研究生: |
吳宇萱 Wu, Yu-Hsuan |
---|---|
論文名稱: |
分子表面修飾的氧化鐵奈米粒子用於穩定人類降鈣素 Surface-decorated iron oxide nanoparticles used in stabilizing human calcitonin |
指導教授: |
杜玲嫻
Tu, Ling-Hsien |
口試委員: |
廖美儀
Liao, Mei-Yi 葉怡均 Yeh, Yi-Chun 杜玲嫻 Tu, Ling-Hsien |
口試日期: | 2022/06/27 |
學位類別: |
碩士 Master |
系所名稱: |
化學系 Department of Chemistry |
論文出版年: | 2022 |
畢業學年度: | 110 |
語文別: | 中文 |
論文頁數: | 64 |
中文關鍵詞: | 人類降鈣素 、氧化鐵奈米粒子 、葉綠素金屬衍生物 、蘇木精 |
英文關鍵詞: | human calcitonin, iron oxide nanoparticles, chlorophyll metal derivatives, hematoxylin |
研究方法: | 實驗設計法 |
DOI URL: | http://doi.org/10.6345/NTNU202201014 |
論文種類: | 學術論文 |
相關次數: | 點閱:122 下載:0 |
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類澱粉蛋白纖維的形成在類澱粉蛋白變性中是重要的關鍵。此外,一些多肽類賀爾蒙,如人類降鈣素 (human calcitonin, hCT) ,因具有調節血鈣水平的作用而聞名,但由於hCT具高度傾向形成類澱粉蛋白纖維,因此作為藥物的潛力有限。
奈米材料在各個科學領域具有許多優勢,通常具有獨特的物理及化學性質,如體積小、表面積大和與其他物質反應時活性極高。最近,研究指出氧化鐵奈米粒子經過適當表面修飾能夠抑制類澱粉蛋白纖維的形成。在這項研究中,我們發現通過不同方法合成的 Fe3O4@Chl/Fe (Fe-C) 、Fe3O4@Chl/Cu (Fe-CCu) 和 Fe3O4@hematoxylin (Fe-SU) 會影響hCT的成核和聚集過程。為了獲取雙重的驗證,我們使用兩種放光區域不同的苯並噻唑螢光探針來監測 hCT 類澱粉蛋白纖維的形成,以避免氧化鐵奈米粒子自身光學特性潛在的干擾,並使用電子顯微鏡觀察樣品終點樣貌。額外使用動態光散射粒徑分析儀輔助監測hCT纖維的形成 。由凝膠電泳來佐證出終點樣品的單體含量,和使用4,4'-二苯胺基-1,1'-聯二萘-5,5'-二磺酸與尼羅紅觀測共培育後的纖維含量。我們證實Fe-C為效果最佳的奈米粒子用作抑制hCT類澱粉蛋白纖維的產生,可使hCT更加穩定,經共培養後依然保留最多蛋白質單體,得以與細胞膜上的受體結合,增加其生物活性。此外,我們發現Fe-C以及Fe-CCu能夠降解預先形成的hCT纖維。我們的目標是希望在 hCT 製劑中添加一種新穎材料,以增加 hCT 作為藥物活性成分的機會。
Accumulation of amyloid fibrils plays an important role in the development of amyloidosis. Besides, some hormone peptide such as human calcitonin (hCT) known for its effect in regulating blood calcium levels has limited pharmaceutical potential due to a high tendency to form amyloid.
Nanotechnology offers many advantages in various fields of science, they usually have unique physicochemical properties such as small size, large surface area, and high reactivity. Recently, research has emphasized that appropriate surface modifications of nanoparticles can inhibit amyloid fibril formation. In this study, we found that Fe3O4@Chl/Fe (Fe-C), Fe3O4@Chl/Cu (Fe-CCu), and Fe3O4@hematoxylin (Fe-SU) synthesized via different approaches influence the nucleation and aggregation process of peptides. To obtain unbiased data, we used two different benzothiazole-based fluorescent probes to monitor hCT amyloid formation to avoid potential interference from the optical properties of nanoparticles. The appearance of hCT samples from the ThT endpoint was observed by transmission electron microscopy. Besides, the time course size distribution of hCT and nanoparticles mixture was monitored by dynamic light scattering. The monomer content from the ThT end product was checked by gel electrophoresis, and the fiber content of the ThT end product was measured using bis-ANS and nile red assay. The intracellular activated cAMP level was measured to confirm that Fe-C can inhibit hCT amyloid formation and major content with monomer type has larger remained. In addition, we found that Fe-C and Fe-CCu can dissociate pre-formed hCT fibrils. Our ultimate goal is to apply new material in hCT formulation to increase the opportunity of hCT to serve as active pharmaceutical ingredients in the medicine.
1. Scarpioni, R.; Ricardi, M.; Albertazzi, V.; De Amicis, S.; Rastelli, F.; Zerbini, L., Dialysis-related amyloidosis: challenges and solutions. Int. J. Nephrol. Renovasc. Dis. 2016, 9, 319-328.
2. Lacerda, S.; Morfin, J. F.; Geraldes, C.; Toth, E., Metal complexes for multimodal imaging of misfolded protein-related diseases. Dalton Trans. 2017, 46 (42), 14461-14474.
3. Benson, M. D.; Buxbaum, J. N.; Eisenberg, D. S.; Merlini, G.; Saraiva, M. J. M.; Sekijima, Y.; Sipe, J. D.; Westermark, P., Amyloid nomenclature 2020: update and recommendations by the International society of amyloidosis (ISA) nomenclature committee. Amyloid 2020, 27 (4), 217-222.
4. Picken, M. M., The pathology of amyloidosis in classification: a review. Acta haematol. 2020, 143 (4), 322-334.
5. Thomas, V. E.; Smith, J.; Benson, M. D.; Dasgupta, N. R., Amyloidosis: diagnosis and new therapies for a misunderstood and misdiagnosed disease. Neurodegener. Dis. Manag. 2019, 9 (6), 289-299.
6. Gorevic, P. D.; Schur, P.; Romain, P. J., Overview of amyloidosis. 2011, 10 (3). Please refer to medilib online system. Retrieved June 27, 2022, from https://www.medilib.ir/uptodate/show/5589
7. Corrections. Am. J. Pathol. 2011, 179 (1), 537-538.
8. Knowles, T. P.; Fitzpatrick, A. W.; Meehan, S.; Mott, H. R.; Vendruscolo, M.; Dobson, C. M.; Welland, M. E., Role of intermolecular forces in defining material properties of protein nanofibrils. Science 2007, 318 (5858), 1900-1903.
9. Geddes, A. J.; Parker, K. D.; Atkins, E. D.; Beighton, E., "Cross-beta" conformation in proteins. J. Mol. Biol. 1968, 32 (2), 343-58.
10. Astbury, W. T.; Dickinson, S.; Bailey, K., The X-ray interpretation of denaturation and the structure of the seed globulins. Biochem. J. 1935, 29 (10), 2351-2360.1.
11. Makin, O. S.; Atkins, E.; Sikorski, P.; Johansson, J.; Serpell, L. C., Molecular basis for amyloid fibril formation and stability. Proc. Natl. Acad. Sci. U. S. A. 2005, 102 (2), 315-20.
12. Röder, C.; Kupreichyk, T.; Gremer, L.; Schäfer, L. U.; Pothula, K. R.; Ravelli, R. B. G.; Willbold, D.; Hoyer, W.; Schröder, G. F., Amyloid fibril structure of islet amyloid polypeptide by cryo-electron microscopy reveals similarities with amyloid beta. bioRxiv. 2020, 2020.02.11.944546.
13. Iadanza, M. G.; Jackson, M. P.; Hewitt, E. W.; Ranson, N. A.; Radford, S. E., A new era for understanding amyloid structures and disease. Nat. Rev. Mol. Cell Biol. 2018, 19 (12), 755-773.
14. Gremer, L.; Scholzel, D.; Schenk, C.; Reinartz, E.; Labahn, J.; Ravelli, R. B. G.; Tusche, M.; Lopez-Iglesias, C.; Hoyer, W.; Heise, H.; Willbold, D.; Schroder, G. F., Fibril structure of amyloid-beta(1-42) by cryo-electron microscopy. Science 2017, 358 (6359), 116-119.
15. Kayed, R.; Lasagna-Reeves, C. A., Molecular mechanisms of amyloid oligomers toxicity. J. Alzheimers Dis. 2013, 33 Suppl 1, S67-78.
16. Kumar, S.; Walter, J., Phosphorylation of amyloid beta (Abeta) peptides - a trigger for formation of toxic aggregates in Alzheimer's disease. Aging (Albany NY) 2011, 3 (8), 803-12.
17. Schutz, A. K.; Hornemann, S.; Walti, M. A.; Greuter, L.; Tiberi, C.; Cadalbert, R.; Gantner, M.; Riek, R.; Hammarstrom, P.; Nilsson, K. P. R.; Bockmann, A.; Aguzzi, A.; Meier, B. H., Binding of polythiophenes to amyloids: structural mapping of the pharmacophore. ACS Chem. Neurosci. 2018, 9 (3), 475-481.
18. Paul Tuck, S.; Layfield, R.; Walker, J.; Mekkayil, B.; Francis, R., Adult Paget's disease of bone: a review. Rheumatology (Oxford) 2017, 56 (12), 2050-2059.
19. 余傑明; 吳岱穎; 楊榮森; 廖振焜; 樓亞洲; 台灣老年醫學暨老年學雜誌, 陳. J., 骨質疏鬆症的藥物治療. 2012, 7 (2), 77-90.
20. Andreotti, G.; Mendez, B. L.; Amodeo, P.; Morelli, M. A.; Nakamuta, H.; Motta, A., Structural determinants of salmon calcitonin bioactivity: the role of the Leu-based amphipathic alpha-helix. J. Biol. Chem. 2006, 281 (34), 24193-203.
21. Maricic, M. J., Oral calcitonin. Curr. Osteoporos. Rep. 2012, 10 (1), 80-5.
22. Li, D.; Liu, C., Structural Diversity of amyloid fibrils and advances in their structure determination. Biochemistry 2020, 59 (5), 639-646.
23. Karsdal, M. A.; Henriksen, K.; Bay-Jensen, A. C.; Molloy, B.; Arnold, M.; John, M. R.; Byrjalsen, I.; Azria, M.; Riis, B. J.; Qvist, P.; Christiansen, C., Lessons learned from the development of oral calcitonin: the first tablet formulation of a protein in phase III clinical trials. J. Clin. Pharmacol. 2011, 51 (4), 460-71.
24. Felsenfeld, A. J.; Levine, B. S., Calcitonin, the forgotten hormone: does it deserve to be forgotten? Clin. Kidney J. 2015, 8 (2), 180-7.
25. Kamihira, M.; Naito, A.; Tuzi, S.; Nosaka, A. Y.; Saitô, H., Conformational transitions and fibrillation mechanism of human calcitonin as studied by high-resolution solid-state 13C NMR. Protein Sci. 2000, 9 (5), 867-877.
26. Andreotti, G.; Vitale, R. M.; Avidan-Shpalter, C.; Amodeo, P.; Gazit, E.; Motta, A., Converting the highly amyloidogenic human calcitonin into a powerful fibril inhibitor by three-dimensional structure homology with a non-amyloidogenic analogue. J. Biol. Chem. 2011, 286 (4), 2707-18.
27. Cudd, A.; Arvinte, T.; Gaines Das, R. E.; Chinni, C.; MacIntyre, I., Enhanced potency of human calcitonin when fibrillation is avoided. J. Pharm. Sci. 1995, 84 (6), 717-719.
28. Kamgar-Parsi, K.; Tolchard, J.; Habenstein, B.; Loquet, A.; Naito, A.; Ramamoorthy, A., Structural biology of calcitonin: from aqueous therapeutic properties to amyloid aggregation. Isr. J. Chem. 2017, 57 (7-8), 634-650.
29. Itoh-Watanabe, H.; Kamihira-Ishijima, M.; Javkhlantugs, N.; Inoue, R.; Itoh, Y.; Endo, H.; Tuzi, S.; Saito, H.; Ueda, K.; Naito, A., Role of aromatic residues in amyloid fibril formation of human calcitonin by solid-state 13C NMR and molecular dynamics simulation. Phys. Chem. Chem. Phys. 2013, 15 (23), 8890-901.
30. Lantz, R.; Busbee, B.; Wojcikiewicz, E. P.; Du, D., Flavonoids with vicinal hydroxyl groups inhibit human calcitonin amyloid formation. Chemistry 2020, 26 (57), 13063-13071.
31. Maier, R.; Kamber, B.; Riniker, B.; Rittel, W., Analogues of human calcitonin: iv. influence of leucine substitutios in positions 12, 16 and 19 on hypocalcaemic activity in the rat. Clin. Endocrinol. (Oxf) 1976, 5 (s1), s327-s332.
32. Kamihira, M.; Oshiro, Y.; Tuzi, S.; Nosaka, A. Y.; Saito, H.; Naito, A., Effect of electrostatic interaction on fibril formation of human calcitonin as studied by high resolution solid state 13C NMR. J. Biol. Chem. 2003, 278 (5), 2859-65.
33. Chen, Y. T.; Hu, K. W.; Huang, B. J.; Lai, C. H.; Tu, L. H., Inhibiting human Calcitonin Fibril Formation with Its most relevant aggregation-resistant analog. J. Phys. Chem. B 2019, 123 (48), 10171-10180.
34. Porat, Y.; Abramowitz, A.; Gazit, E., Inhibition of amyloid fibril formation by polyphenols: structural similarity and aromatic interactions as a common inhibition mechanism. Chem. Biol. Drug Des. 2006, 67 (1), 27-37.
35. Huang, R.; Vivekanandan, S.; Brender, J. R.; Abe, Y.; Naito, A.; Ramamoorthy, A., NMR characterization of monomeric and oligomeric conformations of human calcitonin and its interaction with EGCG. J. Mol. Biol. 2012, 416 (1), 108-20.
36. Lantz, R.; Busbee, B.; Wojcikiewicz, E. P.; Du, D., Effects of disulfide bond and cholesterol derivatives on human calcitonin amyloid formation. Biopolymers 2020, 111 (5), e23343.
37. Valiente-Gabioud, A. A.; Miotto, M. C.; Chesta, M. E.; Lombardo, V.; Binolfi, A.; Fernandez, C. O., Phthalocyanines as molecular scaffolds to block disease-associated protein aggregation. Acc. Chem. Res. 2016, 49 (5), 801-8.
38. Valiente-Gabioud, A. A.; Riedel, D.; Outeiro, T. F.; Menacho-Marquez, M. A.; Griesinger, C.; Fernandez, C. O., Binding modes of phthalocyanines to amyloid beta peptide and their effects on amyloid fibril formation. Biophys. J. 2018, 114 (5), 1036-1045.
39. Priola Suzette, A.; Raines, A.; Caughey Winslow, S., Porphyrin and phthalocyanine antiscrapie compounds. Science 2000, 287 (5457), 1503-1506.
40. Valiente-Gabioud, A. A.; Miotto, M. C.; Chesta, M. E.; Lombardo, V.; Binolfi, A.; Fernández, C. O., Phthalocyanines as molecular scaffolds to block disease-associated protein aggregation. Acc. Chem. Res. 2016 May 17;49(5):801-8. 2016, 49 (5), 801-808.
41. Wu, J.; Zhao, J.; Yang, Z.; Li, H.; Gao, Z., Strong inhibitory effect of heme on hIAPP fibrillation. Chem. Res. Toxicol. 2017, 30 (9), 1711-1719.
42. Hayden, E. Y.; Kaur, P.; Williams, T. L.; Matsui, H.; Yeh, S.-R.; Rousseau, D. L., Heme stabilization of α-synuclein oligomers during amyloid fibril formation. Biochemistry 2015, 54 (30), 4599-4610.
43. Bao, Q.; Luo, Y.; Li, W.; Sun, X.; Zhu, C.; Li, P.; Huang, Z. X.; Tan, X., The mechanism for heme to prevent Abeta(1-40) aggregation and its cytotoxicity. J. Biol. Inorg. Chem. 2011, 16 (5), 809-16.
44. Ye, H.; Zhou, J.; Li, H.; Gao, Z., Heme prevents highly amyloidogenic human calcitonin (hCT) aggregation: a potential new strategy for the clinical reuse of hCT. J. Inorg. Biochem. 2019, 196, 110686.
45. 李昆峰; 高肇鴻; 陳錚誼; 趙啟民; 卓慧如; 科儀新知, 林. J., 磁性奈米粒子於生醫領域之應用. 2006, (153), 61-69.
46. Turcheniuk, K.; Tarasevych, A. V.; Kukhar, V. P.; Boukherroub, R.; Szunerits, S., Recent advances in surface chemistry strategies for the fabrication of functional iron oxide based magnetic nanoparticles. Nanoscale 2013, 5 (22), 10729-52.
47. Yu, J.; Liu, F.; Muhammad, Z. Y.; Hou, Y.-L., Magnetic nanoparticles: chemical synthesis, functionalization and biomedical applications. Adv. Mater. 2013, 40 (10).
48. Gu, H.; Xu, K.; Yang, Z.; Chang, C. K.; Xu, B., Synthesis and cellular uptake of porphyrin decorated iron oxide nanoparticles-a potential candidate for bimodal anticancer therapy. Chem. Commun. (Camb) 2005, (34), 4270-2.
49. Banu, N.; Pavithra, S., A review on the bioactivity of various metallochlorophyllin-a chlorophyll derivative. RJPT 2017, 10 (2).
50. Zhan, R.; Wu, J.; Ouyang, J., In vitro antioxidant activities of sodium zinc and sodium iron chlorophyllins from pine needles. Food Technol. Biotechnol. 2014, 52, 505-510.
51. Lu, Y. C.; Luo, P. C.; Huang, C. W.; Leu, Y. L.; Wang, T. H.; Wei, K. C.; Wang, H. E.; Ma, Y. H., Augmented cellular uptake of nanoparticles using tea catechins: effect of surface modification on nanoparticle-cell interaction. Nanoscale 2014, 6 (17), 10297-306.
52. Chen, Y. W.; Chang, C. W.; Hung, H. S.; Kung, M. L.; Yeh, B. W.; Hsieh, S., Magnetite nanoparticle interactions with insulin amyloid fibrils. Nanotechnology 2016, 27 (41), 415702.
53. Bellova, A.; Bystrenova, E.; Koneracka, M.; Kopcansky, P.; Valle, F.; Tomasovicova, N.; Timko, M.; Bagelova, J.; Biscarini, F.; Gazova, Z., Effect of Fe3O4 magnetic nanoparticles on lysozyme amyloid aggregation. Nanotechnology 2010, 21 (6), 065103.
54. Pradhan, N.; Jana, N. R.; Jana, N. R., Inhibition of protein aggregation by iron oxide nanoparticles conjugated with glutamine- and proline-based osmolytes. ACS Appl. Nano Mater. 2018, 1 (3), 1094-1103.
55. Javdani, N.; Rahpeyma, S. S.; Ghasemi, Y.; Raheb, J., Effect of superparamagnetic nanoparticles coated with various electric charges on alpha-synuclein and beta-amyloid proteins fibrillation process. Int. J. Nanomed. 2019, 14, 799-808.
56. Wang, M.; Kakinen, A.; Pilkington, E. H.; Davis, T. P.; Ke, P. C., Differential effects of silver and iron oxide nanoparticles on IAPP amyloid aggregation. Biomater. Sci. 2017, 5 (3), 485-493.
57. Khaengkhan, P.; Nishikaze, Y.; Niidome, T.; Kanaori, K.; Tajima, K.; Ichida, M.; Harada, S.; Sugimoto, H.; Kamei, K., Identification of an antiamyloidogenic substance from mulberry leaves. Neuroreport 2009, 20 (13), 1214-8.
58. Tu, Y.; Ma, S.; Liu, F.; Sun, Y.; Dong, X., Hematoxylin inhibits amyloid beta-protein fibrillation and alleviates amyloid-induced cytotoxicity. J. Phys. Chem. B 2016, 120 (44), 11360-11368.
59. 詹璦綺. 胡椒酸衍生物用於加速胰島類澱粉蛋白聚集. 國立臺灣師範大學, 台北市, 2021.
60. 陳怡婷. 人類降鈣素雙突變體提升抗纖維化能力及做為胜肽藥物之潛能. 國立臺灣師範大學, 台北市, 2019.
61. Bensakhria, A. High Performance Liquid Chromatography (HPLC), 2017. Please refer to analyticaltoxicology online system. Retrieved June 27, 2022, from https://www.analyticaltoxicology.com/en/high-performance-liquid-chromatography-hplc/.
62. 舒振諼. HPLC high performance liquid chromatography, 2017. Retrieved June 27, 2022, from https://slidesplayer.com/slide/11388700/.
63. 古佩芝. 奈米膠體金輔助雷射脫附游離飛行時間質譜法分析低分子量化合物的研究. 東海大學, 台中市, 2009.
64. SHIMADZU. Crop. Principles of MALDI-TOF mass spectrometry. n. d. Retrieved June 27, 2022, from https://www.shimadzu.cz/lifescience/maldi/princpl1.
65. Leszyk, J. D. J. J. o. b. t. J., Evaluation of the new MALDI matrix 4-chloro-α-cyanocinnamic acid. J. Biomol. Tech. 2010, 21 (2), 81.
66. Shieh, D. B.; Cheng, F. Y.; Su, C. H.; Yeh, C. S.; Wu, M. T.; Wu, Y. N.; Tsai, C. Y.; Wu, C. L.; Chen, D. H.; Chou, C. H., Aqueous dispersions of magnetite nanoparticles with NH3+ surfaces for magnetic manipulations of biomolecules and MRI contrast agents. Biomaterials 2005, 26 (34), 7183-91.
67. TREKINTAL Crop.奈米顆粒-DLS分析原理. n.d. Retrieved June 27, 2022, from https://www.trekintal.com.tw/particle/dynamic-light-scattering/.
68. OTSUKA. Crop. 界達電位粒徑分析儀. n. d. Retrieved June 27, 2022, from. https://www.otsuka-tw.com/product-detail/ELSZ2000/.
69. OTSUKA. Crop. dynamic light scattering spectrophotometer. n. d. Retrieved June 27, 2022, from https://www.otsukael.com/product/detail/productid/23.
70. 黃苡叡. 膠體晶體與反蛋白石結構之製作及工程應用. 國立交通大學, 新竹市, 2010.
71. 百科知識. 分子螢光光譜分析. n. d. Retrieved June 27, 2022, from https://www.easyatm.com.tw/wiki/%E5%88%86%E5%AD%90%E7%86%92%E5%85%89%E5%85%89%E8%AD%9C%E5%88%86%E6%9E%90
72. Hawe, A.; Sutter, M.; Jiskoot, W., Extrinsic fluorescent dyes as tools for protein characterization. Pharm. Res. 2008, 25 (7), 1487-99.
73. Biancalana, M.; Koide, S., Molecular mechanism of thioflavin-T binding to amyloid fibrils. Biochim. Biophys. Acta 2010, 1804 (7), 1405-12.
74. Andreea, S., SDS page protocol. 2020. Retrieved June 27, 2022, from https://www.protocols.io/view/sds-page-q26g7bqj8lwz/v1
75. Kumar, G., Principle and method of silver staining of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Methods Mol. Biol. 2018, 1853, 231-236.
76. Trehan, A.; Rotgers, E.; Coffey, E.; Huhtaniemi, I.; Rivero-Müller, A., Candles, an assay for monitoring GPCR induced cAMP generation in cell cultures. Cell Commun. Signal. 2014, 12, 70.
77. Song, C.; Zhang, S.; Huang, H., Choosing a suitable method for the identification of replication origins in microbial genomes. Front. Microbiol. 2015, 6, 1049.
78. Liu, Y.; Peterson, D. A.; Kimura, H.; Schubert, D., Mechanism of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. J. neurochem. 1997, 69 (2), 581-93.
79. wikipedia. MTT試驗. n. d. Retrieved June 27, 2022, from https://zh.wikipedia.org/wiki/MTT%E8%A9%A6%E9%A9%97.
80. Myers, R. The basic chemistry of hematoxylin. n. d. Retrieved June 27, 2022, from https://www.leicabiosystems.com/zh/knowledge-pathway/the-basic-chemistry-of-hematoxylin/.
81. 沈采玲. 磁性奈米材料用於抑制人類降鈣素聚集. 國立臺灣師範大學, 台北市, 2021.
82. Bhattacharjee, S., DLS and zeta potential - What they are and what they are not? J Control Release 2016, 235, 337-351.