非病毒性載體作為口服基因投遞系統，可攜帶基因片段至細胞以表達具療效之蛋白分子，被視為具有治療慢性病的應用潛力，然而如何提升其低轉染效率一直是尚待努力的課題。本研究以小分子的分枝狀聚乙烯亞胺 (Polyethylenimine)高接枝度地接於幾丁聚醣 (Chitosan)合成出一帶強正電共聚物 (CS-grafted-branched PEI, CS-g-bPEI)。在與帶負電的基因 (Plasmid DNA)片段混合後可形成一奈米微粒，作為非病毒型之口服基因載體。在腸胃道環境下，此奈米微粒可保護DNA不受胃酸破壞以及腸道內的酵素降解，並且有效穿越小腸上皮細胞，達到血液循環中使得口服基因轉染 (Gene transfection)不在局限於腸胃道細胞。在動物實驗中，單次口服攜有胰島素基因之奈米微粒可以使動物在肝臟表現胰島素長達十天，同時穩定的控制糖尿病小鼠之空腹血糖。另外，連續多次的口服投遞此奈米微粒均可穩定且有效的表現胰島素、控制血糖。最後，在長時間的觀察下，動物的血液檢測和病理切片皆沒有發現任何肝臟毒性。本研究證實，CS-g-bPEI奈米微粒為一高效率且安全的口服基因投遞系統，對於需要長時間投藥的慢性疾病有很高的應用潛力。

Oral gene delivery to target systemic cell transfection is an appealing method for treating chronic diseases. In this investigation, a copolymer with a high degree of substitution of branched polyethylenimine on chitosan (CS-g-bPEI) is synthesized as a non-viral vector for the oral delivery of a human insulin plasmid. The as-synthesized CS-g-bPEI copolymer can form nanoparticles (NPs) with plasmids and effectively protects them from gastric acidic denaturation and degradation by intestinal enzyme, mediating the transcytosis of NPs across the epithelial cells, eventually achieving systemic cell transfection. A single dose of orally administered insulin plasmid NPs can result in sustained therapeutic gene expression in diabetic mice over a period of ten days, and the level of expression of insulin suffices to ameliorate their hyperglycemia. Efficient expressions of genes in liver tissues in diabetic mice following the repeated oral administration of test NPs is confirmed, while no evidence of significant hepatotoxicity in test animals is detected, revealing that the repeated oral administration of CS-g-bPEI NPs is an effective strategy for treating a chronic disease such as diabetes mellitus.

目錄 III
圖目錄 V
一、 緒論 1
1.1非病毒性的口服基因載體 1
1.2幾丁聚醣 (Chitosan, CS) 1
1.3聚乙烯亞胺 (Polyethylenimine, PEI) 2
1.4胰島素治療的改進-研究動機與目的 3
1.5實驗架構圖 3
二、材料與方法 5
2.1 CS-g-bPEI的合成、結構鑑定以及化學性質分析 5
2.2 pDNA的純化 6
2.4 CS-g-bPEI/pDNA奈米微粒pH穩定性測試 7
2.5 CS-g-bPEI於酸性pH值及酵素環境中保護pDNA之能力 8
2.6製備Cy3螢光標定的pDNA及Cy5螢光標定的CS-g-bPEI 8
2.7 CS-g-bPEI/pDNA奈米微粒通過Caco-2單層細胞能量依賴性的胞吞作用 9
2.8動物實驗 10
2.9.1糖尿病鼠模型的建立 10
2.9.2 CS-g-bPEI/pDNA奈米微粒於生物體內與肝臟分布與累積 11
2.9.3 肝臟內庫氏細胞與人類胰島素之免疫染色 11
2.9.4 降血糖機轉 12
2.9.5 肝毒性測試 12
三、結果與討論 14
3.1 分析CS-g-bPEI的合成、結構鑑定以及化學性質分析 14
3.2 CS-g-bPEI/pDNA奈米微粒pH穩定性質分析 15
3.3 CS-g-bPEI/pDNA於pH值及酵素降解測試 17
3.4 CS-g-bPEI/pDNA通過Caco-2單層細胞能量依賴性的胞吞作用 17
3.5 CS-g-bPEI/pDNA奈米微粒動物實驗 20
3.5.1 CS-g-bPEI/pDNA奈米微粒於生物體內與肝臟分布與累積 20
3.5.2 人類胰島素基因在生物體器官表現 22
3.5.3 降血糖機轉 23
3.5.4肝毒性測試 24
四、結論 26
五、參考文獻 27

1. M.D. Bhavsar, M.M. Amiji, Oral IL-10 Gene Delivery In A Microsphere-Based Formulation For Local Transfection And Therapeutic Efficacy In Inflammatory Bowel Disease. Gene Ther. 15, 1200–1209 (2008).
2. M.J. O'Neill, L Bourre, S Melgar, CM O'Driscoll, Intestinal delivery of non-viral gene therapeutics: physiological barriers and preclinical models. Drug Discov. Today. 16, 203–218 (2011).
3. K. Bowman, R. Sarkar, S. Raut, K.W. Leong, Gene Transfer To Hemophilia A Mice Via Oral Delivery Of FVIII-chitosan Nanoparticles. J. Control. Release. 132, 252–259 (2008).
4. K. Bowman, K.W. Leong, Chitosan Nanoparticles For Oral Drug And Gene Delivery. Int. J. Nanomedicine. 1, 117–128 (2006).
5. J. Kim, J. Kim, C. Jeong, W.J. Kim, Synergistic Nanomedicine By Combined Gene And Photothermal Therapy. Adv. Drug Deliv. Rev. 98, 99–112 (2016).
6. K. Roy, H.Q. Mao, S.K. Huang, K.W. Leong, Oral Gene Delivery With Chitosan-DNA Nanoparticles Generates Immunologic Protection In A Murine Model Of Peanut Allergy. Nat. Med. 5, 387–391 (1999).
7. C. Kriegel, H. Attarwala, M. Amiji, Multi-Compartmental Oral Delivery Systems For Nucleic Acid Therapy In The Gastrointestinal Tract. Adv. Drug Deliv. Rev. 65, 891–901 (2013).
8. D.W. Pack, A.S. Hoffman, S. Pun, P.S. Stayton, Design And Development Of Polymers For Gene Delivery. Nat. Rev. Drug Discov. 4, 581–93 (2005).
9. H. Chen, S. Cui, Y. Zhao, C. Zhang, S. Zhang, X. Peng, Grafting Chitosan With Polyethylenimine In An Ionic Liquid For Efficient Gene Delivery. PLoS One. 10, 1–17 (2015).
10. H. Lu, Y. Dai, L. Lv, H. Zhao, Chitosan-Graft-Polyethylenimine/DNA Nanoparticles As Novel Non-Viral Gene Delivery Vectors Targeting Osteoarthritis. PLoS One. 9 (2014).
11. M.A. Islam, T.E. Park, B. Singh, S. Maharjan, J. Firdous, M.H. Cho, S.K. Kang, C.H. Yun, Y.J. Choi, C.S. Cho, M. Ariful, T. Eun, B. Singh, S. Maharjan, J. Firdous, M.H. Cho, S.K. Kang, C.H. Yun, Y. Jaie, C.S. Cho, Major Degradable Polycations As Carriers For DNA And siRNA. J. Control. Release. 193, 74–89 (2014).
12. B. Singh, S. Maharjan, T.E. Park, T. Jiang, S.K. Kang, Y.J. Choi, C.S. Cho, Tuning The Buffering Capacity Of Polyethylenimine With Glycerol Molecules For Efficient Gene Delivery: Staying In Or Out Of The Endosomes. Macromol. Biosci. 15, 622–635 (2015).
13. S. Xiang, H. Tong, Q. Shi, J.C. Fernandes, T. Jin, K. Dai, X. Zhang, Uptake Mechanisms Of Non-Viral Gene Delivery. J. Control. Release. 158, 371–378 (2012).
14. L.W. Hsu, Y.C. Ho, E.Y. Chuang, C.T. Chen, J.H. Juang, F.Y. Su, S.M. Hwang, H.W. Sung, Effects Of pH On Molecular Mechanisms Of Chitosan-Integrin Interactions And Resulting Tight-Junction Disruptions. Biomaterials. 34, 784–793 (2013).
15. M. Konishi, K. Kawamoto, M. Izumikawa, H. Kuriyama, T. Yamashita, Hydrophobically Modified Low Molecular Weight Chitosans As Efficient And Nontoxic Gene Delivery Vectors. J. Gene Med. 10, 610–618 (2008).
16. Y. Zhang, Global Non-Viral Gene Transfer To The Primate Brain Following Intravenous Administration. Mol. Ther. 7, 11–18 (2003).
17. W.M. Pardridge, Gene Targeting In Vivo With Pegylated Immunoliposomes. Methods Enzymol. 373, 507–528 (2003).
18. E.Y. Chuang, G.T.H. Nguyen, F.Y. Su, K.J. Lin, C.T. Chen, F.L. Mi, T.C. Yen, J.H. Juang, H.W. Sung, Combination Therapy Via Oral Co-Administration Of Insulin- And Exendin-4-Loaded Nanoparticles To Treat Type 2 Diabetic Rats Undergoing OGTT. Biomaterials. 34, 7994–8001 (2013).
19. A. Kwok, S.L. Hart, Comparative Structural And Functional Studies Of Nanoparticle Formulations For DNA And siRNA Delivery. Nanomedicine Nanotechnology, Biol. Med. 7, 210–219 (2011).
20. A.F. Adler, K.W. Leong, Emerging Links Between Surface Nanotechnology And Endocytosis: Impact On Nonviral Gene Delivery. Nano Today. 5, 553–569 (2010).
21. K. Sonaje, K.J. Lin, S.P. Wey, C.K. Lin, T.H. Yeh, H.N. Nguyen, C.W. Hsu, T.C. Yen, J.H. Juang, H.W. Sung, Biodistribution, Pharmacodynamics And Pharmacokinetics Of Insulin Analogues In A Rat Model: Oral Delivery Using pH-Responsive Nanoparticles Vs. Subcutaneous Injection. Biomaterials. 31, 6849–6858 (2010).
22. J.P.M. Almeida, A.L. Chen, A. Foster, R. Drezek, In Vivo Biodistribution Of Nanoparticles. Nanomedicine (Lond). 6, 815–35 (2011).
23. H. Petry, A. Brooks, A. Orme, P. Wang, P. Liu, J. Xie, P. Kretschmer, H.S. Qian, T.W. Hermiston, R.N. Harkins, Effect Of Viral Dose On Neutralizing Antibody Response And Transgene Expression After AAV1 Vector Re-Administration In Mice. Gene Ther. 15, 54–60 (2008).
24. K. Sonaje, Y.J. Chen, H.L. Chen, S.P. Wey, J.H. Juang, H.N. Nguyen, C.W. Hsu, K.J. Lin, H.W. Sung, Enteric-Coated Capsules Filled With Freeze-Dried Chitosan/Poly(Γ-Glutamic Acid) Nanoparticles For Oral Insulin Delivery. Biomaterials. 31, 3384–3394 (2010).
25. C. Ménez, M. Buyse, C. Dugave, R. Farinotti, G. Barratt, Intestinal Absorption Of Miltefosine: Contribution Of Passive Paracellular Transport. Pharm. Res. 24, 546–554 (2007).