非病毒基因載體依據作用原理，可分成陽離子型及非陽離子型。陽離子型載體主要藉由正電高分子修飾，經由靜電作用力攜帶陰電性基因，進行基因遞送，並具有良好的基因轉染效率，但正電性材料將對細胞產生毒性，並容易在血液中吸附血清蛋白導致聚集，從而降低傳遞效率。反之，非陽離子型載體主要藉由氫鍵、疏水作用力等非靜電作用力與基因形成奈米粒子，此類型材料具有低細胞毒性、高生物可降解性及生物相容性等優點，而本研究目標為發展一高效率與低毒性，並可同時遞送基因與蛋白藥物之非陽離子型傳遞載體。
研究中選用無機鹽磷酸鈣（Calcium phosphate，CaP）與聚乙二醇-聚乳酸-乙醇酸共聚物（Poly（ethylene glycol）-poly（lactic-co-glycolic acid），PEG-PLGA）製備奈米基因傳遞載體（CaP/pDNA/PLGA nanoparticles，CDP）。首先CaP將與DNA經親和力作用形成CaP/DNA複合物，接著加入陽離子脂質DOTAP以反向乳化法包覆CaP/DNA核心，提高DNA於內部核心的穩定性，最終藉由奈米沉澱法，將PEG-PLGA包覆於CaP/DNA外層，形成具良好分散性的CDP。在製程最適化探討中，我們評估不同製程參數，包括：CaP/DNA複合物之Ca2+/PO43-莫耳比、DNA/ Ca2+重量比，以及PEG-PLGA濃度，對於載體粒徑、表面電位以及基因包覆率之影響，並以體外細胞實驗評估其載體毒性以及基因轉染效率。由實驗結果顯示當Ca2+/PO43-莫耳比為100、DNA/ Ca2+重量比為10/3000，以及PEG-PLGA濃度為10 mg/mL，可獲得最佳粒徑、帶負電的表面電位及轉染效率。而與陽離子型基因載體disulfide-crosslinked polyethylenimine （SPEI）系統相比，本研究所開發之CDP具有低細胞毒性，且轉染效率亦不受血清蛋白所影響。
接著本研究進一步探討以CDP作為基因與蛋白質共傳遞的應用潛力。將DNA與蛋白質（本研究中選用αHuman-488及Gaot anti-Mouse IgG作為模式蛋白）混合後與CaP結合經由反向乳化法包入奈米核心中，並搭配奈米沉澱法，製作基因與蛋白質共傳遞載體（CaP/pDNA/protein/PLGA nanoparticles，CDPP）。在細胞攝取實驗中，經流式細胞儀定量，小鼠乳腺癌細胞（4T1）對於CDPP的蛋白質與基因攝取效率分別為97 %與10 %。而基因轉染效率結果亦顯示，CDPP相較於CDP具有高於10倍之基因轉染效率，然詳細的作用機制仍待探討。綜合以上結果，本研究所開發之CDPP在血清環境下，具有高穩定性、分散性、低細胞毒性，並可高效率地將蛋白質與基因遞送至細胞等特性，對於體內共遞送基因與蛋白藥物之應用深具潛力，未來值得以腫瘤動物模型，評估其腫瘤治療甚至體內基因編輯之可行性。

Non-viral gene vectors are classified into cationic and non-cationic types based on the principle of action. Cationic vectors are mainly modified by positively charged polymers, carry negative electricity genes through electrostatic forces for gene delivery. It has good gene transfection efficiency, but positively charged materials will be toxic to the cells, and it is easy to adsorb serum proteins in the blood to cause aggregation, thereby reducing the transfection efficiency. On the contrary, non-cationic vectors mainly form nanoparticles with genes through non-static forces such as hydrogen bonding and hydrophobic forces. This type of material has the advantages of low cytotoxicity, high biodegradability and biocompatibility. The goal in our research is to develop a non-cationic delivery vectoes with high efficiency and low toxicity, which can simultaneously deliver gene and protein drugs.
In this study, our gene delivery carrier （CaP/pDNA/PLGA nanoparticles, CDP） was prepared by using calcium phosphate （CaP） and poly （ethylene glycol） -poly （lactic-co-glycolic acid） （PEG-PLGA）. First, the CaP will interact with DNA to form a CaP / DNA complex. Second, added the cationic lipid DOTAP to coat the CaP / DNA core with the inverse emulsification method to improve the stability of the DNA to the internal core. Finally, the biodegradable polymer PEG-PLGA is coated on the outer layer of the vector by nanoprecipitation to form a gene carrier that has good dispersibility. In the process optimization discussion, we evaluated different process parameters, including: Ca2+/PO43- molar ratio, DNA/Ca2+ weight ratio of CaP / DNA complex, and PEG-PLGA concentration of CDP. We measured the effect of vector size, surface potential and gene encapsulation efficiency when we use different experimental parameters. We also test the toxicity and gene transfection efficiency of our vector via in vitro experiment. The experimental results show that when the Ca2+/PO43- molar ratio is 100, the DNA/Ca2+ weight ratio is 10/3000, and the PEG-PLGA concentration is 10 mg/mL, the optimal particle size, negatively charged surface potential, and conversion can be obtained. Compared with the cationic gene delivery vector such as disulfide cross linked polyethylenimine （SPEI）, the CDP developed in this study has low cytotoxicity and the transfection efficiency is not affected by serum proteins.
This study then explores the potential of using CDP as a gene and protein co-delivery vector. The DNA and protein （αHuman-488 and Gaot anti-Mouse IgG was selected as the model protein in this study） were mixed with CaP to be incorporated into the core via inverse emulsification method, and then through the nanoprecipitation to form the gene and protein co-delivery vector （CaP/pDNA/protein/ PLGA nanoparticles, CDPP）. In cell uptake experiments, the protein and gene uptake efficiency of mouse breast cancer cells （4T1） for CDPP were 97% and 10%, respectively quantified by flow cytometry. The results of gene transfection efficiency shows that CDPP has a gene transfection efficiency that is 10 times higher than CDP, but the detailed mechanism is still to be explored. Based on the above results, the CDPP developed in this study has the characteristics of high stability, dispersion, low cytotoxicity, remain the good transfection efficiency in serum environment, and can efficiently deliver proteins and genes to cells. It has great potential to application the co-delivery vector to delivery gene and protein drugs in vivo, and it is worthwhile to evaluate the feasibility of tumor treatment and even in vivo gene editing with tumor animal models in the future.

摘要 i
Abstract iii
致謝 v
目錄 vi
圖目錄 ix
表目錄 xii
第一章、 緒論 1
1.1. 前言 1
1.2. 研究動機與假說 2
第二章、 文獻回顧 5
2.1. 起源 5
2.2. 促進溶酶體逃脫策略 10
2.2.1. 質子化胺基誘導溶酶體逃脫 11
2.2.2. 蛋白質介導溶酶體逃脫 11
2.2.3. 膜融合 12
2.2.4. 其他 13
2.2.5. 結論 14
2.3. 基因遞送載體常用高分子 14
2.3.1. 藉由靜電吸附力與基因結合形成奈米基因載體 14
2.3.2. 以陽離子型為核心包覆或接枝其他陰電性高分子 20
2.3.3. 非陽離子型高分子包覆 23
2.3.4. 結論 26
2.4. 奈米載體同時遞送基因與其他物質 26
2.4.1. 基因與化療藥物共遞送載體 27
2.4.2. 基因與無機奈米粒子共遞送載體 32
2.4.3. 基因與蛋白質藥物共遞送載體 34
2.4.4. 結論 37
2.5. 總結 37
第三章、 材料方法 38
3.1. 實驗材料 38
3.2. 基因遞送載體製備與分析 39
3.2.1. CaP/DNA奈米基因載體製備 39
3.2.2. 載體基本基因包覆效率鑑定 40
3.2.3. 載體大小、電性及穩定性測試 40
3.2.4. 載體長時間封裝基因效率 41
3.3. 載體應用於體外細胞實驗測試 41
3.3.1. 細胞培養 41
3.3.2. 載體加入與細胞共培養之操作方式 41
3.3.3. 載體細胞毒性測試 42
3.3.4. 載體基因轉染效率鑑定 43
3.4. 基因與抗體共遞送載體製備及定量 43
3.4.1. 共遞送載體製備 43
3.4.2. 載體基因與抗體包覆效率鑑定 43
3.5. 共遞送載體遞與細胞共培養實驗步驟 44
3.6. 螢光影像及流式細胞儀分析基因與抗體共遞送成效 44
3.6.1. 細胞攝取共遞送載體之影像 44
3.6.2. 流式細胞儀定量細胞攝取載體效率 44
3.6.3. 螢光顯微鏡/共軛焦顯微鏡試片製作 45
第四章、 結果討論 46
4.1. 基因遞送載體開發及效果探討 46
4.1.1. TFE存在與否所製備之載體基本性質鑑定及轉染效果測試 46
4.1.2. 改變Ca/PO4 molar ratio增加與DNA的作用力對載體基本性質及轉染效果之影響 55
4.1.3. 降低DNA與Ca重量比對載體基本性質及轉染效果之影響 58
4.1.4. 調整PEG-PLGA濃度對載體基本鑑定及轉染效率之影響 60
4.1.5. 載體劑量依賴性探討 63
4.1.6. 載體長時間保存穩定性測試 64
4.1.7. 結論 65
4.2. 基因與蛋白質共同遞送載體開發及成效探討 66
4.2.1. 探討載體用於共遞送之可能性 66
4.2.2. 基因與蛋白質競爭CaP對兩者包覆率及轉染效率之影響 69
4.2.3. 以流式細胞儀定量細胞對載體的攝取率 71
4.2.4. 溶酶體逃脫影像鑑定 72
4.2.5. 結論 76
第五章、 結論與未來展望 78
參考文獻 79

1. Xiao, X.W., et al., Endogenous Reprogramming of Alpha Cells into Beta Cells, Induced by Viral Gene Therapy, Reverses Autoimmune Diabetes. Cell Stem Cell, 2018. 22(1): p. 78-+.
2. Castano, A., et al., Natural history and therapy of TTR-cardiac amyloidosis: emerging disease-modifying therapies from organ transplantation to stabilizer and silencer drugs. Heart Failure Reviews, 2015. 20(2): p. 163-178.
3. Blaese, R.M., et al., T-Lymphocyte-Directed Gene-Therapy for Ada(-) Scid - Initial Trial Results after 4 Years. Science, 1995. 270(5235): p. 475-480.
4. Foldvari, M., et al., Non-viral gene therapy: Gains and challenges of non-invasive administration methods. Journal of Controlled Release, 2016. 240: p. 165-190.
5. Mountain, A., Gene therapy: the first decade. Trends in Biotechnology, 2000. 18(3): p. 119-128.
6. Zhdanov, R.I., O.V. Podobed, and V.V. Vlassov, Cationic lipid-DNA complexes-lipoplexes-for gene transfer and therapy. Bioelectrochemistry, 2002. 58(1): p. 53-64.
7. Pinnapireddy, S.R., et al., Composite liposome-PEI/nucleic acid lipopolyplexes for safe and efficient gene delivery and gene knockdown. Colloids and Surfaces B-Biointerfaces, 2017. 158: p. 93-101.
8. Yang, S.D., et al., Nucleolin-Targeting AS1411-Aptamer-Modified Graft Polymeric Micelle with Dual pH/Redox Sensitivity Designed To Enhance Tumor Therapy through the Codelivery of Doxorubicin/TLR4 siRNA and Suppression of Invasion. Molecular Pharmaceutics, 2018. 15(1): p. 314-325.
9. Zhou, Z.L., et al., Calcium phosphate-polymer hybrid nanoparticles for enhanced triple negative breast cancer treatment via co-delivery of paclitaxel and miR-221/222 inhibitors. Nanomedicine-Nanotechnology Biology and Medicine, 2017. 13(2): p. 403-410.
10. Jiang, Y., et al., Progress and perspective of inorganic nanoparticle-based siRNA delivery systems. Expert opinion on drug delivery, 2016. 13(4): p. 547-559.
11. Riley, M.K. and W. Vermerris, Recent Advances in Nanomaterials for Gene Delivery-A Review. Nanomaterials, 2017. 7(5).
12. Liu, Y., et al., Brain-targeted co-delivery of therapeutic gene and peptide by multifunctional nanoparticles in Alzheimer's disease mice. Biomaterials, 2016. 80: p. 33-45.
13. Behr, J.P., The proton sponge: A trick to enter cells the viruses did not exploit. Chimia, 1997. 51(1-2): p. 34-36.
14. Cross, K.J., et al., Studies on influenza haemagglutinin fusion peptide mutants generated by reverse genetics. Embo Journal, 2001. 20(16): p. 4432-4442.
15. Mason, A.J., et al., The antibiotic and DNA-transfecting peptide LAH4 selectively associates with, and disorders, anionic lipids in mixed membranes. Faseb Journal, 2006. 20(2): p. 320-322.
16. Zelphati, O. and F.C. Szoka, Jr., Mechanism of oligonucleotide release from cationic liposomes. Proc Natl Acad Sci U S A, 1996. 93(21): p. 11493-8.
17. Selbo, P.K., et al., Photochemical internalization provides time- and space-controlled endolysosomal escape of therapeutic molecules. Journal of Controlled Release, 2010. 148(1): p. 2-12.
18. Deduve, C., et al., Lysosomotropic Agents. Biochemical Pharmacology, 1974. 23(18): p. 2495-+.
19. Wightman, L., et al., Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. Journal of Gene Medicine, 2001. 3(4): p. 362-372.
20. Santos-Carballal, B., et al., Physicochemical and biological characterization of chitosan-microRNA nanocomplexes for gene delivery to MCF-7 breast cancer cells. Scientific Reports, 2015. 5.
21. Sheikh, M.A., et al., Polylysine-modified polyethylenimine (PEI-PLL) mediated VEGF gene delivery protects dopaminergic neurons in cell culture and in rat models of Parkinon's Disease (PD). Acta Biomaterialia, 2017. 54: p. 58-68.
22. Nam, J.P. and J.W. Nah, Target gene delivery from targeting ligand conjugated chitosan-PEI copolymer for cancer therapy. Carbohydrate Polymers, 2016. 135: p. 153-161.
23. Grayson, A.C.R., A.M. Doody, and D. Putnam, Biophysical and structural characterization of polyethylenimine-mediated siRNA delivery in vitro. Pharmaceutical Research, 2006. 23(8): p. 1868-1876.
24. Zhang, L., et al., Low Molecular Weight PEI-Based Vectors via Acid-Labile Ortho Ester Linkage for Improved Gene Delivery. Macromolecular Bioscience, 2016. 16(8): p. 1175-1187.
25. Chang, P.K.C., C.A. Prestidge, and K.E. Bremmell, Interfacial analysis of siRNA complexes with poly-ethylenimine (PEI) or PAMAM dendrimers in gene delivery. Colloids and Surfaces B-Biointerfaces, 2017. 158: p. 370-378.
26. Li, Z.B.A., et al., Targeted delivery of Bcl-2 conversion gene by MPEG-PCL-PEI-FA cationic copolymer to combat therapeutic resistant cancer. Materials Science & Engineering C-Materials for Biological Applications, 2017. 76: p. 66-72.
27. Xu, X.M., R.M. Capito, and M. Spector, Delivery of plasmid IGF-1 to chondrocytes via cationized gelatin nanoparticles. Journal of Biomedical Materials Research Part A, 2008. 84a(1): p. 73-83.
28. 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.
29. Kurosaki, T., et al., Ternary complexes of pDNA, polyethylenimine, and gamma-polyglutamic acid for gene delivery systems. Biomaterials, 2009. 30(14): p. 2846-2853.
30. Li, Y.M., et al., Co-delivery of doxorubicin and tumor-suppressing p53 gene using a PUSS-based star-shaped polymer for cancer therapy. Biomaterials, 2015. 55: p. 12-23.
31. Xia, J.L., et al., Sulfathiazole grafted PEG-PLL as pH-sensitive shielding system for cationic gene delivery. Polymer Bulletin, 2016. 73(12): p. 3503-3511.
32. Pittella, F., et al., Enhanced endosomal escape of siRNA-incorporating hybrid nanoparticles from calcium phosphate and PEG-block charge-conversional polymer for efficient gene knockdown with negligible cytotoxicity. Biomaterials, 2011. 32(11): p. 3106-3114.
33. Vachutinsky, Y., et al., Antiangiogenic gene therapy of experimental pancreatic tumor by sFlt-1 plasmid DNA carried by RGD-modified crosslinked polyplex micelles. Journal of Controlled Release, 2011. 149(1): p. 51-57.
34. Dordelmann, G., et al., Calcium phosphate increases the encapsulation efficiency of hydrophilic drugs (proteins, nucleic acids) into poly(D,L-lactide-co-glycolide acid) nanoparticles for intracellular delivery. Journal of Materials Chemistry B, 2014. 2(41): p. 7250-7259.
35. Xu, B., et al., Combined Tumor- and Neovascular-"Dual Targeting" Gene/Chemo-Therapy Suppresses Tumor Growth and Angiogenesis. Acs Applied Materials & Interfaces, 2016. 8(39): p. 25753-25769.
36. Nicol, F., et al., Poly-L-glutamate, an anionic polymer, enhances transgene expression for plasmids delivered by intramuscular injection with in vivo electroporation. Gene Therapy, 2002. 9(20): p. 1351-1358.
37. Guan, X.W., et al., Codelivery of Antitumor Drug and Gene by a pH-Sensitive Charge-Conversion System. Acs Applied Materials & Interfaces, 2015. 7(5): p. 3207-3215.
38. Sun, T.M., et al., Simultaneous Delivery of siRNA and Paclitaxel via a "Two-in-One" Micelleplex Promotes Synergistic Tumor Suppression. Acs Nano, 2011. 5(2): p. 1483-1494.
39. Wang, K., et al., Structure-Invertible Nanoparticles for Triggered Co-Delivery of Nucleic Acids and Hydrophobic Drugs for Combination Cancer Therapy. Advanced Functional Materials, 2015. 25(22): p. 3380-3392.
40. Koganti, S., et al., In vitro and in vivo evaluation of the efficacy of nanoformulation of siRNA as an adjuvant to improve the anticancer potential of cisplatin. Experimental and Molecular Pathology, 2013. 94(1): p. 137-147.
41. Patil, Y.B., et al., The use of nanoparticle-mediated targeted gene silencing and drug delivery to overcome tumor drug resistance. Biomaterials, 2010. 31(2): p. 358-365.
42. Yadav, S., et al., Evaluations of combination MDR-1 gene silencing and paclitaxel administration in biodegradable polymeric nanoparticle formulations to overcome multidrug resistance in cancer cells. Cancer Chemotherapy and Pharmacology, 2009. 63(4): p. 711-722.
43. Yu, Z.Z., et al., Tumor microenvironment-triggered fabrication of gold nanomachines for tumor-specific photoacoustic imaging and photothermal therapy. Chemical Science, 2017. 8(7): p. 4896-4903.
44. Cen, C.D., et al., Improving Magnetofection of Magnetic Polyethylenimine Nanoparticles into MG-63 Osteoblasts Using a Novel Uniform Magnetic Field. Nanoscale Research Letters, 2019. 14.
45. Goodman, A.M., et al., Near-infrared remotely triggered drug-release strategies for cancer treatment. Proceedings of the National Academy of Sciences of the United States of America, 2017. 114(47): p. 12419-12424.
46. Zhang, Y.H., et al., Simultaneous expression and transportation of insulin by supramolecular polysaccharide nanocluster. Scientific Reports, 2016. 6.
47. Mout, R., et al., Direct Cytosolic Delivery of CRISPR/Cas9-Ribonucleoprotein for Efficient Gene Editing. Acs Nano, 2017. 11(3): p. 2452-2458.
48. Shahbazi, R., et al., Targeted Homology Directed Repair in Blood Stem and Progenitor Cells with Highly Potent CRISPR Nanoformulations. Molecular Therapy, 2018. 26(5): p. 176-176.
49. Ma, D., Enhancing endosomal escape for nanoparticle mediated siRNA delivery. Nanoscale, 2014. 6(12): p. 6415-6425.
50. Frohlich, E., The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. International Journal of Nanomedicine, 2012. 7: p. 5577-5591.
51. Chen, J., et al., Multifunctional Click Hyaluronic Acid Nanogels for Targeted Protein Delivery and Effective Cancer Treatment in Vivo. Chemistry of Materials, 2016. 28(23): p. 8792-8799.