段雙股核醣核酸干擾小體與螢光奈米鑽石及帶電荷聚合體結合,並且施加輕微短時間的離心作用,找到生物分子裝載最佳化之條件。並且利用奈米鑽石的螢光做為細胞追蹤工具,研究螢光奈米鑽石進入細胞並於期內所引發之交互作用。我們也同時使用冷光胜肽標定異體移植口腔癌之小鼠,了解螢光奈米鑽石結合特定雙股短片段核醣核酸在生物系統之作用效果與情形。我們的結果顯示,螢光奈米鑽石可作為極佳的生物分子載體,並證明我們可利用螢光奈米鑽石在無毒性反應下,達到更佳的基因抑制的效果。 此外我們也發現螢光奈米鑽石除了可藉由一般的細胞胞吞作用進入細胞以外,更能夠引發細胞的大胞飲作用。而離心的作用增加粒子與細胞表面接觸的頻率,因而增加活化細胞的胞吞作用,以達到更佳的基因抑制效果。我們藉由深入的探討螢光奈米鑽石與生物系統的交互作用,可望發展出可以追蹤目標細胞的核酸裝載系統,成為多功能奈米載體在癌症治療的應用上極具潛力的明日之星! | zh_TW |
dc.description.abstract | Delivery technique holds the key to successful treatment of cancer and many diseases. For example, gene therapy is a promising method which manipulates the defective genes by delivery nucleotides into cells to treat inherited and acquired diseases that are currently considered incurable. It has been exercising in many clinical trials. RNA interference has been considered one of the most promising therapeutic platforms by introducing siRNA into the cell and switch off certain disease-relating genes. There are many vectors reported to be useful for genetic delivery, however, most of them have the concerns of cytotoxicities due to the component molecule and the induction of long-term damage. For these reasons, an efficient delivery system for siRNA remains to be developed.Type Ib fluorescent nanodiamonds (FNDs) can emit no photobleaching and no photoblinking fluorescence from their nitrogen-vacancy point defects, at a spectral range well suited for long term observation in living cells. They have good biocompatibility and can be easily functionalized for specific or nonspecific binding with biomolecules. In this study, based on the experiences of oral cancer, we propose to use cell and animal based oral cancer models to address the biological responses elicited by FNDs, and to simultaneously assess the best combinations of functionalization and cargo loading for future applications. We combined the FND with siRNA and common transfection reagents, applied with mild centrifugation to explore the gene-inhibition efficiency. In the same time, we studied the uptake mechanism and cellular response to the internalization of bare and cargo loaded FNDs. Besides, we tested the genetic delivery efficiency of FND-optimized siRNA complexes into xenografe bioluminescence-marked oral cancer animal models. From our results, FND showed dramatic improvement in silencing effect with good biocompatibility. And FND may be internalized into cells by macropinocytosis besides normal endocytosis pathways. We found that centrifugation facilitated the entrance of FND by increasing the contact between the particles and cellular surface, thus triggered more macropinocytosis responses to include more siRNA-combined FNDs. We provide the first and fundamental knowledge for a well-grounded development in biomedical applications of the FNDs. This is a powerful and promising achievement for subsequent improvements towards the final achievement of revolutionary new capabilities in the diagnosis, tracing and curing of cancer, as well as the treatment of other difficult diseases. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T05:58:04Z (GMT). No. of bitstreams: 1 ntu-99-R97450005-1.pdf: 5289918 bytes, checksum: 004d7ae3b5f7e6f17c2b9cd63d3a2787 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 口試委員會審定書 I
誌謝 II 中文摘要 VI ABSTRACT VIII CONTENTS X ABBREVIATIONS XV FIGURE CONTENT XVI TABLE CONTENT 1 I. INTRODUCTION 2 1 Gene therapy is a promising application for disease treatment. 2 2 Common vectors used in genetic engineering to date 3 2.1 Virus vectors 3 2.2 Non-viral vectors 3 2.2.1 Liposome-based vector: 4 2.2.2 Polycationic polymer vectors: 5 2.2.3 Metal vectors 6 2.2.4 Carbon-backboned vectors 7 2.2.5 Fluorescent nano-diamond 8 3 Physical strengths to promote the delivery efficiency 9 4 Mechanisms of the internalization of particles 10 4.1 Endocytosis 10 4.1.1 Clathrin-mediated endocytosis: 10 4.1.2 Caveolae-mediated endocytosis: 11 4.1.3 Macropinocytosis 11 5 Premise 13 6 Hypothesis 13 II. MATERIALS AND METHODS 14 1 The Fluorescent nanodiamond 14 2 Cell culture condition 14 3 Transmission electron microscope (TEM) characterization of FNDs 15 4 Particle sizing analysis of FNDs 15 5 Cell viability assay 16 6 Gene transfection 17 6.1 The preparation of transfection complexes. 17 6.2 Comparison of si-Lu transfection activity in different time 18 6.3 Comparison of si-Lu transfection activity in different dosages of siRNA. 19 6.4 Comparison of different dosages of FNDs on transfection activity 20 6.5 Comparison FNDs on transfection activity under different centrifugation time. 21 6.6 Comparison of FNDs-modified/no FNDs-modified transfection activity in 24hrs. 22 6.7 Comparison of FND-mediated siRNA activity in YFP-expressing cells. 24 7 Validation the mRNA expression of endogeneou genes using FND-modified transfection process by q-RT-PCR. 25 8 For different transfection reagents in 96well microplate under the same concentration of si-RNA (1nM ): 28 8.1 INTERFERin: 28 8.2 Lipofectamine 2000: 29 8.3 T-Pro-NTR II: 29 8.4 Arrest-IN: 30 8.5 HiPerFect: 30 8.6 Superfect: 31 9 The observation of FND-modified transfection kinetics in luciferase activity and β-actin mRNA expression levels. 31 10 The confocol microscope analysis of cellular uptake of FND under / without centrifugation. 35 11 The examination of the dosages of endocytosis inhibitors by MTT assay. 35 12 Endocytosis inhibition 36 13 In vitro cellular internalization by TEM. 37 14 Molecular analysis of macropinocytosis activities by western blotting 38 15 Animal experiments for optical tumor imaging analysis. 41 III. RESULTS 43 1 Characterization of type Ib Fluorescent Nanodiamond 43 1.1 The morphology of FND particle is irregular and their main size is about 50nm. 43 1.2 Confirmation of FND biocompatibility. 43 2 Investigation of the cellular internalization of FND 44 2.1 Centrifugation strength facilitated FND entrance 44 2.2 Observation of cellular uptake of FND under the interference of endocytosis inhibitors 45 2.3 Centrifuge helped cellular uptake of FND when the endocytosis is inhibited. 46 2.4 Elimination of the toxic effect of endocytosis inhibitors. 47 2.5 Morphological studies of macropinocytosis in FND uptake by transmission electron microscope 47 2.6 Statistics of the cellular structures in 30 min and 4 hr incubation with FNDs. 48 3 Confirmation of the molecular response of macropinocytosis by western blotting 49 4 Evaluation of the efficiency of FND-involved siRNA delivery 49 4.1 Validation of the luciferase targeting siRNA activity 49 4.2 The inhibition extent of FND-optimized process is dose-dependent. 50 4.3 Centrifugation facilitated the FND-optimized silencing effect even more 51 4.4 FND and centrifuge promote better silencing extent regard and regardless of incubation time 51 4.5 FND-optimized transfection proceeded gene silencing effect in many different types of vectors 53 4.6 FND combined with mild centrifugation is non-toxic to cells under the dosage used in this study 53 5 The detailed investigation of the applications of FND-optimized delivery system 54 5.1 The silencing effect of FND-combined transfection complex is non-gene spicific 54 5.2 FND-optimized transfection process works both in endogenous gene 55 5.3 FND-optimized process make faster and prolonged inhibition effect in both genetic and protein levels. 55 5.4 Investigation of FND- optimized siRNA efficiency in vivo 56 IV. DISCUSSION 58 More experiments are needed to improve the suspension of FNDs in physiological environment 58 The excellent biocompatibility of FND promote the usage in clinical trials 58 The rapid recovery of cellular viabilities attribute to good adjustment of FND in biological environment 59 The aggregation of FND particles in the confocol images after centrifuge revealed the enhanced attachment of particles and the cellular surface by centrifuge 60 The extensiveness of FND may relate with the activation of an unique route of internalization, the macropinocytosis as we observed the macropinocytotic structure in TEM image. 61 The macropinocytosis stimulated further by the action of centrifugation, 61 The secondary lysosome and residual bodies appeared in 4hr may due to the centrifuge enhancement 62 Cells kept uptake FNDs up to at least 4 hrs 62 The FND enters into cells with minimal physical and chemical damage to the cells 63 The siRNA activity was promoted by FNDs 63 FND facilitated the siRNA activity by macropinocytosis which promote the cellular damage by endosomal disruption 64 FND has great potential in the application of tailored therapies 65 FND promoted the transfection efficiency even in vectors usually used in DNA delivery 66 The kinetics of FND-optimized transfection showed the faster and prolonged gene silencing effect 67 Preliminary results in animal models 67 The luciferase siRNA didn’t affect the growth of tumor. 68 β-actin is not the main microfilament involved in macropinocytosis. 68 V. CONCLUSION 70 VI. FUTURE WORK 71 Development of the best formula for FND and carriers to get good suspension and delivery efficiency 71 Monitoring the movement of FND inside and outside the cells and the animal bodies to identify the long term systemic distribution of FND. 71 Test the stability of FND-optimized complex mimic the biological environment 72 Qunatiation of the FND particles inside the cells using flow cytometry 72 Identify the molecular regulation levels of macropinocytosis by western blotting and microarray. 72 Indentify the FND particle influences on human whole genome using microarray analysis. 73 Observation of the siRNA-FND connection by labeling the traceable markers on the siRNA. 73 Validation of the FND- optimized transfection process in different types of cell lines. 74 Observation of the FND/siRNA complex by atomic force microscope (AFM) or electrophoresis 74 VII. REFERENCE 75 VIII. TABLES 81 IX. FIGURES 85 X. APPENDIX 120 | |
dc.language.iso | en | |
dc.title | 發展以螢光奈米鑽石攜帶短干擾核醣核酸之生物醫學應用 | zh_TW |
dc.title | Fluorescent Nanodiamond-Assisted Delivery of siRNAs | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳惠文(Huei-Wen Chen),張富雄(Fu-Hsiung Chang) | |
dc.subject.keyword | 基因治療,螢光奈米鑽石,短干擾核醣核酸, | zh_TW |
dc.subject.keyword | gene therapy,fluorescent nano-diamond,siRNA, | en |
dc.relation.page | 124 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-08-17 | |
dc.contributor.author-college | 牙醫專業學院 | zh_TW |
dc.contributor.author-dept | 口腔生物科學研究所 | zh_TW |
顯示於系所單位: | 口腔生物科學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-99-1.pdf 目前未授權公開取用 | 5.17 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。