臺灣博碩士論文加值系統

一般認為，帶正價電之粒子相較於不帶電或是帶陰電性之粒子容易吸附於細胞膜上被細胞所吞噬，在過去基因傳輸系統的研究中，確實已證實帶正電的粒子或載體效力較佳。然而，透過YC Chung等學者研發出帶陰電性單股核酸之基因載體，能有效達到轉殖之效能，因此，本研究將建立陰電性單股核酸載體於細胞吞噬之機制，藉由添加不同路徑之抑制劑，得知，載體能經由兩種以上之路徑進入細胞，然而，只有caveolar及macropinocytosis pathway能達到基因轉殖之效能，一旦確認載體之路徑不僅能改善未來基因載體之製備過程，亦能避免載體進入lysosomes而大幅提升載體之轉殖效能。
此外，本研究藉由單股核酸包覆於基因轉殖載體之方法，結合工研院提供表面帶有不同官能基之氧化鐵粒子作為模板，製備出以單股核酸組裝外層之陰電性氧化鐵粒子。由於氧化鐵本身特性影響，在包覆多層聚電解質時，顯示細胞毒性會受到所使用陽離子層所調控，經單股核酸包覆後能同時降低細胞毒性並被細胞吞噬；此包覆技術運用至氧化鐵上，可直接製備出不須純化，進一步修飾氧化鐵粒子。此外，將單股核酸包覆氧化鐵粒子與吞噬機制結合，得知經單股核酸包覆後之粒子，即可藉由不同之吞噬路徑進入到細胞內。
因此，本研究成功以單股核酸包覆氧化鐵粒子作為模板，未來可利用核酸互補的性質作為連結標的分子和氧化鐵的架橋，發展出磁光合一的功能或是帶有標的功能之氧化鐵粒子，預期將可應用於臨床磁振造影且同時具有螢光標定追蹤癌細胞之功能。

Generally, particle uptake by cells is mediated by electrostatic interactions of positively charged particles with negatively charged cellular surface, so the cationic polyplexes showed efficiently cellular transfection. However, YC Chung et al. developed a new complex with high transfection efficiency by assembling negatively charged single-stranded oligonucleotides with polycation/DNA complexes. Therefore, this project investigated the cellular uptake pathway of single-stranded oligonucleotides with polycation/DNA complexes. The results of different inhibitors showed these complexes can cellular uptake by several pathways. However, only the caveolae and macropinocytosis pathway would influence gene expression.
Furthermore, iron oxide particles will be investigated by single-stranded oligonucleotide-assembled technique. The results indicate the oligonucleotide-coated iron oxide particles could be uptaken and present low cytotoxicity. This technique of oligonucleotide-assembled iron oxide particles can easily manufacture and without further purification. To integrate the oligonucleotide-coated iron oxide particles and cellular uptake pathway, the results also shows the oligonucleotide-coated iron oxide particles could be internalized by several pathways.
Finally, this project successfully develop a multifunctional iron oxide platform by conjugating oligonucleotides, and also develop the ligand or fluorescent probe by nucleic acid base-paired interaction. Therefore, the multifunctional iron oxide particles can be applied to MRI or fluorescent target to track tumor in vivo.

第1章 緒論 1
第2章 文獻回顧 3
2.1 細胞吞噬機制 3
2.2 奈米材料 5
2.3 奈米氧化鐵 7
第3章 實驗材料與方法 9
3.1 實驗理論與架構 9
3.2 實驗材料 10
3.3 儀器 13
3.4 試劑配製 18
3.5 正電高分子之合成 21
3.6 實驗方法 22
3.6.1 細胞吞噬機制探討 22
3.6.1.1 Temperature-dependent nanocomplex uptake 23
3.6.1.2 抑制劑之毒性檢測 23
3.6.1.3 抑制細胞吞噬之路徑 23
3.6.1.4 Bio-image markers of caveolar and clatherin routing 24
3.6.2 以單股核酸包覆技術製備新型氧化鐵粒子 24
3.6.2.1 單股核酸包覆型之氧化鐵粒子IOC/PAAHIS/C10A20 (IOC-P-O)及ION/P AAHIS/C10A20 (ION-P-O)製備: 24
3.6.2.2 細胞吞噬新型氧化鐵粒子(IOC-P-O、ION-P-O) 25
3.6.2.3 體外細胞毒性測試 25
3.6.2.4 MRI of cells after nanocomplexes internalization 26
3.6.2.5 單股核酸/PAA-HIS/氧化鐵之路徑抑制 26
3.6.3 不同碳數之PAm正電性高分子 26
3.6.3.1 單股核酸包覆型之氧化鐵粒子IOC/PAm/ C10A20 (IOC-PAm- O)製備: 27
3.6.3.2 不同碳數之PAm毒性測試 27
3.6.3.3 細胞吞噬IOC-PAm- O 27
第4章 實驗結果 28
4.1 溫度對細胞吞噬載體之效應 28
4.2 抑制劑之毒性檢測 28
4.3 細胞吞噬之路徑 28
4.3.1 單一抑制劑 28
4.3.2 兩種抑制劑 29
4.3.3 三種抑制劑 29
4.4 單股核酸於細胞吞噬路徑之效應 30
4.5 Markers of caveolar and clatherin routing 30
4.6 單股核酸包覆技術之新型氧化鐵 31
4.7 IOC-P-O、ION-P-O之細胞吞噬與毒性檢測 32
4.8 單股核酸/PAA-HIS/氧化鐵之路徑探討 34
4.9 不同碳數之PAm 34
4.9.1 單股核酸包覆不同碳數PAm之新型氧化鐵 34
4.9.2 PAm之毒性檢測及細胞吞噬 35
第5章 討論 36
5.1 溫度對細胞吞噬載體之效應 36
5.2 單股核酸於細胞吞噬路徑及效應 36
5.3 Markers of caveolar 、 clatherin routing 37
5.4 單股核酸包覆技術之新型氧化鐵 38
5.5 單股核酸/PAA-HIS/氧化鐵之細胞吞噬與毒性檢測 38
5.6 MRI of cell internalization 39
5.7 單股核酸包覆氧化鐵之路徑機制 39
5.8 單股核酸/PAm/氧化鐵之細胞吞噬與毒性檢測 40
第6章 結論 41
第7章 參考文獻 42

1、Esfand, R. and D.A. Tomalia, Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications. Drug Discovery Today, 2001. 6(8): p. 427-436.
2、Kunath, K., et al., Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethylenimine. Journal of Controlled Release, 2003. 89(1): p. 113-125.
3、Nimesh, S., R. Kumar, and R. Chandra, Novel polyallylamine-dextran sulfate-DNA nanoplexes: Highly efficient non-viral vector for gene delivery. International Journal of Pharmaceutics, 2006. 320(1-2): p. 143-149.
4、Pathak, A., et al., Engineered polyallylamine nanoparticles for efficient in vitro transfection. Pharmaceutical Research, 2007. 24(8): p. 1427-1440.
5、Saul, J.M., et al., Multilayer nanocomplexes of polymer and DNA exhibit enhanced gene delivery. Advanced Materials, 2008. 20(1): p. 19-+
6、Jiang, X., et al., Chitosan-g-PEG/DNA complexes deliver gene to the rat liver via intrabiliary and intraportal infusions. The Journal of Gene Medicine, 2006. 8(4): p. 477-487.
7、Kleemann, E., et al., Nano-carriers for DNA delivery to the lung based upon a TAT-derived peptide covalently coupled to PEG-PEI. Journal of Controlled Release, 2005. 109(1-3): p. 299-316.
8、Park, I.K., et al., Galactosylated chitosan-graft-poly(ethylene glycol) as hepatocyte-targeting DNA carrier. Journal of Controlled Release, 2001. 76(3): p. 349-362.
9、Trubetskoy, V.S., et al., Layer-by-layer deposition of oppositely charged polyelectrolytes on the surface of condensed DNA particles. Nucleic Acids Research, 1999. 27(15): p. 3090-3095.
10、Trubetskoy, V.S., et al., Recharging cationic DNA complexes with highly charged polyanions for in vitro and in vivo gene delivery. Gene Therapy, 2003. 10(3): p. 261-271
11、YC Chung et al., Polycation/DNA complexes coated with oligonucleotides for gene delivery. Biomaterials, 2010. 31 p.4194–4203
12、Kim, J., et al., Designed fabrication of a multifunctional polymer nanomedical platform for simultaneous cancer-targeted imaging and magnetically guided drug delivery. Advanced Materials, 2008. 20(3): p. 478-+.
13、Lai, C.W., et al., Iridium-complex-functionalized Fe3O4/SiO2 core/shell nanoparticles: A facile three-in-one system in magnetic resonance imaging, luminescence imaging, and photodynamic therapy. Small, 2008. 4(2): p. 218-224.
14、Lin, P.C., et al., Surface modification of magnetic nanoparticle via Cu(I)-Catalyzed alkyne-azide [2+3] cycloaddition. Organic Letters, 2007. 9(11): p. 2131-2134.
15、Makhluf, S.B.D., et al., Modified PVA-Fe3O4 nanopartictes as protein carriers into sperm cells. Small, 2008. 4(9): p. 1453-1458.
16、Wagner, K., et al., Synthesis of oligonucleotide-functionalized magnetic nanoparticles and study on their in vitro cell uptake. Applied Organometallic Chemistry, 2004. 18(10): p. 514-519.
17、H. Herweijer and J. Wolff, Progress and prospects: naked DNA gene transfer and therapy, Gene Ther., 2003. 10: p. 453–458.
18、T. Niidome and L. Huang, Gene therapy progress and prospects: nonviral vectors, Gene Ther., 2002. 9: p. 1647–1652.
19、O. Boussif, T. Delair, C. Brua, L. Veron, A. Pavirani and H.V.J. Kolbe, Synthesis of polyallylamine derivatives and their use as gene transfer vectors in vitro, Bioconjugate Chem., 1999. 10: p. 877–883.
20、Douglas, K. L., Piccirillo, C. A., and Tabrizian, M. Cell line-dependent internalization pathways and intracellular trafficking determine transfection efficiency of nanoparticle vectors. Eur. J. Pharm. Biopharm, 2008. 68: p. 676–687
21、Chithrami, B. D., and Chan, W. C. W. Elucidating the mechanism of cellular uptake and the removal of proteincoated gold nanoparticles of different sizes and shapes. Nano Letters, 2007. 7 : p.1542–1550.
22、Goncalves C et al., Macropinocytosis of polyplexes and recycling of plasmid via the clathrin-dependent pathway impair the transfection efficiency of human hepatocarcinoma cells. Mol Ther, 2004. 10: p.373-85.
23、Tessa Lu¨hmann et al., Cellular Uptake and Intracellular Pathways of PLL-g-PEG-DNA Nanoparticles. Bioconjugate Chem., 2008, 19, p. 1907–1916
24、Conner, S.D. and S.L. Schmid, Regulated portals of entry into the cell. Nature, 2003. 422(6927): p. 37-44.
25、Luhmann, T., et al., Cellular uptake and intracellular pathways of PLL-g-PEG-DNA nanoparticles. Bioconjugate Chemistry, 2008. 19(9): p. 1907-1916
26、van der Aa M, Huth US, Hafele SY, Schubert R, Oosting RS, Mastrobattista E, et al. Cellular uptake of cationic polymer-DNA complexes via caveolae plays a pivotal role in gene transfection in COS-7 cells. Pharm Res, 2007. 24 : p. 1590-8.
27、Brodsky, F. M., Chen, C. Y., Knuehl, C., Towler, M. C., and Wakeham, D. E..Biological basket weaving: formation and function of clathrin-coated vesicles. Annu.Rev. Cell Dev. Biol., 2001. 17: p. 517 – 568.
28、P.U. Le, I.R. Nabi, Distinct caveolae-mediated endocytic pathways target the golgi apparatus and the endoplasmic reticulum, J. Cell Sci., 2003. 1166: p.1059–1071.
29、Thomsen, P., Roepstorff, K., Stahlhut, M., and van Deurs, B. Caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking. Mol. Biol. Cell, 2002. 13: p. 238–250.
30、Pelkmans, L., and Helenius, A. Endocytosis via caveolae. Traffic, 2002 3: p.311 – 320.
31、Grimmer, S., van Deurs, B., and Sandvig, K.. Membrane ruffling and macropinocytosis in A431 cells require cholesterol. J. Cell Sci., 2002. 115: 2953 – 2962.
32、劉仲明、郭東瀛, 競逐原子世界 奈米材料 2002
33、徐善慧, 競逐原子世界 奈米生物醫學 2002
34、Gupta AK, Naregalkar RR, Vaidya VD, Gupta M. Recent advances on surface engineering of magnetic iron oxide nanoparticles and their biomedical applications. Nanomedicine, 2007. 2 : p.23-39.
35、Weissleder R, Elizondo G, Wittenberg J, Rabito CA, Bengele HH, Josephson L.Ultrasmall superparamagnetic iron oxide: characterization of a new class of contrast materials for MR imaging. Radiology, 1990. 175: p.489–93.
36、Corot C, Robert P, Ide’e JM, Port M. Recent advances in iron oxide nanocrystal technology for medical imaging. Adv Drug Delivery Rev, 2006. 58: p.1471–504.
37、Jung CW. Surface properties of superparamagnetic iron oxide MR contrast agents: ferumoxides, ferumoxtran, ferumoxsil. Magn Reson Imaging, 1995. 13: p.675–91.
38、Jung CW, Jacobs P. Physical and chemical properties of superparamagnetic iron oxide MR contrast agents: ferumoxides, ferumoxtran, ferumoxsil. Magn Reson Imaging, 1995. 13: p.661–74.
39、A. Subtil, A. Hemar, A. Dautry-Varsat, Rapid endocytosis of interleukin 2 receptors when clathrin-coated pit endocytosis is inhibited, J. Cell Sci., 1994. 10712 : p.3461–3468
40、T. Aoki, R. Nomura, T. Fujimoto, Tyrosine phosphorylation of caveolin-1 in the endothelium, Exp. Cell Res., 1999. 2532 : p.629–636.