請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55737
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林文貞(Wen-Jen Lin) | |
dc.contributor.author | Wei-Chi Lee | en |
dc.contributor.author | 李瑋綺 | zh_TW |
dc.date.accessioned | 2021-06-16T04:20:34Z | - |
dc.date.available | 2019-08-21 | |
dc.date.copyright | 2014-10-20 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-19 | |
dc.identifier.citation | Abdelwahed W, Degobert G, Stainmesse S, Fessi H. Freeze-drying of nanoparticles: formulation, process and storage considerations. Adv Drug Deliv Rev. 2006;58(15):1688-713.
Adjei IM, Sharma B, Labhasetwar V. Nanoparticles: cellular uptake and cytotoxicity. Adv Exp Med Biol. 2014;811:73-91. Al-Deen FN, Selomulya C, Williams T. On designing stable magnetic vectors as carriers for malaria DNA vaccine. Colloids Surf B Biointerfaces. 2013;102:492-503. Alai M, Lin WJ. A novel once daily microparticulate dosage form comprising lansoprazole to prevent nocturnal acid breakthrough in the case of gastro-esophageal reflux disease: preparation, pharmacokinetic and pharmacodynamic evaluation. J Microencapsul. 2013;30(6):519-29. Ameringer T, Fransen P, Bean P, Johnson G, Pereira S, Evans RA, et al. Polymer coatings that display specific biological signals while preventing nonspecific interactions. J Biomed Mater Res A. 2011;100A(2):370-9. Amoozgar Z, Yeo Y. Recent advances in stealth coating of nanoparticle drug delivery systems. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2012;4(2):219-33. Balazs DA, Godbey W. Liposomes for Use in Gene Delivery. Journal of Drug Delivery. 2011;2011. Banerji S, Wright AJ, Noble M, Mahoney DJ, Campbell ID, Day AJ, et al. Structures of the Cd44-hyaluronan complex provide insight into a fundamental carbohydrate-protein interaction. Nat Struct Mol Biol. 2007;14(3):234-9. Betancourt T, Byrne JD, Sunaryo N, Crowder SW, Kadapakkam M, Patel S, et al. PEGylation strategies for active targeting of PLA/PLGA nanoparticles. J Biomed Mater Res A. 2009;91(1):263-76. Chang G, Wang J, Zhang H, Zhang Y, Wang C, Xu H, et al. CD44 targets Na(+)/H(+) exchanger 1 to mediate MDA-MB-231 cells' metastasis via the regulation of ERK1/2. Br J Cancer. 2014;110(4):916-27. Choi KY, Saravanakumar G, Park JH, Park K. Hyaluronic acid-based nanocarriers for intracellular targeting: interfacial interactions with proteins in cancer. Colloids Surf B Biointerfaces. 2012;99:82-94. Chung EJ, Cheng Y, Morshed R, Nord K, Han Y, Wegscheid ML, et al. Fibrin-binding, peptide amphiphile micelles for targeting glioblastoma. Biomaterials. 2014;35(4):1249-56. Clegg DO, Reda DJ, Harris CL, Klein MA, O'Dell JR, Hooper MM, et al. Glucosamine, chondroitin sulfate, and the two in combination for painful knee osteoarthritis. N Engl J Med. 2006;354(8):795-808. Conner SD, Schmid SL. Regulated portals of entry into the cell. Nature. 2003;422(6927):37-44. Costa P, Sousa Lobo JM. Modeling and comparison of dissolution profiles. Eur J Pharm Sci. 2001;13(2):123-33. Dufay Wojcicki A, Hillaireau H, Nascimento TL, Arpicco S, Taverna M, Ribes S, et al. Hyaluronic acid-bearing lipoplexes: physico-chemical characterization and in vitro targeting of the CD44 receptor. J Control Release. 2012;162(3):545-52. Duncan R, Richardson SC. Endocytosis and intracellular trafficking as gateways for nanomedicine delivery: opportunities and challenges. Mol Pharm. 2012;9(9):2380-402. Eliyahu H, Servel N, Domb AJ, Barenholz Y. Lipoplex-induced hemagglutination: potential involvement in intravenous gene delivery. Gene Ther. 2002;9(13):850-8. Falcone S, Cocucci E, Podini P, Kirchhausen T, Clementi E, Meldolesi J. Macropinocytosis: regulated coordination of endocytic and exocytic membrane traffic events. J Cell Sci. 2006;119(Pt 22):4758-69. Gentile P, Chiono V, Carmagnola I, Hatton PV. An overview of poly(lactic-co-glycolic) acid (PLGA)-based biomaterials for bone tissue engineering. Int J Mol Sci. 2014;15(3):3640-59. Goodison S, Urquidi V, Tarin D. CD44 cell adhesion molecules. Mol Pathol. 1999;52(4):189-96. Greyner HJ, Wiraszka T, Zhang LS, Petroll WM, Mummert ME. Inducible macropinocytosis of hyaluronan in B16-F10 melanoma cells. Matrix Biol. 2010;29(6):503-10. Hallaj-Nezhadi S, Valizadeh H, Dastmalchi S, Baradaran B, Jalali MB, Dobakhti F, et al. Preparation of chitosan-plasmid DNA nanoparticles encoding interleukin-12 and their expression in CT-26 colon carcinoma cells. J Pharm Pharm Sci. 2011;14(2):181-95. Hanif M, Ranjha NM, Shoaib MH, Mudasser J, Yousuf RI, Khan A, et al. Preparation, characterization and release of verapamil hydrochloride from polycaprolactone/acrylic acid (PCL/AA) hydrogels. Pak J Pharm Sci. 2011;24(4):503-11. Harris JM, Chess RB. Effect of pegylation on pharmaceuticals. Nat Rev Drug Discov. 2003;2(3):214-21. Hashizume H, Baluk P, Morikawa S, McLean JW, Thurston G, Roberge S, et al. Openings between defective endothelial cells explain tumor vessel leakiness. Am J Pathol. 2000;156(4):1363-80. Hattori Y, Yamasaku H, Maitani Y. Anionic polymer-coated lipoplex for safe gene delivery into tumor by systemic injection. J Drug Target. 2013;21(7):639-47. Hermanson GT. Bioconjugate Techniques 2nd ed. San Diego: Academic Press; 2008. p. 121. Ivanov AI. Pharmacological inhibition of endocytic pathways: is it specific enough to be useful? Methods Mol Biol. 2008;440:15-33. Jeong YI, Kim do H, Chung CW, Yoo JJ, Choi KH, Kim CH, et al. Self-assembled nanoparticles of hyaluronic acid/poly(DL-lactide-co-glycolide) block copolymer. Colloids Surf B Biointerfaces. 2012;90:28-35. Jhaveri AM, Torchilin VP. Multifunctional polymeric micelles for delivery of drugs and siRNA. Front Pharmacol. 2014;5:1-26. Jiang D, Liang J, Noble PW. Hyaluronan as an immune regulator in human diseases. Physiol Rev. 2011;91(1):221-64. Jiang G, Park K, Kim J, Kim KS, Hahn SK. Target Specific Intracellular Delivery of siRNA/PEI−HA Complex by Receptor Mediated Endocytosis. Molecular Pharmaceutics. 2009;6(3):727-37. Kapoor M, Burgess DJ. Cellular uptake mechanisms of novel anionic siRNA lipoplexes. Pharm Res. 2013;30(4):1161-75. Karbownik MS, Nowak JZ. Hyaluronan: towards novel anti-cancer therapeutics. Pharmacol Rep. 2013;65(5):1056-74. Katagiri YU, Sleeman J, Fujii H, Herrlich P, Hotta H, Tanaka K, et al. CD44 variants but not CD44s cooperate with beta1-containing integrins to permit cells to bind to osteopontin independently of arginine-glycine-aspartic acid, thereby stimulating cell motility and chemotaxis. Cancer Res. 1999;59(1):219-26. Khalil IA, Kogure K, Akita H, Harashima H. Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacol Rev. 2006;58(1):32-45. Koivusalo M, Welch C, Hayashi H, Scott CC, Kim M, Alexander T, et al. Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling. J Cell Biol. 2010;188(4):547-63. Kumari A, Yadav SK, Yadav SC. Biodegradable polymeric nanoparticles based drug delivery systems. Colloids Surf B Biointerfaces. 2010;75(1):1-18. Li J, Huo M, Wang J, Zhou J, Mohammad JM, Zhang Y, et al. Redox-sensitive micelles self-assembled from amphiphilic hyaluronic acid-deoxycholic acid conjugates for targeted intracellular delivery of paclitaxel. Biomaterials. 2012;33(7):2310-20. Lim KI. Retroviral integration profiles: their determinants and implications for gene therapy. BMB Rep. 2012;45(4):207-12. Liu YS, Chiu CC, Chen HY, Chen SH, Wang LF. Preparation of chondroitin sulfate-g-poly(epsilon-caprolactone) copolymers as a CD44-targeted vehicle for enhanced intracellular uptake. Mol Pharm. 2014;11(4):1164-75. Lo YL, Sung KH, Chiu CC, Wang LF. Chemically conjugating polyethylenimine with chondroitin sulfate to promote CD44-mediated endocytosis for gene delivery. Mol Pharm. 2013;10(2):664-76. Mikami T, Kitagawa H. Biosynthesis and function of chondroitin sulfate. Biochim Biophys Acta. 2013;1830(10):4719-33. Milane L, Duan Z, Amiji M. Development of EGFR-targeted polymer blend nanocarriers for combination paclitaxel/lonidamine delivery to treat multi-drug resistance in human breast and ovarian tumor cells. Mol Pharm. 2011;8(1):185-203. Mingozzi F, High KA. Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat Rev Genet. 2011;12(5):341-55. Mizrahy S, Raz SR, Hasgaard M, Liu H, Soffer-Tsur N, Cohen K, et al. Hyaluronan-coated nanoparticles: the influence of the molecular weight on CD44-hyaluronan interactions and on the immune response. J Control Release. 2011;156(2):231-8. Mohajeri E, Noudeh GD. Effect of Temperature on the Critical Micelle Concentration and Micellization Thermodynamic of Nonionic Surfactants: Polyoxyethylene Sorbitan Fatty Acid Esters. E-Journal of Chemistry. 2012;9(4):2268-74. Mundargi RC, Babu VR, Rangaswamy V, Patel P, Aminabhavi TM. Nano/micro technologies for delivering macromolecular therapeutics using poly(D,L-lactide-co-glycolide) and its derivatives. J Control Release. 2008;125(3):193-209. Murai T, Sougawa N, Kawashima H, Yamaguchi K, Miyasaka M. CD44-chondroitin sulfate interactions mediate leukocyte rolling under physiological flow conditions. Immunol Lett. 2004;93(2-3):163-70. Naor D, Wallach-Dayan SB, Zahalka MA, Sionov RV. Involvement of CD44, a molecule with a thousand faces, in cancer dissemination. Semin Cancer Biol. 2008;18(4):260-7. Ndinguri MW, Zheleznyak A, Lauer JL, Anderson CJ, Fields GB. Application of Collagen-Model Triple-Helical Peptide-Amphiphiles for CD44-Targeted Drug Delivery Systems. J Drug Deliv. 2012;2012:1-13. Nehoff H, Parayath NN, Domanovitch L, Taurin S, Greish K. Nanomedicine for drug targeting: strategies beyond the enhanced permeability and retention effect. Int J Nanomedicine. 2014;9:2539-55. Oh N, Park JH. Endocytosis and exocytosis of nanoparticles in mammalian cells. Int J Nanomedicine. 2014;9(Suppl 1):51-63. Olaku V, Matzke A, Mitchell C, Hasenauer S, Sakkaravarthi A, Pace G, et al. c-Met recruits ICAM-1 as a coreceptor to compensate for the loss of CD44 in Cd44 null mice. Mol Biol Cell. 2011;22(15):2777-86. Ossipov DA. Nanostructured hyaluronic acid-based materials for active delivery to cancer. Expert Opin Drug Deliv. 2010;7(6):681-703. Park JH, Cho HJ, Termsarasab U, Lee JY, Ko SH, Shim JS, et al. Interconnected hyaluronic acid derivative-based nanoparticles for anticancer drug delivery. Colloids Surf B Biointerfaces. 2014;121:380-7. Pathak A, Patnaik S, Gupta KC. Recent trends in non-viral vector-mediated gene delivery. Biotechnol J. 2009;4(11):1559-72. Peach RJ, Hollenbaugh D, Stamenkovic I, Aruffo A. Identification of hyaluronic acid binding sites in the extracellular domain of CD44. J Cell Biol. 1993;122(1):257-64. Ponta H, Sherman L, Herrlich PA. CD44: from adhesion molecules to signalling regulators. Nat Rev Mol Cell Biol. 2003;4(1):33-45. Saez A, Guzman M, Molpeceres J, Aberturas MR. Freeze-drying of polycaprolactone and poly(D,L-lactic-glycolic) nanoparticles induce minor particle size changes affecting the oral pharmacokinetics of loaded drugs. Eur J Pharm Biopharm. 2000;50(3):379-87. Sameti M, Bohr G, Ravi Kumar MN, Kneuer C, Bakowsky U, Nacken M, et al. Stabilisation by freeze-drying of cationically modified silica nanoparticles for gene delivery. Int J Pharm. 2003;266(1-2):51-60. Shim G, Kim M-G, Park JY, Oh Y-K. Application of cationic liposomes for delivery of nucleic acids. Asian Journal of Pharmaceutical Sciences. 2013;8(2):72-80. Song S, Chen F, Qi H, Li F, Xin T, Xu J, et al. Multifunctional tumor-targeting nanocarriers based on hyaluronic acid-mediated and pH-sensitive properties for efficient delivery of docetaxel. Pharm Res. 2014;31(4):1032-45. Stallcup WB, Huang FJ. A role for the NG2 proteoglycan in glioma progression. Cell Adh Migr. 2008;2(3):192-201. Tront JS, Willis A, Huang Y, Hoffman B, Liebermann DA. Gadd45a levels in human breast cancer are hormone receptor dependent. J Transl Med. 2013;11(131):1-6. Vercauteren D, Rejman J, Martens TF, Demeester J, De Smedt SC, Braeckmans K. On the cellular processing of non-viral nanomedicines for nucleic acid delivery: mechanisms and methods. J Control Release. 2012;161(2):566-81. Vigetti D, Karousou E, Viola M, Deleonibus S, De Luca G, Passi A. Hyaluronan: Biosynthesis and signaling. Biochim Biophys Acta. 2014;1840(8):2452-9. Wallach-Dayan SB, Grabovsky V, Moll J, Sleeman J, Herrlich P, Alon R, et al. CD44-dependent lymphoma cell dissemination: a cell surface CD44 variant, rather than standard CD44, supports in vitro lymphoma cell rolling on hyaluronic acid substrate and its in vivo accumulation in the peripheral lymph nodes. J Cell Sci. 2001;114(Pt 19):3463-77. Wang W, Li W, Ma N, Steinhoff G. Non-viral gene delivery methods. Curr Pharm Biotechnol. 2013;14(1):46-60. Wasungu L, Hoekstra D. Cationic lipids, lipoplexes and intracellular delivery of genes. J Control Release. 2006;116(2):255-64. Wegman F, Oner FC, Dhert WJ, Alblas J. Non-viral gene therapy for bone tissue engineering. Biotechnol Genet Eng Rev. 2013;29(1-2):206-20. Williams K, Motiani K, Giridhar PV, Kasper S. CD44 integrates signaling in normal stem cell, cancer stem cell and (pre)metastatic niches. Exp Biol Med (Maywood). 2013;238(3):324-38. Wiranowska M, Ladd S, Moscinski LC, Hill B, Haller E, Mikecz K, et al. Modulation of hyaluronan production by CD44 positive glioma cells. Int J Cancer. 2010;127(3):532-42. Xiang S, Tong H, Shi Q, Fernandes JC, Jin T, Dai K, et al. Uptake mechanisms of non-viral gene delivery. J Control Release. 2012;158(3):371-8. Xie S, Tao Y, Pan Y, Qu W, Cheng G, Huang L, et al. Biodegradable nanoparticles for intracellular delivery of antimicrobial agents. J Control Release. 2014;187C:101-17. Xue HY, Liu S, Wong HL. Nanotoxicity: a key obstacle to clinical translation of siRNA-based nanomedicine. Nanomedicine (Lond). 2014;9(2):295-312. Zhang L, Gong F, Zhang F, Ma J, Zhang P, Shen J. Targeted therapy for human hepatic carcinoma cells using folate-functionalized polymeric micelles loaded with superparamagnetic iron oxide and sorafenib in vitro. Int J Nanomedicine. 2013;8:1517-24. Zhang W, Cheng Q, Guo S, Lin D, Huang P, Liu J, et al. Gene transfection efficacy and biocompatibility of polycation/DNA complexes coated with enzyme degradable PEGylated hyaluronic acid. Biomaterials. 2013;34(27):6495-503. Zhao J, Mi Y, Feng SS. Targeted co-delivery of docetaxel and siPlk1 by herceptin-conjugated vitamin E TPGS based immunomicelles. Biomaterials. 2013;34(13):3411-21. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55737 | - |
dc.description.abstract | 玻尿酸(Hyaluronic acid,HA)和硫酸軟骨素(Chondroitin sulfate,CD)為在人體內細胞外基質中的內生性多醣分子。而此二多醣分子不但擁有相似的化學結構,近年來更因為二者皆為會過度表現在癌細胞外之CD44受器的專一性配體,進而有大量的研究將玻尿酸或硫酸軟骨素用作抗癌藥物或基因遞送的載體。
此研究以帶正電的1,2-二油酰基-3-三甲基銨丙烷(DOTAP)吸附帶負電質體DNA形成帶正電的奈米複合體(D/P),雖其轉染效果佳,但許多研究指出,帶正電的載體進入體循環後,容易引起血液中紅血球凝集,故本研究進一步將聚乙二醇二胺化的聚乳酸-甘醇酸(PLGA-PEG-NH2)接枝在低分子量玻尿酸(LPP)、高分子量玻尿酸(HPP)或硫酸軟骨素(CPP)的末端(直鏈)或側鏈,作為基因遞送的載體,包覆帶正電的奈米複合體分別形成LPP-D/P、HPP-D/P、CPP-D/P、LPPg-D/P、HPPg-D/P、CPPg-D/P,以增加其安全性及標靶遞送。 首先,合成的聚乙二醇二胺化的聚乳酸-甘醇酸接枝玻尿酸或硫酸軟骨素先以核磁共振(1H-NMR)、傅立葉轉換紅外線光譜儀(FT-IR)確認其化學結構及接枝率的定量。以透析方法形成微胞並包覆奈米複合體後,以Zetasizer測所形成的粒徑大小以及表面電位、以凝膠阻滯實驗確認質體DNA的被包覆性,並以PicoGreen reagent進行質體DNA包覆率的定量,最後也進行質體DNA在pH4.0的醋酸緩衝溶液與pH7.4磷酸緩衝溶液的體外釋放試驗,以確認質體DNA的釋放模式。以流式細胞儀篩選出過度表現CD44的細胞株(U87、MDA-MB-231、L929、MCF-7)進行奈米微胞對細胞之轉染試驗,並針對效果較佳的玻尿酸或硫酸軟骨素修飾奈米微胞(LPP-D/P、HPP-D/P、CPP-D/P)進行細胞胞飲途徑的探討,最後進行細胞存活率試驗,以確定奈米微胞對於正常細胞的安全性。 結果顯示,不同比例的LPP、HPP、CPP、LPPg、HPPg、CPPg形成的奈米微胞的粒徑分別在184.4-360.8 nm之間,帶正電的奈米複合體被聚乙二醇二胺化的聚乳酸-甘醇酸接枝玻尿酸或硫酸軟骨素包覆形成奈米微胞後,表面電位皆變為帶負電,因而不會造成紅血球凝集。凝膠阻滯實驗的結果確認直鏈合成聚合物包覆的pDNA不會因電場的影響而釋放出來,而支鏈合成聚合物會受到電場的影響而有pDNA釋放。合成之直鏈聚合物的細胞轉染效果,在MDA-MB-231、MCF-7細胞株中,HPP包覆D/P的轉染效果最佳,而在U87這株細胞則為CPP包覆D/P的轉染效果最佳,而LPP包覆D/P的綠色螢光蛋白轉染效率無論在MDA-MB-231、U87或MCF-7的效果都不太明顯,但是在L929細胞的轉染率卻相當高。合成之枝鏈聚合物的細胞轉染效果,在U87、MDA-MB-231、MCF-7、L929細胞株中,轉染效率皆是以LPPg-D/P最佳,甚至CD44 negative的HepG2對於LPPg-D/P都能有31.25 %之細胞轉染率,間接說明雖然LPPg-D/P轉染率高,但可能不是藉由CD44胞攝入細胞的專一性途徑。 在藉由不同機轉之胞飲抑制劑探討LPP、HPP、CPP進入細胞之途徑實驗中,說明了LPP、HPP在U87細胞中皆為clathrin-mediated endocytosis及macropinocytosis,而CPP在U87僅有clathrin-mediated endocytosis。LPP、HPP、CPP在MDA-MB-231皆為macropinocytosis,而HPP在MDA-MB-231細胞中還有Caveolin-mediated endocytosis一途徑。 細胞存活率試驗,LPP-D/P、HPP-D/P、CPP-D/P對L929的細胞存活率皆大於64.8%,對U87的細胞存活率大於64.7%,而對MDA-MB-231則為大於76.0%,其安全性皆優於商品化的Lipofectamine 2000。 | zh_TW |
dc.description.abstract | Hyaluronic acid (HA) and chondroitin sulfate (CD) are endogenous polysaccharides existed in the extracellular matrix. In recent years, these two endogenous polysaccharides have aroused much interest of scientists because of their specificity of binding to CD44 receptor which is overexpressed in several types of tumors. As a result, a lot of studies are dedicated to develop the HA or CD based drug and gene delivery systems.
Several polymers are synthesized including LHA-b-PEG-PLGA(LPP)、LHA-g-PEG-PLGA(LPPg)、HHA-b-PEG-PLGA(HPP)、HHA-g-PEG-PLGA(HPPg)、CD-b-PEG-PLGA(CPP)、CD-g-PEG-PLGA (CPPg). The structure and conjugation ratio of synthesized polymers were comfirmed by FTIR and 1H-NMR. The positively charged DOTAP was used to complex with the negatively charged plasmid DNA (pDNA) to form lipoplex. The dialysis method was used to encapsulate the D/P into the synthesized polymers to form micelles. The size and zeta potential of the micelles were measured by Zetasizer and the pDNA encapsulation efficiency was quantified by PicoGreen reagent. The in vitro release of the pDNA was conducted in the PBS buffer (pH 7.4) and acetate buffer (pH 4.0) separately. The CD44 overexpressed U87, MDA-MB-231, L929, MCF-7 were screened out by flow cytometry and used for cellular transfection experiment. The endocytosis mechanism of the CD44 tareting micelles was investigated. Finally, the MTT assay was conducted to comfirm the safety of the micelles. The size of the micelles were around 184.4-360.8 nm and the zeta potential was converted to negative after encapsulating D/P into the synthesized polymers. The positively charged D/P led to erythrocytes agglutination but the micelles didn’t because of the negatively charged surface of the micelles. The resuls of the electrophoresis retardation assay comfirmed the micelles formed by LPP, HPP and CPP could retain the pDNA inside the micelles well but LPPg, HPPg and CPPg could not. The transfection efficiencies of the micelles formed by LPP, HPP, and CPP were evaluated by flow cytometry. The HPP micelle showed the best transfection efficiency in MDA-MB-231 cell and MCF-7 cell. The CPP micelle showed the best efficiency in U87 cell. The LPP micelle showed the poorest efficiency in three CD44 overexpressed cancer cell lines U87, MDA-MB-231 and MCF-7 but the best efficiency in mouse fibroblast L929.The transfection efficiency of the micelles formed by LPPg, HPPg, and CPPg were different from LPP, HPP, and CPP micelles. In U87, MDA-MB-231, MCF-7 and L929 cell, the LPPg micelle showed the best transfection efficiency. Although the CD44 expression level of the HepG2 was low, the transfection efficiency of the LPPg was high. This result indirectly indicated that the cellular uptake pathway of the LPPg micelle was not the CD44-meiated pathway. The endocytosis study comfirmed the pathways of LPP, HPP, and CPP micelles. LPP and HPP were uptaken by the pathways of clathrin-mediated endocytosis and macropinocytosis in U87. However, the CPP micelle was uptaken by clathrin-mediated endocytosis. LPP, HPP, and CPP micelles were uptaken by the pathway of macropinocytosis in MDA-MB-231. Besides the macropinocytosis, HPP micelle was also uptaken by caveolin-mediated endocytosis in MDA-MB-231.The MTT assay comfirmed the synthesized polymer formed micelles were safer than the commercial product Lipofectamine 2000. In conclusion, the HPP and CPP micelles could provide a safer and more specific way to deliver gene to cancer cells. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T04:20:34Z (GMT). No. of bitstreams: 1 ntu-103-R01423017-1.pdf: 11806559 bytes, checksum: 44275a81904ac919510eb681275bc377 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 口試委員會審定書 I
致謝 II 中文摘要 III Abstract V 目錄 VII 表目錄 XIII 圖目錄 XVI 第一章 緒論 1 一、 基因療法 1 (一) 病毒載體(Viral vector) 2 (二) 非病毒載體(Non-viral vector) 3 二、 奈米劑型之遞送策略 4 (一) 被動型標的(Passive targeting) 4 (二) 主動型標的(Active targeting) 5 三、 CD44受體 6 四、 玻尿酸(Hyaluronic acid, HA) 8 (一) 玻尿酸之基本結構及體內分佈 8 (二) 玻尿酸之體內生成及分解代謝 8 (三) 玻尿酸受體 11 (四) 以玻尿酸作為遞輸載體之發展 12 五、 硫酸軟骨素(Chondroitin sulfate, CD) 13 六、 聚乳酸-甘醇酸(Poly(lactide-co-glycolide), PLGA) 15 七、 奈米劑型之內吞途徑 (Endocytosis pathway) 17 (一) Clathrin-mediated endocytosis 18 (二) Caveolin-mediated endocytosis 19 (三) Macropinocytosis 20 第二章 試劑與材料介紹 21 一、 玻尿酸(Hyaluronic acid, HA) 21 二、 硫酸軟骨素(Chondroitin Sulfate, CD) 22 三、 聚乳酸-甘醇酸(Poly(lactide-co-glycolide), PLGA) 22 四、 聚乙二醇二胺(Poly(ethylene glycol) bis(amine), PEG diamine) 23 五、 1,2-二油酰基-3-三甲基銨丙烷(1,2-dioleoyl-3-trimethylammonium-propane (chloride salt),DOTAP) 24 六、 質體DNA(pEGFP-N1) 25 第三章 實驗動機與目的 26 第四章 實驗試劑與儀器 27 一、 藥品 27 二、 Plasmid DNA 純化實驗材料 30 三、 細胞實驗材料 31 四、 儀器 33 五、 耗材 35 六、 藥品溶液與緩衝溶液製備 35 第五章 實驗方法 37 一、 HA-PEG-PLGA、CD-PEG-PLGA之合成 40 (一) PLGA接枝PEG-diamine 40 (二) LHA接枝PLGA-PEG-NH2 42 (三) HHA接枝PLGA-PEG-NH2 46 (四) CD接枝PLGA-PEG-NH2 51 (五) 臨界微膠粒濃度(CMC)測試 (Jeong et al., 2012) 56 (六) 分子量評估-膠體滲透層析(GPC) 57 二、 HA-PEG-PLGA、CD-PEG-PLGA包覆DOTAP/pDNA之劑型製備及物性測定 59 (一) DOTAP/pDNA之製備 59 (二) HA-PEG-PLGA、CD-PEG- PLGA包覆DOTAP/pDNA之劑型製備 60 (三) 奈米微胞中pDNA的定量 62 (四) 奈米微胞之粒徑及表面電位分析 62 (五) 奈米微胞之粒徑安定性試驗 63 (六) 凝膠阻滯試驗(Gel retardation assay) 65 (七) 紅血球凝集試驗(Hattori et al., 2013) 67 (八) 奈米微胞之穿透式電子顯微鏡拍攝 67 三、 癌細胞表面CD44受器表現量 68 四、 細胞轉染效率試驗 69 五、 螢光顯微鏡分析 69 六、 玻尿酸或硫酸軟骨素修飾之奈米微胞進入細胞之途徑探討 70 (一) Endocytosis inhibiton assay (Oh and Park, 2014; Wasungu and Hoekstra, 2006; Xiang et al., 2012) 70 (二) CD44受體競爭實驗(Dufay Wojcicki et al., 2012) 72 七、 細胞存活率試驗 74 八、 質體DNA的體外釋放實驗 76 (一) 基因體外釋放實驗質體DNA的含量定量 76 (二) 質體DNA的體外釋放實驗(高立庭, 2012;劉佳雯, 2012) 77 九、 統計分析 80 第六章 實驗結果 81 一、 HA-PEG-PLGA、CD-PEG-PLGA之合成 81 (一) PLGA接枝PEG-diamine 81 (二) LHA接枝PLGA-PEG-NH2 89 (三) HHA接枝PLGA-PEG-NH2 103 (四) CD接枝PLGA-PEG-NH2 120 (五) 臨界微膠粒濃度(CMC)測試 (Jeong et al., 2012) 135 二、 HA-PEG-PLGA、CD-PEG-PLGA包覆DOTAP/pDNA之劑型製備及物性測定 139 (一) DOTAP/pDNA(D/P)之製備 139 (二) HA-PEG-PLGA、CD-PEG- PLGA包覆DOTAP/pDNA之劑型製備 140 三、 癌細胞表面CD44受體表現量 176 四、 細胞轉染效率試驗 179 (一) 不同比例之D/P對細胞的轉染效率 179 (二) LPP、HPP、CPP包覆D/P(5/1)之奈米微胞對不同細胞株的細胞轉染效率 181 (三) LPP、HPP、CPP包覆D/P(5/1)和D/P(1/1)之奈米微胞的細胞轉染效率比較 192 (四) 直鏈聚合物(LPP、HPP、CPP)和支鏈聚合物(LPPg、HPPg、CPPg)包覆D/P(5/1)的細胞轉染率比較 196 五、 螢光顯微鏡分析 208 六、 玻尿酸或硫酸軟骨素修飾奈米微胞進入細胞之途徑探討 209 (一) Endocytosis inhibiton assay 209 (二) CD44受體競爭實驗 215 七、 細胞存活率試驗 218 八、質體DNA的體外釋放實驗 225 (一) 基因體外釋放實驗pDNA含量定量 225 (二) 質體DNA的體外釋放實驗 227 第七章 討論 239 一、HA-PEG-PLGA、CD-PEG-PLGA之合成 239 (一) 直鏈聚合物之合成 239 (二) 支鏈聚合物的合成 240 (三) 臨界微膠粒濃度(CMC)測試 241 二、HA-PEG-PLGA、CD-PEG-PLGA包覆DOTAP/pDNA之劑型製備及物性測定 241 (一) DOTAP/pDNA之製備(D/P) 241 (二) HA-PEG-PLGA、CD-PEG- PLGA包覆DOTAP/pDNA之劑型製備 242 三、 癌細胞表面CD44受體表現量 245 四、 細胞轉染效率試驗 246 (一) 不同比例之D/P對細胞的轉染效率 246 (二) LPP、HPP、CPP包覆D/P(5/1)之奈米微胞對不同細胞株的細胞轉染效率 246 (三) LPP、HPP、CPP包覆D/P(5/1)或D/P(1/1)之奈米微胞的細胞轉染效率比較 248 (四) 直鏈聚合物(LPP、HPP、CPP)或支鏈聚合物(LPPg、HPPg、CPPg)包覆D/P(5/1)的細胞轉染率比較 249 五、 螢光顯微鏡分析 249 六、玻尿酸或硫酸軟骨素修飾奈米微胞進入細胞之途徑探討 250 (一) Endocytosis inhibition assay 250 (二) CD44受體競爭實驗 252 七、細胞存活率試驗 252 八、質體DNA的體外釋放實驗 253 第八章 結論 254 第九章 參考文獻 260 | |
dc.language.iso | zh-TW | |
dc.title | 具CD44標的潛力之奈米微胞基因遞送系統的研究 | zh_TW |
dc.title | Study of Self-Assembled Micelles with CD44 Targeting Potential for Gene Delivery | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 邱士捐(Shih-Jiuan Chiu),方嘉佑(Jia-You Fang) | |
dc.subject.keyword | 玻尿酸,硫酸軟骨素,聚乳酸-甘醇酸,基因遞送,CD44, | zh_TW |
dc.subject.keyword | hyaluronic acid,chondroitin sulfate,poly(lactide-co-glycolide),gene delivery,CD44, | en |
dc.relation.page | 266 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-08-20 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 藥學研究所 | zh_TW |
顯示於系所單位: | 藥學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-103-1.pdf 目前未授權公開取用 | 11.53 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。