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研究生：	陳宜君 Chen, Yi-Chun
論文名稱：	開發以鹼性去磷酸酶為引信之多肽微脂體 Development of Alkaline Phosphatase Responsive Peptidyl Liposome
指導教授：	李賢明 Lee, Hsien-Ming
學位類別：	碩士 Master
系所名稱：	化學系 Department of Chemistry
論文出版年：	2020
畢業學年度：	108
語文別：	中文
論文頁數：	97
中文關鍵詞：	引信響應、奈米藥物、微脂體、鹼性去磷酸酶
英文關鍵詞：	triggered release, nanomedicine, liposome, alkaline phosphatase
DOI URL：	http://doi.org/10.6345/NTNU202001136
論文種類：	學術論文
相關次數：	點閱：56 下載：0
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摘要  i
Abstract iii
目錄 v
圖目錄 ix
表目錄  xiv
縮寫表  xv


Chapter 1 緒論 (Introduction)  1
1-1前言 (Preface)  1

1-2微脂體 (Liposome) 2
1-2-1 磷脂質(Phospholipid)  3
1-2-2 膽固醇(Cholesterol)  4
1-2-3 聚乙二醇化微脂體 (PEGylated Liposome)   5

1-3 微脂體包覆藥物與包覆方法 (Liposomal Drug and Drug Loading Method)  5
1-3-1 被動包覆 (Passive Loading)  6
1-3-2 主動包覆 (Active Loading)  6

1-4 引信響應多肽微脂體藥物 (Triggered-Release Peptidyl Liposomal Drug) 8
1-5 膜活性多肽 (Membrane Active Peptide)  8
1-6 鹼性去磷酸酶 (Alkaline Phosphatase) 9
1-7 實驗動機與目的 (Motivation and Purpose)  10



Chapter 2 實驗方法 (Experimental procedure)  11
2-1 實驗設計 (Experimental Design)  11
2-1-1微脂體組成 (Liposome Composition) 11
2-1-2多肽設計與合成 (Peptide Design and Synthesis)  12
2-1-3多肽微脂體藥物合成 (Peptidyl Liposome Synthesis)  15
2-1-4實驗細胞株的挑選 (Choice of Experimental Cell line)  18

2-2 定性與定量 (Characterization and Quantification)  19
2-2-1微脂體定量 (Liposome Quantification) 19
2-2-2粒徑 (Particle Size)  19
2-2-3界達電位 (Zeta Potential)  20
2-2-4藥物包覆率 (Drug encapsulation Efficiency)  21
2-2-5質譜 (Mass Spectroscopy) 21
2-2-6多肽定量 (Peptide Quantification) 22
2-2-7圓二色光譜 (Circular Dichroism Spectroscopy, CD)  23
2-2-8冷凍電子顯微鏡影像 (Cryo-EM) 24
2-2-9多肽微酯體藥物釋放分析 (Peptidyl Liposome Release Analysis)  25

2-3細胞培養條件 (Incubated Condition)  26
2-3-1細胞基本培養條件 (Usual Incubation Condition)  26
2-3-2細胞去磷酸酶活性分析(ALP Activity Assay)  26
2-3-3高通量顯微鏡細胞實驗 (High Content Microscope Cell Experiment) 27
2-3-4流式細胞儀細胞實驗 (Flow Cytometry Cell Experiment) 27
2-3-5 細胞存活率分析 (MTT Assay) 28



Chapter 3實驗結果與討論 (Result and Discussion) 31
3-1 化學合成多肽之定性 (Characterization of Chemical Synthesis Peptides) 31
3-2 2pY8,19-Mag2與2Y-Mag2定量 (Quantification of 2pY8,19-Mag2 and 2Y-Mag2)  41
3-3多肽的磷酸化數目與其遮蔽破膜活性之分析 (Membrane Disruption Activity Test of Different Phosphorylation Peptides)  44
vii
3-4 去磷酸酶與2pY8,19-Mag2之作用效率分析 (ALP Dephosphorylate Kinetics Analysis)  45
3-5 微脂體定性 (Characterization of liposome)   47
3-6多肽共價鍵結滴定 (Peptide Covalent Conjugation Titration) 49

3-7試管內控制釋放 (In vitro Triggered Release)  50
3-7-1 DSPE-PEG2000含量對多肽微脂體之釋放效率分析 (The Releasing Efficiency of Different Percentage of DSPE-PEG2000 in Peptidyl Liposome)  50
3-7-2共價鍵結不同比例的多肽的微脂體釋放分析 (The Effect of Different Ratio of Covalent Conjugated Peptide on Liposome)  58
3-7-3不同比例之18:1 PE MCC微脂體釋放分析(The Effect of Different Percent 18:1 PE MCC in Liposome) 58
3-7-4 2pY8,19-Mag2與3pY8,15,19-Mag2微脂體釋放比較 (Release of 2pY8,19-Mag2 Liposome and 3pY8,15,19-Mag2 Liposome) 61
3-7-5 2pY8,19-MDL與Scramble-2pY5,14-MDL釋放比較(2pY8,19-MDL comparing with Scramble 2pY5,14-MDL) 61

3-8冷凍電子顯微鏡影像 (Cryo-EM Image)  62
3-8-1不同比例DSPE-PEG2000微脂體之冷凍電子顯微鏡影像-無醋酸鈾正染(Cryo-EM images of Liposome with Different percentage DSPE-PEG2000)  63
3-8-2不同比例DSPE-PEG2000微脂體之冷凍電子顯微鏡影像-醋酸鈾正染(UA staining Cryo-EM images of Liposome with Different percentage DSPE-PEG2000) 67

3-9細胞實驗 (Cell Experiment) 70
3-9-1細胞酵素活性分析 (Cells’ ALP Activity Analysis) 70
3-9-2細胞酵素誘發多肽微脂體藥物釋放分析 (Liposomal Drug triggered release by ALP of Cells)  72
3-9-3藥量與細胞存活率之分析 (MTT assay) 83


Chapter 4 結論 (Conclusion)  85

REFERENCE  87

附錄  91
2pY8,19-Mag2 與 3pY8,15,19-Mag2 微脂體釋放比較(5% DSPE-PEG2000 微脂體)  91
2pY12,19-Melittin與2pY13,16-Epinecidin 1多肽微脂體試管內釋放測試  91
CD結構-軟體分析圖  93
本研究所使用之藥品、儀器  95
                                

1. Torchilin, V. P., Multifunctional nanocarriers. Advanced Drug Delivery Reviews 2012, 64, 302-315.
2. Sun, T.; Zhang, Y. S.; Pang, B.; Hyun, D. C.; Yang, M.; Xia, Y., Engineered Nanoparticles for Drug Delivery in Cancer Therapy. Angewandte Chemie International Edition 2014, 53 (46), 12320-12364.
3. Vieira, D. B.; Gamarra, L. F., Getting into the brain: liposome-based strategies for effective drug delivery across the blood-brain barrier. Int J Nanomedicine 2016, 11, 5381-5414.
4. Lozano, N.; Al-Ahmady, Z. S.; Beziere, N. S.; Ntziachristos, V.; Kostarelos, K., Monoclonal antibody-targeted PEGylated liposome-ICG encapsulating doxorubicin as a potential theranostic agent. International Journal of Pharmaceutics 2015, 482 (1), 2-10.
5. Gregoriadis, G., The carrier potential of liposomes in biology and medicine (second of two parts). N Engl J Med 1976, 295 (14), 765-770.
6. Bangham, A. D.; Horne, R. W., Negative staining of phospholipids and their structural modification by surface-active agents as observed in the electron microscope. Journal of Molecular Biology 1964, 8 (5), 660-IN10.
7. Sessa, G.; Weissmann, G., Incorporation of Lysozyme into Liposomes: A MODEL FOR STRUCTURE-LINKED LATENCY. Journal of Biological Chemistry 1970, 245 (13), 3295-3301.
8. Barenholz, Y., Doxil® — The first FDA-approved nano-drug: Lessons learned. Journal of Controlled Release 2012, 160 (2), 117-134.
9. Grüll, H.; Langereis, S., Hyperthermia-triggered drug delivery from temperature-sensitive liposomes using MRI-guided high intensity focused ultrasound. Journal of Controlled Release 2012, 161 (2), 317-327.
10. Briuglia, M. L.; Rotella, C.; McFarlane, A.; Lamprou, D. A., Influence of cholesterol on liposome stability and on in vitro drug release. Drug delivery and translational research 2015, 5 (3), 231-42.
11. Monteiro, N.; Martins, A.; Reis, R. L.; Neves, N. M., Liposomes in tissue engineering and regenerative medicine. Journal of the Royal Society, Interface 2014, 11 (101), 20140459.
12. De Gier, J.; Mandersloot, J. G.; Van Deenen, L. L. M., Lipid composition and permeability of liposomes. Biochimica et Biophysica Acta (BBA) - Biomembranes 1968, 150 (4), 666-675.
13. Cabanes, A.; Tzemach, D.; Goren, D.; Horowitz, A. T.; Gabizon, A., Comparative study of the antitumor activity of free doxorubicin and polyethylene glycol-coated liposomal doxorubicin in a mouse lymphoma model. Clinical Cancer Research 1998, 4 (2), 499.
14. Paola, M.; Franco, D.; Luigi, C., PEGylation of Proteins and Liposomes: a Powerful and Flexible Strategy to Improve the Drug Delivery. Current Drug Metabolism 2012, 13 (1), 105-119.
15. Prabhu, P.; Shetty, R.; Koland, M.; Vijayanarayana, K.; Vijayalakshmi, K. K.; Nairy, M. H.; Nisha, G. S., Investigation of nano lipid vesicles of methotrexate for anti-rheumatoid activity. Int J Nanomedicine 2012, 7, 177-186.
16. Crosasso, P.; Ceruti, M.; Brusa, P.; Arpicco, S.; Dosio, F.; Cattel, L., Preparation, characterization and properties of sterically stabilized paclitaxel-containing liposomes. Journal of Controlled Release 2000, 63 (1), 19-30.
17. Sercombe, L.; Veerati, T.; Moheimani, F.; Wu, S. Y.; Sood, A. K.; Hua, S., Advances and Challenges of Liposome Assisted Drug Delivery. Front Pharmacol 2015, 6, 286-286.
18. Abraham, T.; Mao, M.; Tan, C., Engineering approaches of smart, bio-inspired vesicles for biomedical applications. Physical Biology 2018, 15 (6), 061001.
19. Drummond, D. C.; Meyer, O.; Hong, K.; Kirpotin, D. B.; Papahadjopoulos, D., Optimizing Liposomes for Delivery of Chemotherapeutic Agents to Solid Tumors. Pharmacological Reviews 1999, 51 (4), 691.
20. Boomer, J. A.; Inerowicz, H. D.; Zhang, Z.-Y.; Bergstrand, N.; Edwards, K.; Kim, J.-M.; Thompson, D. H., Acid-Triggered Release from Sterically Stabilized Fusogenic Liposomes via a Hydrolytic DePEGylation Strategy. Langmuir 2003, 19 (16), 6408-6415.
21. Viricel, W.; Mbarek, A.; Leblond, J., Switchable Lipids: Conformational Change for Fast pH-Triggered Cytoplasmic Delivery. Angewandte Chemie International Edition 2015, 54 (43), 12743-12747.
22. Morgan, C. G.; Bisby, R. H.; Johnson, S. A.; Mitchell, A. C., Fast solute release from photosensitive liposomes: an alternative to ‘caged’ reagents for use in biological systems. FEBS Letters 1995, 375 (1), 113-116.
23. Punnamaraju, S.; You, H.; Steckl, A. J., Triggered Release of Molecules across Droplet Interface Bilayer Lipid Membranes Using Photopolymerizable Lipids. Langmuir 2012, 28 (20), 7657-7664.
24. Mizukami, S.; Kashibe, M.; Matsumoto, K.; Hori, Y.; Kikuchi, K., Enzyme-triggered compound release using functionalized antimicrobial peptide derivatives. Chemical Science 2017, 8 (4), 3047-3053.
25. Skyttner, C., Peptide-Liposome Model Systems for Triggered Release. Linköping University Electronic Press: 2018.
26. Li, Q.-Q.; Chen, P.-G.; Hu, Z.-W.; Cao, Y.; Chen, L.-X.; Chen, Y.-X.; Zhao, Y.-F.; Li, Y.-M., Selective inhibition of cancer cells by enzyme-induced gain of function of phosphorylated melittin analogues. Chemical Science 2017, 8 (11), 7675-7681.
27. Motion, J. P. M.; Nguyen, J.; Szoka, F. C., Phosphatase-Triggered Fusogenic Liposomes for Cytoplasmic Delivery of Cell-Impermeable Compounds. Angewandte Chemie International Edition 2012, 51 (36), 9047-9051.
28. Sharma, U.; Pal, D.; Prasad, R., Alkaline phosphatase: an overview. Indian J Clin Biochem 2014, 29 (3), 269-278.
29. Lim, S. M.; Kim, Y. N.; Park, K. H.; Kang, B.; Chon, H. J.; Kim, C.; Kim, J. H.; Rha, S. Y., Bone alkaline phosphatase as a surrogate marker of bone metastasis in gastric cancer patients. BMC Cancer 2016, 16, 385-385.
30. Ji, S.; Gao, H.; Mu, W.; Ni, X.; Yi, X.; Shen, J.; Liu, Q.; Bao, P.; Ding, D., Enzyme-instructed self-assembly leads to the activation of optical properties for selective fluorescence detection and photodynamic ablation of cancer cells. Journal of Materials Chemistry B 2018, 6 (17), 2566-2573.
31. Zhou, J.; Du, X.; Yamagata, N.; Xu, B., Enzyme-Instructed Self-Assembly of Small d-Peptides as a Multiple-Step Process for Selectively Killing Cancer Cells. Journal of the American Chemical Society 2016, 138 (11), 3813-3823.
32. Shi, J.; Schneider, J. P., De novo Design of Selective Membrane-Active Peptides by Enzymatic Control of Their Conformational Bias on the Cell Surface. Angewandte Chemie International Edition 2019, 58 (39), 13706-13710.
33. Au - Qvit, N.; Au - Kornfeld, O. S., Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation. JoVE 2016, (107), e53589.
34. Maruyama, K., Intracellular targeting delivery of liposomal drugs to solid tumors based on EPR effects. Advanced Drug Delivery Reviews 2011, 63 (3), 161-169.
35. Herz, F., Alkaline phosphatase in KB cells: Influence of hyperosmoiality and prednisolone on enzyme activity and thermostability. Archives of Biochemistry and Biophysics 1973, 158 (1), 225-235.
36. Herz, F.; Kaplan, E.; Fineman, E. L., Regulation of alkaline phosphatase activity in KB cells: Influence of serum. Biochimica et Biophysica Acta (BBA) - General Subjects 1973, 304 (3), 660-668.
37. Domon, B.; Aebersold, R., Mass Spectrometry and Protein Analysis. Science 2006, 312 (5771), 212-217.
38. Bhagavan, N. V., CHAPTER 4 - Three-Dimensional Structure of Proteins. In Medical Biochemistry (Fourth Edition), Bhagavan, N. V., Ed. Academic Press: San Diego, 2002; pp 51-65.
39. Kuete, V.; Karaosmanoğlu, O.; Sivas, H., Chapter 10 - Anticancer Activities of African Medicinal Spices and Vegetables. In Medicinal Spices and Vegetables from Africa, Kuete, V., Ed. Academic Press: 2017; pp 271-297.
40. Zasloff, M., Antimicrobial peptides of multicellular organisms. Nature 2002, 415 (6870), 389-395.
41. Goto, C.; Hirano, M.; Hayashi, K.; Kikuchi, Y.; Hara-Kudo, Y.; Misawa, T.; Demizu, Y., Development of Amphipathic Antimicrobial Peptide Foldamers Based on Magainin 2 Sequence. ChemMedChem 2019, 14 (22), 1911-1916.

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