本研究為探討使用不同數目羥基官能基之聚乙二醇，即 ɑ-methoxy-ω-hydroxy PEG(mPEG45-OH)與 α,ω-bis-hydroxy PEG(HO-PEG45-OH)合成之陽離子高分子對基因之複合效果比較，並作為基因載體運用於基因傳遞，此外本研究使用的基因類型為 pDNA 。本研究以具有雙鍵官能化之乳酸單體(allyl-functional lactide, ALAene)及無官能化之單體(lactide, LA)進行開環聚合，分別為單端鏈段之 mono-mPEG45-b-poly(ALAene0.55-co-LA0.45)44 及雙端鏈段之 di-PEG45-b-poly(ALAene0.54-co-LA0.46)41 。將成功聚合之高分子與2-diethylaminoethanethiol hydrochloride 進行 thiol-ene 點擊化學來賦予兩種高分子正電荷之胺基團，而胺基團之接枝率分別以半取代及全取代作為目標，分別為mono-mPEG45-b-poly(Cationic LA0.28-co-ALAene0.27-co-LA0.45)44 、 mono-mPEG45-b-poly(Cationic LA0.55-co-LA0.45)44 、 di-PEG45-b-poly(Cationic LA0.30-co-ALAene0.24-co-LA0.46)41 及 di-PEG45-b-poly(Cationic LA0.54-co-LA0.46)41 。最終透過核磁共振儀(nuclear magnetic resonance, NMR)及膠體滲透層析儀(gel permeation chromatography, GPC)來確認高分子之結構與分子量。為測試四種高分子對基因之複合性，本研究使用之基因為質體並透過凝膠電泳進行比較。粒徑測試、界面電位分析及穿透式電子顯微鏡(transmission electron microscope, TEM)了解陽離子高分子及基因對粒徑及淨電荷的影響。穩定性測試及降解測試可以觀察在不同環境下複合物的狀態，其中陽離子高分子在不同環境會有不同的降解機制。陽離子聚合物之正電荷會吸附帶負電之蛋白質，因此蛋白質吸附測試可以知道各材料對蛋白質吸附的差異性。細胞毒性測試及基因轉染是本研究中涉及應用之部分，前者透過細胞活性之結果來鑑定材料是否能在細胞中存活及投藥量的極限，後者可以確切得知陽離子高分子是否能作為基因傳遞之載體。最後基因釋放試驗是以轉染效果最好之 di-PEG45-b-poly(Cationic LA0.54-co-LA0.46)41 ，希望透過此實驗了解環境對基因釋放的影響。

In this study, we investigated the effect of cationic polymers synthesized by using polyethylene glycols with different numbers of hydroxyl functional groups, i.e. ɑ-methoxy-ω-hydroxy PEG (mPEG45-OH) and α,ω-bis-hydroxy PEG (HO-PEG45-OH), on gene complexation and their use as gene carriers for gene delivery. In addition, the gene type used in this study is pDNA. The mono-mPEG45-b-poly (ALAene0.55-co-LA0.45)44 and the di-PEG45-b-poly (ALAene0.54-co-LA0.46)41 were obtained by the ring-opening polymerization of lactic acid monomers with double bond functionalization and monomers without functionalization The successful polymerization of the polymer with 2-diethylaminoethanethiol hydrochloride was performed by thiol-ene click chemistry to give the two polymers positively charged amine groups, and the grafting rate of the amine groups was targeted to be semi-substituted and fully substituted, respectively, as mono-mPEG45-b-poly(Cationic LA0.28-co-ALAene0.27-co-LA0.45)44, mono-mPEG45-b-poly(Cationic LA0.55-co-LA0.45)44, di-PEG45-b-poly(Cationic LA0.30-co-ALAene0.24-co-LA0.46)41 and di-PEG45-b-poly(Cationic LA0.30-co-ALAene0.24-co-LA0.46)42. di-PEG45-b-poly(Cationic LA0.54-co-LA0.46)41. Finally, the structures and molecular weights of the polymers were confirmed by magnetic resonance spectroscopy (NMR) and gel permeation chromatography (GPC). In order to test the complexity of the four polymers to the genes, the genes were used as plasmids and compared by gel electrophoresis. Particle size tests, zeta potential analysis and transmission electron microscope (TEM) were performed to understand the effect of cationic polymers and genes on particle size and net charge, and even to understand whether this property persists in the organism for a long time and helps gene delivery. Stability and degradation tests can observe the state of the complexes under different environments, where cationic polymers have different degradation mechanisms in different environments. Cellular toxicity and gene transfection are the applications of this study. The former is to determine the viability of the material in cells and the limit of drug delivery through the results of cell activity, and the latter is to determine whether the cationic polymer can be used as a carrier for gene delivery. The final gene release assay was performed on di-PEG45-b-poly(Cationic LA0.54-co-LA0.46)41, which had the best transfection effect, in order to understand the effect of environment on gene release.

論文審定書………………………………………………………………………i
摘要……………………………………………………………………………...ii
Abstract…………………………………………………………………………iii
目錄……………………………………………………………………………...v
圖次……………………………………………………………………………viii
表次……………………………………………………………………………...x
第一章 緒論…………………………………………………………………….1
1.1 研究背景……………………………….…………………………………...1
1.2 研究動機與目的…………………………….……………………………...2
第二章 文獻回顧………………………………………………………………3
2.1 基因治療…………………………….……………………………………...3
2.1.1 病毒基因載體……………….…………………………………………4
2.1.1.1 線病毒(adeno virus, AV) ……………….………………………4
2.1.1.2 逆轉錄病毒(retrolviral, RV) ……………….…………………..4
2.1.1.3 慢病毒(lentiv virus, LV) ……………….………………………4
2.1.2 非病毒基因載體……………….………………………………………5
2.1.2.1 脂質……………….…………………………………………….5
2.1.2.2 聚合物……………….………………………………………….6
2.2 生物可降解性高分子……………….……………………………………...9
2.2.1 聚乳酸……………….………………………………………………..10
2.3 聚乙二醇……………….………………………………………………….11
第三章 實驗方法……………….……………………………………………..12
3.1 實驗藥品與材料……………….………………………………………….12
3.2 實驗原理……………….………………………………………………….14
3.2.1 開環聚合……………….……………………………………………..14
3.2.2 硫醇-烯點擊化學…….……………………………………………….15
3.2.3 電泳…….……………………………………………………………..16
3.2.4 定量反轉錄 PCR (reverse transcription-quantitive real-time polymerase chain reaction, RT-qPCR) …….………………………………...17
3.3 實驗流程….……………………………………………………………….20
3.3.1 雙鍵官能化乳酸單體合成……………………………………...……20
3.3.2 mono-mPEG45-b-poly(ALAene0.55-co-LA0.45)44共聚物合成...…...21
3.3.3 di-PEG45-b-poly(ALAene0.54-co-LA0.46)41共聚物合成...…………21
3.3.4 mono-mPEG45-b-poly(ALAene0.55-co-LA0.45)44 之正電荷修飾....22
3.3.5 di-PEG45-b-poly(ALAene0.54-co-LA0.46)41 之正電荷修飾...………..22
3.3.6 凝膠電泳測試………………………………………………………...23
3.3.7 粒徑與界面電位分析………………………………………………...23
3.3.8 TEM 分析……………………………………………………………..24
3.3.9 穩定性測試…………………………………………………………...24
3.3.10 降解測試…………………………………………………………….24
3.3.11 蛋白質吸附測試…………………………………………………….24
3.3.12 細胞毒性測試……………………………………………………….24
3.3.13 基因轉染…………………………………………………………….25
3.3.14 基因釋放測試……………………………………………………….25
3.4 實驗測試儀器………………………………………………………………..25
3.4.1 核磁共振儀(NMR) …………………………………………………..25
3.4.2 膠體滲透層析儀(GPC) ………………………………………………25
3.4.3 膠體影像分析儀……………………………………………………...26
3.4.4 微孔板分光光度計…………………………………………………...26
3.4.5 動態光散射粒徑分析儀(DLS) ………………………………………26
3.4.6 穿透式電子顯微鏡(TEM) …………………………………………...26
第四章 結果與討論…………………………………………………………...27
4.1 核磁共振之結構分析……………………………………………………..27
4.2 膠體滲透層析分析………………………………………………………..35
4.3 凝膠電泳測試……………………………………………………………..37
4.4 聚合物/基因複合物之粒徑、界面電位及 TEM 分析………………….38
4.5 穩定性及降解性測試……………………………………………………..40
4.6 蛋白質吸附測試…………………………………………………………..45
4.7 細胞毒性測試……………………………………………………………..46
4.8 基因轉染…………………………………………………………………..49
4.9 基因釋放測試……………………………………………………………..51
第五章 結論…………………………………………………………………...53
第六章 參考資料……………………………………………………………...54

[1] Fang, E.; Liu, X.; Li, M.; Zhang, Z.; Song, L.; Zhu, B.; Wu, X.; Liu, J.; Zhao, D.; Li, Y., Advances in COVID-19 mRNA vaccine development. Signal Transduction and Targeted Therapy 2022, 7 (1), 1-31.
[2] Wirth, T.; Parker, N.; Ylä-Herttuala, S., History of gene therapy. Gene 2013, 525 (2), 162-169.
[3] Knop, K.; Hoogenboom, R.; Fischer, D.; Schubert, U. S., Poly (ethylene glycol) in drug delivery: pros and cons as well as potential alternatives. Angewandte Chemie International Edition 2010, 49 (36), 6288-6308.
[4] Wang, J.; Li, B.; Qiu, L.; Qiao, X.; Yang, H., Dendrimer-based drug delivery systems: history, challenges, and latest developments. Journal of Biological Engineering 2022, 16 (1), 1-12.
[5] Nayerossadat, N.; Maedeh, T.; Ali, P. A., Viral and nonviral delivery systems for gene delivery. Advanced Biomedical Research 2012, 1 (2), 1-27.
[6] Giovannelli, I.; Higginbottom, A.; Kirby, J.; Azzouz, M.; Shaw, P. J., Prospects for gene replacement therapies in amyotrophic lateral sclerosis. Nature Reviews Neurology 2023, 19 (1), 39-52.
[7] Robbins, P. D.; Ghivizzani, S. C., Viral vectors for gene therapy. Pharmacology & Therapeutics 1998, 80 (1), 35-47.
[8] McTaggart, S.; Al-Rubeai, M., Retroviral vectors for human gene delivery. Biotechnology Advances 2002, 20 (1), 1-31.
[9] Lundstrom, K., Latest development in viral vectors for gene therapy. Trends in Biotechnology 2003, 21 (3), 117-122.
[10] de Ilarduya, C. T.; Sun, Y.; Düzgüneş, N., Gene delivery by lipoplexes and polyplexes. European Journal of Pharmaceutical Sciences 2010, 40 (3), 159-170.
[11] Mintzer, M. A.; Simanek, E. E., Nonviral vectors for gene delivery. Chemical Reviews 2009, 109 (2), 259-302.
[12] Park, T. G.; Jeong, J. H.; Kim, S. W., Current status of polymeric gene delivery systems. Advanced Drug Delivery Reviews 2006, 58 (4), 467-486.
[13] Cojocaru, E.; Ghitman, J.; Stan, R., Electrospun-fibrous-architecture-mediated non-viral gene therapy drug delivery in regenerative medicine. Polymers 2022, 14 (13), 2647.
[14] Chen, C. K.; Huang, P. K.; Law, W. C.; Chu, C. H.; Chen, N. T.; Lo, L. W., Biodegradable polymers for gene-delivery applications. International Journal of Nanomedicine 2020, 15, 2131-2150.
[15] Aied, A.; Greiser, U.; Pandit, A.; Wang, W., Polymer gene delivery: overcoming the obstacles. Drug Discovery Today 2013, 18 (21), 1090-1098.
[16] Doppalapudi, S.; Jain, A.; Khan, W.; Domb, A. J., Biodegradable polymers—an overview. Polymers for Advanced Technologies 2014, 25 (5), 427-435.
[17] Nair, L. S.; Laurencin, C. T., Biodegradable polymers as biomaterials. Progress in Polymer Science 2007, 32 (8), 762-798.
[18] Jurak, M.; Wiącek, A. E.; Ładniak, A.; Przykaza, K.; Szafran, K., What affects the biocompatibility of polymers? Advances in Colloid and Interface Science 2021, 294, 102451.
[19] Da Silva, D.; Kaduri, M.; Poley, M.; Adir, O.; Krinsky, N.; Shainsky-Roitman, J.; Schroeder, A., Biocompatibility, biodegradation and excretion of polylactic acid (PLA) in medical implants and theranostic systems. Chemical Engineering Journal 2018, 340, 9-14.
[20] El Habnouni, S.; Darcos, V.; Garric, X.; Lavigne, J. P.; Nottelet, B.; Coudane, J., Mild methodology for the versatile chemical modification of polylactide surfaces: original combination of anionic and click chemistry for biomedical applications. Advanced Functional Materials 2011, 21 (17), 3321-3330.
[21] Ahmad, A.; Banat, F.; Alsafar, H.; Hasan, S. W., An overview of biodegradable poly (lactic acid) production from fermentative lactic acid for biomedical and bioplastic applications. Biomass Conversion and Biorefinery 2022, 1-20.
[22] Nuyken, O.; Pask, S. D., Ring-opening polymerization—an introductory review. Polymers 2013, 5 (2), 361-403.
[23] Duda, A.; Kowalski, A., Thermodynamics and kinetics of ring‐opening polymerization. Handbook of Ring‐Opening Polymerization 2009, 1-51.
[24] Thomas, C. M., Stereocontrolled ring-opening polymerization of cyclic esters: synthesis of new polyester microstructures. Chemical Society Reviews 2010, 39 (1), 165-173.
[25] Lecomte, P.; Jérôme, C., Recent developments in ring-opening polymerization of lactones. Synthetic Biodegradable Polymers 2012, 245, 173-217.
[26] Kamber, N. E.; Jeong, W.; Waymouth, R. M.; Pratt, R. C.; Lohmeijer, B. G. G.; Hedrick, J. L., Organocatalytic ring-opening polymerization. Chemical Reviews 2007, 107 (12), 5813-5840.
[27] Türünç, O.; Meier, M. A. R., The thiol-ene (click) reaction for the synthesis of plant oil derived polymers. European Journal of Lipid Science and Technology 2013, 115 (1), 41-54.
[28] Lowe, A. B., Thiol-ene “click” reactions and recent applications in polymer and materials synthesis. Polymer Chemistry 2010, 1 (1), 17-36.
[29] Brody, J. R.; Kern, S. E., History and principles of conductive media for standard DNA electrophoresis. Analytical Biochemistry 2004, 333 (1), 1-13.
[30] Gebrekidan, S.; Woo, B. H.; DeLuca, P. P., Formulation and in vitro transfection efficiency of poly (D, L-lactideco-glycolide) microspheres containing plasmid DNA for gene delivery. AAPS Pharmscitech 2000, 1 (4), 28.
[31] Garibyan, L.; Avashia, N., Polymerase chain reaction. Journal of Investigative Dermatology 2013, 133 (3), 1-4.
[32] Smith, C. J.; Osborn, A. M., Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiology Ecology 2009, 67 (1), 6-20.
[33] Zhang, X.; Lin, Z. I.; Yang, J.; Liu, G. L.; Hu, Z.; Huang, H.; Li, X.; Liu, Q.; Ma, M.; Xu, Z., Carbon dioxide-derived biodegradable and cationic polycarbonates as a new siRNA carrier for gene therapy in pancreatic cancer. Nanomaterials 2021, 11 (9), 2312.
[34] Petros, R. A.; DeSimone, J. M., Strategies in the design of nanoparticles for therapeutic applications. Nature Reviews Drug Discovery 2010, 9 (8), 615-627.
[35] Rice-Ficht, A. C.; Arenas-Gamboa, A. M.; Kahl-McDonagh, M. M.; Ficht, T. A., Polymeric particles in vaccine delivery. Current Opinion in Microbiology 2010, 13 (1), 106-112.
[36] Ariful Islam, M.; Park, T. E.; Firdous, J.; Li, H. S.; Jimenez, Z.; Lim, M.; Choi, J. W.; Yun, C. H.; Cho, C. S., Essential cues of engineered polymeric materials regulating gene transfer pathways. Progress in Materials Science 2022, 128, 100961.
[37] Bannunah, A. M.; Vllasaliu, D.; Lord, J.; Stolnik, S., Mechanisms of nanoparticle Internalization and transport across an intestinal epithelial cell model: effect of size and surface charge. Molecular Pharmaceutics 2014, 11 (12), 4363-4373.
[38] Mazumdar, S.; Chitkara, D.; Mittal, A., Exploration and insights into the cellular internalization and intracellular fate of amphiphilic polymeric nanocarriers. Acta Pharmaceutica Sinica B 2021, 11 (4), 903-924.
[39] Srijampa, S.; Buddhisa, S.; Ngernpimai, S.; Leelayuwat, C.; Proungvitaya, S.; Chompoosor, A.; Tippayawat, P., Influence of gold nanoparticles with different surface charges on localization and monocyte behavior. Bioconjugate Chemistry 2020, 31 (4), 1133-1143.
[40] Chen, C.-K.; Jones, C. H.; Mistriotis, P.; Yu, Y.; Ma, X.; Ravikrishnan, A.; Jiang, M.; Andreadis, S. T.; Pfeifer, B. A.; Cheng, C., Poly(ethylene glycol)-block-cationic polylactide nanocomplexes of differing charge density for gene delivery. Biomaterials 2013, 34 (37), 9688-9699.
[41] Fife, T. H.; Singh, R.; Bembi, R., Intramolecular General Base Catalyzed Ester Hydrolysis. The Hydrolysis of 2-Aminobenzoate Esters. The Journal of Organic Chemistry 2002, 67 (10), 3179-3183.
[42] Jones, C. H.; Chen, C. K.; Jiang, M.; Fang, L.; Cheng, C.; Pfeifer, B. A., Synthesis of cationic polylactides with tunable charge densities as nanocarriers for effective gene delivery. Molecular Pharmaceutics 2013, 10 (3), 1138-1145.