癌症，又稱為惡性腫瘤，為十大死因之首，目前常見的癌症治療方式有手術、放射及化學治療，其中當腫瘤發生轉移或復發時，化學治療是一種較全面且合適的治療手段。化學治療透過靜脈注射的方式，使藥物隨血液循環至腫瘤區域，然而藥物本身之生物毒性亦會毒殺健康細胞並引發副作用，因此為了降低藥物的副作用，使用藥物載體是一個有效的解決方案。近年來，超音波對比劑微氣泡，外殼由磷脂質材料，中心包覆氟碳化合物構成的微小氣泡，其高度生物相容性被認為是極具潛力的新式藥物載體。許多研究團隊已證實可將不同的化療藥物包覆於微氣泡中，並利用微氣泡會被高聲壓超音波擊破的特性來控制微氣泡釋放藥物，而此過程亦可同步由超音波影像監測，可達成非侵入性、局部藥物釋放控制、診斷治療合一等目的。目前文獻均指出載藥微氣泡需以高聲壓超音波擊破(稱為慣性穴蝕效應)，才可釋放藥物至周遭細胞，但在擊破微氣泡時產生的機械力會造成細胞產生不可回復的損傷甚至死亡。因此，本研究的目的為使用高速螢光顯微系統探討載藥微氣泡於低聲壓超音波照射時的動態行為、釋出的藥物型態，在此聲學條件下藥物進入細胞的機制，以及實際負載化療藥物Doxorubicin後毒殺癌細胞之成效，藉此解開載藥微氣泡透過穩定穴蝕效應釋藥的可行性。
本研究為了觀察藥物釋放動態，將作為模擬藥物的DiI-C18螢光染劑負載於微氣泡上，並在殼層上修釋葉酸分子，使其吸附於於C6神經膠質瘤細胞上。微氣泡穴蝕效應種類和細胞藥物遞送成效分別是由被動式聲學偵測和流式細胞儀評估。超音波照射過程中(頻率：2.1 MHz、聲壓：200-800 kPa、週波數：500、叢集波數： 1)的微氣泡動態是由高速白光(500 kfps)和螢光(40 kfps)顯微成像監測。而由於藥物需要較長時間才能被攝取進細胞，為了確認釋出的微氣泡囊泡是否會被攝取進細胞，利用活細胞觀察腔長時間觀察微氣泡釋藥後，藥物遞送進細胞的結果，並使用流式細胞儀觀察實際在微氣泡上負載化療藥物Doxorubicin後之細胞藥物遞送成效。結果顯示微氣泡發生穩定穴蝕效應與慣性穴蝕效應的聲壓閾值分別為200和600 kPa。流式細胞儀顯示成功細胞藥物遞送的聲壓閥值400 kPa，顯示微氣泡僅透過穩定穴蝕效應即可釋放藥物至細胞。在此聲壓條件下，高速顯微影像顯示超音波照射可立即驅動微氣泡釋放，在微氣泡未發生位移時可釋放出 37.9 ± 12.5 % 的螢光囊泡物質，且未位移及有位移微氣泡分別可以同時增加周圍細胞36.5 ± 5.6 % 和 50.9 ± 3.5 % 的螢光強度，且在局部區域條件下，穩定穴蝕效應具有比慣性穴蝕效應更佳的藥物遞送效果。活細胞顯微成像則顯示此聲學條件亦可驅動藥物從微氣泡遞送至細胞，並達到30 ± 5.1 %的細胞毒殺率。未來工作包含調控聲學參數以控制穩定穴蝕效應下的藥物釋放量、釋放距離、藥物囊泡大小，並進行活體抗癌方面的應用。

Cancer, also known as a malignant tumor, is one of the top ten causes of death. Common cancer treatments included surgery, radiation, and chemotherapy. When tumors have metastasized or recurred, chemotherapy was a more comprehensive and appropriate treatment. Chemotherapy through intravenous injection allowed the drug to circulate the tumor area with the blood. However, the biological toxicity of the drug itself would kill healthy cells and cause side effects. Therefore, in order to reduce the side effects of drugs, the use of drug carriers was an effective solution. Ultrasound contrast agent microbubbles were highly biocompatible and were considered to be a new potential drug carrier. Many research teams have demonstrated that different chemo-drugs would be carried in microbubbles(MBs), and the carried drug could be triggered released upon ultrasound-induce inertial cavitation. This process was monitored by ultrasound images simultaneously, which could achieve the purpose of non-invasive, local drug delivery, diagnosis, and treatment. However, the mechanical force generated from MBs inertial cavitation would result in server cellular damage, even death. Herein, the aim of this study was to verify that the drug release behavior, MBs debris, and the mechanism of cellular uptake of drug-loaded MBs by stable cavitation. Finally, Doxorubicin, a chemo-drug, was used to confirm the effect of cell toxicity, thereby unlocking the feasibility of drug-loaded MBs releasing drugs by stable cavitation.
DiI-C18 was incorporated in the MBs (diameter: 1.6 µm) to model hydrophobic drugs. The outer lipid shell of MBs was conjugated with folate molecular for fixing the distance between drug-loaded MBs and C6 glioma cells. An acoustic-optical system that allowed high-speed fluorescence (40 kfps) microscopic imaging for visualizing the dynamics of MBs-cell drug transportation under ultrasound excitations (frequency = 2.1 MHz, pressure = 200-800 kPa, cycle number = 500, pulse number = 1). The MBs cavitation threshold and successful cellular drug delivery were estimated by passive cavitation detection and flow cytometry, respectively. The live cell microscopy was used to observe the results of drug delivery into cells after ultrasound exposed. The effect of cell drug delivery after loading the chemo-drug Doxorubicin on the MBs was observed using flow cytometry.
The results showed that the threshold of MBs stable cavitation and inertial cavitation were 200 kPa and 600 kPa, respectively. Flow cytometry showed that the acoustic pressure threshold of successful cellular drug delivery was 400 kPa, showing that MBs could release the drug to cells by stable cavitation. Further, the high-speed microscopic imaging data showed that the MBs would release 38 ± 12.5 % of fluorescence vesicles upon 400 kPa of ultrasound sonication, while surrounding cells increased 50.9 ± 3.5% of fluorescence intensity. Live cell microscopy showed that this acoustic condition could drive drug delivery to cells, with the cytotoxicity rate of 31 ± 5.2%. Future work would adjust the acoustic parameters to control the amount of drug released, the distance released, and the size of the drug vesicles by stable cavitation.

摘要
目錄
第一章 緒論 1
1.1癌症 1
1.1.1癌症治療方法 1
1.1.2 化學治療 2
1.1.3 化學治療的缺點 2
1.2 超音波結合微氣泡用於藥物遞送 3
1.2.1超音波對比劑微氣泡 3
1.2.1.1 超音波對比劑微氣泡的發展 3
1.2.1.2 穴蝕效應 3
1.2.2藥物輔以微氣泡用於藥物遞送 4
1.2.3 載藥微氣泡用於藥物遞送 5
1.3 超音波結合負載藥物微氣泡之釋藥機制探討 6
1.4 超音波結合標靶微氣泡之生物效應 11
1.5 細胞攝取 13
1.6研究目的及論文架構 14
第二章 實驗材料與方法 16
2.1 概論 16
2.2 標靶微氣泡之製備 16
2.2.1 葉酸脂質溶液之製備 16
2.2.2 DiI-loaded, folate targeted MBs(DiI-ftMBs) 之薄膜製備 17
2.2.3 Folate-targeted MBs (ftMBs) 之薄膜製備 17
2.2.4 DiI-loaded, NBD-labeled ftMBs (DiI-NBD-ftMBs) 之薄膜製備 18
2.2.5標靶微氣泡之製備 18
2.2.6 DOX-loaded, folate-targeted MBs 之製備 19
2.2.7 Liposomal-DOX (Lipo-DOX) 之製備 19
2.3 微氣泡之特性量測 20
2.3.1 微氣泡之光學定性 20
2.3.2 濃度與粒徑分佈之量測 20
2.3.3 Folate鍵結微氣泡效率之定量 21
2.3.4 微氣泡之穩定度測量 21
2.3.5 被動式穴蝕效應偵測 22
2.3.6 微氣泡囊泡之粒徑分布量測 24
2.3.7 微氣泡之囊泡型態測量 25
2.3.8 微氣泡之囊泡結構 25
2.4 細胞培養 25
2.4.1 細胞株-大鼠腦神經膠質瘤細胞 25
2.4.2 大鼠腦腫瘤神經膠質瘤細胞之繼代 26
2.4.3 細胞藥物遞送效率之實驗 26
2.4.4 細胞膜通透性及活性之實驗 27
2.4.5 細胞藥物致死濃度 28
2.4.6 細胞毒殺死亡率之測試 28
2.5 高速螢光結合多螢光顯微系統實驗 29
2.5.1 高速螢光顯微系統硬體架構 29
2.5.2 微氣泡藥物釋放分析 30
2.5.2.1 微氣泡釋藥量評估 31
2.5.2.2 細胞藥物遞送效率評估 32
2.5.3 多螢光套件顯微系統硬體架構 34
2.5.4 高速螢光系統與超音波照射儀器同步時序 35
2.5.5多螢光顯微系統與超音波照射儀器同步時序 36
2.6 細胞胞吞作用之實驗 37
2.7 統計分析 38
第三章 實驗結果 39
3.1 微氣泡之物化特性 39
3.1.1 微氣泡之光學定性 39
3.1.2 微氣泡之粒徑分析 39
3.1.3 Folate鍵結微氣泡效率之定量 40
3.1.4 微氣泡之穩定度測量 40
3.2 被動式穴蝕效應偵測 41
3.3 微氣泡釋放物分析 43
3.3.1 微氣泡釋放物之粒徑分佈 43
3.3.2 微氣泡釋放物之組成 44
3.3.3 微氣泡釋放物之低溫電子顯微鏡 觀察 45
3.4 細胞實驗 46
3.4.1 細胞藥物遞送效率之實驗 46
3.4.2 細胞膜通透性及活性之實驗 47
3.5 藥物釋放之觀察 48
3.5.1 高速螢光釋藥影像 48
3.5.1.1 未照射超音波之控制組影像 49
3.5.1.2 在 200 kPa超音波照射下之釋藥行為 50
3.5.1.3 在 400 kPa超音波照射下之釋藥行為 51
3.5.1.4 在 600 kPa超音波照射下之釋藥行為 54
3.5.2 微氣泡釋藥量評估 55
3.5.3 藥物遞送效率評估 56
3.6 細胞攝取實驗 58
3.6.1 細胞胞吞作用之實驗 58
3.6.1.1 控制組 58
3.6.1.2 實驗組 60
3.6.2 細胞藥物致死濃度 61
3.6.3 細胞死亡率之實驗 62
第四章 結論與未來展望 64
4.1 結論 64
4.2未來工作 65
4.3 參考文獻 65

1. 行政院衛生署, 102年國民死因結果分析. 2013.
2. Arcamone, F., Doxorubicin: anticancer antibiotics. 2012: Elsevier.
3. Germain, E., et al., Enhancement of doxorubicin cytotoxicity by polyunsaturated fatty acids in the human breast tumor cell line MBA‐MB‐231: Relationship to lipid peroxidation. International journal of cancer, 1998. 75(4): p. 578-583.
4. Olson, R.D. and P.S. Mushlin, Doxorubicin cardiotoxicity: analysis of prevailing hypotheses. The FASEB journal, 1990. 4(13): p. 3076-3086.
5. Gabizon, A. and F. Martin, Polyethylene glycol-coated (pegylated) liposomal doxorubicin. Drugs, 1997. 54(4): p. 15-21.
6. NANDA, N.C., R. Gramiak, and P.M. SHAH, Diagnosis of aortic root dissection by echocardiography. Circulation, 1973. 48(3): p. 506-513.
7. Dijkmans, P., et al., Microbubbles and ultrasound: from diagnosis to therapy. European Journal of Echocardiography, 2004. 5(4): p. 245-246.
8. Chapelon, J.-Y., et al., Ultrasound therapy apparatus delivering ultrasound waves having thermal and cavitation effects. 1997, Google Patents.
9. Morgan, K.E., et al., Experimental and theoretical evaluation of microbubble behavior: Effect of transmitted phase and bubble size. IEEE transactions on ultrasonics, ferroelectrics, and frequency control, 2000. 47(6): p. 1494-1509.
10. Zhu, S. and P. Zhong, Shock wave–inertial microbubble interaction: A theoretical study based on the Gilmore formulation for bubble dynamics. The Journal of the Acoustical Society of America, 1999. 106(5): p. 3024-3033.
11. Postema, M., et al., High‐speed photography during ultrasound illustrates potential therapeutic applications of microbubbles. Medical physics, 2005. 32(12): p. 3707-3711.
12. Lawrie, A., et al., Microbubble-enhanced ultrasound for vascular gene delivery. Gene therapy, 2000. 7(23): p. 2023.
13. Lin, C.-Y., et al., Enhancement of focused ultrasound with microbubbles on the treatments of anticancer nanodrug in mouse tumors. Nanomedicine: Nanotechnology, Biology and Medicine, 2012. 8(6): p. 900-907.
14. Park, J., et al., Evaluation of permeability, doxorubicin delivery, and drug retention in a rat brain tumor model after ultrasound-induced blood-tumor barrier disruption. Journal of Controlled Release, 2017. 250: p. 77-85.
15. Unger, E.C., et al., Therapeutic applications of lipid-coated microbubbles. Advanced drug delivery reviews, 2004. 56(9): p. 1291-1314.
16. Lentacker, I., S.C. De Smedt, and N.N. Sanders, Drug loaded microbubble design for ultrasound triggered delivery. Soft Matter, 2009. 5(11): p. 2161-2170.
17. Lum, A.F., et al., Ultrasound radiation force enables targeted deposition of model drug carriers loaded on microbubbles. Journal of Controlled Release, 2006. 111(1-2): p. 128-134.
18. Klibanov, A.L., Microbubble contrast agents: targeted ultrasound imaging and ultrasound-assisted drug-delivery applications. Investigative radiology, 2006. 41(3): p. 354-362.
19. Lentacker, I., et al., Design and evaluation of doxorubicin-containing microbubbles for ultrasound-triggered doxorubicin delivery: cytotoxicity and mechanisms involved. Molecular Therapy, 2010. 18(1): p. 101-108.
20. Luo, W., et al., Dual-targeted and pH-sensitive doxorubicin prodrug-microbubble complex with ultrasound for tumor treatment. Theranostics, 2017. 7(2): p. 452.
21. Tinkov, S., et al., New doxorubicin-loaded phospholipid microbubbles for targeted tumor therapy: in-vivo characterization. Journal of Controlled Release, 2010. 148(3): p. 368-372.
22. Ting, C.-Y., et al., Concurrent blood–brain barrier opening and local drug delivery using drug-carrying microbubbles and focused ultrasound for brain glioma treatment. Biomaterials, 2012. 33(2): p. 704-712.
23. Yan, F., et al., Paclitaxel-liposome–microbubble complexes as ultrasound-triggered therapeutic drug delivery carriers. Journal of controlled release, 2013. 166(3): p. 246-255.
24. Huynh, E., et al., In situ conversion of porphyrin microbubbles to nanoparticles for multimodality imaging. Nature nanotechnology, 2015. 10(4): p. 325.
25. EC, G., Ultra-high-speed fluorescence imaging. 2012, University of Twente. p. 144.
26. Luan, Y., et al., Lipid shedding from single oscillating microbubbles. Ultrasound Med Biol, 2014. 40(8): p. 1834-46.
27. Ibsen, S., et al., The behavior of lipid debris left on cell surfaces from microbubble based ultrasound molecular imaging. Ultrasonics, 2014. 54(8): p. 2090-2098.
28. De Cock, I., et al., Sonoprinting and the importance of microbubble loading for the ultrasound mediated cellular delivery of nanoparticles. Biomaterials, 2016. 83: p. 294-307.
29. Saul, J.M., et al., Controlled targeting of liposomal doxorubicin via the folate receptor in vitro. Journal of Controlled Release, 2003. 92(1-2): p. 49-67.
30. Van Wamel, A., et al., Vibrating microbubbles poking individual cells: drug transfer into cells via sonoporation. Journal of controlled release, 2006. 112(2): p. 149-155.
31. Kooiman, K., et al., Sonoporation of endothelial cells by vibrating targeted microbubbles. Journal of controlled release, 2011. 154(1): p. 35-41.
32. van Rooij, T., et al., Viability of endothelial cells after ultrasound-mediated sonoporation: Influence of targeting, oscillation, and displacement of microbubbles. Journal of Controlled Release, 2016. 238: p. 197-211.
33. Liu, Y., et al., DNA uptake, intracellular trafficking and gene transfection after ultrasound exposure. Journal of controlled release, 2016. 234: p. 1-9.
34. Rejman, J., et al., Size-dependent internalization of particles via the pathways of clathrin-and caveolae-mediated endocytosis. Biochemical Journal, 2004. 377(1): p. 159-169.
35. Zuhorn, I.S., R. Kalicharan, and D. Hoekstra, Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis. Journal of Biological Chemistry, 2002. 277(20): p. 18021-18028.
36. Kaneda, M.M., et al., Mechanisms of nucleotide trafficking during siRNA delivery to endothelial cells using perfluorocarbon nanoemulsions. Biomaterials, 2010. 31(11): p. 3079-3086.
37. Haworth, K.J. and O.D. Kripfgans. Initial growth and coalescence of acoustically vaporized perfluorocarbon microdroplets. in 2008 IEEE Ultrasonics Symposium. 2008.