臺灣博碩士論文加值系統

癌症為國人十大死因之首已蟬聯多年，在癌症的治療方法普遍為外科手術、放射療法及化學藥物療法，近年來隨著許多研究發現，也有一些新興的療法可以治療癌症並帶來較少的副作用，熱治療為目前相當熱門的研究之一，透過將組織升至42-47°C，癌細胞會死亡而正常細胞則不會。
本研究以合併化學療法和熱療為主題，利用本實驗室所開發的磁性氫氧基磷灰石(magnetic hydroxyapatite, mHAP)做為藥物載體，磁性氫氧基磷灰石為一生物相容性良好的材料，於共沉澱法加入蛋白經由高溫 (800°C) 脫碳後，可形成中孔洞 (mesoporous) 的多孔結構；再透過鐵離子置換鈣離子可合成出中孔洞磁性氫氧基磷灰石，施加交流電感應磁場後，材料的溫度約十分鐘後即可上升至熱療所需溫度42°C，並且透過計算出其特定吸收率 (specific absorption rate) 為182 W/g，且於超導性量子干涉儀之分析結果顯示中孔洞磁性氫氧基磷灰石為超順磁性材料，具有良好發熱效率。利用紅外線光譜分析，硬脂酸確實能達到表面改質的目的，使原本親水性的磁性氫氧基磷灰石的表面變為疏水性，藉此可以透過物理吸附上疏水性的抗癌藥物抑特癌，其載藥效率經由UV/Vis檢測為26.12%，相當於每克的材料可以攜帶21.77毫克的抑特癌。
於小鼠纖維母細胞 (3T3 cell) 之WST-1測試結果顯示中孔洞磁性氫氧基磷灰石具有良好的生物相容性，從WST-1測試結果得知，材料通交流電磁場後於熱療溫度42 - 43°C間會降低約40%的A549肺腺癌細胞之存活率；化療部分顯示抑特癌於第一天就有藥物釋放，並於第三天能隨著劑量上升殺死更多的癌細胞；於LDH試驗結果分析，化療合併熱治療無論第一天或第三天都能比單一療法具有更好的療效，具有加成性效果，並於第三天達到殺死約80 %的癌細胞，可達到雙功能(dual function)的療效。

Cancer is the fatal disease in the world. Clinical cancer treatment can be commonly classified into three categories: surgery, radiation therapy and chemotherapy. With research gradually developing, there is an emerging treatment, hyperthermia, which can reduce the side effect of cancer treatment. Hyperthermia can lead cancer cell to death by rising the temperature between 42-47°C in the body tissue.
Our topic focuses on the combination of hyperthermia and chemotherapy in order to enhance the efficiency of cancer treatments. As a medicine carrier, magnetic hydroxyapatite (mHAP) shows good biocompatibility. By adding egg white in co-precipitation method to form hydroxyapatite (Ca10(PO4)6(OH)2) and then calcinating it at 800°C to remove carbon, we can develop mesoporous hydroxyapatite. After iron ion (Fe2+) substituting the calcium ion (Ca2+) of hydroxyapatite, we develop mesoporous magnetic hydroxyapatite. It has specific absorption rate at about 182 W/g and it can rise to hyperthermia temperature within ten minutes by adding alternating magnetic field. Besides, it also has superparamagnetic property according to SQUID analysis. Since we use anticancer drug Etoposide is hydrophobic, we use stearic acid to change hydrophilic mesoporous magnetic hydroxyapatite into hydrophobic one. Therefore, we can load Etoposide on mesoporous magnetic hydroxyapatite by adsorption. The loading efficiency is 26.12% by UV/Vis analysis.
Mesoporous magnetic hydroxyapatite shows good biocompatibility in 3T3 cell by WST-1 assay. After keeping hyperthermia temperature (42-43°C) for 20 minutes, the cell viability of A549 cells is reduced by 40%. Etoposide release causes cytotoxicity and kills more cancer cells after three days. Evidence shows that combination of hyperthermia and chemotherapy can enhance cancer treatment of hyperthermia or chemotherapy and cause about 80% of cytotoxicity of A549 lung cancer cells after three day treatment. Combination of hyperthermia and chemotherapy can enhance cancer treatment additively and fulfill therapy in dual function.

中文摘要 i
Abstract iii
目錄 v
圖目錄 viii
表目錄 x
第一章 簡介 1
1-1 前言 1
1-2 癌症 3
1-3 癌症治療 4
1-3-1 外科手術 4
1-3-2 放射線療法 5
1-3-3 化學藥物療法 6
1-3-4 免疫療法 7
1-3-5 熱治療 8
1-4 熱療機制 9
1-5 研究目的 12
1-6 癌症熱治療的磁性粒子裝載化療藥物 13
第二章 基本理論 15
2-1 磁性理論 15
2-1-1 磁性來源 15
2-1-2 磁性材料分類 15
2-1-3 發熱機制 18
2-1-4 發熱效率 20
2-2 材料和表面改質 21
2-2-1 氫氧基磷灰石 (hydroxyapatite, HAP) 21
2-2-2 硬脂酸 (stearic acid) 21
2-2-3 抑特癌 (etoposide) 22
第三章 材料與方法 23
3-1 實驗儀器 23
3-2 實驗藥品 24
3-3 材料製備 25
3-3-1 中孔洞磁性氫氧基磷灰石製備 25
3-3-2 中孔洞磁性氫氧基磷灰石的表面改質 26
3-4 材料分析 28
3-4-1 X光粉末繞射儀 (X-Ray diffraction, XRD) 28
3-4-2 粒徑分析儀 (Dynamic light scattering, DLS) 29
3-4-3 掃描式電子顯微鏡 (Scanning Electron Microscopy, SEM) 30
3-4-4 比表面積分析測定儀 31
3-4-5 超導性量子干涉儀 (Superconducting QUantum Interference Device Magnetometer, SQUID) 32
3-4-6 高週波加熱器 33
3-4-7 傅立葉轉換紅外線光譜儀 33
3-4-8 紫外光/可見光光譜儀 (UV/Vis Spectroscopy) 34
3-5 生物相容性測試 35
3-6 細胞毒性測試 36
3-7 體外測試 (in vitro test) 37
第四章 結果與討論 38
4-1 磁性氫氧基磷灰石之性質分析 38
4-1-1 磁性氫氧基磷灰石之X光繞射圖譜 38
4-1-2 磁性氫氧基磷灰石之粒徑分析儀圖譜 40
4-1-3 中孔洞氫氧基磷灰石的型態和孔徑分析 41
4-1-4 中孔洞磁性氫氧基磷灰石磁性分析 43
4-2 中孔洞磁性氫氧基磷灰石裝載藥物之分析 45
4-2-1 中孔洞磁性氫氧基磷灰石表面修飾 45
4-2-2 載藥效率 47
4-3 中孔洞磁性氫氧基磷灰石之體外測試 48
4-3-1 生物相容性測試 48
4-3-2 中孔洞磁性氫氧基磷灰石之熱治療 49
4-3-3 中孔洞磁性氫氧基磷灰石裝載抑特癌之化學治療 50
4-3-4 中孔洞磁性氫氧基磷灰石裝載抑特癌合併熱治療及化學治療 51
第五章 結論 52
第六章 參考文獻 53

1.林宗洲, 林萬哲 “癌症是什麼”, 談起癌症,健康文化事業股份有限公司, 2-5, 1978
2.施益民, 梁偉華 “腫瘤的命名及分類”, 癌症生物學, 藝軒圖書出版社, 59-65, 1991
3.施益民, 梁偉華 “癌症之治療”, 癌症生物學, 藝軒圖書出版社, 277-297, 1991
4.施益民, 梁偉華 “癌症的擴張”, 癌症生物學, 藝軒圖書出版社, 67-85, 1991
5.Rosenberg, S.A., Cell transfer immunotherapy for metastatic solid cancer-what clinicians need to know. Nature Reviews Clinical Oncology, 2011. 8(10): p. 577-585.
6.Sapareto, S.A., et al., Effects of hyperthermia on survival and progression of chinese-hamster ovary cells. Cancer Research, 1978. 38(2): p. 393-400.
7.Wust, P., et al., Hyperthermia in combined treatment of cancer. Lancet Oncology, 2002. 3(8): p. 487-497.
8.Harmon, B.V., et al., Cell-death induced in a murine mastocytoma by 42-47-degrees-C heating invitro - evidence that the form of death changes from apoptosis to necrosis above a critical heat load. International Journal of Radiation Biology, 1990. 58(5): p. 845-858.
9.Kapronbras, C.M., et al., Heat-shock induced tolerance to the embryos effects of hyperthermia and cadmium in mouse embryos invitro. Teratology, 1991. 43(1): p. 83-94.
10.Roti, J.L.R., Cellular responses to hyperthermia (40-46 degrees C): Cell killing and molecular events. International Journal of Hyperthermia, 2008. 24(1): p. 3-15.
11.Kampinga, H.H., et al., Cells overexpressing HSP27 show accelerated recovery from heat-induced nuclear-protein aggregation. Biochemical and Biophysical Research Communications, 1994. 204(3): p. 1170-1177.
12.Song, C.W., Effect of local hyperthermia on blood-flow and microenvironment - a review. Cancer Research, 1984. 44(10): p. 4721-4730.
13.Matsuoka, F., et al., Hyperthermia using magnetite cationic liposomes for hamster osteosarcoma. Biomagn Res Technol, 2004. 2(1): p. 3.
14.Moreira, F.T.C., et al., Smart plastic antibody material (SPAM) tailored on disposable screen printed electrodes for protein recognition: Application to myoglobin detection. Biosensors & Bioelectronics, 2013. 45: p. 237-244.
15.Hall, E. J., Hyperthermia, Radiobiology for the radiologist, 5th ed., 504,
Philadelphia, Lippincott Williams & Wilkins, 2000.
16.Issels, R.D., Hyperthermia adds to chemotherapy. European Journal of Cancer, 2008. 44(17): p. 2546-2554.
17.Vaupel, P., et al., Blood-flow, oxygen and nutrient supply, and metabolic microenvironment of human-tumors - a review. Cancer Research, 1989. 49(23): p. 6449-6465.
18.Streffer, C., Molecular and cellular mechanisms of hyperthermia, Hyperthermia and the therapy of malignant tumors, 61, Berlin; New York: Springer-Verlag, 1987
19.Jordan, A., et al., Endocytosis of dextran and silan-coated magnetite nanoparticles and the effect of intracellular hyperthermia on human mammary carcinoma cells in vitro. Journal of Magnetism and Magnetic Materials, 1999. 194(1-3): p. 185-196.
20.Bergey, E.J., et al., DC magnetic field induced magnetocytolysis of cancer cells targeted by LH-RH magnetic nanoparticles in vitro. Biomedical Microdevices, 2002. 4(4): p. 293-299.
21.Mi, Y., et al., Multimodality treatment of cancer with herceptin conjugated, thermomagnetic iron oxides and docetaxel loaded nanoparticles of biodegradable polymers. Biomaterials, 2012. 33(30): p. 7519-7529.
22.Pradhan, P., et al., Targeted temperature sensitive magnetic liposomes for thermo-chemotherapy. Journal of Controlled Release, 2010. 142(1): p. 108-121.
23.Ito, A., et al., 4-S-Cysteaminylphenol-loaded magnetite cationic liposomes for combination therapy of hyperthermia with chemotherapy against malignant melanoma. Cancer Science, 2007. 98(3): p. 424-430.
24.Wu, H.C., et al., A novel biomagnetic nanoparticle based on hydroxyapatite. Nanotechnology, 2007. 18(16).
25.Hou, C.H., et al., The in vivo performance of biomagnetic hydroxyapatite nanoparticles in cancer hyperthermia therapy. Biomaterials, 2009. 30(23-24): p. 3956-3960.
26.王蔚任 “可分解磁性玻璃於腫瘤熱治療之研究”, 國立台灣大學醫學工程研究所碩士論文, 19-22, 2005
27.Laurent, S., et al., Magnetic fluid hyperthermia: Focus on superparamagnetic iron oxide nanoparticles. Advances in Colloid and Interface Science, 2011. 166(1-2): p. 8-23.
28.Frenkel, J., et al., Spontaneous and induced magnetisation in ferromagnetic bodies. Nature, 1930. 126: p. 274-275.
29.Batlle, X., et al., Finite-size effects in fine particles: magnetic and transport properties. Journal of Physics D-Applied Physics, 2002. 35(6): p. R15-R42.
30.Kumar, C., et al., Magnetic nanomaterials for hyperthermia-based therapy and controlled drug delivery. Advanced Drug Delivery Reviews, 2011. 63(9): p. 789-808.
31.Mornet, S., et al., Magnetic nanoparticle design for medical diagnosis and therapy. Journal of Materials Chemistry, 2004. 14(14): p. 2161-2175.
32.Jordan, A., et al., Magnetic fluid hyperthermia (MFH): Cancer treatment with AC magnetic field induced excitation of biocompatible superparamagnetic nanoparticles. Journal of Magnetism and Magnetic Materials, 1999. 201: p. 413-419.
33.Fortin, J.P., et al., Size-sorted anionic iron oxide nanomagnets as colloidal mediators for magnetic hyperthermia. Journal of the American Chemical Society, 2007. 129(9): p. 2628-2635.
34.Weitschies, W., et al., Mobility of magnetic PEG-nanoparticles in blood, liver and spleen of rats, in Proc. And World Meeting APGI/APV, paris, 1998.
35.Ma, M., et al., Size dependence of specific power absorption of Fe3O4 particles in AC magnetic field. Journal of Magnetism and Magnetic Materials, 2004. 268(1-2): p. 33-39.
36.Uskokovic, V., et al., Nanosized hydroxyapatite and other calcium phosphates: Chemistry of formation and application as drug and gene delivery agents. Journal of Biomedical Materials Research Part B-Applied Biomaterials, 2011. 96B(1): p. 152-191.
37.Yao, J., et al., Hydroxyapatite nanostructure material derived using cationic surfactant as a template. Journal of Materials Chemistry, 2003. 13(12): p. 3053-3057.
38.Li, Y.B., et al., Surface modification of hydroxyapatite by stearic acid: characterization and in vitro behaviors. Journal of Materials Science-Materials in Medicine, 2008. 19(1): p. 19-25.
39.Patton, J.S., et al., Inhaling medicines: delivering drugs to the body through the lungs. Nature Reviews Drug Discovery, 2007. 6(1): p. 67-74.
40.Zhou, R.C., et al., Water-dispersible hydroxyapatite nanoparticles synthesized in aqueous solution containing grape seed extract. Applied Surface Science, 2012. 258(8): p. 3578-3583.
41.Wang, H.L., et al., Preparation of irregular mesoporous hydroxyapatite. Materials Research Bulletin, 2008. 43(6): p. 1607-1614.
42.Zhang, L., et al., Oleic acid coating on the monodisperse magnetite nanoparticles. Applied Surface Science, 2006. 253(5): p. 2611-2617.
43.Sivakumar, M., et al., Preparation, characterization and in vitro release of gentamicin from coralline hydroxyapatite-gelatin composite microspheres. Biomaterials, 2002. 23(15): p. 3175-3181.
44.Wadee, A., et al., Recent advances in the design of drug-loaded polymeric implants for the treatment of solid tumors. Expert Opinion on Drug Delivery, 2011. 8(10): p. 1323-1340.