接枝聚乙二醇甲基丙烯酸酯及四級胺化之聚乙烯亞胺對於基因轉殖效率之影響

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：18

、訪客IP：18.217.212.222

姓名

黃于齊(Yu-Chi Huang) 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

接枝聚乙二醇甲基丙烯酸酯及四級胺化之聚乙烯亞胺對於基因轉殖效率之影響
(Effects of PEGylated and Quaternized Polyethylenimines on Gene Transfection Efficiency)

相關論文

★ 類澱粉胜肽聚集行為之電腦模擬	★ 溶解度參數計算及量測於HPLC純化胜肽程序之最佳化研究
★ 利用恆溫滴定微卡計量測蛋白質分子於溶液中之第二維里係數與自我聚集之行為	★ 利用SPRi探討中性DNA探針相較於一般DNA探針在低鹽雜交環境下之優勢
★ 矽奈米線場效電晶體多點之核酸檢測研究	★ 使用不帶電中性核酸探針於矽奈米線場效電晶體檢測去氧核醣核酸與微核醣核酸之研究
★ 運用nDNA 修飾引子於PCR及qPCR平台以提升專一性之研究	★ 設計中性DNA引子及探針以提升PCR與qPCR專一性之研究
★ 使用中性不帶電去氧核醣核酸探針於矽奈米線場效電晶體檢測微核醣核酸之研究	★ 使用不帶電中性核酸探針於原位雜交技術檢測微核醣核酸之研究
★ 設計不帶電中性核酸探針於矽奈米線場效電晶體來改善富含GC鹼基核醣核酸之檢測專一性	★ 合成5’-MeNPOC-2’-deoxynucleoside p-methoxy phosphoramidite以作為應用於原位合成之新穎性中性核苷酸之研究
★ 立體紙基外泌體核酸萃取裝置應用於檢測不同微環境下癌細胞所釋放之外泌體與外泌體微小核醣核酸之表現量	★ 利用抗原結合區段之抗體片段探針於矽奈米線場效電晶體來改善抗原檢測濃度極限之研究
★ 利用表面電漿共振影像儀驗證最適化之抗非專一性吸附場效電晶體表面於血清環境下之免疫測定	★ 使用混合自組裝單層膜於矽奈米線場效電晶體檢測微小核醣核酸之研究

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]

[檢視]

[下載]

本電子論文使用權限為同意立即開放。
已達開放權限電子全文僅授權使用者為學術研究之目的，進行個人非營利性質之檢索、閱讀、列印。
請遵守中華民國著作權法之相關規定，切勿任意重製、散佈、改作、轉貼、播送，以免觸法。

摘要(中)

聚乙烯亞胺(Polyethyleneimine，PEI)除了是一帶有高正電荷密度的高分子外，也因其具有不同質子化程度的胺基，因而能藉由高正電性有效的以胞飲作用傳送基因質體進入細胞，並協助基因質體逃脫核內體(Endosome)，而達到良好的轉染效率，因而堪稱新一代的非病毒型之基因載體。然而由於PEI之高正電密度易導致血栓或凝血作用的產生，反而限制了其在臨床的應用。為了降低PEI的血栓或凝血作用，本研究將高生物相容性的聚乙二醇甲基丙烯酸酯(Poly(ethylene glycol) methyl ether methacrylate，PEGMA)以Michael addition的方式接枝於PEI上，並調控PEGMA在PEI上的接枝數目(PEGMA/PEI) 比為2, 4, 6, 10, 13, 18。首先我們發現PEGMA/PEI小於10時，PEI-g-PEGMA與質體DNA的複合物具有高結合穩定性且其表面電荷仍為正電。此外若PEGMA/PEI大於4以上則具有低溶血性，這意味著PEGMA/PEI之接枝數目比在4到10時之間為基因轉染之最佳比例。另一方面，我們藉由調控PEGMA/PEI與質體DNA複合物的重量百分比(2, 5, 10, 40 wt%)來找尋最佳化基因轉染的條件，結果發現若PEGMA在PEI上的接枝數目比增加時，由於PEGMA的保護作用，會使得轉染效率的區間會趨往高複合物的重量百分比，但轉染效率並未隨之增加。由我們的實驗結果發現最佳轉染效率是在PEGMA/PEI之接枝數目比在4時，且PEGMA/PEI與質體DNA複合物的重量比在2時，這個條件下的轉染效率比以PEI轉染更高且細胞毒性也低於PEI。
本研究進一步將PEI做不同程度之四級胺化，其四級胺化程度為19.8%、21.8%及26.8%，並期望能藉由調整PEI上之不同質子化胺基程度及N/P比，來降低PEI對細胞之毒性和最佳之轉染濃度。我們藉由調控四級胺化之PEI(QPEI)與質體DNA複合物的電荷比(N/P=5, 10, 30, 35, 40, 70)來找尋最佳化之基因轉染條件。由膠體電泳實驗發現QPEI之包覆能力並未比PEI強，在動態雷射光散射粒徑儀(Dynamic Light Scattering, DLS) 和滴定實驗，發現四級胺化後，由於四級胺之質子化能力降低，導致緩衝能力降低。粒徑大小也由於正電性較強，導致粒徑較PEI大。但在轉染實驗及細胞毒性之檢測，可發現QPEI並沒有什麼細胞毒性在N/P比70以下且四級胺化程度為26.8%在N/P比為40之螢光圖下發現其轉染與PEI相當。

摘要(英)

Polyethyleneimine(PEI), a polymer with highly positive charged density at physiological condition, has been proposed to be an effectively gene delivery carrier for the translocation of plasmid DNA in cell via endocytosis pathway. According to the proton sponge hypothesis, PEI could facilitate the gene to escape from early endosome resulting in high transfection efficiency. However, the higher cytotoxicity and lower blood compatibility of PEI limit its clinical applications; in particular, PEI would cause the blood coagulation and thrombus. Therefore, we conjugated the biocompatible polymer, poly(ethylene glycol) methyl ether methacrylate(PEGMA), with branched PEI (25 KDa) to reduce the mentioned drawbacks of PEI through the Michael addition method. The PEGMA grafting densities were 2, 4, 6, 10, 13, and 18. The measurements of Zeta potential and gel retardation assay revealed that the stable PEI-PEGMA-DNA complexes (weight ratio above 2) with positive charge are formed as the grafting density less than 10, but the stability of complexes decrease as the grafting density up to 13, even 18. Besides, it exhibited low hemolysis if PEGMA/PEI was more than 4. Otherwise, we found that the PEI-PEGMA-DNA complexes exhibit lower toxicity than PEI-DNA complexes, but the PEI-PEGMA-DNA complexes still have cytotoxicity as the weight ratio of PEI-PEGMA-DNA complexes increase. Furthermore, the transfection experiments of EGFP expression showed that effective transfection locates within the different intervals of the weight ratio of PEI-PEGMA-DNA complexes for different grafting ratio of PEGMA in PEI, and the minimum weight ratio of PEI-PEGMA-DNA complexes for transfection would increase with the increase of grafting ratio of PEGMA in PEI. In this study, we have proposed that the optimal transfection efficiency is under the condition of the grafting ratio of PEGMA/PEI being 4 and the weight ratio of PEI-PEGMA-DNA complexes is 2. Consequently, we developed a polymer-based vector with lower cytotoxicity for gene transfection, and the optimal operation windows are also presented.
Furthermore, we investigated the different degrees of quaternization in PEI whose degrees are 19.8%, 21.8%, and 26.8%, respectively. By tuning the composition of different protonated amines and N/P ratio, we intended to decrease the cytotoxicity of PEI and obtain the optimized N/P ratio for gene transfection. On the other hand, we regulated the charge ratio of quaternized PEI (QPEI) over plasmid DNA ( N/P=5, 10, 30, 35, 40, 70) to achieve the optimized gene transfection. The result of gel retardation showed that the stability of QPEI is less than PEI. From the dynamic light scattering (DLS) measurement, we found that the particle diameter of QPEI-DNA complex is larger than that of PEI due to its stronger positive charge. Besides, the buffering effect in QPEI-DNA complex titration decreases owing to the decrease in the protonation of quaternary amine. Nevertheless, QPEI exhibits little cytotoxicity at the N/P ratio less than 70. Under the N/P ratio equaling 40 and 26.8% of the quaternization degree, the transfection efficiency of QPEI is equivalent to PEI from gene transfection and cytotoxicity measurement.

關鍵字(中)

★ 聚乙烯胺
★ 聚乙二醇甲基丙烯酸酯
★ 基因傳送

關鍵字(英)

★ PEI
★ PEGMA
★ Gene Delivery

論文目次

中文摘要..........................................I
Abstract.......................................III
致謝.............................................V
目錄圖...........................................IX
表目錄...........................................XI
第一章緒論.........................................1
第二章文獻回顧....................................4
2.1 基因治療介紹...................................4
2.1.1基因治療之進展.................................4
2.2 基因治療之方法..................................5
2.2.1 病毒型載體法(Viral vector)....................5
2.2.1.1 轉錄病毒(Retrovirus).......................6
2.2.1.2 腺病毒(Adenovirus).........................7
2.2.1.3 相關病毒(Adeno-Associated Virus)............7
2.2.2 病毒型載體法(non-viral vector).................8
2.2.2.1 基因傳送的機制與障礙..........................8
2.2.2.2 電擊方式轉染................................10
2.2.2.3 顯微注射法..................................11
2.2.2.4 粒子轟擊-基因槍法............................11
2.2.2.5 磷酸鈣轉染法................................12
2.2.2.6 脂質載體法..................................14
2.2.2.7 聚陽離子轉染法-Poly(ethylene imine)之相關研究..17
第三章實驗藥品、設備及實驗方法.........................27
3.1 實驗藥品........................................27
3.1.1 質體DNA......................................27
3.1.2 細胞.........................................28
3.1.3 藥品.........................................28
3.2 實驗設備........................................31
3.3實驗方法.........................................32
3.3.1 PEI-g-PEGMA之合成.............................33
3.3.2 PEI四級胺化...................................33
3.3.3 溴化乙錠嵌入檢測法..............................33
3.3.4 膠體電泳檢測 ...................................34
3.3.5 基因載體之粒徑大小及表面電性量測...................34
3.3.6 基因載體之溶血實驗...............................35
3.3.7 將質體DNA轉入E.coli中...........................35
3.3.8 酸鹼滴定實驗 ....................................36
3.3.9 質體DNA純化 ....................................36
3.3.10 細胞培養......................................37
3.3.11 基因載體之轉染.................................39
3.3.12 細胞毒性測試...................................40
第四章 PEGylated PEI載體之基因轉殖效率..................42
4.1共聚高分子分子之合成與鑑定............................42
4.2 基因載體與Plasmid DNA的交互作用分析.................46
4.3 PEI-PEGMA基因載體對於DNA 之包覆能力分析..............48
4.4 載體之粒徑大小與表面電荷結果分析......................50
4.5 基因載體對於紅血球之溶血情形.........................52
4.6 基因載體對於細胞轉染之結果分析........................54
4.7 基因載體對於細胞活性之結果分析........................58
4.8結論..............................................61
第五章 Quaternized PEI載體之基因轉殖效率.................62
5.1高分子分子之改質與鑑定................................62
5.2 QPEI與Plasmid DNA的交互作用分析.....................63
5.3 QPEI基因載體對於DNA 之包覆能力分析....................65
5.4 QPEI/DNA複合物之粒徑大小結果分析.....................67
5.5 QPEI之緩衝能力分析.................................68
5.6 QPEI/DNA複合物對於紅血球之溶血情形...................69
5.7 QPEI/DNA複合物對於細胞轉染之結果分析..................70
5.8 QPEI/DNA複合物對於細胞活性之結果分析..................75
5.9 結論..............................................77
第六章總結與未來展望....................................78
參考資料 79

參考文獻

1.Biasco, L., C. Baricordi, and A. Aiuti, Retroviral Integrations in Gene Therapy Trials. Molecular Therapy, 2012. 20(4): p. 709-716.
2.Lam, J.K.W., et al., Phosphorylcholine-polycation diblock copolymers as synthetic vectors for gene delivery. Journal of Controlled Release, 2004. 100(2): p. 293-312.
3.Li, J., et al., Biodegradable calcium phosphate nanoparticle with lipid coating for systemic siRNA delivery. Journal of Controlled Release, 2010. 142(3): p. 416-421.
4.Oh, S., et al., Efficacy of nonviral gene transfer in the canine brain. Journal of Neurosurgery, 2007. 107(1): p. 136-144.
5.Zhou, J.Y., et al., Intracellular kinetics of non-viral gene delivery using polyethylenimine carriers. Pharmaceutical Research, 2007. 24(6): p. 1079-1087.
6.Petersen, H., et al., Polyethylenimine-graft-poly(ethylene glycol) copolymers: Influence of copolymer block structure on DNA complexation and biological activities as gene delivery system. Bioconjugate Chemistry, 2002. 13(4): p. 845-854.
7.Chollet, P., et al., Side-effects of a systemic injection of linear polyethylenimine-DNA complexes. Journal of Gene Medicine, 2002. 4(1): p. 84-91.
8.Dekie, L., et al., Poly-L-glutamic acid derivatives as vectors for gene therapy. Journal of Controlled Release, 2000. 65(1-2): p. 187-202.
9.Ferruti P, K.S., Ranucci E, Gianasi E, R D., A novel chemical modification of poly-l-lysine reducing toxicity while preserving cationic properties. Proc. Int. Symp. Controlled Release Bioact. Mater., 1997. 24: p. 45-46.
10.Fischer, D., et al., Copolymers of ethylene imine and N-(2-hydroxyethyl)-ethylene imine as tools to study effects of polymer structure on physicochemical and biological properties of DNA complexes. Bioconjugate Chemistry, 2002. 13(5): p. 1124-1133.
11.Tang, M.X. and F.C. Szoka, The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes. Gene Therapy, 1997. 4(8): p. 823-832.
12.Choosakoonkriang, S., et al., Biophysical characterization of PEI/DNA complexes. Journal of Pharmaceutical Sciences, 2003. 92(8): p. 1710-1722.
13.Bhise, N.S., et al., The relationship between terminal functionalization and molecular weight of a gene delivery polymer and transfection efficacy in mammary epithelial 2-D cultures and 3-D organotypic cultures. Biomaterials, 2010. 31(31): p. 8088-8096.
14.Akinc, A., et al., Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis. Journal of Gene Medicine, 2005. 7(5): p. 657-663.
15.Vroman, B., et al., Copolymers of epsilon-caprolactone and quaternized epsilon-caprolactone as gene carriers. J Control Release, 2007. 118(1): p. 136-44.
16.Thomas, M. and A.M. Klibanov, Enhancing polyethylenimine’s delivery of plasmid DNA into mammalian cells. Proc Natl Acad Sci U S A, 2002. 99(23): p. 14640-5.
17.Steven, A., et al.,, Gene transfer into humans-immunotherapy of patients with advenced melanoma using TIL modified by retroviral transduction. The New England Journal of Medicine, 1990. 323: p. 570-578.
18.Zeilfelder, U., et al., The potential of retroviral vectors to cotransfer human endogenous retroviruses (HERVs) from human packaging cell lines. Gene, 2007. 390(1-2): p. 175-179.
19.Deng, X.M., et al., 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, 2004. 311(3): p. 982-988.
20.Daya, S. and K.I. Berns, Gene Therapy Using Adeno-Associated Virus Vectors. Clinical Microbiology Reviews, 2008. 21(4): p. 583-593.
21.Walther, W. and U. Stein, Viral vectors for gene transfer - A review of their use in the treatment of human diseases. Drugs, 2000. 60(2): p. 249-271.
22.Enders, J.F., et al., Adenoviruses: group name proposed for new respiratory-tract viruses. Science, 1956. 124(3212): p. 119-20.
23.Dai, Y.F., et al., Cellular and Humoral Immune-Responses to Adenoviral Vectors Containing Factor-Ix Gene - Tolerization of Factor-Ix and Vector Antigens Allows for Long-Term Expression. Proc Natl Acad Sci U S A, 1995. 92(5): p. 1401-1405.
24.Mittereder, N., et al., Evaluation of the Efficacy and Safety of in-Vitro, Adenovirus-Mediated Transfer of the Human Cystic-Fibrosis Transmembrane Conductance Regulator Cdna. Hum Gene Ther, 1994. 5(6): p. 717-729.
25.Yang, Y.P., et al., Inactivation of E2a in Recombinant Adenoviruses Improves the Prospect for Gene-Therapy in Cystic-Fibrosis. Nature Genetics, 1994. 7(3): p. 362-369.
26.Buning, H., et al., Recent developments in adeno-associated virus vector technology. Journal of Gene Medicine, 2008. 10(7): p. 717-733.
27.Rolland, A.P., From genes to gene medicines: Recent advances in nonviral gene delivery. Critical Reviews in Therapeutic Drug Carrier Systems, 1998. 15(2): p. 143-198.
28.Luo, D. and W.M. Saltzman, Synthetic DNA delivery systems. Nature Biotechnology, 2000. 18(1): p. 33-37.
29.Coster, H.G., A quantitative analysis of the voltage-current relationships of fixed charge membranes and the associated property of "punch-through". Biophys J, 1965. 5(5): p. 669-86.
30.Sale, A.J. and W.A. Hamilton, Effects of high electric fields on micro-organisms. 3. Lysis of erythrocytes and protoplasts. Biochim Biophys Acta, 1968. 163(1): p. 37-43.
31.Sale, A.J.H. and W.A. Hamilton, Effects of high electric fields on microorganisms: I. Killing of bacteria and yeasts. Biochimica et Biophysica Acta (BBA) - General Subjects, 1967. 148(3): p. 781-788.
32.E.Neumann, M.S.-R., Y.Wang, and and P.H.Hofschneider, Gene transfer into mouse lyoma cells by electroporation in high electric fields. The EMBO Journal, 1982. 1: p. 841-845.
33.Fromm, M., L.P. Taylor, and V. Walbot, Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc Natl Acad Sci U S A, 1985. 82(17): p. 5824-8.
34.Potter, H., L. Weir, and P. Leder, Enhancer-dependent expression of human kappa immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc Natl Acad Sci U S A, 1984. 81(22): p. 7161-5.
35.Gao, X., K.S. Kim, and D.X. Liu, Nonviral gene delivery: What we know and what is next. Aaps Journal, 2007. 9(1): p. E92-E104.
36.Escoffre, J.M., et al., What is (Still not) Known of the Mechanism by Which Electroporation Mediates Gene Transfer and Expression in Cells and Tissues. Molecular Biotechnology, 2009. 41(3): p. 286-295.
37.Capecchi, M.R., High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell, 1980. 22(2 Pt 2): p. 479-88.
38.Zhang, Y. and L.C. Yu, Microinjection as a tool of mechanical delivery. Current Opinion in Biotechnology, 2008. 19(5): p. 506-510.
39.Klein, R.M., et al., High-velocity microprojectiles for delivering nucleic acids into living cells. 1987. Biotechnology, 1992. 24: p. 384-6.
40.Williams, R.S., et al., Introduction of Foreign Genes into Tissues of Living Mice by DNA-Coated Microprojectiles. Proc Natl Acad Sci U S A, 1991. 88(7): p. 2726-2730.
41.Yang, N.S., et al., In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment. Proc Natl Acad Sci U S A, 1990. 87(24): p. 9568-72.
42.Mahvi, D.M., et al., Phase I/IB study of immunization with autologous tumor cells transfected with the GM-CSF gene by particle-mediated transfer in patients with melanoma or sarcoma. Hum Gene Ther, 1997. 8(7): p. 875-888.
43.Sun, Y., et al., Vaccination with IL-12 gene-modified autologous melanoma cells: preclinical results and a first clinical phase I study. Gene Therapy, 1998. 5(4): p. 481-490.
44.Trimble, C., et al., Comparison of the CD8+ T cell responses and antitumor effects generated by DNA vaccine administered through gene gun, biojector, and syringe. Vaccine, 2003. 21(25-26): p. 4036-4042.
45.Tahtinen, M., et al., DNA vaccination in mice using HIV-1 nef, rev and tat genes in self-replicating pBN-vector. Vaccine, 2001. 19(15-16): p. 2039-2047.
46.Yoshida, A., et al., Protective CTL response is induced in the absence of CD4(+) T cells and IFN-gamma by gene gun DNA vaccination with a minigene encoding a CTL epitope of Listeria monocytogenes. Vaccine, 2001. 19(30): p. 4297-4306.
47.Jordan, M., A. Schallhorn, and F.M. Wurm, Transfecting mammalian cells: Optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Research, 1996. 24(4): p. 596-601.
48.Jordan, M. and F. Wurm, Transfection of adherent and suspended cells by calcium phosphate. Methods, 2004. 33(2): p. 136-143.
49.Lee, D., K. Upadhye, and P.N. Kumta, Nano-sized calcium phosphate (CaP) carriers for non-viral gene delivery. Materials Science and Engineering B-Advanced Functional Solid-State Materials, 2012. 177(3): p. 289-302.
50.Varga, C.M., T.J. Wickham, and D.A. Lauffenburger, Receptor-mediated targeting of gene delivery vectors: Insights from molecular mechanisms for improved vehicle design. Biotechnology and Bioengineering, 2000. 70(6): p. 593-605.
51.Rangavittal, N., et al., Structural study and stability of hydroxyapatite and beta-tricalcium phosphate: Two important bioceramics. Journal of Biomedical Materials Research, 2000. 51(4): p. 660-668.
52.Chen, Q.Z., et al., Strengthening mechanisms of bone bonding to crystalline hydroxyapatite in vivo. Biomaterials, 2004. 25(18): p. 4243-4254.
53.Poste, G. and D. Papahadjopoulos, Fusion of mammalian cells by lipid vesicles. Methods Cell Biol, 1976. 14: p. 23-32.
54.Felgner, P.L.G., T. R.; Holm, M.; Roman, R.; Chan, H. W.;Wenz, M.; Northrop, J. P.; Ringold, G. M.; Danielsen, M., Lipofection:a highly efficient,lipid-mediated DNA-transfection procedure. Proceedings of the National Academy of Sciences, 1987. 84: p. 7413-7417.
55.Gebeyehu, G.J., J. A.; Ciccarone, V. C.; Hawley-Nelson, P.;Chytil, A., Cationic lipids. U.S. Patent, 1994. 5: p. 334-761.
56.Stamatatos, L., et al., Interactions of cationic lipid vesicles with negatively charged phospholipid vesicles and biological membranes. Biochemistry, 1988. 27(11): p. 3917-25.
57.Behr, J.P., et al., Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA. Proc Natl Acad Sci U S A, 1989. 86(18): p. 6982-6.
58.Wang, C.Y. and L. Huang, Highly efficient DNA delivery mediated by pH-sensitive immunoliposomes. Biochemistry, 1989. 28(24): p. 9508-14.
59.Legendre, J.Y. and F.C. Szoka, Delivery of Plasmid DNA into Mammalian-Cell Lines Using Ph-Sensitive Liposomes - Comparison with Cationic Liposomes. Pharmaceutical Research, 1992. 9(10): p. 1235-1242.
60.Zhou, X.H. and L. Huang, DNA Transfection Mediated by Cationic Liposomes Containing Lipopolylysine - Characterization and Mechanism of Action. Biochimica Et Biophysica Acta-Biomembranes, 1994. 1189(2): p. 195-203.
61.Mintzer, M.A. and E.E. Simanek, Nonviral Vectors for Gene Delivery. Chemical Reviews, 2009. 109(2): p. 259-302.
62.Jones, G.D., et al., THE POLYMERIZATION OF ETHYLENIMINE. The Journal of Organic Chemistry, 1944. 09(2): p. 125-147.
63.Brissault, B., et al., Synthesis of linear polyethylenimine derivatives for DNA transfection. Bioconjugate Chemistry, 2003. 14(3): p. 581-587.
64.Ferrari, S., et al., ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo. Gene Therapy, 1997. 4(10): p. 1100-1106.
65.Baker, A., et al., Polyethylenimine (PEI) is a simple, inexpensive and effective reagent for condensing and linking plasmid DNA to adenovirus for gene delivery. Gene Therapy, 1997. 4(8): p. 773-782.
66.Fischer, D., et al., A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: Effect of molecular weight on transfection efficiency and cytotoxicity. Pharmaceutical Research, 1999. 16(8): p. 1273-1279.
67.Godbey, W.T., K.K. Wu, and A.G. Mikos, Size matters: molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle. Journal of Biomedical Materials Research, 1999. 45(3): p. 268-75.
68.Fischer, D., et al., In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis. Biomaterials, 2003. 24(7): p. 1121-1131.
69.Forrest, M.L., J.T. Koerber, and D.W. Pack, A degradable polyethylenimine derivative with low toxicity for highly efficient gene delivery. Bioconjugate Chemistry, 2003. 14(5): p. 934-940.
70.Peng, Q., Z. Zhong, and R. Zhuo, Disulfide cross-linked polyethylenimines (PEI) prepared via thiolation of low molecular weight PEI as highly efficient gene vectors. Bioconjug Chem, 2008. 19(2): p. 499-506.
71.Suh, J., H.J. Paik, and B.K. Hwang, Ionization of Poly(Ethylenimine) and Poly(Allylamine) at Various Phs. Bioorganic Chemistry, 1994. 22(3): p. 318-327.
72.Boussif, O., et al., A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci U S A, 1995. 92(16): p. 7297-301.
73.Behr, J.P., The proton sponge: A trick to enter cells the viruses did not exploit. Chimia, 1997. 51(1-2): p. 34-36.
74.Godbey, W.T., K.K. Wu, and A.G. Mikos, Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery. Proc Natl Acad Sci U S A, 1999. 96(9): p. 5177-81.
75.Lee, J.H., et al., Quaternized polyamidoamine dendrimers as novel gene delivery system: Relationship between degree of quaternization and their influences. Bulletin of the Korean Chemical Society, 2003. 24(11): p. 1637-1640.
76.Chiag, Y.C., et al., Biofouling Resistance of Ultrafiltration Membranes Controlled by Surface Self-Assembled Coating with PEGylated Copolymers. Langmuir, 2012. 28(2): p. 1399-1407.
77.Chang, Y., et al., A systematic SPR study of human plasma protein adsorption behavior on the controlled surface packing of self-assembled poly(ethylene oxide) triblock copolymer surfaces. Journal of Biomedical Materials Research Part A, 2010. 93A(1): p. 400-408.
78.Papahadjopoulos, D., et al., Sterically Stabilized Liposomes - Improvements in Pharmacokinetics and Antitumor Therapeutic Efficacy. Proc Natl Acad Sci U S A, 1991. 88(24): p. 11460-11464.
79.Ogris, M., et al., PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Therapy, 1999. 6(4): p. 595-605.
80.Kichler, A., Gene transfer with modified polyethylenimines. Journal of Gene Medicine, 2004. 6: p. S3-S10.
81.Sagara, K. and S.W. Kim, A new synthesis of galactose-poly(ethylene glycol)-polyethylenimine for gene delivery to hepatocytes. Journal of Controlled Release, 2002. 79(1-3): p. 271-281.
82.Blessing, T., et al., Different strategies for formation of PEGylated EGF-conjugated PEI/DNA complexes for targeted gene delivery. Bioconjugate Chemistry, 2001. 12(4): p. 529-537.
83.Kou, J.P., et al., Michael Addition of Amines to Activated Alkenes Promoted by Zn/NH4Cl System. Chemical Research in Chinese Universities, 2009. 25(4): p. 461-464.
84.Al-Dosari, M.S. and X. Gao, Nonviral Gene Delivery: Principle, Limitations, and Recent Progress. Aaps Journal, 2009. 11(4): p. 671-681.
85.Lim, Y.B., et al., The inhibition of prions through blocking prion conversion by permanently charged branched polyamines of low cytotoxicity. Biomaterials, 2010. 31(8): p. 2025-2033.
86.Geisse, S., M. Jordan, and F.M. Wurm, Large-scale transient expression of therapeutic proteins in mammalian cells. Methods Mol Biol, 2005. 308: p. 87-98.
87.Singer, V.L., et al., Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation. Analytical Biochemistry, 1997. 249(2): p. 228-238.
88.van de Wetering, P., et al., 2-(dimethylamino)ethyl methacrylate based (co)polymers as gene transfer agents. Journal of Controlled Release, 1998. 53(1-3): p. 145-153.
89.Scaiewicz, V., et al., Use of H19 Gene Regulatory Sequences in DNA-Based Therapy for Pancreatic Cancer. J Oncol, 2010. 2010: p. 178174.

指導教授

陳文逸(Wen-Yih Chen)

審核日期

2013-8-26

推文