臺灣博碩士論文加值系統

本研究合成出三種具有不同功能性的雙親性嵌段共聚高分子：Poly(ɛ-caprolactone)-SS-b-poly[triethylene glycol methacrylate-co- 6-(methacrylamido) hexanoic acid] [PCL-SS-b-P(TEGMA-co-AHA)]、Poly(ɛ-caprolactone)-SS-b-poly[triethylene glycol methacrylate-co- N-(2-(methacrylamido)ethyl) folatic amide] [PCL-SS-b-P(TEGMA-co-FA)]、Poly(ɛ-caprolactone)-b-poly[triethylene glycol methacrylate-co- (2-(1,2,3,4,5-pentaphenyl-1H-silol-1-yloxy) ethyl methacrylate)] [PCL-b-P(TEGMA-co-(PPS-HEMA))] ，於水中自組裝形成混合微胞，針對其奈米結構、環境響應性、藥物包覆釋放、螢光共振能量轉移以及細胞毒性作探討。
Poly(ɛ-caprolactone) (PCL)作為雙親性高分子疏水鏈段形成內核用以包覆疏水性癌症藥物Doxorubicin (DOX)，triethylene glycol methacrylate (TEGMA)作為親水性鏈段及溫度敏感性單體形成微胞外殼而能均勻分散於水溶液中，親疏水鏈段間之雙硫鍵(disulfide)具有氧化還原響應性，修飾於親水鏈段上的不同功能性單體分別為具有酸鹼響應性之6-aminohexanoic acid (AHA)、主動標靶性質之folic acid (FA)以及螢光基團2-(1,2,3,4,5-pentaphenyl-1H-silol-1-yloxy)ethyl methacrylate (PPS-HEMA)。
藉由調整溫度響應性單體TEGMA以及酸鹼響應性單體AHA於高分子中的聚合度，以及三種雙親性共聚高分子 於混合微胞中的混摻比例，使最低臨界溶液溫度(lower critical solution temperature, LCST)於酸性環境低於人體體溫37 °C，而在中性環境時則高於37 °C，因此混合微胞能穩定存在於血液循環中並能於癌症細胞溶酶體釋放藥物：而雙硫鍵在腫瘤細胞質液中會因為較高濃度的還原劑榖光甘肽而斷鍵，造成微胞結構崩解而釋放藥物；FA則可以與Hela cell上的葉酸受體結合，達到主動標靶的功能使微胞能於目標細胞中累積；PPS-HEMA之螢光光譜與DOX之吸收光譜有重疊，且當兩者距離足夠接近會產生Förster Resonance Energy Transfer (FRET)現象，可作為監控藥物包覆與釋放之依據。
在in vitro細胞實驗中，結果顯示空白微胞不具有細胞毒性，而包藥微胞之IC50為10 μg/ml，證明有良好的毒殺細胞效果，配合上其奈米尺寸與低臨界微胞濃度，於藥物載體上具有發展潛力。

In this research, we investigated the nanostructures, stimuli-responsive, drug encapsulation and release, Förster Resonance Energy Transfer (FRET) and cytotoxicity to Hela cells of the multifunctional polymeric mixed micelles. The mixed micelles were co-assembled from three kinds of amphiphilic block copolymers, [PCL-SS-b-P(TEGMA-co-AHA)], [PCL-SS-b-P(TEGMA-co-FA)] and [PCL-b-P(TEGMA-co-(PPS-HEMA))].
Poly(ɛ-caprolactone) (PCL) is the hydrophobic core of micelle to encapsulate hydrophobic drug, Doxorubicin (DOX). Triethylene glycol methacrylate (TEGMA) is thermo-sensitive monomer and also forms the hydrophilic shell of micelle. As a result, micelles could be well-dispersed in aqueous solution. The disulfide bond in polymers has the redox-responsive property . We also introduced pH-sensitive (AHA), active targeting (FA) and fluorescent (PPS-HEMA) moieties to hydrophilic block.
We adjusted the degree of polymerization of TEGMA and AHA and changed the ratio of SSPTAHA in mixed micelles to control the LCST. The LCST should be higher than body temperature (37 °C) in neutral environment but lower than 37 °C when in acidic environment, so the micelles can be steady in blood circulation and release drug when in lysosome of cancer cell. Because the concentration of glutathione tripeptide of cancer cell is higher than normal cell, the disulfide bond is prone to rapid cleavage under a more reductive environment. Folic acid can be combined with the folate receptor which is overexpressed on the surface of cancer cells. The micelles can accumulate in cancer cells more effectively because of the property of active targeting. FRET would occur when the donor was close to the acceptor owing to the spectral overlap of PPS-HEMA and DOX. The FRET phenomenon could be used as a basis for detecting whether the drug is encapsulated or released.
In the in vitro cytotoxicity test, we found that the blank micelles were nontoxic and the drug-loaded micelles could kill the Hela cells effectively. With the nanometer size and low critical micelle concentration, the mixed micelles are promising drug carrier of cancer therapy.

摘要 I
Abstract III
誌謝 XII
流程圖目錄 XVI
表目錄 XVII
圖目錄 XVIII
公式目錄 XXII
第一章、緒論 1
1.1研究背景與文獻回顧 1
1.1.1藥物傳遞系統(Drug Delivery System, DDS) 1
1.1.1.1奈米藥物載體 1
1.1.1.2藥物輸送 4
1.1.1.3藥物釋放 7
1.1.2多功能性奈米微胞 8
1.1.2.1雙親性嵌段共聚高分子(Amphiphilic block copolymers) 8
1.1.2.2環境響應性 11
1.1.2.3螢光材料 25
1.1.2.4微胞製備 35
1.2研究動機與目的 37
第二章、實驗 39
2.1實驗藥品 39
2.2實驗方法 42
2.2.1單體合成 42
2.2.2高分子聚合 45
2.2.3微胞製備 52
2.2.4臨界微胞濃度(critical micelle concentration, CMC)檢測 53
2.2.5 Lower Critical Solution Temperature(LCST)測試 53
2.2.6藥物包覆與釋放 54
2.2.7細胞毒性測試 56
2.3儀器鑑定 58
2.3.1 Gel Permeation Chromatography(GPC) 58
2.3.2 Nuclear Magnetic Resonance (NMR) 59
2.3.3 Dynamic Light Scattering (DLS) 60
2.3.4 Transmission Electron Microscopy(TEM) 60
2.3.5 Ultraviolet-Visilbe Spectroscopy (UV-vis.) 60
2.3.6 Photoluminescence Spectroscopy (PL) 61
2.3.7 Enzyme-Linked Immunosorbent Assay (ELISA) Reader 61
第三章、結果討論 62
3.1聚合與鑑定 62
3.1.1 hydroxyethl 2-bromoisobutyrate (HEBiB)合成 65
3.1.2 hydroxyethyl-2’-(bromoisobutyryl)ethyl disulfide (HO-SS-iBuBr)合成 65
3.1.3 NSMA單體合成 66
3.1.4 PPS-HEMA單體合成 67
3.1.5 poly(ε-caprolactone)(PCL)聚合 68
3.1.6 SS-poly(ε-caprolactone)(SSPCL)聚合 69
3.1.7 雙親性嵌段共聚高分子Poly(ɛ-caprolactone)-SS-b-poly[triethylene glycol methacrylate-co- N-hydroxysuccinimide methacrylate] [PCL-SS-b-P(TEGMA-co-NSMA)](SSPTN)合成 71
3.1.8 雙親性嵌段共聚高分子[PCL-SS-b-P(TEGMA-co-AHA)](SSPTAHA)、[PCL-SS-b-P(TEGMA-co-FA)](SSPTFA)之合成 73
3.1.9 雙親性嵌段共聚高分子[PCL-b-P(TEGMA-co-(PPS-HEMA))](PTAIE)之合成 78
3.2混合微胞的製備與性質鑑定 80
3.2.1溫度敏感性 80
3.2.2氧化還原敏感性 86
3.2.3 CMC性質測試 88
3.3藥物包覆與釋放測試 90
3.3.1藥物包覆 90
3.3.2藥物釋放 95
3.4.1細胞毒性測試 99
第四章、結論與未來工作 104
第五章、參考文獻 106

[1]Mishra, Deepa, Justin R. Hubenak, and Anshu B. Mathur. Nanoparticle systems as tools to improve drug delivery and therapeutic efficacy. Journal of Biomedical Materials Research Part A: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials 101.12 (2013): 3646-3660.
[2]Hu, Che-Ming Jack, and Liangfang Zhang. Nanoparticle-based combination therapy toward overcoming drug resistance in cancer. Biochemical pharmacology 83.8 (2012): 1104-1111.
[3]Miyata, Kanjiro, R. James Christie, and Kazunori Kataoka. Polymeric micelles for nano-scale drug delivery. Reactive and Functional Polymers 71.3 (2011): 227-234.
[4]Sanna, Vanna, Nicolino Pala, and Mario Sechi. Targeted therapy using nanotechnology: focus on cancer. International journal of nanomedicine 9 (2014): 467.
[5]Jain, R. K. Delivery of molecular medicine to solid tumors: lessons from in vivo imaging of gene expression and function. Journal of controlled release 74.1-3 (2001): 7-25.
[6]Nishiyama, Nobuhiro, and Kazunori Kataoka. Nanostructured devices based on block copolymer assemblies for drug delivery: designing structures for enhanced drug function. Polymer Therapeutics II. Springer, Berlin, Heidelberg, 2006. 67-101.
[7]Quintana, Antonio, et al. Design and function of a dendrimer-based therapeutic nanodevice targeted to tumor cells through the folate receptor. Pharmaceutical research 19.9 (2002): 1310-1316.
[8]Ye, Wei-liang, et al. Cellular uptake and antitumor activity of DOX-hyd-PEG-FA nanoparticles. PloS one 9.5 (2014): e97358.
[9]Zuo, Yajun, et al. Chitosan based nanogels stepwise response to intracellular delivery kinetics for enhanced delivery of doxorubicin. International journal of biological macromolecules104 (2017): 157-164.
[10]Torchilin, Vladimir P. Structure and design of polymeric surfactant-based drug delivery systems. Journal of controlled release 73.2-3 (2001): 137-172.
[11]Matyjaszewski, Krzysztof. Atom transfer radical polymerization (ATRP): current status and future perspectives. Macromolecules45.10 (2012): 4015-4039.
[12]Matyjaszewski, Krzysztof, and Jianhui Xia. Atom transfer radical polymerization. Chemical reviews 101.9 (2001): 2921-2990.
[13]Schmaljohann, Dirk. Thermo-and pH-responsive polymers in drug delivery. Advanced drug delivery reviews 58.15 (2006): 1655-1670.
[14]Klaikherd, Akamol, Chikkannagari Nagamani, and S. Thayumanavan. Multi-stimuli sensitive amphiphilic block copolymer assemblies. Journal of the American Chemical Society 131.13 (2009): 4830-4838.
[15]Ward, Mark A., and Theoni K. Georgiou. Thermoresponsive polymers for biomedical applications. Polymers 3.3 (2011): 1215-1242.
[16]De las Heras Alarcón, Carolina, Sivanand Pennadam, and Cameron Alexander. Stimuli responsive polymers for biomedical applications. Chemical Society Reviews 34.3 (2005): 276-285.
[17]Liu, Jingchong, et al. Bioinspired graphene membrane with temperature tunable channels for water gating and molecular separation. Nature communications 8.1 (2017): 2011.
[18]Liu, Ruixue, Michael Fraylich, and Brian R. Saunders. Thermoresponsive copolymers: from fundamental studies to applications. Colloid and Polymer Science 287.6 (2009): 627-643.
[19]Rao, Kummara, Kummari Rao, and Chang-Sik Ha. Stimuli responsive poly (vinyl caprolactam) gels for biomedical applications. Gels 2.1 (2016): 6.
[20]Vihola, Henna, et al. Cytotoxicity of thermosensitive polymers poly (N-isopropylacrylamide), poly (N-vinylcaprolactam) and amphiphilically modified poly (N-vinylcaprolactam). Biomaterials26.16 (2005): 3055-3064.
[21]Porsch, Christian, et al. Thermo-responsive cellulose-based architectures: tailoring LCST using poly (ethylene glycol) methacrylates. Polymer Chemistry 2.5 (2011): 1114-1123.
[22]Lutz, Jean-François, and Ann Hoth. Preparation of ideal PEG analogues with a tunable thermosensitivity by controlled radical copolymerization of 2-(2-methoxyethoxy) ethyl methacrylate and oligo (ethylene glycol) methacrylate. Macromolecules 39.2 (2006): 893-896.
[23]Kanamala, Manju, et al. Mechanisms and biomaterials in pH-responsive tumour targeted drug delivery: a review. Biomaterials 85 (2016): 152-167.
[24]Mura, Simona, Julien Nicolas, and Patrick Couvreur. Stimuli-responsive nanocarriers for drug delivery. Nature materials12.11 (2013): 991.
[25]Strandman, S., and X. X. Zhu. Thermo-responsive block copolymers with multiple phase transition temperatures in aqueous solutions. Progress in Polymer Science 42 (2015): 154-176.
[26]Schilli, Christine M., et al. A new double-responsive block copolymer synthesized via RAFT polymerization: poly (N-isopropylacrylamide)-b lock-poly (acrylic acid). Macromolecules37.21 (2004): 7861-7866.
[27]Chen, Ching-Yi, et al. pH-dependent, thermosensitive polymeric nanocarriers for drug delivery to solid tumors. Biomaterials34.18 (2013): 4501-4509.
[28]Wu, Guoyao, et al. Glutathione metabolism and its implications for health. The Journal of nutrition 134.3 (2004): 489-492.
[29]Schafer, Freya Q., and Garry R. Buettner. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free radical biology and medicine30.11 (2001): 1191-1212.
[30]Coban, T., et al. Glutathione and lipid peroxidation levels in human breast tumors. Neoplasma 45.3 (1998): 151-156.
[31]Koo, Ahn Na, et al. Disulfide-cross-linked PEG-poly (amino acid) s copolymer micelles for glutathione-mediated intracellular drug delivery. Chemical Communications 48 (2008): 6570-6572.
[32]Kamada, Jun, et al. Redox responsive behavior of thiol/disulfide-functionalized star polymers synthesized via atom transfer radical polymerization. Macromolecules 43.9 (2010): 4133-4139.
[33]Lee, Min Hee, et al. Disulfide-cleavage-triggered chemosensors and their biological applications. Chemical reviews 113.7 (2013): 5071-5109.
[34]Huo, Meng, et al. Redox-responsive polymers for drug delivery: from molecular design to applications. Polymer Chemistry 5.5 (2014): 1519-1528.
[35]Wen, Hui-Yun, et al. Rapidly disassembling nanomicelles with disulfide-linked PEG shells for glutathione-mediated intracellular drug delivery. Chemical Communications 47.12 (2011): 3550-3552.
[36]Sun, Jingjing, et al. Doxorubicin delivered by a redox-responsive dasatinib-containing polymeric prodrug carrier for combination therapy. Journal of Controlled Release 258 (2017): 43-55.
[37]Chen, Wei, et al. Redox and pH-responsive degradable micelles for dually activated intracellular anticancer drug release. Journal of controlled release 169.3 (2013): 171-179.
[38]Shimizu, Masaki, and Tamejiro Hiyama. Organic fluorophores exhibiting highly efficient photoluminescence in the solid state. Chemistry–An Asian Journal 5.7 (2010): 1516-1531.
[39]Valeur, Bernard. Molecular fluorescence. digital Encyclopedia of Applied Physics (2003): 477-531.
[40]Sahoo, Harekrushna. Förster resonance energy transfer–A spectroscopic nanoruler: Principle and applications. Journal of Photochemistry and Photobiology C: Photochemistry Reviews12.1 (2011): 20-30.
[41]Sapsford, Kim E., Lorenzo Berti, and Igor L. Medintz. Materials for fluorescence resonance energy transfer analysis: beyond traditional donor–acceptor combinations. Angewandte Chemie International Edition 45.28 (2006): 4562-4589.
[42]Clapp, Aaron R., Igor L. Medintz, and Hedi Mattoussi. Förster resonance energy transfer investigations using quantum‐dot fluorophores. ChemPhysChem 7.1 (2006): 47-57.
[43]Rajdev, Priya, Dipankar Basak, and Suhrit Ghosh. Insights into noncovalently core cross-linked block copolymer micelles by fluorescence resonance energy transfer (FRET) studies. Macromolecules 48.10 (2015): 3360-3367.
[44]Chen, Ko-Jie, et al. Intracellularly monitoring/imaging the release of doxorubicin from pH-responsive nanoparticles using Förster resonance energy transfer. Biomaterials 32.10 (2011): 2586-2592.
[45]Hong, Yuning, Jacky WY Lam, and Ben Zhong Tang. Aggregation-induced emission. Chemical Society Reviews40.11 (2011): 5361-5388.
[46]Luo, Jingdong, et al. Aggregation-induced emission of 1-methyl-1, 2, 3, 4, 5-pentaphenylsilole. Chemical communications 18 (2001): 1740-1741.
[47]Mei, Ju, et al. Aggregation‐induced emission: the whole is more brilliant than the parts. Advanced materials 26.31 (2014): 5429-5479.
[48]Hong, Yuning, Jacky WY Lam, and Ben Zhong Tang. Aggregation-induced emission: phenomenon, mechanism and applications. Chemical communications 29 (2009): 4332-4353.
[49]Qin, Anjun, Jacky WY Lam, and Ben Zhong Tang. Luminogenic polymers with aggregation-induced emission characteristics. Progress in Polymer Science 37.1 (2012): 182-209.
[50]Zhang, Chunqiu, et al. Imaging intracellular anticancer drug delivery by self-assembly micelles with aggregation-induced emission (AIE micelles). ACS applied materials ＆ interfaces 6.7 (2014): 5212-5220.
[51]Lu, Hongguang, et al. Pluronic F127–folic acid encapsulated nanoparticles with aggregation-induced emission characteristics for targeted cellular imaging. RSC Advances 4.35 (2014): 18460-18466.
[52]Gaucher, Geneviève, et al. Block copolymer micelles: preparation, characterization and application in drug delivery. Journal of controlled release 109.1-3 (2005): 169-188.
[53]Yang, Liu, et al. Novel biodegradable polylactide/poly (ethylene glycol) micelles prepared by direct dissolution method for controlled delivery of anticancer drugs. Pharmaceutical research 26.10 (2009): 2332-2342.
[54]Jones, Marie-Christine, and Jean-Christophe Leroux. Polymeric micelles–a new generation of colloidal drug carriers. European journal of pharmaceutics and biopharmaceutics 48.2 (1999): 101-111.
[55]Ai, Xiaoyu, et al. Thin-film hydration preparation method and stability test of DOX-loaded disulfide-linked polyethylene glycol 5000-lysine-di-tocopherol succinate nanomicelles. asian journal of pharmaceutical sciences 9.5 (2014): 244-250.
[56]Gou, MaLing, et al. Curcumin-loaded biodegradable polymeric micelles for colon cancer therapy in vitro and in vivo. Nanoscale3.4 (2011): 1558-1567.
[57]Farokhzad, Omid C., and Robert Langer. Impact of nanotechnology on drug delivery. ACS nano 3.1 (2009): 16-20.
[58]Sutton, Damon, et al. Functionalized micellar systems for cancer targeted drug delivery. Pharmaceutical research 24.6 (2007): 1029-1046.
[59]Sarbu, Traian, et al. Synthesis of hydroxy-telechelic poly (methyl acrylate) and polystyrene by atom transfer radical coupling. Macromolecules 37.26 (2004): 9694-9700.
[60]Khorsand Sourkohi, Behnoush, et al. Biodegradable block copolymer micelles with thiol-responsive sheddable coronas. Biomacromolecules 12.10 (2011): 3819-3825.
[61]Batz, Hans‐Georg, Giselher Franzmann, and Helmut Ringsdorf. Model reactions for synthesis of pharmacologically active polymers by way of monomeric and polymeric reactive esters. Angewandte Chemie International Edition in English 11.12 (1972): 1103-1104.
[62]Chen, Junwu, et al. Synthesis, light emission, nanoaggregation, and restricted intramolecular rotation of 1, 1-substituted 2, 3, 4, 5-tetraphenylsiloles. Chemistry of materials 15.7 (2003): 1535-1546.
[63]Gillies, Elizabeth R., and Jean MJ Fréchet. pH-responsive copolymer assemblies for controlled release of doxorubicin. Bioconjugate chemistry 16.2 (2005): 361-368.
[64]Domínguez, Ana, et al. Determination of critical micelle concentration of some surfactants by three techniques. Journal of chemical education 74.10 (1997): 1227.