研究生: |
陳稼豫 Chia-Yu Chen |
---|---|
論文名稱: |
藉由超音波結合微氣泡促進皮膚細胞藥物傳輸之機制探討 Investigation of The Mechanism of Ultrasound Mediated Microbubbles Cavitation for Transdermal Drug Delivery Enhancement |
指導教授: |
廖愛禾
Ai-Ho Liao |
口試委員: |
江建平
Chien-Ping Chiang 沈哲州 Che-Chou Shen 莊賀喬 Ho-Chiao Chuang 王正康 Jehng-Kang Wang |
學位類別: |
碩士 Master |
系所名稱: |
應用科技學院 - 醫學工程研究所 Graduate Institute of Biomedical Engineering |
論文出版年: | 2019 |
畢業學年度: | 107 |
語文別: | 中文 |
論文頁數: | 65 |
中文關鍵詞: | 超音波 、白蛋白微氣泡 、皮膚 、穴蝕效應 、絲氨酸蛋白酶 |
外文關鍵詞: | Ultrasound, Microbubble, Skin, Cavitation, Matriptase |
相關次數: | 點閱:349 下載:0 |
分享至: |
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報 |
藥物及保養品的導入方法較常見的為超音波,將超音波結合微氣泡增加藥物或保養品的滲透量也是近幾年熱門的經皮傳輸方法,不少研究指出,微氣泡結合超音波作用後能引發穴蝕效應,增加皮膚的通透度以此提升傳輸的藥量,本研究為探討此經皮傳輸於皮膚內的機制。
ㄧ開始先做了超音波的溫度檢測,為確保微氣泡是經由超音波施打後破裂而非熱效應,了解到1分鐘時其溫度上升幅度最小打破效率也最佳,再使用豬皮,找出微氣泡的使用濃度及超音波的施打次數,將豬皮以染劑模擬藥物滲透,超音波結合微氣泡施打後做染劑滲透,經過超音波的施打使微氣泡破裂,引發穴蝕效應,開啟皮膚的通透度,增加染劑的滲透量,無施打超音波其滲透深度為17.67 ± 1.56 μm,以超音波施打1分鐘後其染劑滲透深度為26.02 ± 1.11 μm,施打次數越多其染劑滲透深度越深,控制組與有施打超音波之染劑滲透深度皆有明顯的差異(p <0.001)。
經過豬皮的實驗證實超音波結合微氣泡的促滲效果,找出細胞實驗所使用的超音波參數,以細胞實驗了解經超音波施打後細胞的變化。matriptase為絲胺酸蛋白酶的ㄧ種,與表皮的分化、恆定與傷口癒合等生物調控起重要作用,細胞經過超音波結合微氣泡的作用後,使用西方墨點法觀察matriptase是否有被活化,將蛋白表現做影像強度量化後,可以知道M24的組別中其影像強度至少有74.32 ± 6.65 %的差異,M69的組別中其影像強度則至少有46.44 ± 4.12 %的差異,施打超音波後western蛋白表現所計算出的影像皆有40 %以上的差異,本研究從豬皮實驗及細胞實驗中可以觀察出超音波結合微氣泡的施打後可以開啟細胞間的通道,之後matriptase會修復所打開的通道,因而活化matriptase,同時細胞內合成matriptase的量增加,進而恢復細胞膜上matriptase的量,因此可以回復之前的狀態。
The most common method for introducing drugs and skin care products is ultrasound. Ultrasound mediated microbubbles to increase the penetration of drugs or skin care products is also a popular transdermal delivery method in recent years. Many studies have pointed out that ultrasound mediated microbubbles can induce cavitation effects and increase the permeability of the skin to increase the amount of drug delivered. This study was to investigate the mechanism of this transdermal delivery.
Starting from the temperature detection of the ultrasonic wave, it is ensured that the microbubbles are broken by the ultrasonic instead of the thermal effect. Understand that the temperature rises the smallest at 1 minute and destruction efficiency is also the best. Then use the pigskin to find out the concentration of microbubbles and the number of times the ultrasonic are applied. The pig skin is dyed with Evans blue to simulate drug penetration. After ultrasound mediated microbubbles is applied, the microbubbles are broken to cause cavitation effects, and the permeability of the skin is opened to increase the penetration amount of the dye. The group without ultrasound had a penetration depth of 17.67 ± 1.56 μm. After 1 minute of ultrasound application, the penetration depth of the dye was 26.02 ± 1.11 μm. There was a significant difference in the penetration depth between the Control group and the dyeing agent (p < 0.001).
After confirming the effect of ultrasonic wave combined with the promotion of microbubbles, find out the ultrasonic parameters used in the cell experiment. After confirming the effect of ultrasound mediated microbubble penetration, find out the ultrasonic parameters used in cell experiments. Cellular experiments were used to understand the changes in cells after supersonic application. Matriptase is an enzyme of serine protease, which plays an important role in the biological regulation of epidermal differentiation, constant and wound healing. After the cells were subjected to ultrasound mediated microbubbles effects, western blotting was used to observe whether matriptase was activated. After the protein relative expression was quantified, it was found that the image intensity of the M24 group was at least 74.32 ± 6.65 %, and the M69 group had at least 46.44 ± 4.12 % of the image intensity. After the western protein expression, the calculated images all have more than 40% difference. This study observed that after the application of ultrasound mediated microbubbles, it will activate matriptase. After ultrasound mediated microbubbles, it will affect the expression of proteins in the cells, and then open the permeability of the skin. Ultrasound mediated microbubble can open the channel between cells and activate matriptase.
[1] J. S. Barbieri, K. Wanat, and J. Seykora, "Skin: Basic Structure and Function," in Pathobiology of Human Disease, 2014, pp. 1134-1144.
[2] J. E. Lai-Cheong and J. A. McGrath, "Structure and function of skin, hair and nails," Medicine, vol. 45, no. 6, pp. 347-351, 2017.
[3] A. Baroni, E. Buommino, V. De Gregorio, E. Ruocco, V. Ruocco, and R. Wolf, "Structure and function of the epidermis related to barrier properties," Clin Dermatol, vol. 30, no. 3, pp. 257-62, 2012.
[4] OpenStax, Anatomy & Physiology. OpenStax CNX, Feb 26, 2016 p. Chapter 5. The Integumentary System.
[5] M. Venus, J. Waterman, and I. McNab, "Basic physiology of the skin," Surgery (Oxford), vol. 29, no. 10, pp. 471-474, 2011.
[6] O. Arda, N. Goksugur, and Y. Tuzun, "Basic histological structure and functions of facial skin," Clin Dermatol, vol. 32, no. 1, pp. 3-13, 2014.
[7] M. Machado, T. M. Salgado, J. Hadgraft, and M. E. Lane, "The relationship between transepidermal water loss and skin permeability," Int J Pharm, vol. 384, no. 1-2, pp. 73-7, 2010.
[8] 皮膚科王修含醫師, "超音波傳遞速率:聲音速度與介質的關係皮膚科," Available: http://www.skin168.net/2012/02/blog-post.html, 2012.
[9] 李嘉明、李玉華, "新超音波醫學1:醫用超音波的基礎," 合計圖書出版社, 2003.
[10] LightYear, "Ultrasound range diagram," Available: https://commons.wikimedia.org/wiki/File:Ultrasound_range_diagram.svg, 2007.
[11] 皮膚科王修含醫師, "醫用超音波(超聲波)簡介," Available: http://www.skin168.net/2012/02/blog-post.html, 2013.
[12] N. J. Hangiandreou, "AAPM/RSNA Physics Tutorial for Residents: Topics in US," B-mode US: Basic Concepts and New Technology1, vol. 23, no. 4, 2003.
[13] 王嘉弘, "高效率超音波驅動電路設計在生醫應用之研究," 碩士, 生物醫學科技研究所, 國立暨南國際大學, 南投縣, 2006.
[14] L. D. Johns, "Nonthermal effects of therapeutic ultrasound: the frequency resonance hypothesis," Journal of athletic training, vol. 37, no. 3, p. 293, 2002.
[15] P. Greillier, C. Bawiec, F. Bessière, and C. Lafon, "Therapeutic Ultrasound for the Heart: State of the Art," Irbm, vol. 39, no. 4, pp. 227-235, 2018.
[16] V. Sboros, "Response of contrast agents to ultrasound," Adv Drug Deliv Rev, vol. 60, no. 10, pp. 1117-36, 2008.
[17] M. J. Blomley, J. C. Cooke, E. C. Unger, M. J. Monaghan, and D. O. Cosgrove, "Microbubble contrast agents: a new era in ultrasound," Bmj, vol. 322, no. 7296, pp. 1222-1225, 2001.
[18] S. Sirsi and M. Borden, "Microbubble Compositions, Properties and Biomedical Applications," Bubble Sci Eng Technol, vol. 1, no. 1-2, pp. 3-17, 2009.
[19] M. W. G. a. K. S. Suslick, "Air-filled proteinaceous microbubbles: synthesis of an echo-contrast agent.," Proc Natl Acad Sci US A., vol. 88, pp. 7708-7710, 1991.
[20] S. Tinkov, R. Bekeredjian, G. Winter, and C. Coester, "Microbubbles as ultrasound triggered drug carriers," Journal of pharmaceutical sciences, vol. 98, no. 6, pp. 1935-1961, 2009.
[21] J. Wischhusen and F. Padilla, "Ultrasound-Targeted Microbubble Destruction (UTMD) for Localized Drug Delivery into Tumor Tissue," Irbm, vol. 40, no. 1, pp. 10-15, 2019.
[22] M. Cheng, F. Li, T. Han, A. C. H. Yu, and P. Qin, "Effects of ultrasound pulse parameters on cavitation properties of flowing microbubbles under physiologically relevant conditions," Ultrason Sonochem, vol. 52, pp. 512-521, 2019.
[23] N. Wallace, S. Dicker, P. Lewin, and S. P. Wrenn, "Inertial cavitation threshold of nested microbubbles," Ultrasonics, vol. 58, pp. 67-74, 2015.
[24] M. Guedra, C. Cornu, and C. Inserra, "A derivation of the stable cavitation threshold accounting for bubble-bubble interactions," Ultrason Sonochem, vol. 38, pp. 168-173, 2017.
[25] P. Muleki Seya, C. Desjouy, J. C. Bera, and C. Inserra, "Hysteresis of inertial cavitation activity induced by fluctuating bubble size distribution," Ultrason Sonochem, vol. 27, pp. 262-267, 2015.
[26] M. Wang and Y. Zhou, "Numerical investigation of the inertial cavitation threshold by dual-frequency excitation in the fluid and tissue," Ultrason Sonochem, vol. 42, pp. 327-338, 2018.
[27] V. Meidan and B. B. Michniak-Kohn, "Ultrasound-based Technology for Skin Barrier Permeabilization," in Handbook of Non-Invasive Drug Delivery Systems: Elsevier, 2010, pp. 119-133.
[28] S. R. Sirsi and M. A. Borden, "Advances in ultrasound mediated gene therapy using microbubble contrast agents," Theranostics, vol. 2, no. 12, p. 1208, 2012.
[29] K. J. L. D, "Effect of ultrasound on transdermal drug delivery to rats and guinea pigs," The Journal of Clinical Investigation, vol. 83, no. 2, pp. 2074-2078, 1989.
[30] 陳思嘉 and 李百祺, "靶向超音波於血栓溶解之研究," 臺灣大學生醫電子與資訊學研究所學位論文, pp. 1-64, 2009.
[31] 謝依峻, "組織背景抑制於諧波對比劑偵測," 2007.
[32] N. D. Rawlings and A. J. Barrett, "Evolutionary families of peptidases," Biochemical Journal, vol. 290, no. 1, pp. 205-218, 1993.
[33] L. Hedstrom, "Serine protease mechanism and specificity," Chemical reviews, vol. 102, no. 12, pp. 4501-4524, 2002.
[34] K. List, T. H. Bugge, and R. Szabo, "Matriptase: potent proteolysis on the cell surface," Mol Med, vol. 12, no. 1-3, pp. 1-7, 2006.
[35] P. Ovaere, S. Lippens, P. Vandenabeele, and W. Declercq, "The emerging roles of serine protease cascades in the epidermis," Trends in biochemical sciences, vol. 34, no. 9, pp. 453-463, 2009.
[36] W. Appel, "Chymotrypsin: molecular and catalytic properties," Clinical biochemistry, vol. 19, no. 6, pp. 317-322, 1986.
[37] L. B. Evnin, J. R. Vásquez, and C. S. Craik, "Substrate specificity of trypsin investigated by using a genetic selection," Proceedings of the National Academy of Sciences, vol. 87, no. 17, pp. 6659-6663, 1990.
[38] Y. E. Shi, J. Torri, L. Yieh, A. Wellstein, M. E. Lippman, and R. B. Dickson, "Identification and characterization of a novel matrix-degrading protease from hormone-dependent human breast cancer cells," Cancer research, vol. 53, no. 6, pp. 1409-1415, 1993.
[39] C.Y. Lin, J. Anders, M. Johnson, Q. A. Sang, and R. B. Dickson, "Molecular cloning of cDNA for matriptase, a matrix-degrading serine protease with trypsin-like activity," Journal of Biological Chemistry, vol. 274, no. 26, pp. 18231-18236, 1999.
[40] C. Kim, Y. Cho, C. H. Kang, M. G. Kim, H. S. Lee, E. G. Cho, D. Park, "Filamin is essential for shedding of the transmembrane serine protease, epithin," EMBO Rep, vol. 6, no. 11, pp. 1045-51, 2005.
[41] C. Y. Lin, J. Anders, M. Johnson, and R. B. Dickson, "Purification and characterization of a complex containing matriptase and a Kunitz-type serine protease inhibitor from human milk," Journal of Biological Chemistry, vol. 274, no. 26, pp. 18237-18242, 1999.
[42] T. M. Antalis, M. S. Buzza, K. M. Hodge, J. D. Hooper, and S. Netzel-Arnett, "The cutting edge: membrane-anchored serine protease activities in the pericellular microenvironment," Biochem J, vol. 428, no. 3, pp. 325-46, 2010.
[43] C. Y. Lin, I. C. Tseng, F. P. Chou, S. F. Su, Y.-W. Chen, M. D. Johnson, R. B. Dickson, "Zymogen activation, inhibition, and ectodomain shedding of matriptase," Frontiers in bioscience: a journal and virtual library, vol. 13, pp. 621-635, 2008.
[44] T. S. H. Kataoka, T. Kawaguchi, R. Hamasuna, H. Itoh, N. Kitamura, K. Miyazawa, M. Koono, "Hepatocyte growth factor activator inhibitor type 1 is a specific cell surface binding protein of hepatocyte growth factor activator (HGFA) and regulates HGFA activity in the pericellular microenvironment," Journal of Biological Chemistry, vol. 275, no. 51, pp. 40453-40462, 2000.
[45] C. Benaud, M. Oberst, J. P. Hobson, S. Spiegel, R. B. Dickson, and C. Y. Lin, "Sphingosine 1-phosphate, present in serum-derived lipoproteins, activates matriptase," J Biol Chem, vol. 277, no. 12, pp. 10539-46, 2002.
[46] H. X. I. C. Tseng, F. P. Chou, G. Li, A. P. Vazzanno, J. P. Y. Kao, M. D. Johnson, C. Y. Lin, "Matriptase activation, an early cellular response to acidosis," Journal of Biological Chemistry, vol. 285, no. 5, pp. 3261-3270, 2010.
[47] I. J. T. J. K. Wang, T. J. Lo, S. Moore, Y. H. Yeo, Y. C. Teng, M. Kaul, C. C. Chen, A. H. Zuo, F. P. Chou, X. Yang, I. C. Tseng, M D. Johnson, C. Y Lin, "Matriptase autoactivation is tightly regulated by the cellular chemical environments," PLoS One, vol. 9, no. 4, p. e93899, 2014.
[48] C. C. H. K. List, R. Szabo, W. Chen, S. M. Wahl, W. Swaim, L. H. Engelholm, N Behrendt, TH Bugge,, "Matriptase/MT-SP1 is required for postnatal survival, epidermal barrier function, hair follicle development, and thymic homeostasis," Oncogene, vol. 21, no. 23, p. 3765, 2002.
[49] B. M. C. S. Netzel-Arnett, R. Szabo, C. Y. Lin, L. M. Chen, K. X. Chai, T.-M. Antalis, TH Bugge, K. List, "Evidence for a matriptase-prostasin proteolytic cascade regulating terminal epidermal differentiation," Journal of Biological Chemistry, vol. 281, no. 44, pp. 32941-32945, 2006.
[50] 陳奇雍, "人類皮膚對紫外線的反應,以及其中 matriptase所扮演的角色," 國防醫學院, 生命科學研究所, 博士 2017.
[51] A. H. Liao, H. C. Ho, Y. C. Lin, H. K. Chen, and C. H. Wang, "Effects of microbubble size on ultrasound-induced transdermal delivery of high-molecular-weight drugs," PloS one, vol. 10, no. 9, p. e0138500, 2015.
[52] E. Stride, "Physical principles of microbubbles for ultrasound imaging and therapy," Cerebrovascular Diseases, vol. 27, no. Suppl. 2, pp. 1-13, 2009.
[53] J. C. Stockert, A. Blazquez-Castro, M. Canete, R. W. Horobin, and A. Villanueva, "MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets," Acta Histochem, vol. 114, no. 8, pp. 785-96, 2012.