利用電場控制導電性高分子以進行基因於聚電解質多層膜的組裝

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：11

、訪客IP：18.219.39.211

姓名

鄭晏蓉(Yan-rong Zheng) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

利用電場控制導電性高分子以進行基因於聚電解質多層膜的組裝
(The assembly of polyelectrolyte multilayer regulated by electrical control using conductive polymer)

相關論文

★ 利用穿膜胜肽改善帶正電高分子之轉染效率	★ 利用導電高分子聚吡咯為基材以電刺激促進幹細胞分化
★ 以電刺激增進骨髓基質細胞骨分化之最佳化探討	★ 以短鏈胜肽接枝聚乙烯亞胺來進行基因輸送應用之研究
★ 電紡絲製備褐藻酸鈉/聚己內酯之奈米複合纖維進行原位轉染	★ 電場對於複合奈米絲進行原位基因傳送之影響
★ 利用電場調控聚電解質多層膜的釋放以應用於基因輸送	★ 發展載藥電紡聚乳酸/多壁奈米碳管/聚乙二醇纖維
★ 利用寡聚精胺酸促進去氧寡核苷酸輸送	★ 利用聚己內酯/褐藻酸鈉之複合電紡絲擴增癌症幹細胞
★ 以二元體形式之Indolicidin 應用於去氧寡核苷酸之輸送	★ Indolicidin之色胺酸殘基對於轉染效率的影響
★ Indolicidin之二聚體形式對輸送去氧寡核?酸的影響	★ 搭建可提供電刺激與機械刺激之生物反應器
★ 硬脂基化的Indolicidin作為傳送質體去氧核酸的非病毒載體	★ 開發促進傷口癒合之複合敷料

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

至系統瀏覽論文 ( 永不開放)

摘要(中)

在本研究中，我們利用外加電場來輔助疊層組裝，控制聚電解質高分子
於基材上的吸附及其後的釋放行為。使用導電性高分子聚吡咯為基材，分
別在堆疊DNA 或幾丁聚醣時施加電場，以進行電泳沉積。並使用幾丁聚醣
酶將多層膜降解後，以分別定量在不同層數時DNA 及幾丁聚醣的堆疊量。
從吸附量的結果顯示，幾丁聚醣在低電場下(0.1V~0.2V)的疊層組裝確實增
加了DNA 與chitosan 的吸附，然而在0.5V 時吸附量卻開始有下降的趨勢，
推測是此時的電壓已到達水中溶氧的還原電位，所產生的氫氧根離子使溶
液產生pH值變化，因此減低了溶液中聚電解質的帶電量。而隨著電壓上升，
電解所造成的pH 改變及吸附減少的現象也更加明顯，然而當電壓5V 時，
可能pH 上升的幅度已超過而溶液中的幾丁聚醣的pKa 值，因此反而導致幾
丁聚醣析出而增加沉積量。而DNA 也有類似的現象，即在低電場下
(0.1V~0.2V)疊層組裝會增加吸附，但是隨著電壓增加，水開始氧化產生氫
離子，導致pH 值下降而造成DNA吸附減少。接著將通電組裝薄膜進行DNA
釋放實驗，發現DNA 通電所造成吸附量上升的多層膜，其釋放出來的DNA
質量也會較多。幾丁聚醣雖然也有類似的現象，但釋放提升程度並不如DNA
通電所帶來的效益。值得一提的是，針對電壓5V 擁有幾丁聚醣通電組中最
高的釋放量，這可能是因為幾丁聚醣是以析出的方式吸附於多層膜，導致
多層膜的結構鬆散，進而增加了DNA 的釋放量。最後將通電組裝薄膜進行
原子力顯微鏡(AFM)拍攝，觀察通電後薄膜表面粗糙度的變化，發現在DNA
低電壓的處理可以使膜表面趨於平滑，這可能是因為通電後膜上的電荷使
得高分子吸附更加的密合。然而在高電壓下，膜表面則會變得粗糙，推測
聚電解質由於pH 值的改變而減少帶電量，進而使其構型傾向蜷曲而導致膜
的粗糙度增加。然而幾丁聚醣的通電處理對於粗糙度並無明顯的趨勢，這
可能是因為與DNA 相比，幾丁聚醣分子量過小，因此電泳吸附對於膜表面
的影響不大。

摘要(英)

To regulate the adsorption and the following release of polyelectrolyte on
substrate, we developed external electric field to assist layer-by-layer (LbL)
assembly in this study. Conductive polymer, polypyrrole, was utilized as the
substrate, and DNA as well as chitosan was applied to deposit on the surfaces.
To elucidate the effect of electricophoretic deposition, the electric field was
solely administrated to chitosan or DNA adsorption. Chitosanase was used to
degrade polyelectrolyte multilayers (PEMs) at different bilayer numbers, and the
adsorbed chitosan and DNA may thus be quantified. The adsorption results
demonstrated that LbL assembled DNA and chitosan can both be augmented
under low electric field (0.1~0.2V). However, for the groups using electrical
field during chitosan deposition, the improvement began to reduce at 0.5 V. It
should be due to that the voltage approach to the reduction potential of dissolved
oxygen in solution, and the increased hydroxyl ions decreased the charges on
chitosan molecules to inhibit their adsorption. But when the voltage increased to
5V, the adsorption of chitosan was increased. It probably due to that the
electrolysis significantly increased pH value to close to the pKa of chitosan,
which chitosan precipitation on PEMs. The electrical field treatment during
DNA deposition also revealed similar trends that using low voltage can increase
deposition. However, the oxidization occurred when the voltage was high which
resulted in proton release to decrease pH. Therefore, the charged density of DNA
was decreased which decline DNA adsorption to PEMs. Then we soaked PEMs
to PBS to determine their DNA release efficiency. Because electric-assisted LbL
during DNA deposition highly increased DNA adsorption, their releases were
thus also improved. However, this trend was not obvious to the chitosan groups.
Interestingly, the group using 5V during chitosan deposition demonstrated
highest release. It was consistent to our deduction that 5V led chitosan
participation, which weakened the stability of PEM that the DNA can be
released easily from loose structure. Finally, these films were illustrated by
atomic force microscopy (AFM). When films were treated low voltage (0.1V
and 0.2V) for chitosan deposition, surfaces were smoothened, suggesting that
surface potential of substrate increased the contact of polyelectrolyte to surfaces
that the defects of PEMs can thus be reduced. However, using high voltage
increased the roughness of the films. It should be due to that the reduction of
charge density of polylelectrolyte on PEM caused their configuration as
coiled-form and thus the microstructure of PEMs was not as smooth as those
using low voltages. In contrast, for electric field-treated DNA deposition, there
was no obvious trend between voltage and roughness. We deduced that chitosan
was extremely small compared to DNA, so the surface roughness mainly
depended on DNA but not chitosan.

關鍵字(中)

★ 疊層組裝
★ 電場
★ 聚吡咯
★ 基因
★ 幾丁聚醣

關鍵字(英)

★ Layer-by-Layer assembly
★ Electric field
★ Polypyrrole
★ DNA
★ chitosan

論文目次

摘要 .................................................................................................................... I
Abstract ............................................................................................................. III
致謝 ................................................................................................................... V
目錄 .................................................................................................................. VI
圖目錄 .............................................................................................................. IX
表目錄 ................................................................................................................ I
第一章序論 ..................................................................................................... 1
1-1 背景 .................................................................................................... 1
1-2 實驗目的 ............................................................................................ 3
第二章文獻回顧 .............................................................................................. 4
2-1 組織工程 ............................................................................................ 4
2-1-1 基因傳遞.................................................................................. 5
2-2 疊層組裝 ............................................................................................ 6
2-2-1 疊層組裝原理與製備 .............................................................. 6
2-2-2 疊層組裝於基因傳遞之應用 .................................................. 8
2-2-3 影響疊層組裝的因素 ............................................................ 10
2-2-4 通電疊層組裝 ........................................................................ 14
2-2-5 通電對吸附的影響 ................................................................ 18
2-3 聚吡咯(Polypyrrole) ......................................................................... 22
2-4 幾丁聚醣 .......................................................................................... 23
2-4-1 幾丁聚醣來源 ........................................................................ 23
2-4-2 幾丁聚醣性質 ........................................................................ 23
2-4-3 幾丁聚醣於生醫材料之應用 ................................................ 24
2-5 Chitosanase ..................................................................................... 25
2-5-1 Chitosanase應用 ................................................................. 26
2-6 紫外光光譜儀 ................................................................................... 27
2-6-1 紫外光光譜儀 ........................................................................ 27
2-6-2 紫外光光譜儀原理 ................................................................ 27
2-7 原子力顯微鏡 ................................................................................... 29
2-7-1 原子力顯微鏡原理 ................................................................ 29
2-7-2 原子力顯微鏡操作模式 ........................................................ 30
2-7-3 原子力顯微鏡於疊層組裝之應用 ......................................... 31
第三章實驗方法 ............................................................................................ 32
3-1 實驗架構 .......................................................................................... 32
3-2 實驗藥品與儀器 ............................................................................... 33
3-2-1 藥品 ....................................................................................... 33
3-2-2 儀器 ....................................................................................... 33
3-3 試藥製備 .......................................................................................... 34
3-3-1 DNA cloning .......................................................................... 34
3-3-2 陽離子水溶液 ........................................................................ 35
3-3-3 陰離子水溶液 ........................................................................ 35
3-3-4 Polypyrrole 製備 ................................................................. 36
3-3-5 Chitosanase 溶液 ................................................................ 36
3-4 LbL 製備 ......................................................................................... 37
3-4-1 傳統式疊層組裝(未通電場) .................................................. 38
3-4-2 外加電場疊層組裝 ................................................................ 38
3-4-2-1 DNA通電 ..................................................................... 38
3-4-2-2 Chitosan通電 ............................................................... 38
3-5 吸附實驗 .......................................................................................... 40
3-5-1 DNA吸附量檢測 .................................................................. 40
3-5-2 chitosan吸附量檢測 ........................................................... 40
3-5-3 釋放實驗................................................................................ 40
3-6 原子力顯微鏡分析 ........................................................................... 41
3-7 循環伏安法 ....................................................................................... 41
第四章結果與討論 ........................................................................................ 42
4-1 全反射傅立葉轉換紅外線光譜儀分析 ............................................ 42
4-2 通電對聚電解質吸附質量的影響 .................................................... 45
4-2-1 chitosan通電對DNA與chitosan吸附量的影響 .................. 46
4-2-2 DNA通電對DNA與chitosan吸附量的影響 ...................... 50
4-3 聚電解質吸附性質-循環伏安法 ...................................................... 54
4-4 通電吸附對釋放的影響 ................................................................... 56
4-5 原子力顯微鏡分析 ........................................................................... 61
第五章結論 ................................................................................................... 65
參考文獻 ......................................................................................................... 67

參考文獻

1. Oragui, E., et al., A new technique of endoprosthetic replacement for osteosarcoma of proximal femur with intra-articular extension. International Journal of Surgery Case Reports, 2013. 4(1): p. 101-4.
2. Sooraj, Y.S., et al., Chromoblastomycosis in a renal allograft recipient. Indian Journal of Nephrology, 2013. 23(3): p. 235-6.
3. Bell, E., Tissue engineering: a perspective. Journal of Cellular Biochemistry, 1991. 45(3): p. 239-41.
4. Griffith, L.G., Emerging design principles in biomaterials and scaffolds for tissue engineering. Annals of the New York Academy of Sciences, 2002. 961: p. 83-95.
5. Freed, L.E., et al., Frontiers in tissue engineering. In vitro modulation of chondrogenesis. Clinical Orthopaedics and Related Research, 1999(367 Suppl): p. S46-58.
6. Ditto, A.J., et al., In vivo gene delivery with l-tyrosine polyphosphate nanoparticles. Molecular Pharmaceutics, 2013. 10(5): p. 1836-44.
7. Hutmacher, D.W., Scaffolds in tissue engineering bone and cartilage. Biomaterials, 2000. 21(24): p. 2529-43.
8. Mansouri, S., et al., Chitosan-DNA nanoparticles as non-viral vectors in gene therapy: strategies to improve transfection efficacy. European Journal of Pharmaceutics and Biopharmaceutics, 2004. 57(1): p. 1-8.
9. Ariga, K., et al., Layer-by-layer assembly for drug delivery and related applications. Expert Opinion in Drug Delivery, 2011. 8(5): p. 633-44.
10. Lu, Z.Z., et al., Biodegradable polycation and plasmid DNA multilayer film for prolonged gene delivery to mouse osteoblasts. Biomaterials, 2008. 29(6): p. 733-41.
11. Jewell, C.M., et al., Multilayered polyelectrolyte films promote the direct and localized delivery of DNA to cells. Journal of Controlled Release, 2005. 106(1-2): p. 214-23.
12. Lvov, Y., et al., Assembly of Multicomponent Protein Films by Means of Electrostatic Layer-by-Layer Adsorption. Journal of American Chemical Society, 1995. 117: p. 6117-6123.
13. Madihally, S.V., et al., Porous chitosan scaffolds for tissue engineering. Biomaterials, 1999. 20(12): p. 1133-42.
14. Ko, Y.H., et al., Electric-Field-Assisted Layer-by-Layer Assembly of Weakly Charged
68
Polyelectrolyte Multilayers. Macromolecules, 2011. 44: p. 2866-2872.
15. Guillaume-Gentil, O., et al., Chemically Tunable Electrochemical Dissolution of Noncontinuous Polyelectrolyte Assemblies: An In Situ Study Using ecAFM. ACS Applied Material & Interfaces, 2010. 2(12): p. 3525-3531.
16. Svirskis, D., et al., Electrically switchable polypyrrole film for the tunable release of progesterone. Therapeutic delivery, 2013. 4(3): p. 307-313.
17. Tassel, P.V., Polyelectrolyte adsorption and layer-by-layer assembly: Electrochemical control. Current Opinion in Colloid & Interface Science, 2012. 17: p. 106-113.
18. Bearinger, J.P., et al., Electrochemical optical waveguide lightmode spectroscopy (EC-OWLS): A pilot study using evanescent-field optical sensing under voltage control to monitor polycationic polymer adsorption onto indium tin oxide (ITO)-coated waveguide chips. Biotechnology and Bioengineering, 2003. 82(4): p. 465-473.
19. Marcotte, E., et al., Crystallization of a chitosanase from Streptomyces N174. Journal of Molecular Biology, 1993. 232(3): p. 995-6.
20. LEVER, M., New Reaction for Calorimetric Determination of Carbohydrates. Analytical Biochemistry, 1972. 47: p. 273-279.
21. Yunoki, S., et al., Effects of increased collagen-matrix density on the mechanical properties and in vivo absorbability of hydroxyapatite-collagen composites as artificial bone materials. Biomedical Materials, 2011. 6(1): p. 1-10.
22. Shive, M.S., et al., Biodegradation and biocompatibility of PLA and PLGA microspheres. Advanced Drug Delivery Reviews, 1997. 28(1): p. 5-24.
23. Macri, L., et al., Growth factor binding to the pericellular matrix and its importance in tissue engineering. Advanced Drug Delivery Reviews, 2007. 59(13): p. 1366-81.
24. Manni, L., et al., Nerve growth factor: basic studies and possible therapeutic applications. Growth Factors, 2013.
25. Kaplitt, M.G., et al., Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain. Nature Genetics, 1994. 8(2): p. 148-54.
26. Zhu, Y., et al., Layer-by-layer assembly to modify poly(l-lactic acid) surface toward improving its cytocompatibility to human endothelial cells. Biomacromolecules, 2003. 4(2): p. 446-52.
27. Ai, H., et al., Biomedical applications of electrostatic layer-by-layer nano-assembly of polymers, enzymes, and nanoparticles. Cell Biochemistry and Biophysics, 2003. 39(1): p. 23-43.
28. Decher, G., Fuzzy nanoassemblies: Toward layered polymeric multicomposites.
69
Science, 1997. 277(5330): p. 1232-1237.
29. Decher, G., et al., New nanocomposite films for biosensors: layer-by-layer adsorbed films of polyelectrolytes, proteins or DNA. Biosensors and Bioelectronics, 1994. 9(9-10): p. 677-684.
30. Pei, R., et al., Assembly of alternating polycation and DNA multilayer films by electrostatic layer-by-layer adsorption. Biomacromolecules, 2001. 2(2): p. 463-8.
31. Yamauchi, F., et al., Layer-by-layer assembly of cationic lipid and plasmid DNA onto gold surface for stent-assisted gene transfer. Biomaterials, 2006. 27(18): p. 3497-3504.
32. Hu, Y., et al., Surface mediated in situ differentiation of mesenchymal stem cells on gene-functionalized titanium films fabricated by layer-by-layer technique. Biomaterials, 2009. 30(21): p. 3626-3635.
33. Yamauchi, F., et al., Layer-by-layer assembly of poly(ethyleneimine) and plasmid DNA onto transparent indium-tin oxide electrodes for temporally and spatially specific gene transfer. Langmuir, 2005. 21(18): p. 8360-8367.
34. Yuan, W., et al., pH-controlled construction of chitosan/alginate multilayer film: characterization and application for antibody immobilization. Langmuir, 2007. 23(26): p. 13046-52.
35. Yoo, D., et al., Controlling bilayer composition and surface wettability of sequentially adsorbed multilayers of weak polyelectrolytes. Macromolecules, 1998. 31(13): p. 4309-4318.
36. Shiratori, S.S., et al., pH-dependent thickness behavior of sequentially adsorbed layers of weak polyelectrolytes. Macromolecules, 2000. 33(11): p. 4213-4219.
37. Aytar, B.S., et al., Rapid Release of Plasmid DNA from Surfaces Coated with Polyelectrolyte Multilayers Promoted by the Application of Electrochemical Potentials. Acs Applied Materials & Interfaces, 2012. 4(5): p. 2726-2734.
38. 陳勇任, 利用疊層組裝控制DNA吸附與釋放之行為. 國立中央大學, 2011: p. 41-79.
39. Porcel, C., et al., From exponential to linear growth in polyelectrolyte multilayers. Langmuir, 2006. 22(9): p. 4376-83.
40. Garza, J.M., et al., Multicompartment films made of alternate polyelectrolyte multilayers of exponential and linear growth. Langmuir, 2004. 20(17): p. 7298-302.
41. Michel, A., et al., Layer by layer self-assembled polyelectrolyte multilayers with embedded phospholipid vesicles obtained by spraying: Integrity of the vesicles. Langmuir, 2005. 21(17): p. 7854-7859.
70
42. Picart, C., et al., Molecular basis for the explanation of the exponential growth of polyelectrolyte multilayers. Proceedings of the National Academy of Sciences of the United States of America, 2002. 99(20): p. 12531-12535.
43. Boulmedais, F., et al., Controlled electrodissolution of polyelectrolyte multilayers: A platform technology towards the surface-initiated delivery of drugs. Advanced Functional Materials, 2006. 16(1): p. 63-70.
44. Dieguez, L., et al., Electrochemical tuning of the stability of PLL/DNA multilayers. Soft Matter, 2009. 5(12): p. 2415-2421.
45. Zhang, G.J., et al., Effects of External Electric Field on Film Growth, Morphology, and Nanostructure of Polyelectrolyte and Nanohybrid Multilayers onto Insulating Substrates. Langmuir, 2011. 27(6): p. 2093-2098.
46. Ngankam, A.P., et al., In situ layer-by-layer film formation kinetics under an applied voltage measured by optical waveguide lightmode spectroscopy. Langmuir, 2005. 21(13): p. 5865-5871.
47. Bocchi, V., et al.,, On pyrrole oxidation with hydrogen peroxide. Tetrahedron, 1970. 26(17): p. 4073-4082.
48. Lee, J.W., et al., Carboxylic acid-functionalized conductive polypyrrole as a bioactive platform for cell adhesion. Biomacromolecules, 2006. 7(6): p. 1692-5.
49. Gilmore, K.J., et al., Skeletal muscle cell proliferation and differentiation on polypyrrole substrates doped with extracellular matrix components. Biomaterials, 2009. 30(29): p. 5292-304.
50. Rinaudo, M., Chitin and chitosan: Properties and applications. Progress in Polymer Science, 2006. 31(7): p. 603-632.
51. Suh, J.K.F., et al., Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. Biomaterials, 2000. 21(24): p. 2589-2598.
52. Zakhem, E., et al., Chitosan-based scaffolds for the support of smooth muscle constructs in intestinal tissue engineering. Biomaterials, 2012. 33(19): p. 4810-4817.
53. Li, Z., et al., Chitosan-alginate as scaffolding material for cartilage tissue engineering. Journal of Biomedical Materials Research. Part A., 2005. 75(2): p. 485-93.
54. Ma, J., et al., A preliminary in vitro study on the fabrication and tissue engineering applications of a novel chitosan bilayer material as a scaffold of human neofetal dermal fibroblasts. Biomaterials, 2001. 22(4): p. 331-6.
55. Boucher, I., et al., Purification and Characterization of a Chitosanase from Streptomyces N174. Applied Microbiology and Biotechnology, 1992. 38(2): p.
71
188-193.
56. Somashekar, D., et al., CHITOSANASES -PROPERTIES AND APPLICATIONS : A REVIEW. Bioresource Technology, 1996. 55: p. 35-45.
57. Yoon, N.Y., et al., Acetylcholinesterase inhibitory activity of novel chitooligosaccharide derivatives. Carbohydrate Polymers, 2009. 78(4): p. 869-872.
58. Hunger, M., et al., In situ IR, NMR, EPR, and UV/Vis spectroscopy: Tools for new insight into the mechanisms of heterogeneous catalysis. Angewandte Chemie-International Edition, 2001. 40(16): p. 2954-2971.
59. Sukhorukov, G.B., et al., Assembly of polyelectrolyte multilayer films by consecutively alternating adsorption of polynucleotides and polycations. Thin Solid Films, 1996. 284: p. 220-223.
60. Ultraviolet-Visible (UV-Vis) Spectroscopy – Principle http://pharmaxchange.info/press/2011/12/ultraviolet-visible-uv-vis-spectroscopy-principle/
61. Binnig, G., et al., Atomic force microscope. Physical Review Letters, 1986. 56(9): p. 930-933.
62. Hansma, P.K., et al., Scanning tunneling microscopy and atomic force microscopy: application to biology and technology. Science, 1988. 242(4876): p. 209-16.
63. 科榮股份有限公司http://www.scientek.com.tw/ct/index.php
64. Jing, G.Y., et al., Surface effects on elastic properties of silver nanowires: Contact atomic-force microscopy. Physical Review B, 2006. 73(23).
65. Ara, M., et al., Non-contact atomic force microscopy using silicon cantilevers covered with organic monolayers via silicon-carbon covalent bonds. Nanotechnology, 2004. 15(2): p. S65-S68.
66. Zhong, Q., et al., Fractured Polymer Silica Fiber Surface Studied by Tapping Mode Atomic-Force Microscopy. Surface Science, 1993. 290(1-2): p. L688-L692.
67. Cho, C., et al., Electric Field Induced Morphological Transitions in Polyelectrolyte Multilayers. Acs Applied Materials & Interfaces, 2013. 5(11): p. 4930-4936.

指導教授

胡威文(Wei-wen Hu)

審核日期

2013-8-19

推文