研發新頴核酸載體平台於調控基因表現與抑制之應用

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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：2 下載：0
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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.

                                

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