利用電場調控聚電解質多層膜的釋放 以應用於基因輸送

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：5

、訪客IP：3.144.102.239

姓名

鍾坤達(Kun-da Chung) 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

利用電場調控聚電解質多層膜的釋放以應用於基因輸送
(The manipulation of gene delivery from PEI/DNA multilayers by the application of electrochemical potentials)

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	★ 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	★ 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)

摘要
在本次實驗中，利用聚乙烯亞胺(Polyethylenimine)和DNA在具有生物相容性的導電基材聚吡咯(Polypyrrole) 上面進行疊層組裝，並將所形成多層膜之結構進行通電釋放。從釋放實驗結果顯示以固定縱向通電的方式可以有效的從膜中釋放出PEI和DNA，在進一步的探討中，電壓成為了主要影響釋放的參數，通予越大電壓，釋放出的PEI和DNA的比例就越高。對於增加通電時間，也有類似的效果。進一步將通電過後的多層膜，以原子力顯微鏡去拍攝，比較有通電與沒通電之結果，可以發現，通電過後膜的表面粗糙度有了明顯的上升，通電越大，粗糙度相對就越大，可能原因是由於膜的厚度不均，因電場和電化學作用下，導致膜比較薄區域，越容易溶解，導致膜高低起伏會隨之增加。最後將所釋放的DNA及PEI進行轉染實驗，可發現通電組其轉染效率高於無通電組，證實所釋放的DNA及PEI，可以有效地被細胞所攝取並提升了轉染效率。

摘要(英)

Abstract
In this study, polyethylenimine (PEI) and DNA were deposited onto conductive polypyrrole (PPy) substrate using layer-by-layer assembly. The formed polyelectrolyte multilayers (PEMs) films were released under electric field. The release results suggested that the electric field perpendicular to the substrate can efficiently release PEI and DNA from PEMs. Voltages were the main factors controlled the PEMs disassembly that higher potential released more PEI and DNA. The treatment period demonstrated similar effects. The surface morphologies of PEMs after electric field treatment were observed using atomic force microscopy (AFM). Compared to the nontreated group, the roughness of the films significantly increased after treating electric field, which was more obvious as the potential was high. These results suggested that the polyelectrolytes may be easily released from thin regions of the films due to the driving force of electric field as well as the dissolution resulted from electrochemical reaction on electrodes. Finally, the release DNA and PEI were used for transfection. The released material from the films with electric field treatment demonstrated superior transgene expression, suggesting that the electric assisted gene delivery from PEMs should be beneficial to gene delivery.

關鍵字(中)

★ 疊層組裝
★ 電場
★ 釋放
★ 聚吡咯
★ 基因
★ 聚乙烯亞胺

關鍵字(英)

★ Layer-by-Layer
★ Electric field
★ release
★ Polypyrrole
★ DNA
★ Polyethylenimine

論文目次

目錄
摘要 I
Abstract II
目錄 III
圖目錄 VI
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
2-2基因治療 5
2-2-1基因治療的載體 5
2-3疊層組裝 8
2-3-1疊層組裝的原理 8
2-3-2疊層組裝的製備 8
2-3-3疊層組裝的在生物上的應用 9
2-3-4通電對疊層組裝釋放的影響 11
2-4聚吡咯(Polypyrrole) 21
2-5聚乙烯亞胺(Polyethylenimine) 22
2-5-1聚乙烯亞胺於基因傳遞之應用 22
2-6紫外光光譜儀 24
2-6-1紫外光光譜儀 24
2-6-2紫外光光譜儀原理 24
2-7原子力顯微鏡 27
2-7-1原子力顯微鏡原理 27
2-7-2原子力顯微鏡操作模式 28
第三章實驗方法 29
3-1實驗架構 29
3-2實驗藥品與儀器 30
3-2-1藥品 30
3-2-2儀器 30
3-3試藥製備 32
3-3-1 DNA cloning 32
3-3-2陽離子水溶液 33
3-3-3陰離子水溶液 33
3-3-4 Polypyrrole 製備 34
3-3-5 TNBSA溶液 34
3-4疊層組裝製備(LBL) 34
3-4-1基材製備 34
3-4-2疊層組裝 35
3-5 釋放實驗 36
3-5-1 DNA釋放量檢測 36
3-5-2 PEI釋放量檢測 36
3-6 HEK293T細胞轉染實驗 37
3-7 原子力顯微鏡分析 37
3-8 掃描式電子顯微鏡分析 37
第四章結果與討論 38
4-1 通電對聚電解質釋放影響 38
4-1-1 不同電場方向對聚電解質釋放影響 38
4-1-2 不同電流對聚電解質釋放影響 41
4-1-3 不同電壓對聚電解質釋放影響 43
4-1-4 通電不同時間對聚電解質釋放影響 46
4-2原子力顯微鏡探和掃描式電子顯微鏡探討表面結構變化 49
4-3細胞轉染 53
第五章結論 55
第六章參考文獻 56

參考文獻

第六章參考文獻
1. Anderson, J.M. and M.S. Shive, Biodegradation and biocompatibility of PLA and PLGA microspheres. Advanced Drug Delivery Reviews, 2012. 64: p. 72-82.
2. Friess, W., Collagen--biomaterial for drug delivery. European Journal of Pharmaceutics and Biopharmaceutics, 1998. 45(2): p. 113-36.
3. Laemmli, U.K., Characterization of DNA condensates induced by poly(ethylene oxide) and polylysine. Proceedings of the National Academy of Sciences, 1975. 72(11): p. 4288-92.
4. Aytar, B.S., M.R. Prausnitz, and D.M. Lynn, Rapid release of plasmid DNA from surfaces coated with polyelectrolyte multilayers promoted by the application of electrochemical potentials. ACS Appl Mater Interfaces, 2012. 4(5): p. 2726-34.
5. Diéguez, L., et al., Electrochemical tuning of the stability of PLL/DNA multilayers. Soft Matter, 2009. 5(12): p. 2415.
6. Guillaume-Gentil, O., et al., Chemically tunable electrochemical dissolution of noncontinuous polyelectrolyte assemblies: an in situ study using ecAFM. ACS Appl Mater Interfaces, 2010. 2(12): p. 3525-31.
7. 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.
8. Cho, C., et al., Electric field induced morphological transitions in polyelectrolyte multilayers. ACS Appl Mater Interfaces, 2013. 5(11): p. 4930-6.
9. Guillaume-Gentil, O., et al., Global and local view on the electrochemically induced degradation of polyelectrolyte multilayers: from dissolution to delamination. Soft Matter, 2010. 6(17): p. 4246-4254.
10. Langer, R. and J.P. Vacanti, Tissue engineering. Science, 1993. 260(5110): p. 920-6.
11. Yasuhiko Tabata, M.Y., and Yoshito Ikada, Biodegradable hydrogels for bone regeneration
through growth factor release. Pure and Applied Chemistry, 1998. 70(6): p. p1277-82.
12. Tabata, Y., The importance of drug delivery systems in tissue engineering. Pharmaceutical Science & Technology Today, 2000. 3(3): p. 80-89.
13. Chen, D., R. Sung, and J.S. Bromberg, Gene therapy in transplantation. Transplant Immunology, 2002. 9(2–4): p. 301-314.
14. Volpers, C. and S. Kochanek, Adenoviral vectors for gene transfer and therapy. The Journal of Gene Medicine, 2004. 6 Suppl 1: p. S164-71.
15. Park, T.G., J.H. Jeong, and S.W. Kim, Current status of polymeric gene delivery systems. Advanced Drug Delivery Reviews 2006. 58(4): p. 467-86.
16. P L Felgner, et al., Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proceedings of the National Academy of Sciences, 1987. 84(21): p. 7413-17.
17. Jin, L., et al., Current progress in gene delivery technology based on chemical methods and nano-carriers. Theranostics, 2014. 4(3): p. 240-55.
18. Decher, G., Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites. Science, 1997. 277(5330): p. 1232-1237.
19. Kotov, N.A., I. Dekany, and J.H. Fendler, Layer-by-Layer Self-Assembly of Polyelectrolyte-Semiconductor Nanoparticle Composite Films. The Journal of Physical Chemistry, 1995. 99(35): p. 13065-13069.
20. Lvov, Y., et al., Ultrathin films of charged polysaccharides assembled alternately with linear polyions. J Biomater Sci Polym Ed, 1998. 9(4): p. 345-55.
21. Müller, M., Orientation of α-Helical Poly(l-lysine) in Consecutively Adsorbed Polyelectrolyte Multilayers on Texturized Silicon Substrates. Biomacromolecules, 2001. 2(1): p. 262-269.
22. Lvov, Y., G. Decher, and G. Sukhorukov, Assembly of thin films by means of successive deposition of alternate layers of DNA and poly(allylamine). Macromolecules, 1993. 26(20): p. 5396-5399.
23. Lvov, Y., K. Ariga, and T. Kunitake, Layer-by-Layer Assembly of Alternate Protein/Polyion Ultrathin Films. Chemistry Letters, 1994. 23(12): p. 2323-2326.
24. Yoo, P.J., et al., Spontaneous assembly of viruses on multilayered polymer surfaces. Nature Materials, 2006. 5(3): p. 234-240.
25. Eugenia Kharlampieva, V. Kozlovskaya, and S.A. Sukhishvili, Layer-by-Layer Hydrogen-Bonded Polymer Films: From Fundamentals to Applications. General & Introductory Materials Scienc, 2009. 21(30): p. 3053-65.
26. Brynda, E. and M. Houska, Multiple Alternating Molecular Layers of Albumin and Heparin on Solid Surfaces. Journal of Colloid and Interface Science, 1996. 183(1): p. 18-25.
27. Anzai, J.-i., et al., Layer-by-Layer Construction of Multilayer Thin Films Composed of Avidin and Biotin-Labeled Poly(amine)s. Langmuir, 1998. 15(1): p. 221-226.
28. Kotov, N.A., Layer-by-layer self-assembly: The contribution of hydrophobic interactions. Nanostructured Materials, 1999. 12(5–8): p. 789-796.
29. Lojou, E. and P. Bianco, Buildup of polyelectrolyte-protein multilayer assemblies on gold electrodes. Role of the hydrophobic effect. Langmuir, 2004. 20(3): p. 748-55.
30. Anzai, J.-i. and N. Nakamura, Preparation of active avidin films by a layer-by-layer deposition of poly(vinyl sulfate) and avidin on a solid surface. Journal of the Chemical Society, Perkin Transactions 2, 1999(11): p. 2413-2414.
31. Anzai, J.-i., T. Hoshi, and N. Nakamura, Construction of Multilayer Thin Films Containing Avidin by a Layer-by-Layer Deposition of Avidin and Poly(anion)s. Langmuir, 2000. 16(15): p. 6306-6311.
32. Boura, C., et al., Endothelial cells grown on thin polyelectrolyte mutlilayered films: an evaluation of a new versatile surface modification. Biomaterials, 2003. 24(20): p. 3521-3530.
33. M. K. Gheith, et al., Single-Walled Carbon Nanotube Polyelectrolyte Multilayers and Freestanding Films as a Biocompatible Platform for Neuroprosthetic Implants. General & Introductory Materials Science, 2005. 17(22): p. 2663-70.
34. Jungwoo Lee , S. Shanbhag, and N.A. Kotov, Inverted colloidal crystals as three-dimensional microenvironments for cellular co-cultures. J. Mater. Chem.,, 2006. 16: p. 3358-64.
35. Jensen, A.W., et al., Photohydrolysis of Substituted Benzyl Esters in Multilayered Polyelectrolyte Films. Macromolecules, 2004. 37(11): p. 4196-4200.
36. Zhang, J., L.S. Chua, and D.M. Lynn, Multilayered Thin Films that Sustain the Release of Functional DNA under Physiological Conditions. Langmuir, 2004. 20(19): p. 8015-8021.
37. Yamauchi, F., K. Kato, and H. Iwata, 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. 8360-7.
38. Balabushevich, N.G., et al., Loading the multilayer dextran sulfate/protamine microsized capsules with peroxidase. Biomacromolecules, 2003. 4(5): p. 1191-7.
39. Wang, F., et al., Selective electrodissolution of inorganic ions/DNA multilayer film for tunable DNA release. Journal of Materials Chemistry, 2009. 19(2): p. 286-291.
40. Angelatos, A.S., B. Radt, and F. Caruso, Light-Responsive Polyelectrolyte/Gold Nanoparticle Microcapsules. The Journal of Physical Chemistry B, 2005. 109(7): p. 3071-3076.
41. Petrov, A.I., A.V. Gavryushkin, and G.B. Sukhorukov, Effect of Temperature, pH and Shell Thickness on the Rate of Mg2+ and Ox2- Release from Multilayered Polyelectrolyte Shells Deposited onto Microcrystals of Magnesium Oxalate. The Journal of Physical Chemistry B, 2002. 107(3): p. 868-875.
42. Lu, Z., et al., Magnetic Switch of Permeability for Polyelectrolyte Microcapsules Embedded with Co@Au Nanoparticles. Langmuir, 2005. 21(5): p. 2042-2050.
43. Hu, S.-H., et al., Controlled Rupture of Magnetic Polyelectrolyte Microcapsules for Drug Delivery. Langmuir, 2008. 24(20): p. 11811-11818.
44. Van Tassel, P.R., Polyelectrolyte adsorption and layer-by-layer assembly: Electrochemical control. Current Opinion in Colloid & Interface Science, 2012. 17(2): p. 106-113.
45. Aytar, B.S., M.R. Prausnitz, and D.M. Lynn, Rapid Release of Plasmid DNA from Surfaces Coated with Polyelectrolyte Multilayers Promoted by the Application of Electrochemical Potentials
ACS Appl Mater Interfaces, 2012. 4(5): p. 2726-34.
46. Kurrat, R., et al., Instrumental improvements in optical waveguide light mode spectroscopy for the study of biomolecule adsorption. Review of Scientific Instruments, 1997. 68(5): p. 2172-2176.
47. Picart, C., et al., Measurement of film thickness up to several hundreds of nanometers using optical waveguide lightmode spectroscopy. Biosensors and Bioelectronics, 2004. 20(3): p. 553-61.
48. Gabi, M., et al., Influence of applied currents on the viability of cells close
to microelectrodesw . Integr Biol (Camb), 2009. 1(1): p. 108-15.
49. Schlenoff, J.B. and S.T. Dubas, Mechanism of Polyelectrolyte Multilayer Growth: Charge Overcompensation and Distribution. Macromolecules, 2001. 34(3): p. 592-598.
50. Gabi, M., et al., Influence of applied currents on the viability of cells close to microelectrodes. Integr Biol (Camb), 2009. 1(1): p. 108-15.
51. Wang, X., et al., Evaluation of biocompatibility of polypyrrole in vitro and in vivo. J Biomed Mater Res A, 2004. 68(3): p. 411-22.
52. Schmidt, C.E., et al., Stimulation of neurite outgrowth using an electrically conducting polymer. Proceedings of the National Academy of Sciences, 1997. 94(17): p. 8948-53.
53. Wong, J.Y., R. Langer, and D.E. Ingber, Electrically conducting polymers can noninvasively control the shape and growth of mammalian cells. Proceedings of the National Academy of Sciences, 1994. 91(8): p. 3201-4.
54. Boussif, O., et al., A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proceedings of the National Academy of Sciences, 1995. 92(16): p. 7297-301.
55. Jing, G.Y., et al., Surface effects on elastic properties of silver nanowires: Contact atomic-force microscopy. Physical Review B, 2006. 73(23): p. 235409.
56. Poon, C.Y. and B. Bhushan, Comparison of surface roughness measurements by stylus profiler, AFM and non-contact optical profiler. Wear, 1995. 190(1): p. 76-88.
57. 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.
58. Vázquez, E., et al., Construction of Hydrolytically-Degradable Thin Films via Layer-by-Layer Deposition of Degradable Polyelectrolytes. Journal of the American Chemical Society, 2002. 124(47): p. 13992-13993.
59. 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.
60. Benjaminsen, R.V., et al., The possible "proton sponge " effect of polyethylenimine (PEI) does not include change in lysosomal pH. Molecular Therapy, 2013. 21(1): p. 149-57.

指導教授

胡威文

審核日期

2014-8-20

推文