抗血管治療是以阻斷腫瘤不成熟血管，使氧氣與養分無法運輸至腫瘤組織內，進而達到抑制腫瘤的治療效果。然若以現行之抗血管治療，則會因腫瘤周圍血管多為成熟且具有穩定結構使其無法被治療，產生存活邊緣效應，造成局部腫瘤復發。而以超音波刺激微氣泡產生之慣性穴蝕效應可造成血管破裂，達到抗血管之療效。由文獻中得知血管破裂效率會與慣性穴蝕效應劑量成正比，因此若藉由調控超音波參數提高慣性穴蝕效應劑量，則可能擊破腫瘤周圍血管。此外，若再搭配化療藥物，除了可使其累積在腫瘤周圍組織中，補償抗血管治療對腫瘤周圍產生之抗性，也可使其經由破裂血管滲透至腫瘤組織提升藥物滲透量。故本研究藉由調控超音波參數提升抗血管治療效率和藥物滲透，來提高整體療效。本研究使用自製之微氣泡(1.12±0.08 µm)進行體外慣性穴蝕效應劑量之測量，用以評估與抗血管效應之相關性。並以載有窗型觀測腔之小鼠透過血管破裂效率和藥物滲透量來評估適當超音波參數，以應用後續實質腫瘤之治療。在實質腫瘤治療之部分，以2 MHz、1000週期數、2 Hz脈衝重複頻率和7 MPa聲壓並注射2×107微氣泡進行超音波照射，同時搭配自製之微脂體阿黴素(160 µg/mouse)進行結合治療。透過窗型觀測腔實驗，發現以3、5、7和9 MPa的聲壓照射時，血管直徑介於21-30 µm分別有0、33、50和50%的機率被擊破，並且血管破裂大小與慣性穴蝕效應劑量之相關指數為0.931，因此選用7 MPa進行實質腫瘤之結合治療。在療效部分，腫瘤中心和周圍血流灌注分別下降至27±3%和27±4% (p>0.05)，而血管密度則分別下至23±14%和24±5% (p>0.05)。由此結果可知結合治療可均勻地治療腫瘤中心和周圍，並相較於控制組有74±2.4%的腫瘤生長抑制效果(p<0.05)。因此，本研究證實了使用適當之超音波參數，能有效提升血管破裂效率和藥物滲透，減少存活邊緣效應並提升活體存活率。

Anti-vascular therapy disrupts tumor immature vessels to restrict oxygen and nutrient transport for tumor growth inhibition. However, tumor peripheral vessels with mature and stable structure show treatment resistance in current anti-vascular therapy, called the viable rim effect, to induce local tumor recurrence. Ultrasound stimulated microbubble can damage vascular endothelial cells to accomplish anti-vascular therapy by microbubble inertial cavitation. Since previous studies reported that the efficiency of vascular disruption was proportional to the microbubble inertial cavitation dose, which provided a potential way to disrupt tumor peripheral vessels by regulating ultrasound parameters. Furthermore, chemotherapy was applied to compensate the treatment resistance of anti-vascular therapy via drug accumulation in tumor periphery, the disrupted tumor vessels can also improve drug penetration in tumor center. Therefore, our study investigated the efficiency of anti-vascular effect and drug penetration to improve therapeutic efficacy by regulating ultrasound stimulated microbubbles. The in vitro inertial cavitation dose of home-made microbubbles (1.12±0.08 µm) was measured to evaluate the correlation in anti-vascular effect. Window chamber mice model was applied to define the optimal ultrasound parameters by assessing the efficiency of vascular disruption and drug penetration. The optimal parameters of ultrasound (2-MHz, 1000-cycle, pulse repetition frequency of 2 Hz, 7 MPa, and 2x107 microbubbles/mouse) and liposomal-doxorubicin (160 µg/mouse) were applied for combination therapy in solid tumor model. The efficiency of vascular disruption at 3, 5, 7, and 9 MPa showed 0, 33, 50, and 50% at the vessel size of 21-30 µm. The correlation coefficient was 0.931 between the in vitro inertial cavitation dose of microbubbles and disrupted vessel size. Comparison the treatment efficacy in tumor center and periphery, blood perfusion was reduced to 27±3% and 27±4% (p>0.05), and vessel density was decreased to 23±14% and 24±5% (p>0.05). The uniform treatment in both tumor center and periphery by combination therapy inhibited 74±2.4% of tumor growth relative to the control group (p<0.05). Therefore, our study confirmed that regulating ultrasound parameters can improve the efficiency of anti-vascular therapy and drug penetration to reduce the viable rim effect and inhibit tumor growth.

摘要
目錄
第一章 緒論 P.1
1.1 惡性腫瘤 P.1
1.1.1. 腫瘤微環境 (Tumor microenvironment)- P.1
1.1.2. 腫瘤血管 P.2
1.1.3. 腫瘤治療與困境 P.3
1.2 抗血管治療 (Anti-vascular therapy) P.5
1.2.1. 血管破裂劑(Vascular disrupting agents, VDAs) P.5
1.2.2. 結合治療 P.6
1.3 超音波 P.7
1.3.1. 超音波對比劑微氣泡 P.7
1.3.2. 超音波與物質之作用 P.8
1.3.3. 超音波抗血管作用 P.10
1.4 阿黴素(Doxorubicin, DOX) P.11
1.4.1. 微脂體阿黴素 (Lipo-Doxorubicin, Lipo-DOX) P.12
1.5 研究目的與內容 P.12
第二章 實驗材料與方法 P.14
2.1 概論 P.14
2.2 超音波對比劑微氣泡之製備 P.14
2.2.1. 粒徑量測 P.15
2.2.2. 穩定度測量 P.15
2.2.3. 擊破閥值測量 P.16
2.2.4. 慣性穴蝕效應劑量測量 P.19
2.2.5. 微氣泡體外超音波實驗之參數統整 P.21
2.3 微脂體阿黴素之製備 P.21
2.3.1. 粒徑測量 P.22
2.3.2. 載藥量測量 P.22
2.4 細胞培養 P.23
2.4.1. 小鼠攝護腺癌細胞之繼代 P.24
2.5 窗型觀測腔(Window chamber) P.24
2.5.1. 窗型觀測腔架設與測量組別 P.24
2.5.2. 滲漏螢光強度 P.29
2.5.3. 血管效應評估 P.29
2.5.4. 有效時間內微氣泡造成之血管效應評估 P.30
2.6 實質腫瘤治療 P.31
2.6.1. 實質腫瘤治療架設 P.31
2.6.2. 灌注影像分析 P.34
2.6.3. 組織切片 P.34
2.7 治療成效 P.36
2.8 統計分析 P.36
第三章 結果與討論 P.38
3.1 微氣泡之物化性質 P.38
3.1.1 粒徑分析 P.38
3.1.2 體外穩定性 P.38
3.1.3 微氣泡擊破閥值測量 P.39
3.1.4 慣性穴蝕效應劑量分析 P.41
3.2 微脂體阿黴素之特性 P.42
3.2.1 粒徑分析 P.42
3.2.2 載藥量效率 P.43
3.3 窗型觀測腔實驗結果 P.43
3.3.1 超音波聲壓評估 P.43
3.3.2 微氣泡濃度評估 P.48
3.3.3 超音波週期評估 P.51
3.3.4 有效時間內微氣泡造成的血管效應 P.53
3.3.5 窗型觀測腔與慣性穴蝕效應之相關性 P.55
3.4 實質腫瘤治療 P.56
3.4.1 腫瘤內血管灌注測量 P.57
3.4.2 組織切片結果 P.61
3.5 治療成效 P.66
第四章 結論與未來工作 P.71
4.1 結論 P.71
4.2 未來工作 P.72
4.2.1 微氣泡於組織中之治療 P.72
4.2.2 微氣泡載附藥體 P.73
4.2.3 原位腫瘤治療 P.73
參考資料 P.75

[1] 行政院衛生福利部, "104年死因統計結果分析," 2015.
[2] T. L. Whiteside, "The tumor microenvironment and its role in promoting tumor growth," Oncogene, vol. 27, pp. 5904-5912, Oct 6 2008.
[3] A. K. Iyer, G. Khaled, J. Fang, and H. Maeda, "Exploiting the enhanced permeability and retention effect for tumor targeting," Drug Discovery Today, vol. 11, pp. 812-818, Sep 2006.
[4] Y. J. Ho and C. K. Yeh, "Concurrent anti-vascular therapy and chemotherapy in solid tumors using drug-loaded acoustic nanodroplet vaporization," Acta Biomater, vol. 49, pp. 472-485, Feb 2017.
[5] H. Maeda, J. Wu, T. Sawa, Y. Matsumura, and K. Hori, "Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review," J Control Release, vol. 65, pp. 271-84, Mar 01 2000.
[6] C. H. Heldin, K. Rubin, K. Pietras, and A. Ostman, "High interstitial fluid pressure - an obstacle in cancer therapy," Nat Rev Cancer, vol. 4, pp. 806-13, Oct 2004.
[7] J. Ehling, B. Theek, F. Gremse, S. Baetke, D. Mockel, J. Maynard, et al., "Micro-CT imaging of tumor angiogenesis: quantitative measures describing micromorphology and vascularization," Am J Pathol, vol. 184, pp. 431-41, Feb 2014.
[8] A. Raza, M. J. Franklin, and A. Z. Dudek, "Pericytes and vessel maturation during tumor angiogenesis and metastasis," American Journal of Hematology, vol. 85, pp. 593-598, Aug 2010.
[9] J. Holash, S. J. Wiegand, and G. D. Yancopoulos, "New model of tumor angiogenesis: dynamic balance between vessel regression and growth mediated by angiopoietins and VEGF," Oncogene, vol. 18, pp. 5356-5362, Sep 20 1999.
[10] A. Abramsson, P. Lindblom, and C. Betsholtz, "Endothelial and nonendothelial sources of PDGF-B regulate pericyte recruitment and influence vascular pattern formation in tumors," Journal of Clinical Investigation, vol. 112, pp. 1142-1151, Oct 2003.
[11] P. Lai, C. Tarapacki, W. T. Tran, A. El Kaffas, J. Lee, C. Hupple, et al., "Breast tumor response to ultrasound mediated excitation of microbubbles and radiation therapy in vivo," Oncoscience, vol. 3, pp. 98-108, 2016.
[12] Y. J. Ho, T. C. Wang, C. H. Fan, and C. K. Yeh, "Current progress in antivascular tumor therapy," Drug Discov Today, Jun 16 2017.
[13] M. S. Gee, W. N. Procopio, S. Makonnen, M. D. Feldman, N. M. Yeilding, and W. M. Lee, "Tumor vessel development and maturation impose limits on the effectiveness of anti-vascular therapy," Am J Pathol, vol. 162, pp. 183-93, Jan 2003.
[14] E. El-Emir, G. M. Boxer, I. A. Petrie, R. W. Boden, J. L. Dearling, R. H. Begent, et al., "Tumour parameters affected by combretastatin A-4 phosphate therapy in a human colorectal xenograft model in nude mice," Eur J Cancer, vol. 41, pp. 799-806, Mar 2005.
[15] D. W. Siemann and W. Shi, "Dual targeting of tumor vasculature: combining Avastin and vascular disrupting agents (CA4P or OXi4503)," Anticancer Res, vol. 28, pp. 2027-31, Jul-Aug 2008.
[16] P. Nathan, M. Zweifel, A. R. Padhani, D. M. Koh, M. Ng, D. J. Collins, et al., "Phase I trial of combretastatin A4 phosphate (CA4P) in combination with bevacizumab in patients with advanced cancer," Clin Cancer Res, vol. 18, pp. 3428-39, Jun 15 2012.
[17] B. G. Siim, A. E. Lee, S. Shalal-Zwain, F. B. Pruijn, M. J. McKeage, and W. R. Wilson, "Marked potentiation of the antitumour activity of chemotherapeutic drugs by the antivascular agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA)," Cancer Chemother Pharmacol, vol. 51, pp. 43-52, Jan 2003.
[18] P. R. Wachsberger, R. Burd, N. Marero, C. Daskalakis, A. Ryan, P. McCue, et al., "Effect of the tumor vascular-damaging agent, ZD6126, on the radioresponse of U87 glioblastoma," Clin Cancer Res, vol. 11, pp. 835-42, Jan 15 2005.
[19] P. Hinnen and F. A. Eskens, "Vascular disrupting agents in clinical development," Br J Cancer, vol. 96, pp. 1159-65, Apr 23 2007.
[20] Y. Shaked and R. S. Kerbel, "Antiangiogenic strategies on defense: On the possibility of blocking rebounds by the tumor vasculature after chemotheraphy," Cancer Research, vol. 67, pp. 7055-7058, Aug 1 2007.
[21] I. M. Subbiah, D. J. Lenihan, and A. M. Tsimberidou, "Cardiovascular toxicity profiles of vascular-disrupting agents," Oncologist, vol. 16, pp. 1120-30, 2011.
[22] S. Vaezy, V. Y. Fujimoto, C. Walker, R. W. Martin, E. Y. Chi, and L. A. Crum, "Treatment of uterine fibroid tumors in a nude mouse model using high-intensity focused ultrasound," American Journal of Obstetrics and Gynecology, vol. 183, pp. 6-11, Jul 2000.
[23] F. Wu, Z. B. Wang, W. Z. Chen, W. Wang, Y. Gui, M. Zhang, et al., "Extracorporeal high intensity focused ultrasound ablation in the treatment of 1038 patients with solid carcinomas in China: an overview," Ultrason Sonochem, vol. 11, pp. 149-54, May 2004.
[24] N. C. Nanda, R. Gramiak, and P. M. Shah, "Diagnosis of Aortic Root Dissection by Echocardiography," Circulation, vol. 48, pp. 506-513, 1973.
[25] P. A. Dijkmans, L. J. Juffermans, R. J. Musters, A. van Wamel, F. J. ten Cate, W. van Gilst, et al., "Microbubbles and ultrasound: from diagnosis to therapy," Eur J Echocardiogr, vol. 5, pp. 245-56, Aug 2004.
[26] P. J. Frinking, A. Bouakaz, J. Kirkhorn, F. J. Ten Cate, and N. de Jong, "Ultrasound contrast imaging: current and new potential methods," Ultrasound Med Biol, vol. 26, pp. 965-75, Jul 2000.
[27] A. L. Klibanov, T. I. Shevchenko, B. I. Raju, R. Seip, and C. T. Chin, "Ultrasound-triggered release of materials entrapped in microbubble-liposome constructs: a tool for targeted drug delivery," J Control Release, vol. 148, pp. 13-7, Nov 20 2010.
[28] D. L. Miller and R. A. Gies, "Gas-body-based contrast agent enhances vascular bioeffects of 1.09 MHz ultrasound on mouse intestine," Ultrasound Med Biol, vol. 24, pp. 1201-8, Oct 1998.
[29] D. L. Miller and J. Quddus, "Diagnostic ultrasound activation of contrast agent gas bodies induces capillary rupture in mice," Proc Natl Acad Sci U S A, vol. 97, pp. 10179-84, Aug 29 2000.
[30] C. F. Caskey, S. M. Stieger, S. Qin, P. A. Dayton, and K. W. Ferrara, "Direct observations of ultrasound microbubble contrast agent interaction with the microvessel wall," J Acoust Soc Am, vol. 122, pp. 1191-200, Aug 2007.
[31] S. Mitragotri, "Healing sound: the use of ultrasound in drug delivery and other therapeutic applications," Nat Rev Drug Discov, vol. 4, pp. 255-60, Mar 2005.
[32] National Council on Radiation Protection and Measurements., National Council on Radiation Protection and Measurements. Scientific Committee 66 on Biological Effects of Ultrasound., and National Council on Radiation Protection and Measurements., Exposure criteria for medical diagnostic ultrasound : I, Criteria based on thermal mechanisms : recommendations of the National Council on Radiation Protection and Measurements. Bethesda, MD: The Council, 1992.
[33] P. M. Corry, W. J. Spanos, E. J. Tilchen, B. Barlogie, H. T. Barkley, and E. P. Armour, "Combined ultrasound and radiation therapy treatment of human superficial tumors," Radiology, vol. 145, pp. 165-9, Oct 1982.
[34] D. L. Miller and R. M. Thomas, "Ultrasound Contrast Agents Nucleate Inertial Cavitation in-Vitro," Ultrasound in Medicine and Biology, vol. 21, pp. 1059-1065, 1995.
[35] L. A. Crum and G. T. Reynolds, "Sonoluminescence Produced by Stable Cavitation," Journal of the Acoustical Society of America, vol. 78, pp. 137-139, 1985.
[36] J. H. Hwang, J. Tu, A. A. Brayman, T. J. Matula, and L. A. Crum, "Correlation between inertial cavitation dose and endothelial cell damage in vivo," Ultrasound Med Biol, vol. 32, pp. 1611-9, Oct 2006.
[37] J. Tu, T. J. Matula, A. A. Brayman, and L. A. Crum, "Inertial cavitation dose produced in ex vivo rabbit ear arteries with Optison (R) by 1-MHz pulsed ultrasound," Ultrasound in Medicine and Biology, vol. 32, pp. 281-288, Feb 2006.
[38] S. M. Graham, R. Carlisle, J. J. Choi, M. Stevenson, A. R. Shah, R. S. Myers, et al., "Inertial cavitation to non-invasively trigger and monitor intratumoral release of drug from intravenously delivered liposomes," J Control Release, vol. 178, pp. 101-7, Mar 28 2014.
[39] D. L. Miller, "Overview of experimental studies of biological effects of medical ultrasound caused by gas body activation and inertial cavitation," Prog Biophys Mol Biol, vol. 93, pp. 314-30, Jan-Apr 2007.
[40] D. M. Skyba, R. J. Price, A. Z. Linka, T. C. Skalak, and S. Kaul, "Direct in vivo visualization of intravascular destruction of microbubbles by ultrasound and its local effects on tissue," Circulation, vol. 98, pp. 290-3, Jul 28 1998.
[41] J. H. Hwang, J. Tu, A. A. Brayman, T. J. Matula, and L. A. Crum, "Correlation between inertial cavitation dose and endothelial cell damage in vivo," Ultrasound in Medicine and Biology, vol. 32, pp. 1611-1619, Oct 2006.
[42] Z. Liu, S. Gao, Y. Zhao, P. Li, J. Liu, P. Li, et al., "Disruption of tumor neovasculature by microbubble enhanced ultrasound: a potential new physical therapy of anti-angiogenesis," Ultrasound Med Biol, vol. 38, pp. 253-61, Feb 2012.
[43] D. E. Goertz, M. Todorova, O. Mortazavi, V. Agache, B. Chen, R. Karshafian, et al., "Antitumor effects of combining docetaxel (taxotere) with the antivascular action of ultrasound stimulated microbubbles," PLoS One, vol. 7, p. e52307, 2012.
[44] J. F. Wang, Z. L. Zhao, S. X. Shen, C. X. Zhang, S. C. Guo, Y. K. Lu, et al., "Selective depletion of tumor neovasculature by microbubble destruction with appropriate ultrasound pressure," International Journal of Cancer, vol. 137, pp. 2478-2491, Nov 15 2015.
[45] M. S. Lesniak, U. Upadhyay, R. Goodwin, B. Tyler, and H. Brem, "Local delivery of doxorubicin for the treatment of malignant brain tumors in rats," Anticancer Research, vol. 25, pp. 3825-3831, Nov-Dec 2005.
[46] K. J. Chen, H. F. Liang, H. L. Chen, Y. C. Wang, P. Y. Cheng, H. L. Liu, et al., "A Thermoresponsive Bubble-Generating Liposomal System for Triggering Localized Extracellular Drug Delivery," Acs Nano, vol. 7, pp. 438-446, Jan 2013.
[47] R. Duncan, "Polymer conjugates for tumour targeting and intracytoplasmic delivery. The EPR effect as a common gateway?," Pharm Sci Technolo Today, vol. 2, pp. 441-449, Nov 1999.
[48] K. J. Chen, H. F. Liang, H. L. Chen, Y. Wang, P. Y. Cheng, H. L. Liu, et al., "A thermoresponsive bubble-generating liposomal system for triggering localized extracellular drug delivery," ACS Nano, vol. 7, pp. 438-46, Jan 22 2013.
[49] J. J. Chen, S. Y. Fu, C. S. Chiang, J. H. Hong, and C. K. Yeh, "A preclinical study to explore vasculature differences between primary and recurrent tumors using ultrasound Doppler imaging," Ultrasound Med Biol, vol. 39, pp. 860-9, May 2013.
[50] M. Todorova, V. Agache, O. Mortazavi, B. Chen, R. Karshafian, K. Hynynen, et al., "Antitumor effects of combining metronomic chemotherapy with the antivascular action of ultrasound stimulated microbubbles," Int J Cancer, vol. 132, pp. 2956-66, Jun 15 2013.
[51] G. Bergers and S. Song, "The role of pericytes in blood-vessel formation and maintenance," Neuro Oncol, vol. 7, pp. 452-64, Oct 2005.
[52] C. K. Yeh and S. Y. Su, "Effects of acoustic insonation parameters on ultrasound contrast agent destruction," Ultrasound Med Biol, vol. 34, pp. 1281-91, Aug 2008.
[53] A. Yildirim, R. Chattaraj, N. T. Blum, and A. P. Goodwin, "Understanding Acoustic Cavitation Initiation by Porous Nanoparticles: Toward Nanoscale Agents for Ultrasound Imaging and Therapy," Chem Mater, vol. 28, pp. 5962-5972, Aug 23 2016.
[54] D. K. Chang, C. Y. Chiu, S. Y. Kuo, W. C. Lin, A. Lo, Y. P. Wang, et al., "Antiangiogenic targeting liposomes increase therapeutic efficacy for solid tumors," J Biol Chem, vol. 284, pp. 12905-16, May 08 2009.
[55] Y. J. Chang, C. H. Chang, C. Y. Yu, T. J. Chang, L. C. Chen, M. H. Chen, et al., "Therapeutic efficacy and microSPECT/CT imaging of 188Re-DXR-liposome in a C26 murine colon carcinoma solid tumor model," Nucl Med Biol, vol. 37, pp. 95-104, Jan 2010.
[56] H. Chen, A. A. Brayman, M. R. Bailey, and T. J. Matula, "Blood vessel rupture by cavitation," Urol Res, vol. 38, pp. 321-6, Aug 2010.
[57] B. Helfield, X. C. Chen, S. C. Watkins, and F. S. Villanueva, "Biophysical insight into mechanisms of sonoporation," Proceedings of the National Academy of Sciences of the United States of America, vol. 113, pp. 9983-9988, Sep 6 2016.
[58] Z. Z. Fan, H. Y. Liu, M. Mayer, and C. X. Deng, "Spatiotemporally controlled single cell sonoporation," Proceedings of the National Academy of Sciences of the United States of America, vol. 109, pp. 16486-16491, Oct 9 2012.