利用疊層組裝控制DNA吸附與釋放之行為｜國立中央大學博碩士論文系統

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研究生：	陳勇任 Yung-jen Chen
論文名稱：	利用疊層組裝控制DNA吸附與釋放之行為 Control of DNA Adsorption and Releasing Behaviors by Layer-by-Layer Assembly Technique
指導教授：	胡威文 Wei-wen Hu
口試委員:
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程與材料工程學系 Department of Chemical & Materials Engineering
畢業學年度：	99
語文別：	中文
論文頁數：	96
中文關鍵詞：	幾丁聚醣、疊層組裝、基因傳送
外文關鍵詞：	gene delivery, layer- by-layer -assembly, chitosan
相關次數：	點閱：8 下載：0
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本實驗主要利用疊層組裝(layer-by layer assembly)將幾丁聚醣
(chitosan)與質體 DNA 以靜電作用力的方式在基材表面做堆疊。製備好的電雙層以傅立葉轉換紅外線光譜儀與接觸角分析電雙層的化學
與物理性質，並利用紫外光光譜與石英晶體微量天秤來量化基材上的
幾丁聚醣與質體 DNA 之吸附量，藉以討論不同幾丁聚醣分子量及其
水溶液之 pH 值對 DNA 吸附量與釋放量的影響。我們發現分子量 10k Da 幾丁聚醣與質體 DNA 之疊層組裝有較好的基因釋放效果。以此條件之所製備之幾丁聚醣-DNA 基材應用於細胞轉染，以人類胚胎腎臟細胞株(HEK 293T)作為轉染的對象。MTS 測試證實幾丁聚醣並未明顯影響細胞的活性。製備時隨著幾丁聚醣水溶液 pH 值與電雙層層數的增加，對於轉染細胞的轉染基因效果以及表達時間均較好的效果，原因是 pH 6 的製備條件下，會吸附較多的幾丁聚醣分子，提升與 DNA形成複合體進入到細胞內的機會，而較多的層數也可以延長基因轉染的時間長度。此外，相較於 DNA-幾丁聚醣合成的奈米粒子，疊層組裝的轉染可以延長 2~3 天的基因表現。這些結果均顯示疊層組裝可成為基因傳送的有效方法。

Layer-by-layer (LBL) assembly technique was used to immobilize chitosan and plasmid DNA on material surface using electrostatic forces.
The chemistry and physical properties of polyelectrolyte multilayer (PEM) was analyzed by FT-IR and contact angle analysis. UV spectrometry and quartz crystal microbalance (QCM ) were applied to quantify the absorbing amounts of DNA and chitosan. Different molecular weights of
chitosan and the pH value of chitosan solutions were examined to determine their effects to the DNA adsorption and release profiles.
Because 10k Da chitosan had the highest DNA releasing ability, it was used to prepare PEM for transfecting human embryonic kidney 293T cells (HEK 293T cells). MTS assay suggested that chitosan did not reduce the cell viability. Increasing bilayer numbers and pH values of
chitosan solutions may enhance cell transfection efficiencies and elongate gene expression. It may be due to that more chitosan molecules adsorbed on substrates during LBL assembly at pH 6 than at pH 4 conditions. More
chitosan molecules may result in more complex formation and thus facilitate gene delivery. Furthermore, higher bilayer numbers prolonged gene expression. Compared to the cells transfected by DNA-chitosan nanoparticles, cells in situ transfected on PEM demonstrated an improved and extended gene expression. These results suggested that the LBL
method should be a potential strategy to control gene delivery.

摘要 ........................................................................................................... III
目錄 ............................................................................................................ V
第一章 序論 ............................................................................................... 1
1-1 背景 ............................................................................................ 1
1-2 實驗目的 .................................................................................... 3
1-3 實驗架構 .................................................................................... 4
第二章 文獻回顧..................................................................................... 6
2-1 組織工程 .................................................................................... 6
2-2 疊層組裝 .................................................................................. 10
2-2-1 疊層組裝的原理 ............................................................ 10
2-2-2 疊層組裝的製備 ............................................................ 10
2-2-3 疊層組裝於醫療上的應用 ............................................ 11
2-3 幾丁聚醣 .................................................................................. 13
2-3-1 幾丁聚醣來源 ................................................................ 13
2-3-2 幾丁聚醣的性質 ............................................................ 14
2-3-3 幾丁聚醣在醫療上的應用 ............................................ 15
2-4 石英晶體微量天秤 .................................................................. 17
2-4-1 石英晶體微量天秤 ........................................................ 17
VI
2-4-2 石英晶體特性 ................................................................ 17
2-4-3 石英晶體微天秤檢測原理 ............................................ 19
2-5 紫外光光譜儀 ............................................................................ 23
2-5-1 紫外光光譜儀 ................................................................ 23
2-5-2 紫外光光譜儀原理 ........................................................ 23
第三章 實驗 ............................................................................................. 26
3-1 實驗藥品與儀器 ........................................................................ 26
3-1-1 藥品 ................................................................................ 26
3-1-2 儀器 ................................................................................ 27
3-2 試藥製備 .................................................................................... 28
3-2-1 DNA cloning .................................................................. 28
3-2-2 陽離子水溶液 ................................................................ 29
3-2-3 陰離子水溶液 ................................................................ 29
3-3 材料化學表面分析 .................................................................... 30
3-3-1 FT-IR全光譜 ................................................................. 30
3-3-2 接觸角分析 .................................................................... 32
3-4 吸附實驗 .................................................................................... 33
3-4-1 UV-vis全光譜觀測 ....................................................... 33
3-4-2 石英晶體微量天秤觀測 ................................................ 35
3-5 釋放實驗 .................................................................................... 37
VII
3-6 HEK293T細胞轉染實驗............................................................ 38
3-6-1 LBL組製備 ................................................................... 38
3-6-2 NPs對照組製備 ............................................................ 38
3-6-3 MTS細胞毒性測試 ....................................................... 40
第四章 結果與討論 ................................................................................ 41
4-1傅立葉轉換紅外線光譜儀分析 ................................................. 41
4-2 接觸角分析 ................................................................................ 43
4-3吸附時間對DNA吸附量之影響 .............................................. 44
4-4 DNA濃度與體積對吸附量之影響 ........................................... 45
4-5幾丁聚醣分子量與其水溶液pH值對DNA吸附量與釋放量的影響 .................................................................................................... 47
4-5-1 吸附 ............................................................................... 47
4-5-2 釋放 ............................................................................... 52
4-6層數對吸附量與釋放行為之影響 ............................................. 56
4-7細胞存活率測試(MTS assay) ..................................................... 57
4-8 Chitosan水溶液pH值對細胞轉染之影響 ............................... 59
4-9 疊層組裝之轉染途徑 ................................................................ 61
第五章 結論 ............................................................................................. 64
參考文獻 ................................................................................................... 92

                                

1.Nishida, J. and T. Shimarnura, Methods of reconstruction for bone defect after tumor excision: A review of alternatives. Medical Science Monitor, 2008. 14(8): p. RA107-RA113.
2.Niederauer, G.G., D.R. Lee, and S. Sankaran, Bone grafting in arthroscopy and sports medicine. Sports Medicine and Arthroscopy Review, 2006. 14(3): p. 163-168.
3.Paul, C.N., Skin grafting in burns. Wounds-a Compendium of Clinical Research and Practice, 2008. 20(7): p. 199-202.
4.Mooney, D.J. and A.G. Mikos, Growing new organs. Scientific American, 1999. 280(4): p. 60-65.
5.Krebsbach, P.H., et al., Gene therapy-directed osteogenesis: BMP-7-transduced human fibroblasts form bone in vivo. Human Gene Therapy, 2000. 11(8): p. 1201-1210.
6.Hu, W.W., et al., Bone Regeneration in Defects Compromised by Radiotherapy. Journal of Dental Research, 2010. 89(1): p. 77-81.
7.Rinaudo, M., G. Pavlov, and J. Desbrieres, Influence of acetic acid concentration on the solubilization of chitosan. Polymer, 1999. 40(25): p. 7029-7032.
8.Illum, L., Chitosan and its use as a pharmaceutical excipient. Pharmaceutical Research, 1998. 15(9): p. 1326-1331.
9.Madihally, S.V. and H.W.T. Matthew, Porous chitosan scaffolds for tissue engineering. Biomaterials, 1999. 20(12): p. 1133-1142.
10.Richert, L., et al., Layer by layer buildup of polysaccharide films: Physical chemistry and cellular adhesion aspects. Langmuir, 2004. 20(2): p. 448-458.
11.Roy, K., et al., Oral gene delivery with chitosan-DNA nanoparticles generates immunologic protection in a murine model of peanut allergy. Nature Medicine, 1999. 5(4): p. 387-391.
12.Ariga, K., J.P. Hill, and Q.M. Ji, Layer-by-layer assembly as a versatile bottom-up nanofabrication technique for exploratory research and realistic application. Physical Chemistry Chemical Physics, 2007. 9(19): p. 2319-2340.
13.Ariga, K., J.P. Hill, and Q.M. Ji, Biomaterials and Biofunctionality in Layered Macromolecular Assemblies. Macromolecular Bioscience, 2008. 8(11): p. 981-990.
14.Bieker, P. and M. Schönhoff, Linear and Exponential Growth Regimes of Multilayers of Weak Polyelectrolytes in Dependence on pH. Macromolecules, 2010. 43(11): p. 5052-5059.
15.Song, Z.J., et al., Layer-by-Layer Buildup of Poly(L-glutamic acid)/Chitosan Film for Biologically Active Coating. Macromolecular Bioscience, 2009. 9(3): p. 268-278.
16.Yoo, D., S.S. Shiratori, and M.F. Rubner, Controlling bilayer composition and surface wettability of sequentially adsorbed multilayers of weak polyelectrolytes. Macromolecules, 1998. 31(13): p. 4309-4318.
17.Shiratori, S.S. and M.F. Rubner, pH-dependent thickness behavior of sequentially adsorbed layers of weak polyelectrolytes. Macromolecules, 2000. 33(11): p. 4213-4219.
18.Sukhorukov, G.B., J. Schmitt, and G. Decher, Reversible swelling of polyanion/polycation multilayer films in solutions of different ionic strength. Berichte Der Bunsen-Gesellschaft-Physical Chemistry Chemical Physics, 1996. 100(6): p. 948-953.
19.Shin, H., S. Jo, and A.G. Mikos, Biomimetic materials for tissue engineering. Biomaterials, 2003. 24(24): p. 4353-4364.
20.Vacanti, J.P. and R. Langer, Tissue engineering: the design and fabrication of living replacement devices for surgical reconstruction and transplantation. Lancet, 1999. 354: p. SI32-SI34.
21.Langer, R. and J.P. Vacanti, TISSUE ENGINEERING. Science, 1993. 260(5110): p. 920-926.
22.Macri, L., D. Silverstein, and R.A.F. Clark, Growth factor binding to the pericellular matrix and its importance in tissue engineering. Advanced Drug Delivery Reviews, 2007. 59(13): p. 1366-1381.
23.Lee, S.H. and H. Shin, Matrices and scaffolds for delivery of bioactive molecules in bone and cartilage tissue engineering. Advanced Drug Delivery Reviews, 2007. 59(4-5): p. 339-359.
24.Tabata, Y., M. Yamamoto, and Y. Ikada, Biodegradable hydrogels for bone regeneration through growth factor release. Pure and Applied Chemistry, 1998. 70(6): p. 1277-1282.
25.Franceschi, R.T., Biological approaches to bone regeneration by gene therapy. Journal of Dental Research, 2005. 84(12): p. 1093-1103.
26.Jiang, Z.L., et al., Local high-capacity adenovirus-mediated mCTLA4lg and mCD40lg expression prolongs recombinant gene expression in skeletal muscle. Molecular Therapy, 2001. 3(6): p. 892-900.
27.Hannallah, D., et al., Gene therapy in orthopaedic surgery. Journal of Bone and Joint Surgery-American Volume, 2002. 84A(6): p. 1046-1061.
28.Gardlik, R., et al., Vectors and delivery systems in gene therapy. Medical Science Monitor, 2005. 11(4): p. RA110-RA121.
29.Mao, H.Q., et al., Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency. Journal of Controlled Release, 2001. 70(3): p. 399-421.
30.Gaucher, G., R.H. Marchessault, and J.C. Leroux, Polyester-based micelles and nanoparticles for the parenteral delivery of taxanes. Journal of Controlled Release, 2010. 143(1): p. 2-12.
31.Criscione, J.M., et al., Self-assembly of pH-responsive fluorinated dendrimer-based particulates for drug delivery and noninvasive imaging. Biomaterials, 2009. 30(23-24): p. 3946-3955.
32.Decher, G., Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites. Science, 1997. 277(5330): p. 1232-1237.
33.Cai, K., Y. Hu, and K.D. Jandt, Surface engineering of titanium thin films with silk fibroin via layer-by-layer technique and its effects on osteoblast growth behavior. Journal of Biomedical Materials Research Part A, 2007. 82A(4): p. 927-935.
34.Cai, K., et al., Surface modification of titanium thin film with chitosan via electrostatic self-assembly technique and its influence on osteoblast growth behavior. Journal of Materials Science: Materials in Medicine, 2007. 19(2): p. 499-506.
35.Zhu, H., et al., Protein electrostatic self-assembly on poly(DL-lactide) scaffold to promote osteoblast growth. Journal of Biomedical Materials Research, 2004. 71B(1): p. 159-165.
36.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. 214-223.
37.Hu, W.W., et al., Localized viral vector delivery to enhance in situ regenerative gene therapy. Gene Ther, 2007. 14(11): p. 891-901.
38.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.
39.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.
40.Blacklock, J., et al., Gene delivery in vitro and in vivo from bioreducible multilayered polyelectrolyte films of plasmid DNA. Biomaterials, 2009. 30(5): p. 939-950.
41.Jessel, N., Multiple and time-scheduled in situ DNA delivery mediated by beta-cyclodextrin embedded in a polyelectrolyte multilayer. Proceedings of the National Academy of Sciences, 2006. 103(23): p. 8618-8621.
42.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.
43.Takanori Sannan, K.K., and Yoshio Iwakura Studies on Chitin, Effect of Deacetylation on Solubility Makromol. Chem, 1976. 177(3589-3600 ).
44.Hirano, S., et al., Wet spun chitosan-collagen fibers, their chemical N-modifications, and blood compatibility. Biomaterials, 2000. 21(10): p. 997-1003.
45.Yang, T.-L. and T.-H. Young, The specificity of chitosan in promoting branching morphogenesis of progenitor salivary tissue. Biochemical and Biophysical Research Communications, 2009. 381(4): p. 466-470.
46.Chandy, T. and C.P. Sharma, CHITOSAN - AS A BIOMATERIAL. Biomaterials Artificial Cells and Artificial Organs, 1990. 18(1): p. 1-24.
47.Kofuji, K., et al., The controlled release of a drug from biodegradable chitosan gel beads. Chemical & Pharmaceutical Bulletin, 2000. 48(4): p. 579-581.
48.Falk, B., S. Garramone, and S. Shivkumar, Diffusion coefficient of paracetamol in a chitosan hydrogel. Materials Letters, 2004. 58(26): p. 3261-3265.
49.Senel, S., et al., Chitosan films and hydrogels of chlorhexidine gluconate for oral mucosal delivery. International Journal of Pharmaceutics, 2000. 193(2): p. 197-203.
50.Liu, L.-S., et al., Controlled release of interleukin-2 for tumour immunotherapy using alginate/chitosan porous microspheres. Journal of Controlled Release, 1997. 43(1): p. 65-74.
51.Kim, T.H., et al., Galactosylated chitosan/DNA nanoparticles prepared using water-soluble chitosan as a gene carrier. Biomaterials, 2004. 25(17): p. 3783-3792.
52.Brannon-Peppas, L. and J.O. Blanchette, Nanoparticle and targeted systems for cancer therapy. Advanced Drug Delivery Reviews, 2004. 56(11): p. 1649-1659.
53.羅晏如, 幾丁物質的生物活性即其應用. 國立海洋大學食品科學系碩士學位論文, 2008.
54.Chen, H.M., M.L. Lin, and S.H. Chen, Fabrication of piezoelectric biochips with self-assembled alkanethiol layer and hydrocoating. Journal of the Chinese Institute of Chemical Engineers, 2003. 34(1): p. 151-160.
55.Yokoyama, K., et al., Highly sensitive quartz crystal immunosensors for multisample detection of herbicides. Analytica Chimica Acta, 1995. 304(2): p. 139-145.
56.Caruso, F., et al., Quartz crystal microbalance study of DNA immobilization and hybridization for nucleic acid sensor development. Analytical Chemistry, 1997. 69(11): p. 2043-2049.
57.Konig, B. and M. Gratzel, DETECTION OF HUMAN T-LYMPHOCYTES WITH A PIEZOELECTRIC IMMUNOSENSOR. Analytica Chimica Acta, 1993. 281(1): p. 13-18.
58.Chen, S.-H., et al., A method of layer-by-layer gold nanoparticle hybridization in a quartz crystal microbalance DNA sensing system used to detect dengue virus. Nanotechnology, 2009. 20(21): p. 215501.
59.吳宗正, 壓電晶體生物感測器之研究與其應用. 國立台灣大學農業化學研究所博士論文, 1990.
60.O''Sullivan, C.K. and G.G. Guilbault, Commercial quartz crystal microbalances - theory and applications. Biosensors and Bioelectronics, 1999. 14(8-9): p. 663-670.
61.施任青, 以石英晶體微天秤法偵測金奈米標識之IgG時訊號之增強. 國立中山大學化學系研究所碩士學位論文, 2002.
62.J. Curie , P.C., Developpement, par pression, de l"electricite polaire dans les cristaux hemiedres a faces inclinees. Comp. Rend., 1880. 91(294).
63.Czanderna, C.L.a.A.W., Applications of piezoelectric quartz crystal microbalances. 1984.
64.Buttry, D.A. and M.D. Ward, Measurement of Interfacial Processes at Electrode Surfaces with the Electrochemical Quartz Crystal Microbalance. Chemical Reviews, 1992. 92.
65.劉世隆, 以位置篩選法合成組合化學胜太庫應用於氣體辨識材料之研究. 國立東華大學生物技術研究所碩士學位論文, 2002.
66.Chen, G.-P., et al., Layer-by-layer assembly of poly(p-xylyleneviologen)-DNA multilayers and their electrochemical properties. Materials Science and Engineering: C, 2009. 29(3): p. 925-929.
67.Liu, Y. and N.F. Hu, Loading/release behavior of (chitosan/DNA)(n) layer-by-layer films toward negatively charged anthraquinone and its application in electrochemical detection of natural DNA damage. Biosensors & Bioelectronics, 2007. 23(5): p. 661-667.
68.Wang, F., et al., Layer-by-layer assembly of biologically inert inorganic ions/DNA multilayer films for tunable DNA release by chelation. Journal of Controlled Release, 2008. 132(1): p. 65-73.
69.Saurer, E.M., et al., Assembly of erodible, DNA-containing thin films on the surfaces of polymer microparticles: Toward a layer-by-layer approach to the delivery of DNA to antigen-presenting cells. Acta Biomaterialia, 2009. 5(3): p. 913-924.
70.Mincheva, R., et al., Hydrogels from chitosan crosslinked with poly(ethylene glycol) diacid as bone regeneration materials. E-Polymers, 2004.
71.Malins, D.C., et al., A cancer DNA phenotype in healthy prostates, conserved in tumors and adjacent normal cells, implies a relationship to carcinogenesis. Proceedings of the National Academy of Sciences of the United States of America, 2005. 102(52): p. 19093.
72.Porcel, C., et al., From Exponential to Linear Growth in Polyelectrolyte Multilayers. Langmuir, 2006. 22(9): p. 4376-4383.
73.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.
74.Mansouri, S., et al., Characterization of folate-chitosan-DNA nanoparticles for gene therapy. Biomaterials, 2006. 27(9): p. 2060-2065.
75.Pannier, A., Controlled release systems for DNA delivery. Molecular Therapy, 2004. 10(1): p. 19-26.
76.Cai, K., et al., Cell-Specific Gene Transfection from a Gene-Functionalized Poly(d,l-lactic acid) Substrate Fabricated by the Layer-by-Layer Assembly Technique. Angewandte Chemie International Edition, 2008. 47(39): p. 7479-7481.

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