臺灣博碩士論文加值系統

本論文主要討論如何合成及製造一維新穎奈米物質，並探討一維奈米物質的特殊性質，進而將其運用在活體細胞的研究。本論文主要的目標有三：一、發展新型一維奈米物質之製造技術，使其具有高深寬比、特殊功能性、大面積且規則性排列之特性。二、研究一維奈米物質的特殊功能性及其在活體細胞研究之應用。三、探討一維奈米物質可能的應用領域，如藥物傳遞方面。

由於陽極氧化鋁具多孔性及易製備之特性，長久以來陽極氧化鋁一直被當成合成奈米物質的模板。新近的研究顯示，利用陽極氧化鋁，可製成大面積且孔洞具周期性之奈米模板。陽極氧化鋁不僅可單獨成為模板，亦可長在基材表面，藉以在基材表面合成一維奈米物質。此外，商品化的陽極氧化鋁薄膜，也常被用來合成一維奈米物質，利用電化學方式，奈米金屬棒可在陽極氧化鋁奈米模板內被合成。變換不同的電鍍液，多截不同金屬的奈米棒可被合成出來，藉由表面修飾，可將奈米金屬棒修飾成具有功能之材料。

利用上述所合成具有功能性之奈米金棒，我們研究不同深寬比之奈米金棒在纖維母細胞與子宮頸癌細胞中的毒性。在奈米金棒溶液中，細胞增殖及細胞死亡的反應可使用MTT試劑測得。改變金棒表面之化學性質，我們可得知何種表面修飾毒性較低，可用在活細胞系統中。本論文研究發現，寬度為200奈米、長度為數微米具有功能性之奈米金棒，可以在兩種細胞株體內自由進出。其毒性量測結果與奈米金棒上之化學官能基有關，在低奈米金棒濃度下，血清吸附的奈米金棒對細胞毒性最小(細胞存活率大於90%)。然而被11- mercaptoundecanoic acid 分子修飾的奈米金棒，其毒性很大(細胞存活率約在60%)，不同長度的奈米金棒的毒性有相同的反應趨勢。此外，我們也觀察到在細胞中，較短奈米金棒的吸收效率，比長的奈米金棒還多。因此，在考慮有效吸收之奈米金棒後，我們認為，在細胞體內長奈米金棒的毒性比短奈米金棒還要高。

本論文還探討奈米金棒作為生物探針的可能性，利用帶有正電荷離子之表面修飾(aminothiol)，DNA及探針分子可被奈米金棒帶進活體細胞內。利用顯微鏡觀察，我們可獲得奈米金棒週遭的環境，因此我們認為奈米金棒可當成生物探針，自由進出活體細胞中，且因單獨奈米金棒不會聚集於細胞體內，非常適合用來研究奈米物質進入細胞之路徑機制等相關性質。

奈米金棒的藥物傳遞性質亦是本論文的探討重點之ㄧ，利用正電荷離子修飾(aminothiol)的奈米金棒，可藉由靜電力吸附DNA分子，並將DNA分子運送至纖維母細胞與子宮頸癌細胞中，進行轉移感染實驗。我們研究的結果顯示，使用200奈米寬、5微米長的奈米金棒，單一奈米金棒上可攜帶約1pg的藥物進行基因傳遞，相對於市面上所販售的轉染試劑，我們發現奈米金棒不但相對安全，在活體細胞系統中也有較高的轉染效率。因此，我們認為，如能利用多截且為不同金屬之奈米棒，我們可將不同的官能基修飾在不同截的金屬表面，使其同時可以攜帶藥物及作為生物探針使用，此種多功之奈米棒，對於研究藥物如何進入到活細胞體內的機制，有很好的運用潛力。

In this thesis, the fabrication of one-dimensional functional nanomaterials has been established and the applications of one-dimensional functional nanomaterials for the study of living cells have also been investigated. The main goals for this study are i) to develop new techniques for synthesizing and fabricating one-dimensional nanomaterials with very high aspect ratio, unique functionalities, and preferably in an ordered fashion over a large area on various substrates; ii) to utilize the one-dimensional nanomaterials as the probe for the living cells; iii) to explore the possibility of using the one dimensional nanomaterials as the drug carriers.
To synthesize one-dimensional nanomaterials with very high aspect ratio, the anodic aluminum oxides (AAOs) were used as the templates owing to their simple fabrication processes and porous nature. Recent developments in the fabrication techniques of AAOs not only made it possible to fabricate large-area well-ordered AAO membranes, but also allowed such membranes to be grown on silicon and glass substrates. In the past few years, a two-step AAO fabrication technique for producing well-ordered AAOs has been developed. Using both commercial and home-made AAO membranes, nanowires with various composition and aspect ratios have been prepared by electrochemical deposition process. These nanowires were further modified with different functional groups on the surfaces. The cytotoxicity of various surface functionalized gold nanowires with different aspect ratios has been investigated by MTT assays for two cell lines, fibroblast and HeLa. It was found that the functionalized gold nanowires with 200 nm diameter and length up to a few micrometers could be readily internalized by both types of cells regardless of the surface functionalization. However, the cytotoxicity of the gold nanowires was measured to depend on their surface modification. The serum coated gold nanowires were the least toxic surface coating because of the presence of various proteins on the surfaces of gold nanowires. In contrast, more than 50% of cells were damaged in the presence of the mercapto acid modified gold nanowires even at very low concentration (103 nanowire/ml). The nanowires with different aspect ratio exhibited the same cytotoxicity within experimental error. However, the uptake efficiency for the shorter nanowires was found to be higher than the longer ones. Therefore, we concluded that the internalized nanowires with higher aspect ratio were more toxic to the cells than the nanowires with lower aspect ratio. The positively charged aminothiol modified gold nanowires have been employed to deliver both plasmid DNA and probe molecules into cells without compromising the viability of cells. The local environment of the individual nanowires inside the cells has been obtained by monitoring the fluorescence signal from the probe molecules on the nanowires.
To explore the possibility of using nanowires as the drug carriers, the aminothiol modified gold nanowires have been used as vectors for the delivery of plasmid DNA into two different types of mammalian cells: 3T3 and HeLa. It was measured that the positively charged gold nanowires with a diameter of 200 nm and a length around 5 �慆 were capable of carrying 1 pg of plasmid DNA per nanowire into cells. Compared with other transfection reagents, the gold nanowires exhibited the highest transfection efficiency while almost no cytotoxicity was observed. In addition, it has been shown that individual nanowires can be visualized with sub-micrometer resolution, which may allow the use of functionalized multi-segment nanowires as the local probes for the investigation of the microenvironment inside the cells.

摘要...................................................... i
Abstract …………………………………………………………………..…….…………. iii
誌謝 ………………………………………………………………………..….………...... vi
Content ……………………………………………………………………..….…………. vii
Figure list ……………………………………………………………………………..……..x

Chapter 1 Introduction ………………………………………………………….……...1
Reference ….……………………………………………………….….………5

Chapter 2 Fabrication of AAO Templates ………………………………………….… 8
2.1 Ordered AAO template …………………………………………………….…8
2.2 Fabrication of AAO template ………………………………………………..10
2.3 Results and discussion ……………………………………………………….11
2.4 Conclusions ………………………………………………………………….13
Reference …………………………………………………………….14

Chapter 3 Synthesis of Nanowires …………………….………………………...….……23
3.1 Introduction ………………………………………………………...………...23
3.2 Experimental ………………………………………………………..….…….25
3.2.1 Preparation of nanowires ………………………………………..……….….25
3.2.2 Pulse electrodeposition ………………………………………..……….……25
3.3 Results and discussion …………………………………….…….….…..26
3.4 Conclusions …..................…………………………………….….…….29
Reference ……………………………………………………………..……30

Chapter 4 Functionalization and Characterization of Nanowires ………….…….….40
4.1 Introduction ………………………………………………...………..40
4.2 Experimental………………………………………………....….…….42
4.3 Results and discussion………………………………………….……..43
4.4 Conclusions ……………….........………………………...….…...45
Reference ……………………………………………………………….…..46

Chapter 5 Cytotoxicity of Nanowires …………………………………..…….………51
5.1 Introduction ………………………………………………...………..51
5.1.1 Magnetic nanoparticles ………………………………………..……..……..51
5.1.2 Semiconducting nanoparticles ………………………………….…….……52
5.1.3 Gold nanoparticles …………………………………………….……….…..53
5.2 Experimental ……………...…………………………………………………54
5.2.1 Cell culture………………………………………………………….……….. 54
5.2.2 Cytotoxicity of surface-modified nanowires …………………………………54
5.2.3 The uptake of nanowires……………………….……………………………..55
5.3 Result and discussion……………………………………………….……..….56
5.3.1 The cytotoxicity of the nanowires …………………………….………56
5.3.2 The uptake of nanowires ……………………………………………………..58
5.4 Conclusion……………………………………………………………….59
Reference ……………………………………………………………..60

Chapter 6 Nanowires as live cell probes ………………………………………….…..…65
6.1 Introduction……………………………………………….…………....65
6.2 Experimental ……………………………………………….……….....68
6.2.1 Preparation of cells for confocal analysis……………………..….68
6.2.2 Transfection experiment ………………………………….………….68
6.2.3 Intracellular behavior monitored by the Lysosensor yellow/Blue dye.........69
6.3 Results and discussion………………………………………………69
6.4 Conclusion……………………………………………………….…….75
Reference…………………………………………………………………...76

Chapter 7 Nanowire for Gene Delivery ……………………………………….……..….86
7.1 Introduction…………………………………… ………………….……….....86
7.2 Nanomterials as non-viral transfection reagents ………………….……….....87
7.2.1 The cationic lipids …………………...………………………………….…....89
7.2.2 The cationic polymer …………………………………..…………….………90
7.2.3 The biodegradable polymer …..……………………………………….……..91
7.2.4 Metallic nanoparticles ………..………….…..…………………….…………92
7.2.5 Other nanomaterials ……………..……….…………………….………….....94
7.3 Experimental procedures for measuring the transfection efficiency
of gold nanowires …………………………………………….…………..…..95
7.3.1 Plasmid preparation ………...…………………………………………..….…95
7.3.2 AgNi segment binding ………………………...…………………………..…96
7.3.3 Cell culture and transfection …………………..…………………………..…97
7.3.4 Gel electrophoresis……………………………………………………..99
7.4 Results and Discussions ………………………………………………..99
7.4.1 Characterization of nanowires ……………………………………………...99
7.4.2 Binding efficiency of nanowire ……………………………………….……100
7.4.3 Transfection efficiency of nanowires …………………………….……….101
7.5 Conclusion ………………………………………………………………..104
Reference …………………………………………………................……105

Publication ……………………………………………………………………….…116
Curriculum vitae ……………………………………………………………………117

1. Handbook of Nanostructured Materials and Nanotechnology, (H.S. Nalwa, Editor), Academic Press, New York.
2. J. W. Nam, C. S. Thaxton and C. A. Mirkin, Science 2003, 301, 1884.
3. Y. Xiao, F. Patolsky, E. Katz, J. F. Hainfeld and I. Willner Science 2003, 299, 1877 .
4. X. Zhao, L. R. Hilliard, S. H. Mechery, Y. Wang, R. P. Bagwe, S. Jin and W. Tan, Proc. Natl. Acad. Sci. USA 2004, 101, 15027.
5. E. Hutter and J. H. Fendler, Adv. Mater. 2004, 16, 1685.
6. X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir and S. Weiss, Science 2005, 307, 538.
7. I. L. Medintz, H. T. Uyeda, E. R. Goldman and H. Mattoussi, Nat. Mater. 2005, 4, 435.
8. X. Gao, L. Yang, J. A. Petros, F. F. Marshall, J. W. Simons and S. Nie, Curr. Opin. Biotechnol. 2005, 16, 63.
9. C. Sonnichsen, B. M. Reinhard, J. Liphardt and A. P. Alivisatos, Nat. Biotechnol. 2005, 23, 741.
10. A. M. Derfus, W. C. W. Chan and S. N. Bhatia, Nano Lett. 2004, 4, 11.
11. A. Hoshino, K. Fujioka, T. Oku, M. Suga, Y. F. Sasaki, T. Ohta, M. Yasuhara, K. Suzuki and K. Yamamoto, Nano Lett. 2004, 4, 2163.
12. C. Kirchner, T. Liedl, S. Kudera, T. Pellegrino, A. M. Javier, H. E. Gaub, S. Stolzle and W. J. Parak, Nano Lett. 2005, 5, 331.
13. B. D. Chithrani, A. A. Ghazani and W. C. W. Chan, Nano Lett. 2006, 6, 662.
14. C. M. Goodman, C. D. McCusker, T. Yilmaz and V. M. Rotello, Bioconjugate Chem. 2004, 15, 897.
15. E. E. Connor, J. Mwamuka, A. Gole, C. J. Murphy and M. D. Wyatt, Small 2005, 1, 325.
16. H. Takahashi, Y. Niidome, T. Niidome, K. Kaneko, H. Kawasaki and S. Yamada, Langmuir 2006, 22, 2.
17. G. Han, C. T. Martin and V. M. Rotello, Chem. Biol. Drug Des. 2006, 67, 78.
18. C. C. Lin, Y. C. Yeh, C. Y. Yang, C. L. Chen, G. F. Chen, C. C. Chen and Y. C. Wu, J. Am. Chem. Soc. 2002, 124, 3508.
19. J. Wang, Small 2005, 1, 1036.
20. E. Katz and I. Willner, Angew. Chem. Int. Ed. 2004, 43, 6042.
21. H. Shen, J. Tan and W. M. Saltzman, Nat. Mater. 2004, 3, 569.
22. A. K. Salem, P. C. Searson and K. W. Leong, Nat. Mater. 2003, 2, 668.
23. K. K. Sandhu, C. M. McIntosh, J. M. Joseph, S. W. Smith and V. M. Rotello, Bioconjugate Chem. 2002, 13, 3.
24. M. Thomas and A. M. Klibanov, Proc. Natl. Acad. Sci. USA, 2003, 100, 9138.
25. N. L. Rosi, D. A. Giljohann, C. S. Thaxton, A. K. R. Lytton-Jean, M. S. Han and C. A. Mirkin, Science, 2006, 312, 1027.
26. A. G. Tkachenko, H. Xie, D. Coleman, W. Golmm, J. Ryan, M. F. Anderson, S. Franzen and D. L. Feldheim, J. Am. Chem. Soc. 2003, 125, 4700.
27. C. D. Keating and M. J. Natan, Adv. Mater. 2003, 15, 451.
28. A. Hultgren, M. Tanase, C. S. Chen, G. J. Meyer and D. H. Reich, J. Appl. Phys. 2003, 93, 7554.
29. M. Tanase E.J. Felton, D.S. Gray, A. Hultgren, C.S. Chen and D.H. Reich, Lab Chip 2005, 5, 598.