研發新頴核酸載體平台於調控基因表現與抑制之應用

簡易檢索 / 詳目顯示

回結果列表

研究生：	曾士傑 Tseng, S-Ja
論文名稱：	研發新頴核酸載體平台於調控基因表現與抑制之應用 Development of Nucleic-Acid Carrier Platform for Gene Expression and Silencing
指導教授：	湯學成 Tang, Shiue-Cheng
口試委員:
學位類別：	博士 Doctor
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2010
畢業學年度：	98
語文別：	中文
論文頁數：	87
中文關鍵詞：	生物可分解高分子、細胞毒性、核酸傳遞、基因表現、基因抑制
外文關鍵詞：	biodegradable polymer, cytotoxicity, nucleic acid delivery, gene expression, gene silencing
相關次數：	點閱：3 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

天然細胞外間質(extracellular matrices, ECM)已廣泛應用於組織工程與再生醫學。在市售的ECM之中，以豬體中取出的小腸黏膜組織下層(small intestinal submucosa, SIS)對於燒燙傷修補和組織重建具有明顯療效。由於SIS中含有負電之醣胺素分子，故本研究藉由靜電作用力讓帶正電之核酸傳遞載體(polymer/DNA奈米複合體)吸附於SIS上，將SIS與核酸傳遞結合以作為調控基因表現之平台。當奈米複合體與細胞接觸時，可將所包覆之DNA攜帶入細胞中，進行特定基因表現。以生醫材料平台調控基因傳遞至標的細胞，具有專一性且可以大幅提升基因傳遞效率，不管是組織工程或疾病治療均可使用這個平台。平台建立後，需要與低毒性之高分子核酸傳遞載體結合。本研究藉由現今已廣泛應用於組織工程之聚(酯-胺基甲酸酯) (poly(ester urethane), PEU)與聚乙二醇(poly(ethylene glycol), PEG)合成出生物可分解性、毒性低且溶於水之聚(胺基-酯-乙二醇-胺基甲酸酯) (poly(amino-ester-glycol-urethane), PaEGU)，其可與核酸物質自組裝成高度專一性帶且正電之PaEGU/DNA或PaEGU/siRNA奈米複合體，使核酸傳遞入細胞中並抵擋生物體內酵素將其分解，亦可將大幅提升核酸傳遞效率。以建立新穎核酸傳遞之平台，應用於組織工程或疾病治療。

Naturally occurring extracellular matrices (ECMs) have shown great potential in clinical applications as tissue substrates to facilitate tissue repair and regeneration. Among the commercialized ECMs, porcine small intestinal submucosa (SIS) has been used in patients for wound treatment and soft tissue reconstruction. However, there have been no reports exploring the electrostatic properties of SIS as a substrate to control localized nucleic acid delivery. We have demonstrated that the negatively charged glycosaminoglycan (GAG) content in SIS was able to associate with the cationic polymer/DNA polyplexes through electrostatic adsorption and led to transfection upon cellular adhesion. However, a major challenge to the development of localized nucleic acid delivery is the design of suitable vectors with low cytotoxicity. Poly(ester urethane) (PEU) is a class of biodegradable polymer that has been applied as tissue-engineering scaffolds with minimal cytotoxicity in vitro and in vivo. We have developed a method of incorporating tertiary amines and poly(ethylene glycol) (PEG) into PEU to synthesize soluble poly(amino ester glycol urethane) (PaEGU) as a novel platform for nucleic acid delivery. PaEGU can condense DNA or siRNA into nano-scale polyplexes to enter cells through endocytosis, which can be a useful tool to work with ECMs for localized gene expression and silencing applications.

目錄      I
圖目錄    III
表目錄    VII
附錄目錄       VIII

第一章    文獻回顧
1-1. 核酸傳遞之發展與應用    2
1-1.1. 基因治療(gene therapy)    2
1-1.2. 基因抑制(gene silencing)    4
1-2. 核酸傳遞載體系統    6
1-2.1. 病毒型載體(viral vector)    6
1-2.2. 非病毒型載體(non-viral vector)    6
1-2.3. 聚(酯-胺基甲酸酯) (poly(ester urethane), PEU)    7
1-3. 生醫材料支架調控核酸傳遞之局部系統    8
1-3.1. 細胞外間質(extracellular matrix, ECM)    10
1-3.2. 小腸黏膜組織下層(small intestinal submucosa, SIS)    10
1-4. 實驗動機    14
第二章  實驗方法
2-1. 材料    17
2-2. 細胞培養    18
2-3. SIS調控polymer/DNA奈米複合體用於基因傳遞    18
2-3.1. SIS組成中負電荷分子醣胺素之定性分析與表面電位實驗    18
2-3.2. 測量SIS吸附PEI/DNA奈米複合體之實驗    20
2-3.3. SIS吸附PEI/DNA奈米複合體用於基因傳遞之實驗    21
2-3.4. SIS吸附PEI/DNA奈米複合體用於基因傳遞之專一性實驗    23
2-4. PaEGU/DNA奈米複合體用於基因傳遞    25
2-4.1. 高分子之合成    25
2-4.2. PaEGU之水解與細胞毒性實驗    28
2-4.3. PaEGU/DNA奈米複合體之表面電位、粒徑、電泳和核酸限制內切酶實驗    29
2-4.4. PaEGU/DNA奈米複合體之基因表現實驗    29
2-5. PaEGU/siRNA奈米複合體用於基因抑制    31
2-5.1. siRNA之序列    31
2-5.2. PaEGU/siRNA奈米複合體之表面電位、粒徑和電泳分析    31
2-5.3. PaEGU/siRNA奈米複合體之重量比最佳化實驗    32
2-5.4. HT-1080具有luciferase和EGFP表現之實驗    33
2-5.5. PaEGU/siRNA奈米複合體之基因抑制效果之實驗    34
2-5.6. PaEGU/siRNA奈米複合體之細胞毒性測試與解離實驗    34
第三章  結果與討論
3-1. SIS調控polymer/DNA奈米複合體用於基因傳遞    36
3-1.1.存在於SIS之負電分子醣胺素其表面電位與定性分析    36
3-1.2. PEI/DNA奈米複合體之表面電位和粒徑分佈    36
3-1.3. SIS吸附PEI/DNA奈米複合體之效率    37
3-1.4. SIS吸附PEI/DNA奈米複合體用於基因傳遞之效率    38
3-2. PaEGU/DNA奈米複合體用於基因傳遞    45
3-2.1. 高分子之結構鑑定    45
3-2.2. PaEGU之水解與酸鹼緩衝能力    46
3-2.3. 高分子之細胞毒性    47
3-2.4. PaEGU/DNA奈米複合體之表面電位、粒徑、電泳與核酸限制內切酶分析    47
3-2.5. PaEGU/DNA奈米複合體之基因表現    49
3-3. PaEGU/siRNA奈米複合體用於基因抑制    64
3-3.1. PaEGU/siRNA奈米複合體之表面電位、粒徑與電泳分析    64
3-3.2. PaEGU/siRNA奈米複合體之最佳化重量比    65
3-3.3. PaEGU/siRNA奈米複合體之基因抑制效果    65
3-3.5. PaEGU/siRNA奈米複合體之細胞毒性測試與解離    67
第四章  結論
參考文獻
附錄

圖目錄
Figure 1-1. Schematic of electrostatic immobilization of polymer/DNA or polymer/siRNA complexes on small intestinal submucosa (SIS) for tissue substrate-mediated gene expression or silcencing.    15

Figure 2-1. A schematic diagram of the device for measuring the surface zeta potential of SIS (a streaming potential method).    19

Figure 2-2. Chemical structure of branched polyethyleneimine (PEI) for cationic polyplexes preparation.    20

Figure 2-3. (A), (B) and (C) Schematics of the plasmids Luc-EGFP dual reporter plasmid, EGFP reporter plasmid and LacZ reporter plasmid used in this research.    22

Figure 2-4. The two models of small intestinal submucosa (SIS) as tissue substrate-mediated PEI/DNA polyplexes transfection.    25

Figure 2-5. Synthesis schemes for the test transfection reagent poly(amino ester glycol urethane) (PaEGU).    25

Figure 2-6. Synthesis schemes for the control polymer poly(amino ester urethane) (PaEU).    26

Figure 2-7. Synthesis schemes for the control polymer poly(amino ester) (PaE).    27

Figure 2-8. The structure of siRNA sense sequences.    31

Figure 2-9. Schematics of the plasmids pAAV-luciferase-EGFP and pSV40-Puro.    33

Figure 3-1. (A) The surface zeta potential of SIS at different pH. (B) Toluidine blue staining of SIS to reveal GAG molecules.    40

Figure 3-2. (A) Zeta potential and (B) average diameter of PEI/DNA polyplexes at different PEI/DNA mass ratios.    41

Figure 3-3. (A) Density of immobilized PEI/DNA polyplexes on SIS at different PEI/DNA mass ratios. (B) and (C) Fluorescence micrographs of blank SIS (control) and SIS adsorbed with PEI/DNA polyplexes, respectively. (D) An enlarged micrograph of the location around the arrow-pointed vessel shown in (C).    42

Figure 3-4. (A) Luciferase activity of SIS-mediated transfection at different PEI/DNA mass ratios. (B) Assay of mitochondrial activity (an MTS assay) of HT-1080 fibroblasts on SIS conjugated with PEI/DNA at different mass ratios.    43

Figure 3-5. (A) and (B) Fluorescence micrograph of SIS-mediated transfection with PEI/DNA at mass ratio = 2/1. (C) Gross image of X-gal staining of blank SIS (control, left well) and SIS adsorbed with PEI/DNA (2/1, w/w, right well) using □-galactosidase as the reporter. (D) Phase-contrast micrograph of the stained SIS shown in the right well of (C). (E) Relative transfection efficiency when PEI/DNA loaded SIS and cells were separated by a polyester NetwellTM membrane (pore size = 0.5 mm, indirect contact) or by direct seeding of HT-1080 cells onto the loaded SIS (direct contact).    44

Figure 3-6. FT-IR spectra of poly(ethylene glycol) acrylate and PEGamine-diol (A), 1,4-diisocyanatobutane and PaEGU (B), 2-hydroxyethyl acrylate and HEamine-diol (C), 1,4-diisocyanatobutane and PaEU (D) and 1,4-butanediol diacrylate and PaE (E).    55

Figure 3-7. Gross images of the pure PaEU and PaEGU polymers (A) and the PaEU-water and PaEGU-water mixtures (B).    56

Figure 3-8. (A) Hydrolytic degradation of PaEGU and PaE incubated in PBS buffer at pH 7.4 and 37 °C. (B) Acid-base titration profiles of the polymers and the NaCl control solution.    57


Figure 3-9. Assay of mitochondrial activity (an MTS assay) after treatment with PaEGU or PaE.    58

Figure 3-10. Zeta-potential (A) and size of polymer/DNA complexes (B) at different polymer/DNA mass ratios (w/w).    59

Figure 3-11. DNA gel retardation and restriction endonuclease protection assays of PaEGU (A) and PaE (B).    60

Figure 3-12. (A) EGFP-positive cells (%) of PaEGU- and PaE-mediated transfection. (B) Fluorescence intensities of PaEGU- and PaE-mediated transfection. (C) Fluorescence micrograph of PaEGU/DNA (80/1, w/w) mediated transfection of HT-1080 cells using EGFP as the reporter. (D) Phase-contrast micrograph of X-gal staining of PaEGU/DNA (80/1, w/w) mediated transfection of HT-1080 cells using β-galactosidase as the reporter.    62

Figure 3-13. (A) Fluorescence and (B) phase contrast micrographs of PaEGU/DNA (80/1, w/w) mediated transfection of human embryonic kidney 293 (HEK293) cells using enhanced green fluorescent protein (EGFP) as the reporter. (C) Fluorescence micrograph of PaEGU/DNA (80/1, w/w) mediated transfection of Chinese hamster ovary (CHO-K1) cells using EGFP as the reporter. (D) Phase-contrast micrograph of X-gal staining of PaEGU/DNA (80/1, w/w) mediated transfection of human Caco-2 enterocytes using β-galactosidase as the reporter.    63

Figure 3-14. Size (A) and zeta-potential (B) of PaEGU/siRNA polyplexes at different PaEGU/siRNA mass ratios (w/w).    69

Figure 3-15. Gel retardation analysis of the PaEGU/siRNA polyplexes.    70

Figure 3-16. Alexa Fluor 488-positive cells (%) of PaEGU-mediated (A), PEI-mediated (B), and LipofectamineTM RNAiMAX-mediated (C) transfection.    72

Figure 3-17. Fluorescence micrograph of PaEGU-mediated transfection using Alexa Fluor 488 labeled siRNA as the reporter.    73

Figure 3-18. Kinetics of luciferase silencing via delivery of PaEGU/anti-luciferase siRNA (A), PEI/anti-luciferase siRNA (B), and LipofectamineTM RNAiMAX/anti-luciferase siRNA polyplexes (C). (D) Recombinant HT-1080 cells were transfected with PaEGU/anti-luciferase siRNA polyplexes at the indicated dose for luciferase silencing.    75

Figure 3-19. EGFP silencing via delivery of PaEGU/anti-EGFP siRNA polyplexes. (A) Micrographs of untreated recombinant HT-1080 cells which constitutively expressed EGFP. (B) Micrographs of recombinant HT-1080 cells transfected with PaEGU/anti-EGFP siRNA polyplexes (80/1, w/w) to suppress EGFP expression.    76

Figure 3-20. (A) Assessment of mitochondrial activity (an MTS assay) after treatment with PaEGU/siRNA or PEI/siRNA polyplexes. (B) Dissociation kinetics of PaEGU/siRNA and PEI/siRNA polyplexes in PBS at 37 oC. The Alexa Fluor 488-siRNA was used as the reporter. (C) Comparative cytotoxicity of PaEGU/siRNA and LipofectamineTM RNAiMAX/siRNA polyplexes.    78

Figure 4-1. The commercial value and clinical applications of polymer/DNA or polymer/siRNA complexes on small intestinal submucosa (SIS) for tissue substrate-mediated gene expression or silcencing.    80

表目錄
Table 1-1. Therapeutic targets of RNAi tested in vivo.    5

Table 1-2. In vivo studies involving therapeutically relevant genes delivered with polymeric systems.    9

Table 1-3. Partial list of commercially available devices composed of extracellular matrix.    13

Table 3-1. 1H-NMR data of the synthesized poly(amino ester glycol urethane), PaEGU (A), poly(amino ester urethane), PaEU (B) and poly(amino ester), PaE (C).    51

Table 3-2. 13C-NMR data of the synthesized poly(amino ester glycol urethane), PaEGU (A), poly(amino ester urethane), PaEU (B) and poly(amino ester), PaE (C).    52






附錄目錄
Publications in the fields of biomaterials-mediated gene delivery and biomaterial imaging.
1. Tseng SJ, Lee YH, Chen ZH, Lin HH, Lin CY, and Tang SC*. Integration of Optical Clearing and Optical Sectioning Microscopy for Three Dimensional Imaging of Natural Biomaterial Scaffolds in Thin Sections. Journal of Biomedical Optics. 14(4), Article Number: 044004 (9pp), July/August 2009.

2. Tseng SJ, Chuang CJ and Tang SC*. Electrostatic Immobilization of DNA Polyplexes on Small Intestinal Submucosa for Tissue Substrate-Mediated Transfection. Acta Biomaterialia. 4: p799-807, 2008.

3. Tseng SJ and Tang SC*. Development of Poly(amino ester glycol urethane)/siRNA Polyplexes for Gene Silencing. Bioconjugate Chemistry. 18(5): p1383-1390, 2007.

4. Tseng SJ and Tang SC*. Synthesis and Characterization of a Novel Transfection Reagent Poly(amino ester glycol urethane). Biomacromolecules. 8(1): p50-58, 2007.

                                

[1] Rosenberg S., Aebersold P., Cornetta K., Kasid A., Morgan R., Karson E., Lotze M., Yang J., Topalian S., Merino M., Culver K., Miller D., Blaese R., and Anderson F. (1990) Gene transfer into humans – immunotherapy of patients with advanced melanoma, using TIL modified by retroviral transduction. N Engl J Med 323, 570–578
[2] Culver K., Anderson F., and Blaese R. (1991) Lymphocyte gene therapy. Hum Gene Ther 2, 107–109.
[3] Lown, J. W. (1988) Development of Sequence Specific DNA Effectors - Potential Gene Probes for Diagnosis and Therapy. Anticancer Research 8, 1114-1114.
[4] Watts, S. (1990) DNA Gun on Target for Gene-Therapy. New Scientist 127, 34-34.
[5] Fire, A., Xu, S. Q., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811.
[6] Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498.
[7] Shelton, C. A., and Bowerman, B. (1998) Examining gene function in Caenorhabditis elegans embryos using RNA interference. Developmental Biology 198, 175-175.
[8] Rao, M. K., and Wilkinson, M. F. (2006) Tissue-specific and cell type-specific RNA interference in vivo. Nature Protocols 1, 1494-1501.
[9] Ma, Y., Chan, C. Y., and He, M. L. (2007) RNA interference and antiviral therapy. World Journal of Gastroenterology 13, 5169-5179.
[10] Couzin, J. (2003) RNA interference: New screen nets 'Hedgehog' genes. Science 299, 1961-1961.
[11] Couzin, J. (2003) RNA interference - Mini RNA molecules shield mouse liver from hepatitis. Science 299, 995-995.
[12] Sun, X. G., Rogoff, H. A., and Li, C. J. (2008) Asymmetric RNA duplexes mediate RNA interference in mammalian cells. Nature Biotechnology 26, 1379-1382.
[13] Shan, G., Li, Y. J., Zhang, J. L., Li, W. D., Szulwach, K. E., Duan, R. H., Faghihi, M. A., Khalil, A. M., Lu, L. H., Paroo, Z., Chan, A. W. S., Shi, Z. J., Liu, Q. H., Wahlestedt, C., He, C., and Jin, P. (2008) A small molecule enhances RNA interference and promotes microRNA processing. Nature Biotechnology 26, 933-940.
[14] Gruber, J. J., Zatechka, D. S., Sabin, L. R., Yong, J., Lum, J. J., Kong, M., Zong, W. X., Zhang, Z. X., Lau, C. K., Rawlings, J., Cherry, S., Ihle, J. N., Dreyfuss, G., and Thompson, C. B. (2009) Ars2 Links the Nuclear Cap-Binding Complex to RNA Interference and Cell Proliferation. Cell 138, 328-339.
[15] Uprichard, S. L. (2005) The therapeutic potential of RNA interference. Febs Letters 579, 5996-6007.
[16] Zeilfelder, U., Frank, O., Sparacio, S., Schon, U., Bosch, V., Seifarth, W., and Leib-Mosch, C. (2007) The potential of retroviral vectors to cotransfer human endogenous retroviruses (HERVs) from human packaging cell lines. Gene 390, 175-179.
[17] Deng, X. M., Szabo, S., Khomenko, T., Jadus, M. R., and Yoshida, M. (2004) Gene therapy with adenoviral plasmids or naked DNA of vascular endothelial growth factor and platelet-derived growth factor accelerates healing of duodenal ulcer in rats. Journal of Pharmacology and Experimental Therapeutics 311, 982-988.
[18] Flotte, T. R., Schwiebert, E. M., Zeitlin, P. L., Carter, B. J., and Guggino, W. B. (2005) Correlation between DNA transfer and cystic fibrosis airway epithelial cell correction after recombinant adeno-associated virus serotype 2 gene therapy. Human Gene Therapy 16, 921-928.
[19] Zuber, G., Dauty, E., Nothisen, M., Belguise, P., and Behr, J. P. (2001) Towards synthetic viruses. Advanced Drug Delivery Reviews 52, 245-253.
[20] Anderson, D. G., Peng, W. D., Akinc, A., Hossain, N., Kohn, A., Padera, R., Langer, R., and Sawicki, J. A. (2004) A polymer library approach to suicide gene therapy for cancer. Proceedings of the National Academy of Sciences of the United States of America 101, 16028-16033.
[21] Martin, B., Sainlos, M., Aissaoui, A., Oudrhiri, N., Hauchecorne, M., Vigneron, J. P., Lehn, J. M., and Lehn, P. (2005) The design of cationic lipids for gene delivery. Current Pharmaceutical Design 11, 375-394.
[22] Wolff, J. (2005) Nonviral vectorology: In a good place. Molecular Therapy 11, 333-333.
[23] Brown, M. D., Gray, A. I., Tetley, L., Santovena, A., Rene, J., Schatzlein, A. G., and Uchegbu, I. F. (2003) In vitro and in vivo gene transfer with poly(amino acid) vesicles. Journal of Controlled Release 93, 193-211.
[24] Fu, H. L., Cheng, S. X., and Zhuo, R. X. (2009) Biodegradable Polymers for Controlled Release of Gene Delivery Systems. Acta Polymerica Sinica, 97-103.
[25] Arote, R. B., Jere, D., Jiang, H. L., Kim, Y. K., Choi, Y. J., and Cho, C. S. (2009) Biodegradable poly(ester amine)s for gene delivery applications. Biomedical Materials 4, 1-4.
[26] Tseng, S. J., Tang, S. C., Shau, M. D., Zeng, Y. F., Cherng, J. Y., and Shih, M. F. (2005) Structural characterization and buffering capacity in relation to the transfection efficiency of biodegradable polyurethane. Bioconjugate Chemistry 16, 1375-1381.
[27] Kim, I. S., Lee, S. K., Park, Y. M., Lee, Y. B., Shin, S. C., Lee, K. C., and Oh, I. J. (2005) Physicochemical characterization of poly(L-lactic acid) and poly(D,L-lactide-co-glycolide) nanoparticles with polyethylenimine as gene delivery carrier. International Journal of Pharmaceutics 298, 255-262.
[28] Andrianov, A. K. (2005) Water-soluble biodegradable polyphosphazenes: Emerging systems for biomedical applications. Abstracts of Papers of the American Chemical Society 230, U4157-U4158.
[29] Khatri, K., Goyal, A. K., Gupta, P. N., Mishra, N., and Vyas, S. P. (2008) Plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B. International Journal of Pharmaceutics 354, 235-241.
[30] Azzam, T., Raskin, A., Makovitzki, A., Brem, H., Vierling, P., Lineal, M., and Domb, A. J. (2002) Cationic polysaccharides for gene delivery. Macromolecules 35, 9947-9953.
[31] Jere, D., Jiang, H. L., Arote, R., Kim, Y. K., Choi, Y. J., Cho, M. H., Akaike, T., and Chot, C. S. (2009) Degradable polyethylenimines as DNA and small interfering RNA carriers. Expert Opinion on Drug Delivery 6, 827-834.
[32] Akinc, A., Thomas, M., Klibanov, A. M., and Langer, R. (2005) Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis. Journal of Gene Medicine 7, 657-663.
[33] Borkenhagen, M., Stoll, R. C., Neuenschwander, P., Suter, U. W., and Aebischer, P. (1998) In vivo performance of a new biodegradable polyester urethane system used as a nerve guidance channel. Biomaterials 19, 2155-2165.
[34] Guan, J. J., Sacks, M. S., Beckman, E. J., and Wagner, W. R. (2002) Synthesis, characterization, and cytocompatibility of efastomeric, biodegradable poly(ester-urethane)ureas based on poly(caprolactone) and putrescine. Journal of Biomedical Materials Research 61, 493-503.
[35] Li, X., Loh, X. J., Wang, K., He, C. B., and Li, J. (2005) Poly(ester urethane)s consisting of poly[(R)-3-hydroxybutyrate] and poly(ethylene glycol) as candidate biomaterials: Characterization and mechanical property study. Biomacromolecules 6, 2740-2747.
[36] Riboldi, S. A., Sampaolesi, M., Neuenschwander, P., Cossu, G., and Mantero, S. (2005) Electrospun degradable polyesterurethane membranes: potential scaffolds for skeletal muscle tissue engineering. Biomaterials 26, 4606-4615.
[37] Stankus, J. J., Guan, J. J., Fujimoto, K., and Wagner, W. R. (2006) Microintegrating smooth muscle cells into a biodegradable, elastomeric fiber matrix. Biomaterials 27, 735-744.
[38] Lee, H., Cusick, R. A., Browne, F., Kim, T. H., Ma, P. X., Utsunomiya, H., Langer, R., and Vacanti, J. P. (2002) Local delivery of basic fibroblast growth factor increases both angiogenesis and engraftment of hepatocytes in tissue-engineered polymer devices. Transplantation 73, 1589-1593.
[39] Yoon, J. J., Chung, H. J., Lee, H. J., and Park, T. G. (2006) Heparin-immobilized biodegradable scaffolds for local and sustained release of angiogenic growth factor. Journal of Biomedical Materials Research Part A 79A, 934-942.
[40] Hubbell, J. A. (2006) Matrix-bound growth factors in tissue repair. Swiss Medical Weekly 136, 387-391.
[41] Shea, L. D., Smiley, E., Bonadio, J., and Mooney, D. J. (1999) DNA delivery from polymer matrices for tissue engineering. Nature Biotechnology 17, 551-554.
[42] Jang, J. H., and Shea, L. D. (2003) Controllable delivery of non-viral DNA from porous scaffolds. Journal of Controlled Release 86, 157-168.
[43] Segura, T., Chung, P. H., and Shea, L. D. (2005) DNA delivery from hyaluronic acid-collagen hydrogels via a substrate-mediated approach. Biomaterials 26, 1575-1584.
[44] Jang, J. H., Rives, C. B., and Shea, L. D. (2005) Plasmid delivery in vivo from porous tissue-engineering scaffolds: Transgene expression and cellular Transfection. Molecular Therapy 12, 475-483.
[45] Bengali, Z., and Shea, L. D. (2005) Gene delivery by immobilization to cell-adhesive substrates. Mrs Bulletin 30, 659-662.
[46] Pannier, A. K., and Shea, L. D. (2004) Controlled release systems for DNA delivery. Molecular Therapy 10, 19-26.
[47] Fang, J. M., Zhu, Y. Y., Smiley, E., Bonadio, J., Rouleau, J. P., Goldstein, S. A., McCauley, L. K., Davidson, B. L., and Roessler, B. J. (1996) Stimulation of new bone formation by direct transfer of osteogenic plasmid genes. Proceedings of the National Academy of Sciences of the United States of America 93, 5753-5758.
[48] Takakura, Y., Mahato, R. I., and Hashida, M. (1998) Extravasation of macromolecules. Advanced Drug Delivery Reviews 34, 93-108.
[49] Langer, R. (1998) Drug delivery and targeting. Nature 392, 5-10.
[50] Scherer, F., Schillinger, U., Putz, U., Stemberger, A., and Plank, C. (2002) Nonviral vector loaded collagen sponges for sustained gene delivery in vitro and in vivo. Journal of Gene Medicine 4, 634-643.
[51] Krebs, M. D., Jeon, O., and Alsberg, E. (2009) Localized and Sustained Delivery of Silencing RNA from Macroscopic Biopolymer Hydrogels. Journal of the American Chemical Society 131, 9204-9206.
[52] Kleinman, H. K., Philp, D., and Hoffman, M. P. (2003) Role of the extracellular matrix in morphogenesis. Current Opinion in Biotechnology 14, 526-532.
[53] Lutolf, M. P., and Hubbell, J. A. (2005) Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nature Biotechnology 23, 47-55.
[54] Brown-Etris, M., Cutshall, W. D., and Hiles, M. C. (2002) A new biomaterial derived from small intestine submucosa and developed into a wound matrix device. Wounds-a Compendium of Clinical Research and Practice 14, 150-166.
[55] Badylak, S. F. (2007) The extracellular matrix as a biologic scaffold material. Biomaterials 28, 3587-3593.
[56] Wissink, M. J. B., Beernink, R., Poot, A. A., Engbers, G. H. M., Beugeling, T., van Aken, W. G., and Feijen, J. (2000) Improved endothelialization of vascular grafts by local release of growth factor from heparinized collagen matrices. Journal of Controlled Release 64, 103-114.
[57] Sakiyama-Elbert, S. E., and Hubbell, J. A. (2000) Controlled release of nerve growth factor from a heparin-containing fibrin-based cell ingrowth matrix. Journal of Controlled Release 69, 149-158.
[58] Ishihara, M., Ono, K., Ishikawa, K., Hattori, H., Saito, Y., Yura, H., Akaike, T., Ozeki, Y., Tanaka, S., Mochizuki, H., and Kurita, A. (2000) Enhanced ability of heparin-carrying polystyrene (HCPS) to bind to heparin-binding growth factors and to inhibit growth factor-induced endothelial cell growth. Journal of Biochemistry 127, 797-803.
[59] Ishihara, M., Sato, M., Hattori, H., Saito, Y., Yura, H., Ono, K., Masuoka, K., Kikuchi, M., Fujikawa, K., and Kurita, A. (2001) Heparin-carrying polystyrene (HCPS)-bound collagen substratum to immobilize heparin-binding growth factors and to enhance cellular growth. Journal of Biomedical Materials Research 56, 536-544.
[60] Guan, J., Stankus, J. J., and Wagner, W. R. (2007) Biodegradable elastomeric scaffolds with basic fibroblast growth factor release. Journal of Controlled Release 120, 70-78.
[61] Son, J. S., Park, K., Oh, S., Kim, J. J., and Han, D. K. (2008) Preparation and characterization of porous heparin-immobilized PLGA scaffolds using novel solid hydrogen peroxide for cartilage regeneration. Tissue Engineering and Regenerative Medicine 5, 528-534.
[62] Princz, M. A., and Sheardown, H. (2008) Heparin-modified dendrimer cross-linked collagen matrices for the delivery of basic fibroblast growth factor (FGF-2). Journal of Biomaterials Science-Polymer Edition 19, 1201-1218.
[63] Bae, K., Moon, C., Lee, Y., and Park, T. (2009) Intracellular Delivery of Heparin Complexed with Chitosan-g-Poly(Ethylene Glycol) for Inducing Apoptosis. Pharmaceutical Research 26, 93-100.
[64] Hurst, R. E., and Bonner, R. B. (2001) Mapping of the distribution of significant proteins and proteoglycans in small intestinal submucosa by fluorescence microscopy. Journal of Biomaterials Science-Polymer Edition 12, 1267-1279.
[65] Fievet, P., Sbai, M., Szymczyk, A., and Vidonne, A. (2003) Determining the zeta-potential of plane membranes from tangential streaming potential measurements: effect of the membrane body conductance. Journal of Membrane Science 226, 227-236.
[66] Mockel, D., Staude, E., Dal-Cin, M., Darcovich, K., and Guiver, M. (1998) Tangential flow streaming potential measurements: Hydrodynamic cell characterization and zeta potentials of carboxylated polysulfone membranes. Journal of Membrane Science 145, 211-222.
[67] Tang, S. C., Sambanis, A., and Sibley, E. (2005) Proteasome modulating agents induce rAAV2-mediated transgene expression in human intestinal epithelial cells. Biochemical and Biophysical Research Communications 331, 1392-1400.
[68] Adams, M. E., and Muir, H. (1981) The Glycosaminoglycans of Canine Menisci. Biochemical Journal 197, 385-389.
[69] Lv, H. T., Zhang, S. B., Wang, B., Cui, S. H., and Yan, J. (2006) Toxicity of cationic lipids and cationic polymers in gene delivery. Journal of Controlled Release 114, 100-109.
[70] Lim, Y. B., Choi, Y. H., and Park, J. S. (1999) A self-destroying polycationic polymer: Biodegradable poly(4-hydroxy-L-proline ester). Journal of the American Chemical Society 121, 5633-5639.
[71] Lim, Y. B., Kim, C. H., Kim, K., Kim, S. W., and Park, J. S. (2000) Development of a safe gene delivery system using biodegradable polymer, poly[alpha-(4-aminobutyl)-L-glycolic acid]. Journal of the American Chemical Society 122, 6524-6525.
[72] Yang, T. F., Chin, W., Cherng, J., and Shau, M. (2004) Synthesis of novel biodegradable cationic polymer: N,N-diethylethylenediamine polyurethane as a gene carrier. Biomacromolecules 5, 1926-1932.
[73] Behr, J. P. (1997) The proton sponge: A trick to enter cells the viruses did not exploit. Chimia 51, 34-36.
[74] Belguise-Valladier, P., and Behr, J. P. (2001) Nonviral gene delivery: Towards artificial viruses. Cytotechnology 35, 197-201.
[75] Forrest, M. L., Meister, G. E., Koerber, J. T., and Pack, D. W. (2004) Partial acetylation of polyethylenimine enhances in vitro gene delivery. Pharmaceutical Research 21, 365-371.
[76] Sonawane, N. D., Szoka, F. C., and Verkman, A. S. (2003) Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes. Journal of Biological Chemistry 278, 44826-44831.
[77] Lynn, D. M., and Langer, R. (2000) Degradable poly(beta-amino esters): Synthesis, characterization, and self-assembly with plasmid DNA. Journal of the American Chemical Society 122, 10761-10768.
[78] Akinc, A., Anderson, D. G., Lynn, D. M., and Langer, R. (2003) Synthesis of poly(beta-amino ester)s optimized for highly effective gene delivery. Bioconjugate Chemistry 14, 979-988.
[79] Jon, S., Anderson, D. G., and Langer, R. (2003) Degradable poly(amino alcohol esters) as potential DNA vectors with low cytotoxicity. Biomacromolecules 4, 1759-1762.
[80] Kim, T. I., Seo, H. J., Choi, J. S., Yoon, J. K., Baek, J. U., Kim, K., and Park, J. S. (2005) Synthesis of biodegradable cross-linked poly(beta-amino ester) for gene delivery and its modification, inducing enhanced transfection efficiency and stepwise degradation. Bioconjugate Chemistry 16, 1140-1148.
[81] Wu, D. C., Liu, Y., Jiang, X., Chen, L., He, C. B., Goh, S. H., and Leong, K. W. (2005) Evaluation of hyperbranched poly(amino ester)s of amine constitutions similar to polyethylenimine for DNA delivery. Biomacromolecules 6, 3166-3173.
[82] Tseng, S. J., and Tang, S. C. (2007) Synthesis and characterization of the novel transfection reagent poly(amino ester glycol urethane). Biomacromolecules 8, 50-58.
[83] Ignowski, J. M., and Schaffer, D. V. (2004) Kinetic analysis and modeling of firefly luciferase as a quantitative reporter gene in live mammalian cells. Biotechnology and Bioengineering 86, 827-834.
[84] Boussif, O., Lezoualch, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J. P. (1995) A Versatile Vector for Gene and Oligonucleotide Transfer into Cells in Culture and in-Vivo - Polyethylenimine. Proceedings of the National Academy of Sciences of the United States of America 92, 7297-7301.
[85] Pun, S. H., Bellocq, N. C., Liu, A. J., Jensen, G., Machemer, T., Quijano, E., Schluep, T., Wen, S. F., Engler, H., Heidel, J., and Davis, M. E. (2004) Cyclodextrin-modified polyethylenimine polymers for gene delivery. Bioconjugate Chemistry 15, 831-840.
[86] Grzelinski, M., Urban-Klein, B., Martens, T., Lamszus, K., Bakowsky, U., Hobel, S., Czubayko, F., and Aigner, A. (2006) RNA interference-mediated gene silencing of pleiotrophin through polyethylenimine-complexed small interfering RNAs in vivo exerts antitumoral effects in glioblastoma xenografts. Human Gene Therapy 17, 751-766.
[87] Mao, S. R., Neu, M., Germershaus, O., Merkel, O., Sitterberg, J., Bakowsky, U., and Kissel, T. (2006) Influence of polyethylene glycol chain length on the physicochemical and biological properties of poly(ethylene imine)-graft-poly(ethylene glycol) block copolymer/SiRNA polyplexes. Bioconjugate Chemistry 17, 1209-1218.

全文公開日期本全文未授權公開 (校內網路)
全文公開日期本全文未授權公開 (校外網路)
全文公開日期本全文未授權公開 (國家圖書館：臺灣博碩士論文系統)

簡易檢索 / 詳目顯示

相關論文