本研究利用高度生物相容性且具聚電解質 (polyelectrolytes, PEs) 性質的團聯性高分子 methoxy poly(ethylene glycol)-b-poly(methacrylic acid-co-histamine methacrylamine) (mPEG-b-P(MAA-co-HMA)) 與兼具生物可降解性的高分子 poly(lactic-co-glycolic acid) (PLGA) 於水溶液中與疏水性光熱治療藥物IR780自組裝形成載藥奈米微胞 (hybrid micelles)，使其對癌細胞進行光熱治療及探討其膠體性質。而本研究所開發之mPEG-b-P(MAA-co-HMA) 為具有雙電荷的高分子，製備出的奈米載藥微粒，由粒徑測定儀顯示，此奈米微粒不僅具備良好的生理穩定性並能針對周遭環境酸鹼值產生應答。一旦其所處環境酸鹼值降低至pH 6.0~6.3 (近似於腫瘤環境)，奈米微粒表面除了電荷轉換外，還有微粒構型上的改變。由液態穿透式電顯、粒徑測定儀及疏水螢光探針等鑑定分析其中的交互作用力。最後，由細胞實驗的結果表明，所開發之奈米微粒於為酸環境下的應答，有利於提升IR780的光熱治療效果。

Highly biocompatible block copolymer Methoxy poly(ethylene glycol)-b-poly (methacrylic acid-co-histamine methacrylamine) (mPEG-b-P(MAA-co-HMA)) with polyelectrolyte properties and biodegradable polymer Poly(lactic-co-glycolic acid) (PLGA) are utilized to form a self-assembled hybrid micelles encapsulating a hydrophobic IR-780 iodide, a photothermal agent in this research with a view to achieving the near-infrared laser-triggered photothermal ablation of cancer cells and exploring the colloid properties. The mPEG-b-P(MAA-co-HMA) which we synthesize is a zwitterionic polymer and the particles prepared by this polymer are very stable in physiological environment and are pH-responsive to tumor microenviroment on the basis of the result of particle size analyzers(DLS, SLS). Once the pH of environment decreases to 6.0~6.3, the nanoparticles switch their surface charges as well as transform their structure. We also use liquid sample TEM, particle size analyzer and fluorescent probes to analyze their interactions. In conclusion, according to in vitro experiments, we proved that pH-responsive property can enhance the effect of photothermal therapy.

目錄
Abstract-------3
摘要-------4
一、研究動機-------5
二、文獻回顧-------7
2.1癌症治療-------7
2.1.1 熱治療-------7
2.1.2 光熱治療藥物IR780-------10
2.1.3奈米藥物載體傳遞系統-------11
2.2高分子材料簡介-------12
2.2.1 雙性高分子-------12
2.2.2雙性高分子微胞-------13
2.2.3 Imidazole contain polymer 於藥物傳遞系統之應用-------15
2.2.4聚乙二醇之性質介紹-------17
2.2.5聚電解質簡介-------18
三、實驗方法與步驟-------21
3.1 實驗藥品-------21
3.2 實驗儀器-------23
3.3 高分子合成與鑑定-------24
3.3.1 Methoxy poly(ethylene glycol) - carbonyl di imidazole (mPEG-CDI)之合成與鑑定-------24
3.3.2 Poly(methacrylic acid) (PMAA) 之合成與鑑定-------24
3.3.3 Methoxy poly(ethylene glycol)-b-poly(methacrylic acid) (mPEG-b-PMAA)之合成與鑑定-------24
3.3.4 Methoxy poly(ethylene glycol)-b-poly(methacrylic acid-co-histamine methacrylamine) (mPEG-b-P(MAA-co-HMA))之合成與鑑定-------25
3.3.5高分子等電點測定-------25
3.4 奈米微粒製備與性質探討-------26
3.4.1 奈米微粒製備-------26
3.4.2奈米微粒的藥物裝載量-------26
3.4.3奈米微粒的表面電荷分析-------27
3.4.4奈米微粒的粒徑分布-------28
3.4.5奈米微粒膠體及IR 780光學穩定性-------28
3.4.6液相穿透式電子顯微鏡影像-------29
3.4.7 穿透式電子顯微鏡影像-------31
3.4.8 熱場發射掃描式電子顯微鏡-------32
3.4.9穩態螢光光譜儀測定-------32
3.4.10奈米微粒經近紅外光雷射照射升溫實驗-------32
3.5 體外細胞實驗-------33
3.5.1 細胞來源及適合之培養環境-------33
3.5.2 配置細胞培養液與磷酸鹽緩衝溶液-------33
3.5.3 細胞繼代-------33
3.5.4 細胞計數-------34
3.5.5 螢光顯微鏡觀察-------34
3.5.6 細胞毒性分析-------35
3.5.7細胞內 IR780 吞噬含量分析-------36
3.5.8細胞胞內 IR780 升溫效率評估-------37
四、結果與討論-------37
4.1 高分子合成與鑑定-------37
4.1.1 mPEG-CDI與PMAA 合成與鑑定-------37
4.1.2 mPEG-b-PMAA 與 mPEG-b-P(MAA-co-HMA) 合成與鑑定-------38
4.1.3高分子等電點檢測-------39
4.2 奈米粒子特性分析-------40
4.2.1奈米微粒性質及其酸鹼應答之分析-------40
4.4.2奈米微粒所搭載之光熱藥物性質分析-------47
4.3 細胞實驗-------50
4.3.1載藥奈米微粒於微酸環境下的細胞吞噬評估-------50
4.5.2載藥奈米微粒於微酸環境下的細胞光熱升溫效果評估-------51
五、結論-------53
六、參考文獻-------54

[1] Grothey, A., et al., Survival of patients with advanced colorectal cancer improves with the availability of fluorouracil-leucovorin, irinotecan, and oxaliplatin in the course of treatment. Journal of Clinical Oncology, 2004, 22(7), 1209-1214.
[2] Thorn, C.F., et al., Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenet Genomics, 2011, 21(7), 440-446.
[3] Cascinu, S., et al., Neuroprotective effect of reduced glutathione on oxaliplatin-based chemotherapy in advanced colorectal cancer: a randomized, double-blind, placebo-controlled trial. Journal of Clinical Oncology, 2002, 20(16), 3478-3483.
[4] Kwon, G.S. and T. Okano, Polymeric micelles as new drug carriers. Advanced Drug Delivery Reviews, 1996, 21(2), 107-116.
[5] Chu, K.F. and D.E. Dupuy, Thermal ablation of tumours: biological mechanisms and advances in therapy. Nature Reviews Cancer, 2014, 14(3), 199-208.
[6] Van der Zee, J., Heating the patient: a promising approach? Annals of Oncology, 2002, 13(8), 1173-1184.
[7] Huang, X.H., et al., Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. Journal of the American Chemical Society, 2006, 128(6), 2115-2120.
[8] Gobin, A.M., et al., Near-infrared resonant nanoshells for combined optical imaging and photothermal cancer therapy. Nano Letters, 2007, 7(7), 1929-1934.
[9] Huang, X.H., et al., Plasmonic photothermal therapy (PPTT) using gold nanoparticles. Lasers in Medical Science, 2008, 23(3), 217-228.
[10] Loo, C., et al., Immunotargeted nanoshells for integrated cancer imaging and therapy. Nano Letters, 2005, 5(4), 709-711.
[11] Robinson, J.T., et al., Ultrasmall Reduced Graphene Oxide with High Near-Infrared Absorbance for Photothermal Therapy. Journal of the American Chemical Society, 2011, 133(17), 6825-6831.
[12] Xu, L.G., et al., Conjugated polymers for photothermal therapy of cancer. Polymer Chemistry, 2014, 5(5), 1573-1580.
[13] Pais-Silva C., de Melo-Diogo D., Correia I.J., IR780-loaded TPGS-TOS micelles for breast cancer photodynamic therapy. European Journal of Pharmaceutics and Biopharmaceutics, 2017, 113, 108-117.
[14] Guo, F., et al., Smart IR780 Theranostic Nanocarrier for Tumor-Specific Therapy: Hyperthermia-Mediated Bubble-Generating and Folate-Targeted Liposomes. ACS Applied Materials & Interfaces, 2015, 7(37), 20556-20567.
[15] Guo, F., et al., The mitochondria-targeted and IR780-regulated theranosomes for imaging and enhanced photodynamic/photothermal therapy. RSC Advances, 2016, 6(14), 11070-11076.
[16] Wang, K.K., et al., Self-assembled IR780-loaded transferrin nanoparticles as an imaging, targeting and PDT/PTT agent for cancer therapy. Scientific Reports, 2016, 6.
[17] Luke, D.R., et al., Effects of Cyclosporine on the Isolated Perfused Rat-Kidney. Transplantation, 1987, 43(6), 795-799.
[18] Jain, R.K., Delivery of molecular and cellular medicine to solid tumors. Advanced Drug Delivery Reviews, 2012, 64, 353-365.
[19] Peer, D., et al., Nanocarriers as an emerging platform for cancer therapy. Nature Nanotechnology, 2007, 2(12), 751-760.
[20] Ferrari, M., Cancer nanotechnology: opportunities and challenges. Nature Reviews Cancer, 2005, 5(3), 161-171.
[21] Torchilin, V.P., Multifunctional, stimuli-sensitive nanoparticulate systems for drug delivery. Nature Reviews Drug Discovery, 2014, 13(11), 813-827.
[22] Lee, C.T., Huang, C.P., Lee, Y.D., Preparation of Amphiphilic Poly(L-lactide)-graft-Chondroitin Sulfate Copolymer Self-aggregates and Its Aggregation Behavior. Biomacromolecules, 2006, 7(4), 1179-1186.
[23] Christine, M., et al., A New Double-Responsive Block Copolymer Synthesized via RAFT Polymerization: Poly(N-isopropylacrylamide)-block-poly(acrylic acid). Macromolecules, 2004, 37(21), 7861-7866.
[24] Zhang, L., Lin, J., Lin S., Self-Assembly Behavior of Amphiphilic Block Copolymer/Nanoparticle Mixture in Dilute Solution Studied by Self-Consistent-Field Theory/Density Functional Theory. Macromolecules, 2007, 40(15), 5582-5592.
[25] Zhang, W., et al., Thermoresponsive core–shell–corona micelles of poly(ethyleneglycol)-b-poly(N-isopropylacrylamide)-b-polystyrene. Polymer, 2006, 47(24), 8203-8209.
[26] Liu, X., et al., Bowl-Shaped Aggregates from the Self-Assembly of an Amphiphilic Random Copolymer of Poly(styrene-co-methacrylic acid). Macromolecules, 2005, 38(16), 6749–6751.
[27] Comments off on Polymers and macromolecules, PHARMACY, Aug 14, 2016.
[28] Gao, W. P., et al., Controlling Vesicle Formation via Interpolymer Hydrogen-Bonding Complexation between Poly(ethylene oxide)-block-polybutadiene and Poly(acrylic acid) in Solution. Macromolecules, 2006, 39(14), 4894-4898.
[29] Harada, A., Kataoka K., Chain Length Recognition: Core-Shell Supramolecular Assembly from Oppositely Charged Block Copolymers. Science, 1999, 283(5398), 65-67.
[30] Harada, A., Kataoka K., Formation of Polyion Complex Micelles in an Aqueous Milieu from a Pair of Oppositely-Charged Block Copolymers with Poly(ethylene glycol) Segments. Macromolecules, 1995, 28(15), 5294-5299.
[31] Rijcken, C., et al., Triggered destabilisation of polymeric micelles and vesicles by changing polymers polarity: An attractive tool for drug delivery. Journal of Controlled Release, 2007, 120(3), 131-148.
[32] Bader, H., Ringsdorf, H., Schmidt B., Watersoluble polymers in medicine. Angewandte Chemie, 1984, 123(1), 457-485.
[33] Yokoyama, M., et al., Characterization and anticancer activity of the micelle-forming polymeric anticancer drug adriamycin-conjugated poly(ethylene glycol)-poly(aspartic acid) block copolymer. Cancer Research, 1990, 50(6), 1693-1700.
[34] Allen, C., Maysinger, D., Eisenberg, A., Nano-engineering block copolymer aggregates for drug delivery. Colloids and Surfaces B: Biointerfaces, 1999, 16(1-4), 3-27.
[35] Kataoka, K., et al., Spontaneous Formation of Polyion Complex Micelles with Narrow Distribution from Antisense Oligonucleotide and Cationic Block Copolymer in Physiological Saline. Macromolecules, 1996, 29(26), 8556-8557.
[36] Thuemann, A. F., Beyermann, J., Kukula, H., Poly(ethylene oxide)-b-poly(L-lysine) Complexes with Retinoic Acid. Macromolecules, 2000, 33(16), 5906-5911.
[37] Lee, E. S., et al., Poly(L-histidine)-PEG block copolymer micelles and pH-induced destabilization. Journal of Controlled Release, 2003, 90(3), 363-374.
[38] Benjaminsen, R. V., et al., The Possible "Proton Sponge" Effect of Polyethylenimine (PEI) Does Not Include Change in Lysosomal pH. Molecular Therapy, 2013, 21(1), 149-157.
[39] Li, S. L., et al., pH-responsive biocompatible fluorescent polymer nanoparticles based on phenylboronic acid for intracellular imaging and drug delivery. Nanoscale, 2014, 6(22), 13701-13709.
[40] Vivek, R., et al., pH-responsive drug delivery of chitosan nanoparticles as Tamoxifen carriers for effective anti-tumor activity in breast cancer cells. Colloids and Surfaces B-Biointerfaces, 2013, 111, 117-123.
[41] He, Q. J., et al., A pH-responsive mesoporous silica nanoparticles-based multi-drug delivery system for overcoming multi-drug resistance. Biomaterials, 2011, 32(30), 7711-7720.
[42] Gao, W. W., Chan, J. M. , and Farokhzad, O. C., pH-Responsive Nanoparticles for Drug Delivery. Molecular Pharmaceutics, 2010, 7(6), 1913-1920.
[43] Lee, E. S., et al., Super pH-sensitive multifunctional polymeric micelle for tumor pHe specific TAT exposure and multidrug resistance. Journal of Controlled Release, 2008. 129(3), 228-236.
[44] Lee, E. S., et al., Tumor pH-responsive flower-like micelles of poly(L-lactic acid)-b-poly (ethylene glycol)-b-poly(L-histidine). Journal of Controlled Release, 2007, 123(1), 19-26.
[45] Lee, E. S., Na, K., Bae, Y. H., Super pH-Sensitive Multifunctional Poymeric Micelle. NANO LETTERS, 2005, 5(2), 325-329.
[46] Lee, E. S., Na, K., Bae, Y. H., Polymeric micelle for tumor pH and folate-mediated targeting. Journal of Controlled Release, 2002, 91(1-2), 103-113.
[47] Zhu, X., et al., Imidazole-modified porphyrin as a pH-responsive sensitizer for cancer photodynamic therapy. Chemical Communications, 2011, 47(37), 10311-10313.
[48] Zalipsky, S., Functionalized Poly(ethylene glycols) for Preparation of Biologically Relevant Conjugates. Bioconjugate Chemistry, 1995, 6(2), 150–165.
[49] Zalipsky, S., Chemistry of polyethylene glycol conjugates with biologically active molecules. Advanced Drug Delivery Reviews, 1995, 16(2–3), 157-182.
[50] Merril, E. W., Salzman, E. W., Polyethylene oxide as a biomaterial. ASAIO Journal, 1983, 6, 60-64.
[51] Dreborg, S., Akerblom, B., Immunotherapy with monomethoxypolyethylene glycol modified allergens. Critical Reviews™ in Therapeutic Drug Carrier Systems, 1990, 6(4), 315-365.
[52] Yamaoka, T., Tabata, Y., Ikada, Y., Distribution and tissue uptake of poly(ethylene glycol) with different molecular weights after intravenous administration to mice. Journal of Pharmaceutical Sciences, 1994, 83(4), 601-606.
[53] Blu, G., Cevc, G., Liposomes for the sustained drug release in vivo. Biochimica et Biophysica Acta, 1990, 1029(1), 91-97.
[54] Antonsen, K. P., Hoffman, A. S., Harris, J. M. (ed), Poly(ethylene glycol) : Chemistry – Biotechnical and Biomedical Applications. Plenum Press, 1992, 15-28.
[55] Jeon, S. I., et al., Protein—surface interactions in the presence of polyethylene oxide: I. Simplified theory. Journal of Colloid and Interface Science, 1991, 142(1), 149-158.
[56] Polson, A., et al., The fractional of protein mixtures by linear polymers of high molecular weight. Biochimica et Biophysica Acta, 1964, 82, 463-475.
[57] Zeppezauer, M., Brishammar, S., Protein precipitation by uncharged water-soluble polymers. Biochimica et Biophysica Acta, 1965, 94(2), 581-583.
[58] Chun, P. W., Fried, M., Ellis, E. E., Use of water-soluble polymers for the isolation and purification of human immunoglobulins. Analytical Biochemistry, 1967, 19(3), 481-497.
[59] Albertsson, P. A., Partition of Cell Particles and Macromolecules, 3rd, ed. Wiley: New York, 1986.
[60] Abuchowski, A., et al., Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol. Journal of Biological Chemistry, 1977, 252(11), 3578-3581.
[61] Hurwitz, E., et al., Inhibition of tumor growth by poly(ethylene glycol) derivatives of anti-ErbB2 antibodies. Cancer Immunology, Immunotherapy, 2000, 49(4-5), 226-234.
[62] Veronese, F. M., Peptide and protein PEGylation: a review of problems and solutions. Biomaterials, 2001, 22(5), 405-417.
[63] Bentley, D. M., Zhao, X., PCT Int. Appl., 2001, WO 01/00246 A2, 21pp.
[64] Scott, R. A., Peppas, N. A., Highly crosslinked, PEG-containing copolymers for sustained solute delivery. Biomaterials, 1999, 20(1), 1371-1380.
[65] Fares, H. M., Zatz, J. L., Mechanism of polyethylene glycol-8\/SMDI copolymer in controlled delivery of topically applied drugs. Journal of Cosmetic Science, 1999, 50(3), 133-146.
[66] Dunn, S. E., et al. Polystyrene-Poly (Ethylene Glycol) (PS-PEG2000) Particles as Model Systems for Site Specific Drug Delivery. 2. The Effect of PEG Surface Density on the in Vitro Cell Interaction and in Vivo Biodistribution. Pharmaceutical Research, 1994, 11(7), 1016-1022.
[67] Carelli, V., et al., Evaluation of a silicone based matrix containing a crosslinked polyethylene glycol as a controlled drug delivery system for potential oral application. Journal of Controlled Release, 1995, 33(1), 153-162.
[68] Zalipsky, S., Gilon, C., Zilkha, A., Attachment of drugs to polyethylene glycols. European Polymer Journal, 1983, 19(12), 1177-1183.
[69] Meka, V. S., et al., A comprehensive review on polyelectrolyte complexes. Drug Discovery Today, 2017, 22(11), 1697-1706.
[70] Li, Q. L., et al., Chitosan-phosphorylated chitosan polyelectrolyte complex hydrogel as an osteoblast carrier. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2007, 82(2), 481-486.
[71] Dragan, S., et al., Polyelectrolyte complexes. I. Synthesis and characterization of some insoluble polyanion-polycation complexes. Journal of Polymer Science, Part A: Polymer Chemistry, 1996, 34(17), 3485-3494.
[72] Dautzenberg, H., Light scattering studies on polyelectrolyte complexes. Macromolecular Symposia, 2000, 162(1), 1-22.
[73] Hartig, S.M., et al., Multifunctional nanoparticulate polyelectrolyte complexes. Pharmaceutical Research, 2007, 24(12), 2353-2369.
[74] Dai, Z., et al., Polyelectrolyte complexes based on chitosan and poly (L-glutamic acid). Polymer International, 2007, 56(9), 1122-1127.
[75] Wu, H. D., et al., Development of a chitosan–polyglutamate based injectable polyelectrolyte complex scaffold. Carbohydrate Polymers, 2011, 85(2), 318-324.
[76] Lankalapalli, S., and Kolapalli, V. R. M., Polyelectrolyte complexes: A review of their applicability in drug delivery technology. Indian Journal of Pharmaceutical Sciences, 2009, 71(5), 481-487.