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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 謝銘鈞(Shieh Ming-Jium) | |
| dc.contributor.author | Yu-Chi Chiou | en |
| dc.contributor.author | 邱鈺棋 | zh_TW |
| dc.date.accessioned | 2021-06-07T17:53:30Z | - |
| dc.date.copyright | 2012-08-18 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-18 | |
| dc.identifier.citation | Reference
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McLeod, Human Carboxylesterase 2 Is Commonly Expressed in Tumor Tissue and Is Correlated with Activation of Irinotecan. Clinical Cancer Research, 2002. 8(8): p. 2605-2611. 16. Sanghani, S.P., S.K. Quinney, T.B. Fredenburg, Z. Sun, W.I. Davis, D.J. Murry, O.W. Cummings, D.E. Seitz, and W.F. Bosron, Carboxylesterases Expressed in Human Colon Tumor Tissue and Their Role in CPT-11 Hydrolysis. Clinical Cancer Research, 2003. 9(13): p. 4983-4991. 17. Wu, W., R. Li, X. Bian, Z. Zhu, D. Ding, X. Li, Z. Jia, X. Jiang, and Y. Hu, Covalently Combining Carbon Nanotubes with Anticancer Agent: Preparation and Antitumor Activity. ACS Nano, 2009. 3(9): p. 2740-2750. 18. Sharpe, M.A., D.C. Marcano, J.M. Berlin, M.A. Widmayer, D.S. Baskin, and J.M. Tour, Antibody-Targeted Nanovectors for the Treatment of Brain Cancers. ACS Nano, 2012. 6(4): p. 3114-3120. 19. Berlin, J.M., T.T. Pham, D. Sano, K.A. Mohamedali, D.C. Marcano, J.N. Myers, and J.M. Tour, Noncovalent Functionalization of Carbon Nanovectors with an Antibody Enables Targeted Drug Delivery. ACS Nano, 2011. 5(8): p. 6643-6650. 20. Wang, R., C. Mikoryak, E. Chen, S. Li, P. Pantano, and R.K. Draper, Gel Electrophoresis Method to Measure the Concentration of Single-Walled Carbon Nanotubes Extracted from Biological Tissue. Analytical Chemistry, 2009. 81(8): p. 2944-2952. 21. Peng, C.L., P.S. Lai, F.H. Lin, S. Yueh-Hsiu Wu, and M.J. Shieh, Dual chemotherapy and photodynamic therapy in an HT-29 human colon cancer xenograft model using SN-38-loaded chlorin-core star block copolymer micelles. Biomaterials, 2009. 30(21): p. 3614-25. 22. Mukherjee, S., R.N. Ghosh, and F.R. Maxfield, Endocytosis. Physiological Reviews, 1997. 77(3): p. 759-803. 23. Kam, N.W.S., T.C. Jessop, P.A. Wender, and H. Dai, Nanotube molecular transporters: internalization of carbon nanotube-protein conjugates into mammalian cells. Journal of the American Chemical Society, 2004. 126(22): p. 6850-6851. 24. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15846 | - |
| dc.description.abstract | 單壁奈米碳管作為奈米藥物載體結合喜樹鹼衍生物SN-38以及單株抗體爾必得舒Erbitux(c225) ，並探討以單壁奈米碳管作為藥物載體的藥物釋放、細胞途徑以及標靶治療之療效。
本論文中針對不同的表皮生長因子接受器(EGFR)表現量，選擇了三株大腸直腸癌細胞，分別是HCT116、HT29及SW620。其EGFR表現量的表現程度多寡:HCT116大於 HT29大於 SW620。此藥物載體針對這三株大腸直腸癌細胞皆能抑制50%以上的細胞存活率，並且EGFR表現程度愈高、愈能夠降低癌細胞存活率，其中EGFR表現量最高的HCT116之細胞存活率只剩10%。此奈米碳管藥物載體在一般生理環境pH 7.4 的藥物釋放率(20%)遠小於在h-CE (human carboxylesterase enzyme)羧酸酯酶中(80%) 及三株癌細胞的萃取蛋白中(60%)。此藥物載體利用C225主動標靶EGFR表現程度高之癌細胞，並藉由h-CE2 酶的調控在細胞內部大量釋放，達到控制藥物釋放(drug control release)。 | zh_TW |
| dc.description.abstract | Single-walled carbon nanotubes (SWNTs) combined with chemotherapeutic drug 7-Ethyl-10-hydroxy-camptothecin (SN-38) and monoclonal antibody Erbitux (C225) . The carriers were designed to explore the specific binding ability, anti-proliferation ability against to colorectal cancer cell lines and drug control release. The monoclonal antibody Erbitux (C225) bind to EGFR specifically. Therefore the subjects here were 3 kinds of colorectal cancer cell lines with different level of EGFR expression. EGFR expression level of these cells is: HCT116>HT29>SW620. SWNT25/pyCPY carrier reduce more than 50% cellular viability to all these 3 kinds of cells. Moreover the cell viability of EGFR over-expressing cell HCT116 is only 10%. The cellular viability is lower while the cell’s EGFR expression is higher. The anti-proliferation ability of SWNT25/py38 carrier is EGFR-depending and EGFR-Targeting. The drug control release process was designed to utilizing Human Carboxylesterase enzyme (hCE) to detach the SN-38 from SWNTs .This drug-release process is supposed by hCE would broke the ester bond link the SN-38 and Pyrene. The percentage of SN38 releasing from SWNTs carrier in physical environment pH7.4 buffer(20%) is much lower than cell lysate(60%) and hCE(80%). The SWNT25/py38 carrier using C225 specifically bind to EGFR expressing cell and releasing abundant SN38 to kill the cells. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-07T17:53:30Z (GMT). No. of bitstreams: 1 ntu-101-R99548014-1.pdf: 3413041 bytes, checksum: 7a889c5376f78ae6730a0152702b4fba (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 口試委員會審定書 I
謝誌 II 中文摘要 III Abstract IV Contents V List of figures IX Chapter 1 Introduction 1 Chapter 2 Experimental design and method 4 2.1.1 Materials 4 2.2 Synthesis experimental method 5 2.2.1 Oxidizations and characterization of SWNTs 5 2.2.2 Funtionalization of SWNTs with PEG6000 6 2.2.3 Synthesis of Py38 conjugation 6 2.2.4 SN38 Drug Loading of SWNTs 7 2.2.5 Functionalization of SWNTs with C225 8 2.2.6 Combing SWNT-carrier with fluorescence marker 8 2.3 Characterization of SWNTs carriers 9 2.3.1 Transmission electron microscopy (TEM) 9 2.3.2 Concentration of SNWT 9 2.3.3 Drug loading of SN38 9 Fluorescence and UV-vis-NIR absorbance spectra of SWNT-carriers 10 2.3.4 Characterization of SWNT25/py38 carriers 10 SDS-PAGE 10 BCA protein assay 11 The gold conjugated goat-anti-human IgG secondary antibody 12 2.4 In vitro experiments 12 2.4.1 Cell line and Cell culture 12 2.4.2 EGFR expression 13 Western blot 13 Flow cytometry 13 2.4.3 Human Carboxylesterase enzyme expression 14 Revers-Transcription PCR 14 Western blot 15 2.4.4 In vitro cytotoxicity assay 15 MTT assay 15 Trypan blue assay(Dye exclusion assay) 16 Apoptosis assay 17 2.5 In vitro experiment: cellular uptake 18 2.5.1 Cellular uptake of SN-38 carried by SWNT-Carriers 18 2.5.2 Low temperature incubation and ATP depletion incubation 19 2.5.3 Hypertonic incubation and K+ depletion incubation 20 2.5.4 Flow cytometry. 20 2.5.5 Confocal microscopy 21 2.6 Drug release profile 21 2.7 hCE-2 breaking the ester bond of Py-38 22 2.8 Statistical Methods 22 Chapter 3 Results and discussion 23 3.1 Characterizations of SWNT-COOH and SWNT-PEG6000 23 3.1.1 EA (Elemental Analyzer) 23 3.1.2 1H-NMR spectrum of SWNT-PEG6000 23 3.1.3 FT-IR (Fourier Transform Infrared Spectrophotometer) 23 3.2 Characterizations of py-38 24 3.2.1 1H-NMR spectrum of py-38 24 3.2.2 Cellular cytotoxicity of py-38 24 3.3 Fluorescence and UV-vis-NIR absorbance spectra of SWNT-carrier 25 3.4 Drug content (%) of SWNT25/py38 and SNWT/py38 26 BCA -protein assay and C225 Drug content(%) of SWNT25/py38 27 SDS-page 27 The gold conjugated goat-anti-human IgG secondary antibody 28 3.5 Size distribution of SWNT-carriers 28 3.6 In vitro experiment 29 3.6.1 EGFR expression 29 3.6.2 hCE-2 expression 30 3.7 In vitro drug release profile 30 3.8 Cellular uptake 32 3.8.1 Confocal image and flow cytometry of 4 ℃ incubation 32 3.8.2 Cellular uptake of SN-38 carried by SWNT-Carriers 32 3.8.3 Low temperature incubation and ATP depletion incubation 34 3.8.4 Hypertonic incubation and K+ depletion incubation 35 3.9 Confocal microscopy 36 Tracking the SWNT25/py38 with lysotracker 36 SN38 drug release from SWNT25/py38 36 3.10 In vitro cytotoxicity 38 Cell cytotoxicity of SWNT-COOH and SWNT-PEG6000 38 Cell cytotoxicity of SNWT25/py38, SWNT/py38 and c225 for 72h 39 Cell cytotoxicity of Short time incubation for 2h 39 SWNT25/38 Pretreating with c225 and incubated with c225 together 40 Apoptosis assay 40 Chapter 4 Conclusion 42 Figure : 43 Reference 74 List of figures Figure 1. SWNT-carrier synthesis scheme 43 Figure 2. The hypothetical pathway of SWNT-carrier inside cells. 43 Figure 3. Chemical equation of Synthesis of Py-38 conjugation 44 Figure 4. A symbolize chemical formulation of SWNT-carrier modified with a fluorescence marker. 44 Figure 5. 1H-NMR spectrum of SWNT-PEG6000 45 Figure 6. Elemental analysis of SWNT-PEG,SWNT-COOH and SWNT 45 Figure 7. FT-IR spectrum of SWNT and SWNT-PEG6000. 46 Figure 8. The absorption of different concentration SWNT-PEG at 800nm was chosen to construct a calibration curve 46 Figure 9. 1H-NMR spectrum of (A) Pyrene, (B)SN-38 and (C) Py-38. 47 Figure 10. Cyto-toxicity of 500ng/ml Pyrene, SN-38 and Py-38 48 Figure 11. (A)The absorbance spectrum of SWNT-PEG/py38, SWNT-PEG and SN-38 free drug and (B) fluorescence spectrum of SWNT-PEG/py38 and SN-38 free drug 49 Figure 12. The composition and drug content (%)of SWNT/py38 (A) and the composition and drug content (%)of SWNT25/py38 (B) 49 Figure 13. The BCA protein assay of SWNT-C225/pySN38 and SWNT-PEG. 50 Figure 14. SDS-PAGE assay of SWNT carrier. 50 Figure 15. The TEM image of SWNT25/py38 with the 15nm gold conjugated goat-anti-human IgG secondary antibody was binding with the C225 antibody on SWNT sidewall. 51 Figure 16. TEM-EDX image of SWNT25/Py38. 51 Figure 17. TEM observations show that size distribution of (A)SWNT-COOH, (B) SWNT-PEG, (C) SWNT/py-38 and (D) SWNT25/py-38 52 Figure 18. The western blot for the EGFR expression of HCT116 ,HT29 and SW620. 53 Figure 19. The EGFR expression of HCT116, HT29 and SW620 analysis by using anti-human EGFR antibody and detected by Flow cytometry. 53 Figure 20. PCR analysis of hCE-1 and hCE-2 DNA expression in HCT116, HT29 and SW620.The result showed all of these 3kinds of colon cancer cell expressed hCE-2 DNA but lack of hCE-1. 54 Figure 21. Western blot analysis of hCE-2 enzyme expression if HCE116, HT29 and SW620.The result confirmed all these 3 kinds of cells expressed hCE-2 enzyme. 54 Figure 22. The drug release profile of SWNT25/py38. The environmental conditions were pH7.4, pH5 and 1unit hCE-2 in pH7.4. 55 Figure 23. The drug release profile of SWNT25/py38. The environmental conditions are HCT116 cell lysate, HT29 cell lysate and SW620 cell lysate all in the pH7.4 PBS surrounding. And the control environmental condition is ph7.4 PBS. 55 Figure 24. The drug releasing profile of SWNT25/py38 analysed by HPLC. 56 Figure 25. The Confocal image of SWNT25/38-rhodamine co-localized in cell. The HCT116, HT29 and SW620 are incubated for 10min in 4℃. 57 Figure 26. The flow cytometry data of SWNT25/38-rhodamine co-localized in cell. The HCT116, HT29 and SW620 are incubated for 5 min and 10min in 4℃. 57 Figure 27. The cellular uptake of (A)HCT116, (B) HT29 and (C) SW620 in 1h, 3h, 6h, 24h and 48h. 58 Figure 28. The cellular uptake of SWNT-C225/Py38 for HCT116,HT29 and SW630 in 0h,1h, 3h, 6h, 24h and 48h. 59 Figure 29. The cellular uptake of SWNT25/38-rhodamine treated to HCT116, HT29 and SW620 in 6h and analysis by flow cytometry. 59 Figure 30. The cyto-toxicity of low temperature incubation(4 ℃), ATP depletion incubation(NaN3), hypertonic incubation(Sucrose), and K+ depletion incubation(HEPES) in 2.5 hours. 60 Figure 31. The uptake here is defined as the SN38 fluorescence normalized with cell protein. The (A) HCT116 (B) HT29 and (C)SW620 cellular fluorescence uptake date obtained after incubated in 37℃, 37℃ with pretreated sodium azide and 4℃. 61 Figure 32. The uptake here is defined as the SN38 fluorescence normalized with cell protein. The (A) HCT116 (B) HT29 and (C)SW620 cellular fluorescence uptake date obtained after incubated in K+ depletion incubation(operated by HEPES) and hypertonic incubation (operated by sucrose). 62 Figure 33. Confocal microscope images of cellular uptake by fluorescent SWNTs (red) with HCT116, HT29 and SW620 cells. After 6 hours incubation, the SWNT was co-localized with a lysosomal marker (lysotracker green). Hoechst stain for nucleus. 63 Figure 34. Tracking of the SN-38 releasing from SWNT carrier inside the HCT116 cell. 64 Figure 35. Tracking of the SN-38 releasing from SWNT carrier inside the HT29 cell. 65 Figure 36. Tracking of the SN-38 releasing from SWNT carrier inside the SW620cell. 66 Figure 37. The cyto-toxicity of (A)HCT116, (B)HT29 and (C) SW620 after treated 1μg/ml~50μg /ml of SWNT-COOH for 72h. 67 Figure 38. The cyto-toxicity of (A)HCT116, (B)HT29 and (C) SW620 after treated 1μg/ml~50μg /ml of SWNT-PEG6000 for 72h. 68 Figure 39. The cyto-toxicity of SWNT25/py38, SWNT/py38 and C225 incubated with HCT116, HT29 and SW620 for 72h. 69 Figure 40. The cyto-toxicity of SWNT/py38 and C225 incubated with HCT116, HT29 and SW620 for 2hours and transfer fresh medium for continuing cultured for 72h. 69 Figure 41. The cyto-toxicity of C225, SWNT25/py38, SWNT25/py38 pretreated with C225, and SWNT/py38 with C225. The various condition incubated with (A) HCT116, (B) HT29 and (C)SW620 for 72hours. 70 Figure 42. The cyto-toxicity of SN38 and SN38 with C225 treated to cells. The various condition incubated with (A) HCT116, (B) HT29 and (C) SW620 for 72hours. 71 Figure 43. The cyto-toxicity of SWNT and SWNT-C225 treated to cells. The various condition incubated with (A) HCT116, (B) HT29 and (C) SW620 for 72hours. 72 Figure 44. The Annexin V-FITC/PI staining assay of SWNT25/py38, and C225 incubated with(A) HCT116,(B) HT29 and (C)SW620 for 72h analysis by flow cytometry. 73 | |
| dc.language.iso | en | |
| dc.subject | h-CE | zh_TW |
| dc.subject | 喜樹鹼衍生物SN-38 | zh_TW |
| dc.subject | 單壁奈米碳管 | zh_TW |
| dc.subject | 單株抗體Erbitux(c225) | zh_TW |
| dc.subject | 羧酸酯酶 | zh_TW |
| dc.title | 單壁奈米碳管結合化療藥物SN-38與單株抗體
作為大腸癌標靶治療之藥用載體 | zh_TW |
| dc.title | Combining Single-walled Carbon Nanotubes With
Anticancer Agents”SN-38” and EGFR antibody for Colorectal Cancer Targeting Chemotherapeutics | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 賴秉杉,張富雄,駱俊良,羅采月 | |
| dc.subject.keyword | 單壁奈米碳管,喜樹鹼衍生物SN-38,單株抗體Erbitux(c225),羧酸酯酶,h-CE, | zh_TW |
| dc.subject.keyword | Carbon nanotube,SN-38,Erbitux,Carboxylesterase enzyme, | en |
| dc.relation.page | 75 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2012-08-18 | |
| dc.contributor.author-college | 工學院 | zh_TW |
| dc.contributor.author-dept | 醫學工程學研究所 | zh_TW |
| 顯示於系所單位： | 醫學工程學研究所 | |
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