臺灣博碩士論文加值系統

本研究目的主要為製備同時具有緩慢釋放生長因子與超音波觸發藥物釋放特性的複合藥物載體。研究中以微脂體、褐藻酸鹽為主要材料，分別擔負親水性藥物的包覆與超音波的觸發釋控及緩慢釋放兩種功能。實驗中以螢光顯微鏡與穿透式電子顯微鏡分析證實磷脂質結構，並以共軛焦顯微鏡驗證藥物包覆於複合載體內。之後利用多功能微量盤式分析儀測量包覆於微脂體中藥物之包覆效率並繪製藥物釋放曲線，探討載體緩慢釋放與施予不同物理治療用超音波條件下， 藥物釋放曲線的改變。最後藉由細胞與動物實驗來驗證載體內生長因子仍具效用

In this research work, the drug carrier alginate liposome composite (ALC) beads capable of being ultrasound triggered and sustained release have been studied. ALC beads were made of two different structures, in which hydrogel was to provide sustained releases and liposome ultrasound responses. The ALC beads were characterized by utilizing fluorescent microscopy, confocal microscopy, transmission electron microscopy (TEM) and multimode micro-plate readers. The effects of ultrasound response and sustained release were investigated. It was found that the proposed composite carrier could sustain release growth factor and could be efficiently and remotely triggered by using therapeutic ultrasound. Furthermore, in vitro / vivo experiments confirmed that the growth factor in ALC beads could still work.

致謝 I
中文摘要 III
ABSTRACT IV
目錄 V
圖目錄 IX
表目錄 XI
第一章 緒論 1
1.1 前言 1
1.2研究背景與動機 2
第二章 文獻回顧 1
2.1 骨 1
2.1.1 骨組織 1
2.1.1.1 骨組織結構 2
2.1.1.2軟、硬骨之介紹 4
2.1.1.3 骨之種類 8
2.1.2 骨修復與骨生成 9
2.1.2.1 骨組織的修復 (Bone healing) 9
2.1.3 生長因子 13
2.1.3.1 骨形成蛋白 (Bone Morphogenetic Proteins) 介紹 14
2.1.3.2 包覆BMPs之藥物傳輸系統 17
2.1.3.3 攜載蛋白質藥物的方法 21
2.2 微脂體 (liposome) 23
2.2.1 微脂體構造 23
2.2.2 微脂體的種類 24
2.2.3 使用微脂體的優點 26
2.2.4 微脂體的製備方法 27
2.2.5 影響微脂體包覆效率的因素 29
2.2.6 微脂體的純化 30
2.2.7 微脂體的相變化 31
2.3 褐藻酸鹽 (Alginate) 34
2.4 超音波介紹 36
2.4.1 醫療用超音波 36
2.4.2 超音波之效應 39
2.4.2.1超音波之機械效應 39
2.4.2.2 超音波之氣穴效應 40
2.4.2.3 超音波之熱效應 42
2.5 研究目的 42
第三章 實驗藥品與儀器設備 44
3.1 實驗藥品 44
3.2 儀器設備 45
第四章 實驗方法與步驟 47
4.1 材料的製備 47
4.1.1 微脂體製備 47
4.1.2 微脂體褐藻酸水膠球(ALC beads)製備 47
4.2檢測 48
4.2.1穿透式電子顯微鏡 (TEM) 48
4.2.2 掃描式電子顯微鏡 (SEM) 48
4.2.3 雷射掃描式共軛焦顯微鏡 (Confocal) 49
4.2.4 多功能盤式分析儀 49
4.3 藥物包覆與釋放 49
4.3.1 藥物包覆 49
4.3.1.1 檢量線的製備 50
4.3.1.2 藥物包覆的測量 51
4.3.2 藥物釋放 51
4.3.3 超音波觸發 52
4.4.4 超音波熱效應 52
4.4 細胞實驗 52
4.4.1 老鼠前驅骨細胞株(MC3T3-E1)培養 52
4.4.2. 細胞存活率試驗 53
4.4.3 ALP (Alkaline phosphatase) 54
4.4.4 細胞內蛋白質含量測試 54
4.4.5 Alizarin red 染色 55
4.5 動物實驗 55
4.5.1 骨外成骨 55
4.5.2 骨磨損成骨 57
4.5.3 組織切片 58
第五章 結果與討論 61
5.1 微脂體 (liposome) 61
5.1.1 微脂體之結構 61
5.1.2 微脂體之載藥 64
5.2 ALC beads 之結構 67
5.3 不同參數之超音波熱效應 70
5.4 ALC 複合球之藥物釋放與超音波觸發釋放 72
5.4.1 ALC複合球載有DFITC之藥物釋放與超音波觸發釋放 73
5.4.2 ALC複合球載有BMP-2之藥物釋放與超音波觸發釋放 74
5.4.3 ALC 複合球載DFITC於不同頻率超音波之藥物釋放 76
5.5 細胞實驗 77
5.5.1 超音波對細胞存活率的影響 77
5.5.2 載藥ALC 複合球對於細胞存活率的影響 81
5.5.3 ALC beads 對於骨細胞分化的影響 82
5.6 動物實驗 84
5.6.1 組織染色 85
第六章 結論 89
參考文獻 90

圖目錄
圖 2- 1皮質骨的顯微結構 (a) 皮質骨的三維結構圖 (b) 哈氏系統(Haversian system) 部分結構圖 (c) 哈氏系統(Haversian system) 顯微結構圖。 4
圖 2- 2 各式軟骨圖 (a) 透明軟骨 (hyaline cartilage) (b) 彈性軟骨(elastic cartilage) (c) 纖維軟骨 (fibrocartilage) 6
圖 2- 3 膜內骨化過程 7
圖 2- 4 軟骨內骨化過程 8
圖 2- 5 骨缺陷修復之階段示意圖 13
圖 2- 6 應用於骨修復之生長因子載體鍵結方式 22
圖 2- 7 微脂體構造圖親水性藥物包覆於微脂體中間，疏水性藥物包覆於脂質層中。 24
圖 2- 8 不同微脂體結構示意圖 25
圖 2- 9磷脂質脂肪酸鍊結構 32
圖 2- 10 微脂體經由溫度觸發控釋示意圖 33
圖 2- 11 褐藻酸鹽結構 34
圖 2- 12 褐藻酸鹽與鈣離子膠體化示意圖 35
圖 2- 13 可應用於治療之超音波圖 38
圖 2- 14超音波之三大效應 39

圖 4- 1 BMP2 ELISA 測試流程圖 51
圖 4- 2動物實驗 (骨外成骨)流程圖 56
圖 4- 3動物實驗 (骨磨損成骨) 58
圖 4- 4 細胞實驗H&;E 染色流程圖 60

圖 5- 1以乙醇注射法製備出之MLV型微脂體之穿透式電子顯微鏡圖 (a) 多顆微脂體聚集 (b) 單顆微脂體 *右上角黑框中為微脂體之脂質層 62
圖 5- 2 微脂體之掃描式電子顯微鏡圖 63
圖 5- 3 微脂體之DLS測量圖 63
圖 5- 4 UV燈下載有螢光染劑Dextran-FITC 之微脂體離心上清液 65
圖 5- 5微脂體包覆螢光染劑Dextran-FITC 之共軛焦顯微鏡圖 (a) 白光下 (b) 螢光下 (c) 合併 65
圖 5- 6 螢光染劑 Dextran-FITC 檢量線 (R2= 0.99549) 66
圖 5- 7生長因子 BMP-2 檢量線 (R2= 0.99542) 66
圖 5- 8載有微脂體ALC beads 圖(黑線長度為 2cm) 68
圖 5- 9 ALC beads之掃描式電子顯微鏡圖 (a) 褐藻酸鹽水膠無微脂體 (b) 褐藻酸水膠含微脂體 (紅圈處) 69
圖 5- 10載有螢光染劑之ALC 複合球之共軛焦顯微鏡3D圖 70
圖 5- 11 ALC 複合球載螢光染劑DFITC之藥物釋放曲線 74
圖 5- 12 ALC 複合球載生長因子BMP-2之藥物釋放曲線 75
圖 5- 13 載有螢光染劑DFITC 之ALC複合球於不同超音波頻率觸發下之藥物釋放曲線。 77
圖 5- 14 不同功率超音波下細胞存活率 79
圖 5- 15 不同超音波觸發時間下細胞存活率 80
圖 5- 16 載有BMP-2之ALC 複合球對於細胞存活率的影響 81
圖 5- 17 骨細胞 MC 3T3-E1 Alizarin Red S 染色顯微鏡圖 (a) 載BMP-2之 ALC 複合球 (b) 無載藥之ALC 複合球 (c) 控制組 (d) 實驗結果巨觀照。(黃色箭頭為鈣離子堆積處) 83
圖 5- 18 動物實驗配置圖。(1) 包覆BMP2之ALC beads加超音波。(2) 包覆BMP2之ALC beads。(3) 無包覆BMP2之ALC beads加超音波。(4) BMP2 溶液加超音波。 85
圖 5- 19 HE 染色圖 86
圖 5- 20 HE染色與Alizarin Red S 染色比較圖 (a)(b) 包覆BMP2之ALC beads施予超音波 (c)(d) 包覆BMP2之ALC beads (e)(f) 無包覆BMP2之ALC beads施予超音波 88

表目錄
表 2- 1骨缺陷自然修復之各階段 12
表 2- 2 BMPs 發現及歷史 16
表 2- 3 BMPs家族因子與其功能 17
表 2- 4常見包覆BMPs載體 20
表 2- 5不同結構微脂體粒徑分布 26
表 2- 6微脂體的相變化 31
表 2- 7常見磷脂質相轉變溫度(Tc) 33

表 5- 1三種模擬藥物比較 64
表 5- 2不同藥物於微脂體包覆 67
表 5- 3 不同參數之超音波熱效應 71

1. Laurencin, C., et al., Bone graft substitutes. Expert Rev Med Devices, 2006. 3(1): pp. 49-57.
2. Belthur, M.V., et al., Bone graft harvest using a new intramedullary system. Clin Orthop Relat Res, 2008. 466(12): pp. 2973-80.
3. Kim, D.H., et al., Prospective study of iliac crest bone graft harvest site pain and morbidity. Spine J, 2009. 9(11): pp. 886-92.
4. Schwartz, C.E., et al., Prospective evaluation of chronic pain associated with posterior autologous iliac crest bone graft harvest and its effect on postoperative outcome. Health Qual Life Outcomes, 2009. 7: pp. 49.
5. Sen, M.K. and T. Miclau, Autologous iliac crest bone graft: should it still be the gold standard for treating nonunions? Injury, 2007. 38 Suppl 1: pp. S75-80.
6. Nauth, A., et al., Growth factors and bone regeneration: how much bone can we expect? Injury, 2011. 42(6): pp. 574-579.
7. Lieberman, J.R., et al., The role of growth factors in the repair of bone. Biology and clinical applications. J Bone Joint Surg Am, 2002. 84-A(6): pp. 1032-44.
8. Helm, G.A., et al, Bone graft substitutes for the promotion of spinal arthrodesis. Neurosurg Focus, 2001. 10(4): pp. E4.
9. Orcel, P., et al., Formation of multinucleated cells with osteoclast precursor features in human cord monocytes cultures. Anat Rec, 1990. 226(1): pp. 1-9.
10. Kenley, R.A., et al., Biotechnology and bone graft substitutes. Pharm Res, 1993. 10(10): pp. 1393-401.
11. Urist, M.R., Bone: Formation by Autoinduction. Science, 1965. 150(3698): pp. 893-899.
12. McGovern, et al., Can Bone Morphogenetic Protein Binding Peptide Increase Efficiency of Bone Formation? Spine 2010. 35(17).
13. SETTI S. RENGACHARY, M.D., Bone morphogenetic proteins: basic concepts. Neurosurgical Focus, 2002. 13.
14. Boden, S.D.M.S.,et al., The use of an osteoinductive growth factor for lumbar spinal fusion. Part II:study of dose, carrier, and species. spine 1995. 20(24).
15. Friedlaender, G.E., et al., Osteogenic protein-1 (bone morphogenetic protein-7) in the treatment of tibial nonunions. J Bone Joint Surg Am, 2001. 83-A Suppl 1(Pt 2): pp. S151-8.
16. Martin, G.J., Jr., et al., Posterolateral intertransverse process spinal arthrodesis with rhBMP-2 in a nonhuman primate: important lessons learned regarding dose, carrier, and safety. J Spinal Disord, 1999. 12(3): pp. 179-86.
17. Alanay, A., et al., The adjunctive effect of a binding peptide on bone morphogenetic protein enhanced bone healing in a rodent model of spinal fusion. Spine (Phila Pa 1976), 2008. 33(16): pp. 1709-13.
18. Luginbuehl, V., et al., Localized delivery of growth factors for bone repair. Eur J Pharm Biopharm, 2004. 58(2): pp. 197-208.
19. Haidar, Z.S., et al., Delivery of recombinant bone morphogenetic proteins for bone regeneration and repair. Part B: Delivery systems for BMPs in orthopaedic and craniofacial tissue engineering. Biotechnol Lett, 2009. 31(12): pp. 1825-35.
20. Institute of Pharmaceutical Sciences, S.F.I.o.T.Z.E.Z., Zurich, Switzerland, Localized delivery of growth factors for bone repair. European Journal of Pharmaceutics and Biopharmaceutics, 2004. 58.
21. Bangham, A.D., et al., Diffusion of univalent ions across the lamellae of swollen phospholipids. Journal of Molecular Biology, 1965. 13(1): pp. 238-IN27.
22. Sessa, G. and G. Weissmann, Phospholipid spherules (liposomes) as a model for biological membranes. J Lipid Res, 1968. 9(3): pp. 310-8.
23. Weinstein, J.N. and L.D. Leserman, Liposomes as drug carriers in cancer chemotherapy. Pharmacol Ther, 1984. 24(2): pp. 207-33.
24. Institute, M.D.A.H.a.T., Liposomes as Carriers of Antimicrobial Agents. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,, 1987. 31.
25. Litchfield, et al., Highly sensitive immunoassays based on use of liposomes without complement. Clin Chem, 1984. 30(9): pp. 1441-5.
26. O'Connell, J.PP., et al., A highly sensitive immunoassay system involving antibody-coated tubes and liposome-entrapped dye. Clin Chem, 1985. 31(9): pp. 1424-6.
27. Wiedmer, S.K., et al., Liposomes as carriers in electrokinetic capillary chromatography. Electrophoresis, 2000. 21(15): pp. 3191-8.
28. Mizushima, Y., et al., Use of lipid microspheres as a drug carrier for antitumour drugs. Journal of Pharmacy and Pharmacology, 1986. 38(2): pp. 132-134.
29. Law, S.L., et al., Properties of Human Placental Alkaline-Phosphatase Entrapped in Liposomes. Drug Development and Industrial Pharmacy, 1987. 13(7): pp. 1239-1255.
30. Zalutsky, M.R., et al., Use of liposomes as carriers for radiation synovectomy. Int J Rad Appl Instrum B, 1988. 15(2): pp. 151-6.
31. Sunamoto, J., et al., A newly developed immunoliposome — an egg phosphatidylcholine liposome coated with pullulan bearing both a cholesterol moiety and an IgMs fragment. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1987. 898(3): pp. 323-330.
32. Kato, A., et al., Effect of egg yolk lecithin on transdermal delivery of bunazosin hydrochloride. J Pharm Pharmacol, 1987. 39(5): pp. 399-400.
33. Das, N., et al., Plant glycosides in a liposomal drug-delivery system. Biochem J, 1987. 247(2): pp. 359-61.
34. Steerenberg, PP.A., et al., Liposomes as drug carrier system for cis-diamminedichloroplatinum (II). II. Antitumor activity in vivo, induction of drug resistance, nephrotoxicity and Pt distribution. Cancer Chemother Pharmacol, 1988. 21(4): pp. 299-307.
35.Available from: https://sitn.hms.harvard.edu/sitnflash_wp/2011/06/materials-for-drug-delivery/.
36. Okada, H. and H. Toguchi, Biodegradable microspheres in drug delivery. Crit Rev Ther Drug Carrier Syst, 1995. 12(1): pp. 1-99.
37. Zalipsky, S., Synthesis of an end-group functionalized polyethylene glycol-lipid conjugate for preparation of polymer-grafted liposomes. Bioconjugate Chemistry, 1993. 4(4): pp. 296-299.
38. Harasym, T.O., et al., Poly(ethylene glycol)-modified phospholipids prevent aggregation during covalent conjugation of proteins to liposomes. Bioconjug Chem, 1995. 6(2): pp. 187-94.
39. Huang, C.-H., Phosphatidylcholine vesicles. Formation and physical characteristics. Biochemistry, 1969. 8(1): pp. 344-352.
40. Olson, F., et al., Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1979. 557(1): pp. 9-23.
41. Pick, U., Liposomes with a large trapping capacity prepared by freezing and thawing of sonicated phospholipid mixtures. Archives of Biochemistry and Biophysics, 1981. 212(1): pp. 186-194.
42. Kasahara, M. and PP.C. Hinkle, Reconstitution and purification of the D-glucose transporter from human erythrocytes. J Biol Chem, 1977. 252(20): pp. 7384-90.
43. Fan, M., et al., Preparation of salidroside nano-liposomes by ethanol injection method and in vitro release study. European Food Research and Technology, 2007. 227(1): pp. 167-174.
44. Pons, M., et al., Liposomes obtained by the ethanol injection method. International Journal of Pharmaceutics, 1993. 95(1-3): pp. 51-56.
45. Jaafar-Maalej, C., et al., Ethanol injection method for hydrophilic and lipophilic drug-loaded liposome preparation. J Liposome Res, 2010. 20(3): pp. 228-43.
46. Domazou, A.S. and PP.L. Luisi, Size distribution of spontaneously formed liposomes by the alcohol injection method. J Liposome Res, 2002. 12(3): pp. 205-20.
47. Batzri, S. and E.D. Korn, Single bilayer liposomes prepared without sonication. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1973. 298(4): pp. 1015-1019.
48. Deamer, D. and A.D. Bangham, Large volume liposomes by an ether vaporization method. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1976. 443(3): pp. 629-634.
49. Deamer, D.W., Preparation and Properties of Ether-Injection Liposomes. Annals of the New York Academy of Sciences, 1978. 308(1 Liposomes and): pp. 250-258.
50. Szoka, F. and D. Papahadjopoulos, Procedure for Preparation of Liposomes with Large Internal Aqueous Space and High Capture by Reverse-Phase Evaporation. Proceedings of the National Academy of Sciences of the United States of America, 1978. 75(9): pp. 4194-4198.
51. Lasic, D.D., Novel applications of liposomes. Trends Biotechnol, 1998. 16(7): pp. 307-21.
52. Lasic, D.D. Liposomes：from Physics to Applications. 1993: Elsevier Science Publishers B.V.
53. 楊淑萍, 三團聯共聚物及鎂離子對微脂粒物理穩定性之影響及其機制探討. 中央大學 碩論, 2000.
54. Taylor, K.M.G. and R.M. Morris, Thermal analysis of phase transition behaviour in liposomes. Thermochimica Acta, 1995. 248: pp. 289-301.
55. de Smet, M., et al., Temperature-sensitive liposomes for doxorubicin delivery under MRI guidance. J Control Release, 2010. 143(1): pp. 120-7.
56. Georg Jensen, M., et al., Acute effect of alginate-based preload on satiety feelings, energy intake, and gastric emptying rate in healthy subjects. Obesity (Silver Spring), 2012. 20(9): pp. 1851-8.
57. Kashima, K. and M. Imai, Advanced Membrane Material from Marine Biological Polymer and Sensitive Molecular-Size Recognition for Promising Separation Technology. 2012.
58. Lutz, H.a.B., Manual of diagnostic ultrasound.
59. Speed, C.A., Therapeutic ultrasound in soft tissue lesions. Rheumatology, 2001. 40(12): pp. 1331-1336.
60. Frenkel, V., Ultrasound mediated delivery of drugs and genes to solid tumors. Adv Drug Deliv Rev, 2008. 60(10): pp. 1193-208.
61. Mitragotri, S., Healing sound: the use of ultrasound in drug delivery and other therapeutic applications. Nat Rev Drug Discov, 2005. 4(3): pp. 255-60.
62. Frenkel, V., et al., Ultrasound-facilitated transport of silver chloride (AgCl) particles in fish skin. J Control Release, 2000. 68(2): pp. 251-61.
63. Lauterborn, W., et al., Acoustic cavitation, bubble dynamics and sonoluminescence. Ultrason Sonochem, 2007. 14(4): pp. 484-91.
64. Newman, et al., Gene therapy progress and prospects: ultrasound for gene transfer. Gene Ther, 2007. 14(6): pp. 465-75.
65. Miller, M.W., et al., A review of in vitro bioeffects of inertial ultrasonic cavitation from a mechanistic perspective. Ultrasound Med Biol, 1996. 22(9): pp. 1131-54.
66. Mast, T.D., et al., Ultrasound monitoring of in vitro radio frequency ablation by echo decorrelation imaging. J Ultrasound Med, 2008. 27(12): pp. 1685-97.
67. Holland, C.K. and R.E. Apfel, Thresholds for transient cavitation produced by pulsed ultrasound in a controlled nuclei environment. J Acoust Soc Am, 1990. 88(5): pp. 2059-69.
68. Weiss, PP., Shrimps Spew Bubbles as Hot as the Sun. Science News, 2001. 160(14): pp. 213.
69. Injury Assessment and Rehabilitation part 2 chapter 7 Therapeutic Modalities.
70. Cambier, K.D.H.E.W.D., Therapeutic ultrasound: Temperature increase at different depths by different modes in a human cadaver. Journal of Rehabilitation Medicine, 2001. 33(5): pp. 212-215.