新式超音波藥物載體—相變液滴，外部為脂質殼層，可乘載多種抗癌藥物、內部為液態全氟化合物，於體內可以穩定存在，可透過聲學激發液滴汽化(Acoustic Droplet Vaporization，ADV)進行藥物釋放，對於藥物傳遞相當具有潛力。然而，目前並沒有任何文獻關於相變液滴於瞬間的藥物釋放動力學，這是由於ADV的過程僅為微秒等級，且於傳統白光視野下難以觀察藥物。因此，本研究目的是透過架設一套高速螢光顯微系統，可使得相變液滴於ADV瞬間的藥物動態視覺化，並探討藥物脫離相變液滴的原因、脫離後的藥物於空間上的分佈、以及聲學參數對於釋藥距離、以及釋放量的影響，進而藉此解開相變液滴的藥物釋放機制。
相變液滴的藥物釋放必須高於特定強度的聲學參數(至少9 MPa)才會啟動。而相變液滴的釋放特徵方面，本研究發現所釋放的藥物主要累積於相變液滴與環境的接觸面，這也暗示了相變液滴作為標靶治療的潛力。相變液滴的釋放機制以超高速螢光進行攝影，探討聲學參數對於螢光影像的差異，且透過程式使得影像特徵數值化後，發現聲壓對於脂質動態分佈移動幅度具有正相關、12 MPa所造成的移動幅度為8 MPa的3.09倍，推測是因為具有方向性的匯聚流場所影響，並以白光拍攝相變液滴汽化之粒徑變化，證實氣泡的劇烈震動為流場形成的關鍵、12 MPa所造成的氣泡脹縮幅度為8 MPa的3.61倍。綜合上述可解開相變液滴的藥物釋放機制 : 透過聲學激發液滴汽化後，氣泡劇烈脹縮引發具方向性的流場，使得脂質交互擠壓，進而連帶著脂溶性藥物脫離載體。最後探討粒徑與聲壓對於釋放影響，發現不論是在釋放量、抑或釋放距離，粒徑越小或聲壓越高的組別，皆展現出相對較高的效率，並再度透過高速攝影液滴汽化之粒徑變化，證實氣泡脹縮的幅度為影響釋放效率的主因。

The new ultrasound drug carrier, phase-change droplet (PCD), with liquid perfluorocarbon in lipid shell, and steady in the circulation, was proposed by R. E. Apfel in 1998. PCDs were promising for local drug delivery due to their ability to undergo acoustic droplet vaporization (ADV) under ultrasound excitations. However, no study has investigated the transient dynamics of drug release, since ADV occurred on microsecond scale and drugs were hardly identified in conventional bright field microscopy. Here, we established a high-speed fluorescence imaging system to visualize the process of drug release during ADV. The aim of the study was to find out the way of drug release from PCDs, the distribution of the released drug, the effect between acoustic parameters and release number, and finally the mechanism of drug release from PCDs.
Drug release from PCDs must be triggered by acoustic parameter higher than specific level (at least 9 MPa). At the pressures higher than ADV threshold, the shedding drug were observed only on contacted wall between droplets and tube. To figure out the mechanism of drug release from PCDs, the process was captured by high-speed fluorescence imaging system. From quantified images, acoustic pressure had effect on lipid dynamic on bubble shell, which was resulted from directionally converging flow. Moreover, the bright-field images were quantified to verify the effects of the evolution of bubble resulting in converging flow. In summary, the mechanism of drug release from PCDs was that ADV triggered the evolution of bubble, which resulting in converging flow. The flow induced the effect of lipid bending and shedding with drugs. Finally, small PCDs or high pressure induced the high efficiency on either release number or release distance. Through high-speed images, it was proved that the release efficiency was related to evolution of bubble.

第一章 緒論
1.1 癌症 1
1.1.1 癌症治療 2
1.1.2 癌症治療瓶頸 2
1.1.3 藥物載體 3
1.2 傳統超音波藥物載體__微氣泡 4
1.3 新式超音波藥物載體__相變液滴 4
1.3.1 相變液滴之性質 5
1.3.2 相變液滴之物理治療 7
1.3.3 高速影像應用於物理治療之機制探討 9
1.3.4 相變液滴之化學治療 14
1.3.4.1 螢光技術應用於藥物傳遞 15
1.3.5 高速影像應用於化學治療之機制探討限制 17
1.3.5.1 高速螢光顯微系統 18
1.4 研究動機及論文架構 21
第二章 實驗材料與方法 23
2.1. 概論 23
2.2. 相變液滴之製備 23
2.2.1粒徑與濃度分佈量測 25
2.2.2染劑濃度最佳化 25
2.3 高速螢光顯微系統架設 26
2.3.1 實驗硬體架設 26
2.3.2 雷射於系統可行性 28
2.3.2.1 光源強度量測 28
2.3.2.2 焦斑寬度控制與量測 29
2.3.3 儀器同步時序 31
2.4 相變液滴釋放分析 32
第三章 實驗結果 34
3-1 相變液滴粒徑分佈 34
3-2 螢光染劑濃度最佳化 35
3-3 相變液滴釋放特徵 37
3-3-1 相變液滴藥物釋放視覺化 37
3-3-1-1 白光與螢光對比 37
3-3-1-2 聲壓影響釋放 39
3-3-2 釋放面與管壁交互關係 40
3-3-2-1 觀察面與管壁平行 41
3-3-2-2 觀察面與管壁垂直 42
3-4 相變液滴釋放產物分析 45
3-5 相變液滴釋放機制探討 49
3-5-1 相變液滴汽化之超高速螢光影像 50
3-5-1-1 低聲壓效應 50
3-5-1-2 高聲壓效應 52
3-5-1-3 側面影像 53
3-5-2 相變液滴汽化之表面匯聚流定量 54
3-5-2-1 匯聚流定量方式 54
3-5-2-2 匯聚流定量結果 56
3-5-3 匯聚流的動力來源 57
3-5-4 相變液滴之藥物釋放機制 60
3-6 相變液滴釋放與聲學參數探討 62
3-6-1 聲壓與粒徑對於釋放的影響 62
3-6-2 釋放量定量 64
3-6-3 釋放距離定量 64
3-6-4 釋放效率受粒徑之影響 65
3-6-5 釋放效率受粒徑及聲壓共同調控之機制 66
第四章 結論與未來展望 69
4.1 結論 69
4.2 未來展望 71
參考文獻 72

[1] 行政院衛生署, "102年國民死因結果分析," 2013.
[2] M. D. Lavigne, S. S. Pennadam, J. Ellis, C. Alexander, and D. C. Gorecki, "Enhanced gene expression through temperature profile-induced variations in molecular architecture of thermoresponsive polymer vectors," Journal of Gene Medicine, vol. 9, pp. 44-54, Jan 2007.
[3] D. Schmaljohann, "Thermo- and pH-responsive polymers in drug delivery," Advanced Drug Delivery Reviews, vol. 58, pp. 1655-1670, Dec 30 2006.
[4] R. A. Frimpong, S. Fraser, and J. Z. Hilt, "Synthesis and temperature response analysis of magnetic-hydrogel nanocomposites," Journal of Biomedical Materials Research Part A, vol. 80A, pp. 1-6, Jan 2007.
[5] G. H. Hsiue, C. H. Wang, C. L. Lo, C. H. Wang, J. P. Li, and J. L. Yang, "Environmental-sensitive micelles based on poly(2-ethyl-2-oxazoline)-b-poly(L-lactide) diblock copolymer for application in drug delivery," International Journal of Pharmaceutics, vol. 317, pp. 69-75, Jul 6 2006.
[6] H. L. Liu, C. H. Fan, C. Y. Ting, and C. K. Yeh, "Combining Microbubbles and Ultrasound for Drug Delivery to Brain Tumors: Current Progress and Overview," Theranostics, vol. 4, pp. 432-444, 2014.
[7] S. Tinkov, C. Coester, S. Serba, N. A. Geis, H. A. Katus, G. Winter, and R. Bekeredjian, "New doxorubicin-loaded phospholipid microbubbles for targeted tumor therapy: In-vivo characterization," Journal of Controlled Release, vol. 148, pp. 368-372, Dec 20 2010.
[8] J. A. Ketterling and R. E. Apfel, "Experimental validation of the dissociation hypothesis for single bubble sonoluminescence," Physical Review Letters, vol. 81, pp. 4991-4994, Nov 30 1998.
[9] P. A. Dayton, S. K. Zhao, S. H. Bloch, P. Schumann, K. Penrose, T. O. Matsunaga, R. Zutshi, A. Doinikov, and K. W. Ferrara, "Application of ultrasound to selectively localize nanodroplets for targeted imaging and therapy," Molecular Imaging, vol. 5, pp. 160-174, Jul 2006.
[10] N. Reznik, R. Williams, and P. N. Burns, "Investigation of Vaporized Submicron Perfluorocarbon Droplets as an Ultrasound Contrast Agent," Ultrasound in Medicine and Biology, vol. 37, pp. 1271-1279, Aug 2011.
[11] C. Y. Lin and W. G. Pitt, "Acoustic Droplet Vaporization in Biology and Medicine," Biomed Research International, 2013.
[12] M. Zhang, M. L. Fabiilli, K. J. Haworth, F. Padilla, S. D. Swanson, O. D. Kripfgans, P. L. Carson, and J. B. Fowlkes, "Acoustic Droplet Vaporization for Enhancement of Thermal Ablation by High Intensity Focused Ultrasound," Academic Radiology, vol. 18, pp. 1123-1132, Sep 2011.
[13] O. D. Kripfgans, C. M. Orifici, P. L. Carson, K. A. Ives, O. P. Eldevik, and J. B. Fowlkes, "Acoustic droplet vaporization for temporal and spatial control of tissue occlusion: A kidney study," Ieee Transactions on Ultrasonics Ferroelectrics and Frequency Control, vol. 52, pp. 1101-1110, Jul 2005.
[14] M. J. Schwartz, E. B. Smith, D. W. Trost, and E. D. Vaughan, "Renal artery embolization: clinical indications and experience from over 100 cases," Bju International, vol. 99, pp. 881-886, Apr 2007.
[15] N. Reznik, M. Seo, R. Williams, E. Bolewska-Pedyczak, M. Lee, N. Matsuura, J. Gariepy, F. S. Foster, and P. N. Burns, "Optical studies of vaporization and stability of fluorescently labelled perfluorocarbon droplets," Physics in Medicine and Biology, vol. 57, pp. 7205-7217, Nov 7 2012.
[16] D. S. Li, O. D. Kripfgans, M. L. Fabiilli, J. B. Fowlkes, and J. L. Bull, "Formation of toroidal bubbles from acoustic droplet vaporization," Applied Physics Letters, vol. 104, Feb 10 2014.
[17] O. D. Kripfgans, M. L. Fabiilli, P. L. Carson, and J. B. Fowlkes, "On the acoustic vaporization of micrometer-sized droplets," Journal of the Acoustical Society of America, vol. 116, pp. 272-281, Jul 2004.
[18] K. J. Haworth and O. D. Kripfgans, "Initial Growth and Coalescence of Acoustically Vaporized Perfluorocarbon Microdroplets," 2008 Ieee Ultrasonics Symposium, Vols 1-4 and Appendix, pp. 623-626, 2008.
[19] Z. Z. Wong, O. D. Kripfgans, A. Qamar, J. B. Fowlkes, and J. L. Bull, "Bubble evolution in acoustic droplet vaporization at physiological temperature via ultra-high speed imaging," Soft Matter, vol. 7, pp. 4009-4016, 2011.
[20] C. H. Wang, S. T. Kang, Y. H. Lee, Y. L. Luo, Y. F. Huang, and C. K. Yeh, "Aptamer-conjugated and drug-loaded acoustic droplets for ultrasound theranosis," Biomaterials, vol. 33, pp. 1939-1947, Feb 2012.
[21] N. Reznik, O. Shpak, E. C. Gelderblom, R. Williams, N. de Jong, M. Versluis, and P. N. Burns, "The efficiency and stability of bubble formation by acoustic vaporization of submicron perfluorocarbon droplets," Ultrasonics, vol. 53, pp. 1368-1376, Sep 2013.
[22] O. Shpak, T. J. A. Kokhuis, Y. Luan, D. Lohse, N. de Jong, B. Fowlkes, M. Fabiilli, and M. Versluis, "Ultrafast dynamics of the acoustic vaporization of phase-change microdroplets," Journal of the Acoustical Society of America, vol. 134, pp. 1610-1621, Aug 2013.
[23] P. S. Sheeran, T. O. Matsunaga, and P. A. Dayton, "Phase-transition thresholds and vaporization phenomena for ultrasound phase-change nanoemulsions assessed via high-speed optical microscopy," Physics in Medicine and Biology, vol. 58, pp. 4513-4534, Jul 7 2013.
[24] N. Rapoport, K. H. Nam, R. Gupta, Z. G. Gao, P. Mohan, A. Payne, N. Todd, X. Liu, T. Kim, J. Shea, C. Scaife, D. L. Parker, E. K. Jeong, and A. M. Kennedy, "Ultrasound-mediated tumor imaging and nanotherapy using drug loaded, block copolymer stabilized perfluorocarbon nanoemulsions," Journal of Controlled Release, vol. 153, pp. 4-15, Jul 15 2011.
[25] G. A. Orr, P. Verdier-Pinard, H. McDaid, and S. B. Horwitz, "Mechanisms of Taxol resistance related to microtubules," Oncogene, vol. 22, pp. 7280-7295, Oct 20 2003.
[26] E. S. Hauck, S. Zou, K. Scarfo, M. H. Nantz, and J. G. Hecker, "Whole Animal In Vivo Imaging After Transient, Nonviral Gene delivery to the Rat Central Nervous System," Molecular Therapy, vol. 16, pp. 1857-1864, Nov 2008.
[27] H. A. Lankes, C. N. Zanghi, K. Santos, C. Capella, C. M. P. Duke, and S. Dewhurst, "In vivo gene delivery and expression by bacteriophage lambda vectors," Journal of Applied Microbiology, vol. 102, pp. 1337-1349, May 2007.
[28] J. C. Alexander, S. Browne, A. Pandit, and Y. Rochev, "Biomaterial Constructs for Delivery of Multiple Therapeutic Genes: A Spatiotemporal Evaluation of Efficacy Using Molecular Beacons," Plos One, vol. 8, Jun 3 2013.
[29] M. L. Fabiilli, K. J. Haworth, I. E. Sebastian, O. D. Kripfgans, P. L. Carson, and J. B. Fowlkes, "Delivery of Chlorambucil Using an Acoustically-Triggered Perfluoropentane Emulsion," Ultrasound in Medicine and Biology, vol. 36, pp. 1364-1375, Aug 2010.
[30] S. K. Price P, "Treatment of Cancer," 2002.
[31] Y. F. Huang, D. H. Shangguan, H. P. Liu, J. A. Phillips, X. L. Zhang, Y. Chen, and W. H. Tan, "Molecular Assembly of an Aptamer-Drug Conjugate for Targeted Drug Delivery to Tumor Cells," Chembiochem, vol. 10, pp. 862-868, Mar 23 2009.
[32] C. Cheng, O. Trzcinski, and L. C. Doering, "Fluorescent labeling of dendritic spines in cell cultures with the carbocyanine dye "Dil"," Frontiers in Neuroanatomy, vol. 8, May 9 2014.
[33] S. R. Sirsi, C. Fung, S. Garg, M. Y. Tianning, P. A. Mountford, and M. A. Borden, "Lung Surfactant Microbubbles Increase Lipophilic Drug Payload for Ultrasound-Targeted Delivery," Theranostics, vol. 3, pp. 409-419, 2013.
[34] Y. Luan, G. Lajoinie, E. Gelderblom, I. Skachkov, A. F. W. van der Steen, H. J. Vos, M. Versluis, and N. De Jong, "Lipid Shedding from Single Oscillating Microbubbles," Ultrasound in Medicine and Biology, vol. 40, pp. 1834-1846, Aug 2014.
[35] D. l. E. Gelderblom, N. De Jong, "Ultra-high-speed fluorescence imaging," Dissertation, 2012.
[36] A. L. Klibanov, K. W. Ferrara, M. S. Hughes, J. H. Wible, J. K. Wojdyla, P. A. Dayton, K. E. Morgan, and G. H. Brandenburger, "Direct video microscopic observation of the dynamic effects of medical ultrasound on ultrasound contrast microspheres," Investigative Radiology, vol. 33, pp. 863-870, Dec 1998.
[37] M. J. Benchimol, M. J. Hsu, C. E. Schutt, D. J. Hall, R. F. Mattrey, and S. C. Esener, "Phospholipid/carbocyanine dye-shelled microbubbles as ultrasound-modulated fluorescent contrast agents," Soft Matter, vol. 9, pp. 2384-2388, 2013.
[38] S. Baoukina, L. Monticelli, H. J. Risselada, S. J. Marrink, and D. P. Tieleman, "The molecular mechanism of lipid monolayer collapse," Proceedings of the National Academy of Sciences of the United States of America, vol. 105, pp. 10803-10808, Aug 5 2008.