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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 謝銘鈞 | |
dc.contributor.author | Tang-Min Lin | en |
dc.contributor.author | 林塘閔 | zh_TW |
dc.date.accessioned | 2021-06-07T17:56:18Z | - |
dc.date.copyright | 2012-08-22 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-08-14 | |
dc.identifier.citation | [1] H. Kikuchi, M.S. Pino, M. Zeng, S. Shirasawa, D.C. Chung, Oncogenic KRAS and BRAF differentially regulate hypoxia-inducible factor-1alpha and -2alpha in colon cancer. Cancer research 69 (2009) 8499-8506.
[2] M. Jhawer, S. Goel, A.J. Wilson, C. Montagna, Y.H. Ling, D.S. Byun, S. Nasser, D. Arango, J. Shin, L. Klampfer, L.H. Augenlicht, R. Perez-Soler, J.M. Mariadason, PIK3CA mutation/PTEN expression status predicts response of colon cancer cells to the epidermal growth factor receptor inhibitor cetuximab. Cancer research 68 (2008) 1953-1961. [3] A. Citri, Y. Yarden, EGF-ERBB signalling: towards the systems level. Nature reviews. Molecular cell biology 7 (2006) 505-516. [4] S. Van Schaeybroeck, J.N. Kyula, A. Fenton, C.S. Fenning, T. Sasazuki, S. Shirasawa, D.B. Longley, P.G. Johnston, Oncogenic Kras promotes chemotherapy-induced growth factor shedding via ADAM17. Cancer research 71 (2011) 1071-1080. [5] Cetuximab for Metastatic Colorectal Cancer. New England Journal of Medicine 361 (2009) 95-97. [6] P. Matar, F. Rojo, R. Cassia, G. Moreno-Bueno, S. Di Cosimo, J. Tabernero, M. Guzman, S. Rodriguez, J. Arribas, J. Palacios, J. Baselga, Combined epidermal growth factor receptor targeting with the tyrosine kinase inhibitor gefitinib (ZD1839) and the monoclonal antibody cetuximab (IMC-C225): superiority over single-agent receptor targeting. Clinical cancer research : an official journal of the American Association for Cancer Research 10 (2004) 6487-6501. [7] R.B. Corcoran, H. Ebi, A.B. Turke, E.M. Coffee, M. Nishino, A.P. Cogdill, R.D. Brown, P.D. Pelle, D. Dias-Santagata, K.E. Hung, K.T. Flaherty, A. Piris, J.A. Wargo, J. Settleman, M. Mino-Kenudson, J.A. Engelman, EGFR-mediated re-activation of MAPK signaling contributes to insensitivity of BRAF mutant colorectal cancers to RAF inhibition with vemurafenib. Cancer discovery 2 (2012) 227-235. [8] A. Lievre, J.B. Bachet, D. Le Corre, V. Boige, B. Landi, J.F. Emile, J.F. Cote, G. Tomasic, C. Penna, M. Ducreux, P. Rougier, F. Penault-Llorca, P. Laurent-Puig, KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer research 66 (2006) 3992-3995. [9] M.S. Boguski, F. McCormick, Proteins regulating Ras and its relatives. Nature 366 (1993) 643-654. [10] T.M. Brand, D.L. Wheeler, KRAS mutant colorectal tumors: Past and present. Small GTPases 3 (2012) 5-10. [11] H. Zhu, Z.Y. Liang, X.Y. Ren, T.H. Li, Small interfering RNAs targeting mutant K-ras inhibit human pancreatic carcinoma cells growth in vitro and in vivo. Cancer Biology & Therapy 5 (2006) 1693-1698. [12] P.D. Zamore, T. Tuschl, P.A. Sharp, D.P. Bartel, RNAi: Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals. Cell 101 (2000) 25-33. [13] J.B. Fleming, G.L. Shen, S.E. Holloway, M. Davis, R.A. Brekken, Molecular consequences of silencing mutant K-ras in pancreatic cancer cells: justification for K-ras-directed therapy. Molecular cancer research : MCR 3 (2005) 413-423. [14] S.M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, T. Tuschl, Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411 (2001) 494-498. [15] A.E. Felber, B. Castagner, M. Elsabahy, G.F. Deleavey, M.J. Damha, J.C. Leroux, siRNA nanocarriers based on methacrylic acid copolymers. Journal of controlled release : official journal of the Controlled Release Society 152 (2011) 159-167. [16] T. Friedmann, R. Roblin, Gene Therapy for Human Genetic Disease? Science 175 (1972) 949-955. [17] Toufik Abbas-Terki, William Blanco-Bose, Nicole Déglon, William Pralong, and Patrick Aebischer. Human Gene Therapy. December 2002, 13(18): 2197-2201. doi:10.1089/104303402320987888. [18] S. Martino, I. di Girolamo, R. Tiribuzi, F. D'Angelo, A. Datti, A. Orlacchio, Efficient siRNA delivery by the cationic liposome DOTAP in human hematopoietic stem cells differentiating into dendritic cells. Journal of biomedicine & biotechnology 2009 (2009) 410260. [19] X.-B. Xiong, A. Lavasanifar, Traceable Multifunctional Micellar Nanocarriers for Cancer-Targeted Co-delivery of MDR-1 siRNA and Doxorubicin. ACS Nano 5 (2011) 5202-5213. [20] C. Boyer, G. Boutevin, J.J. Robin, B. Boutevin, Study of the telomerization of dimethylaminoethyl methacrylate (DMAEMA) with mercaptoethanol. Application to the synthesis of a new macromonomer. Polymer 45 (2004) 7863-7876. [21] Y.C. Chen, L.C. Liao, P.L. Lu, C.L. Lo, H.C. Tsai, C.Y. Huang, K.C. Wei, T.C. Yen, G.H. Hsiue, The accumulation of dual pH and temperature responsive micelles in tumors. Biomaterials 33 (2012) 4576-4588. [22] D.J. McClements, S.R. Dungan, Factors that affect the rate of oil exchange between oil-in-water emulsion droplets stabilized by a nonionic surfactant: droplet size, surfactant concentration, and ionic strength. The Journal of Physical Chemistry 97 (1993) 7304-7308. [23] C.-L. Peng, P.-S. Lai, F.-H. Lin, S. Yueh-Hsiu Wu, 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 30 (2009) 3614-3625. [24] E. Yancheva, D. Paneva, V. Maximova, L. Mespouille, P. Dubois, N. Manolova, I. Rashkov, Polyelectrolyte Complexes between (Cross-linked) N-Carboxyethylchitosan and (Quaternized) Poly[2-(dimethylamino)ethyl methacrylate]: Preparation, Characterization, and Antibacterial Properties. Biomacromolecules 8 (2007) 976-984. [25] M.J. McCall, H. Diril, C.F. Meares, Simplified method for conjugating macrocyclic bifunctional chelating agents to antibodies via 2-iminothiolane. Bioconjugate Chemistry 1 (1990) 222-226. [26] K. Hu, J. Li, Y. Shen, W. Lu, X. Gao, Q. Zhang, X. Jiang, Lactoferrin-conjugated PEG–PLA nanoparticles with improved brain delivery: In vitro and in vivo evaluations. Journal of Controlled Release 134 (2009) 55-61. [27] C.-L. Peng, L.-Y. Yang, T.-Y. Luo, P.-S. Lai, S.-J. Yang, W.-J. Lin, M.-J. Shieh, Development of pH sensitive 2-(diisopropylamino)ethyl methacrylate based nanoparticles for photodynamic therapy. Nanotechnology 21 (2010) 155103. [28] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65 (1983) 55-63. [29] C.-L. Peng, Y.-H. Shih, P.-C. Lee, T.M.-H. Hsieh, T.-Y. Luo, M.-J. Shieh, Multimodal Image-Guided Photothermal Therapy Mediated by 188Re-Labeled Micelles Containing a Cyanine-Type Photosensitizer. ACS Nano 5 (2011) 5594-5607. [30] S.M. Henry, M.E.H. El-Sayed, C.M. Pirie, A.S. Hoffman, P.S. Stayton, pH-Responsive Poly(styrene-alt-maleic anhydride) Alkylamide Copolymers for Intracellular Drug Delivery. Biomacromolecules 7 (2006) 2407-2414. [31] Y.-C. Chung, W.-Y. Hsieh, T.-H. Young, Polycation/DNA complexes coated with oligonucleotides for gene delivery. Biomaterials 31 (2010) 4194-4203. [32] J. Gao, W. Liu, Y. Xia, W. Li, J. Sun, H. Chen, B. Li, D. Zhang, W. Qian, Y. Meng, L. Deng, H. Wang, J. Chen, Y. Guo, The promotion of siRNA delivery to breast cancer overexpressing epidermal growth factor receptor through anti-EGFR antibody conjugation by immunoliposomes. Biomaterials 32 (2011) 3459-3470. [33] S. Lin, F. Du, Y. Wang, S. Ji, D. Liang, L. Yu, Z. Li, An Acid-Labile Block Copolymer of PDMAEMA and PEG as Potential Carrier for Intelligent Gene Delivery Systems. Biomacromolecules 9 (2007) 109-115. [34] H. Mok, S.H. Lee, J.W. Park, T.G. Park, Multimeric small interfering ribonucleic acid for highly efficient sequence-specific gene silencing. Nat Mater 9 (2010) 272-278. [35] S.Y. Lee, M.S. Huh, S. Lee, S.J. Lee, H. Chung, J.H. Park, Y.K. Oh, K. Choi, K. Kim, I.C. Kwon, Stability and cellular uptake of polymerized siRNA (poly-siRNA)/polyethylenimine (PEI) complexes for efficient gene silencing. Journal of controlled release : official journal of the Controlled Release Society 141 (2010) 339-346. [36] H. Mok, T.G. Park, Self-crosslinked and reducible fusogenic peptides for intracellular delivery of siRNA. Biopolymers 89 (2008) 881-888. [37] M.-J. Shieh, C.-L. Peng, P.-J. Lou, C.-H. Chiu, T.-Y. Tsai, C.-Y. Hsu, C.-Y. Yeh, P.-S. Lai, Non-toxic phototriggered gene transfection by PAMAM-porphyrin conjugates. Journal of Controlled Release 129 (2008) 200-206. [38] S. Motala-Timol, D. Jhurry, Synthesis of PDMAEMA–PCL–PDMAEMA triblock copolymers. European Polymer Journal 43 (2007) 3042-3049. [39] S. Guo, Y. Qiao, W. Wang, J. Xing, L. Deng, A. Dong, J. Xu, Synthesis and properties of Polycaprolactone-graft-poly(2-(dimethylamino)ethyl methacrylate-co-methoxy polyethylene glycol monomethacrylate) as non-viral gene vector. Polymers for Advanced Technologies 22 (2011) 1925-1930. [40] C. Zhu, S. Jung, S. Luo, F. Meng, X. Zhu, T.G. Park, Z. Zhong, Co-delivery of siRNA and paclitaxel into cancer cells by biodegradable cationic micelles based on PDMAEMA–PCL–PDMAEMA triblock copolymers. Biomaterials 31 (2010) 2408-2416. [41] S. Dai, Z. Li, Enzymatic Preparation of Novel Thermoplastic di-Block Copolyesters Containing Poly[(R)-3-hydroxybutyrate] and Poly(ϵ-Caprolactone) Blocks via Ring-Opening Polymerization. Biomacromolecules 9 (2008) 1883-1893. [42] Zhang, W., He, J., Liu, Z., Ni, P. and Zhu, X. (2010), Biocompatible and pH-responsive triblock copolymer mPEG-b-PCL-b-PDMAEMA: Synthesis, self-assembly, and application. J. Polym. Sci. A Polym. Chem. [43] C.L. Peng, H.M. Tsai, S.J. Yang, T.Y. Luo, C.F. Lin, W.J. Lin, M.J. Shieh, Development of thermosensitive poly(n-isopropylacrylamide-co-((2-dimethylamino) ethyl methacrylate))-based nanoparticles for controlled drug release. Nanotechnology 22 (2011) 265608. [44] Jean-Christophe Olivier, Ramon Huertas, Hwa Jeong Lee, Frederic Calon and William M. Pardridge,Synthesis of Pegylated Immunonanoparticles, Volume 19, Number 8 (2002), 1137-1143, Pharmaceutical Research [45] Q. Gu, J.Z. Xing, M. Huang, C. He, J. Chen, SN-38 loaded polymeric micelles to enhance cancer therapy. Nanotechnology 23 (2012) 205101. [46] L.Y. Qiu, Y.H. Bae, Self-assembled polyethylenimine-graft-poly(ε-caprolactone) micelles as potential dual carriers of genes and anticancer drugs. Biomaterials 28 (2007) 4132-4142. [47] D. Balin-Gauthier, J.P. Delord, P. Rochaix, V. Mallard, F. Thomas, I. Hennebelle, R. Bugat, P. Canal, C. Allal, In vivo and in vitro antitumor activity of oxaliplatin in combination with cetuximab in human colorectal tumor cell lines expressing different level of EGFR. Cancer chemotherapy and pharmacology 57 (2006) 709-718. [48] A.J. Convertine, C. Diab, M. Prieve, A. Paschal, A.S. Hoffman, P.H. Johnson, P.S. Stayton, pH-Responsive Polymeric Micelle Carriers for siRNA Drugs. Biomacromolecules 11 (2010) 2904-2911. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15954 | - |
dc.description.abstract | 近年來,惡性腫瘤已為國人十大死因之首,然而,大腸癌位居惡性腫瘤耗發率之前三名,可見對於大腸癌之診斷與治療之重要性,為了實現更良好的大腸癌治療,高分子奈米藥物載體被廣泛運用在藥物傳輸系統來治療癌症,利用雙親性高分子包覆化療藥物或光感藥物,進行癌細胞毒殺並減緩單純施予藥物對正常細胞之毒性與副作用產生,然而現今之藥物載體並非僅限於藥物傳輸,基因治療與標靶治療也於近年來蓬勃發展。奈米藥物載體之優點為,可利用藥物包覆減低非作用目標之細胞攝取,再者,奈米級載體可藉由其粒徑極小之特性產生增強通透性與延遲效應(EPR effects)進而達成在腫瘤組織之被動標靶(passive targeting)的特性,增強藥物累積量與減少藥物之使用量,除此之外,利用核糖核酸干擾技術(RNAi)抑制特定基因之表現,對於某些疾病有良好的治癒能力,合併基因治療與化學治療於癌症治癒上可以達到加成效應也為現今熱門研究的方向,特別的是,標靶治療為現今癌症治療之方法之一,對於惡性腫瘤有顯著抑制增長的成果,將標靶藥物修飾於奈米藥物載體外圍並整合基因與化學治療之奈米藥物載體,可以相對增加癌症細胞攝取藥物之專一性,並增強毒殺腫瘤之能力,形成多功能之藥物載體。
在人類大腸癌細胞(HCT-116)中,其K-ras基因為突變型,導致其長期處於活性狀態,不受上游上皮生長因子受體(EGFR)之控制使其下游蛋白持續磷酸化,進而造成癌細胞增生與轉移,因此,本篇碩士論文將利用帶正電之高分子p(DMAEMA-co-PEGMEA)-b-PCL與mal-PEG-b-PCL以特定比例形成混和型奈米微胞載體,並利用靜電作用吸附帶負電之K-ras siRNA,以及利用疏水端性質包覆化療藥物SN-38,再者,將奈米微胞表面共價鍵結標靶藥物Erbitux®以達成具有標靶能力同時合併基因與化學治療之多功能型奈米藥物微胞載體,在物理性質方面,粒徑大小位於200-300nm左右,藥物包覆率也可達成60%以上,在臨界微胞濃度值方面,表現此奈米微胞在大量稀釋環境中很穩定並且在於含有血球與蛋白之環境中,安全無虞,除此之外,在細胞攝取量、細胞毒性與轉染效率等觀察,可發現多功能性微胞皆有較佳之治療能力,即為一高潛力之奈米藥物載體,對於癌症治癒之療程與方法有所增進,並希望對於生物醫學領域有些許貢獻。 | zh_TW |
dc.description.abstract | Recently, cancer has been the top reasons of people’s death and colorectal cancer would be the most common diseases of all human cancers, so that the examinations and therapies of colon cancers would be an important thing. For better ways of curing cancers, polymeric nanoparticles were widely used in the field of drug delivery systems. For example, amphiphilic micelles were carried out to encapsulate hydrophobic anticancer drugs or photosensitizers to reduce the cytotoxicity and side effects to normal cells when anticancer drugs were administered directly. The advantages of nano-carriers could be generalized in the following reasons. Nano-sized vectors could reach enhanced permeability and retention (EPR) effects in tumor cells. The passive target causes of accumulations of drugs at tumor sites and could reduce the amount of anticancer drugs used. However, drug delivery systems not only includes drug delivery but gene therapy which is one of the popular methods and well developed in the decades. According to the reasons, if combine chemotherapy with gene therapy, maybe the abilities of killing cancers would be additive. Another, targeting therapy is one widely used way of curing cancers recently and was found to be an efficient method. Hence, we could integrate targeting molecules by surface modifications of micelles and mult-functional nano-medicine was made to reach specific killing of cancers.
In human colorectal cancer cells, HCT-116 cells, K-ras gene was observed mutant, leading to its staying in an activated state all the time. They become uncontrollable to its up-streams such as EGFR and make down-stream proteins continuously phosphorylated. Cells will keep migrating, surviving, and proliferating. Hence, in this study, positively charged polymers p(DMAEMA-co-PEGMEA)-b-PCL were synthesized and its mixture with mal-PEG-b-PCL were formed. Moreover, K-ras siRNA was attached on micelles by electrostatic interactions. Hydrophobic anticancer drug, SN-38, was loaded in the core of micelles, and furthermore, the antibody, C225 was conjugated onto the maleimide group of micelles by covalent bonds to reach genetic and chemo-therapies with targeting at the same time. In physical and chemical properties, the sizes were about 200 nm to 300 nm, and the drug encapsulated efficiency would approximately over 60 %. Nevertheless, the critical micelles concentrations (CMC) were all small enough to be stable in mass dilutions. Particularly, the micelles are safe especially in the environments filled with blood cells and proteins. Moreover, the cell viabilities, cell uptake, and even gene transfection efficiencies were all in good results, which make the vectors to be a potential nano-carrier in curing cancers. Hope that the research would reach little progress in the field of bio-medical engineering. | en |
dc.description.provenance | Made available in DSpace on 2021-06-07T17:56:18Z (GMT). No. of bitstreams: 1 ntu-101-R99548036-1.pdf: 6764628 bytes, checksum: 478bdd9d714267d1f4ed338610e828df (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 口試委員會審定書 #
致謝 i 中文摘要 iii ABSTRACT iv CONTENTS vi LIST OF SCHEMES ix LIST OF TABLES x LIST OF FIGURES xi Chapter 1 Introduction 1 Chapter 2 Experiments procedure 4 2.1 Materials 5 2.2 Synthesis of copolymer poly(DMAEMA-co-PEGMEA)-OH 5 2.3 Syntheses of polymers maleimide-PEG-b-PCL and brush like p(DMAEMA-co-PEGMEA)-b-PCL 6 2.4 Characterizations of polymers 7 2.5 Preparations of micelles with/without siRNA attached, SN-38 loaded, and its drug encapsulation efficiency and drug content 9 2.6 In vitro drug release profile of SN-38 10 2.7 Thiolation, preparation and observation of C225 conjugations 11 2.8 Critical micelle concentration and pH sensitivity 12 2.9 Cell culture and in vitro cytotoxicity 12 2.10 Static light scattering and numbers of SN-38 loaded (siRNA attached) per micelle 13 2.11 Internalization of SN-38 loaded micelles 14 2.12 Human red blood cell hemolysis test and erythrocyte agglutination study 15 2.13 Albumin-induced polyplexes aggregation 16 2.14 Gel retardation assay 16 2.15 Heparin Compatibility and Serum stability tests 16 2.16 Flow cytometry analysis 17 2.17 Confocal laser scanning microscopy 18 2.18 In vitro gene transfection efficiency 18 2.19 Western blot analysis 19 2.20 Statistical analysis 20 Chapter 3 Results and discussions 21 3.1 Syntheses and characterizations of mal-PEG-b-PCL and p(DMAEMA-co-PEGMEA)-b-PCL 21 3.2 Drug release profiles 25 3.3 Critical micelle concentration and pH sensitivity 26 3.4 Compositions of micelles 28 3.5 In vitro cytotoxicity 28 3.6 Internalization of SN-38 30 3.7 Gel retardation assay 30 3.8 Safety concerns of micelles in blood and protein environments 31 3.9 Flow cytometry and cellular uptake 34 3.10 Simultaneous delivery of hydrophobic dye and siRNA 35 3.11 Proton sponge effect 35 3.12 Transfection using a luciferase reporter gene 36 3.13 Western blotting of K-ras protein 36 3.14 Cell proliferations 37 3.15 Multiple function micelle 37 Chapter 4 Conclusions 39 REFERENCE 40 SCHEME 47 TABLE 52 FIGURE 58 | |
dc.language.iso | en | |
dc.title | 製備多功能奈米微胞載體應用於K-RAS基因突變之大腸癌細胞結合EGFR標靶性並進行合併基因與化學治療之毒殺與評估 | zh_TW |
dc.title | Synthesis of Brush-like p(DMAEMA-co-PEGMEA)-b-PCL
as K-RAS siRNA and SN-38 Carrier for Dual Genetic and Chemo- therapies with EGFR Targeting in K-RAS Mutation HCT-116 Colon Cancer Model | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 賴秉杉,張富雄,羅彩月,駱俊良 | |
dc.subject.keyword | 基因治療,化療藥物,標靶治療,癌症,奈米藥物載體, | zh_TW |
dc.subject.keyword | SN-38,Cetuximab,K-ras gene,micelle,colon cancer, | en |
dc.relation.page | 87 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2012-08-15 | |
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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