新式超音波藥物載體—相變液滴，外殼層以磷脂質構成，內核包覆液態全氟碳化合物，可乘載多種抗癌藥物，具有高度體內穩定性。透過聲學激發相變液滴汽化(Acoustic Droplet Vaporization，ADV)為常見的使用相變液滴進行腫瘤治療的方式，目前雖有文獻探討相變液滴汽化的機制，卻沒有探討相變液滴汽化對細胞造成的生物效應與釋藥至細胞的相關研究。由於微米等級的相變液滴的相轉變過程極快速、觀察細胞生物效應需要使用長時間活細胞顯微鏡觀測，且需要搭配多種螢光波段與活細胞染劑。因此本研究將結合高速影像與活細胞顯微鏡觀測相變液滴汽化對細胞產生的生物效應(細胞膜通透性、細胞活性)；也將使用高速影像搭配螢光成像探討相變液滴汽化於細胞上的藥物遞送。
研究結果發現ADV對細胞產生的生物效應可分為三種，與細胞和相變液滴之間距有相當大的關係，由遠至近分別是不改變細胞膜通透性和活性與控制組相同(Type A)、增加細胞膜通透性而仍有細胞活性(Type B)、細胞膜發生不可回復之通透性提升且細胞死亡(Type C)。從高速相機觀察到相變液滴汽化時其體積會瞬間急遽大幅膨脹形成氣泡，再縮小至一穩定半徑的氣泡，發現當細胞與相變液滴兩者距離大於相變液滴汽化之最大半徑外，會產生Type A；距離介於相變液滴汽化之最大半徑與穩定半徑間，會產生Type B；距離介於相變液滴汽化之穩定半徑內，會產生Type C。而相變液滴汽化殺死癌細胞的機制有兩種，(1)汽化形成的氣泡與細胞相黏後，破壞細胞膜；(2)汽化瞬間產生的慣性穴蝕效應。如使相變液滴和細胞保持特定的距離，慣性穴蝕效應可安全的提升細胞膜通透性。釋放藥物方面，高速螢光影像顯示藥物是以囊泡的形式從相變液滴釋放，並在相變液滴汽化瞬間<12 μs)即轉移至細胞膜上；雖然與細胞間距越近，釋藥效率越高，但如需維持細胞的存活率，則需使用小粒徑(1.5 μm)之相變液滴並結合高聲壓(8 MPa)之超音波照射才有最佳的釋藥效率。

The novel ultrasound-responsible drug carrier, phase-change droplet, which was composed of liquid perfluorocarbon within lipid shell, with great anticancer drugs loading capability and highly in vivo stability. Acoustic droplet vaporization (ADV) is widely used for cancer treatment. However, no study has elucidated the cellular biological effects resulted from ADV and the transient dynamics of drug release onto cells. Here, we combined high-speed camera imaging and live cell microscopic imaging to investigate the ADV-related cellular biological effects and the drug release onto cells. This study is try to find out the releasing distribution of drug, affecting region of ADV, the relationship between ADV and cellular biological effects, and the mechanism of cellular damage by ADV.
The ADV-induced cellular biological effects were regulated by the droplet-to-cell distance and could be characterized into: (i) Type A, no distinct change in membrane permeability and viability; (ii) Type B, the cells underwent reversible membrane permeability change and remaining viability; (iii) Type C, the cells were nonviable and underwent irreversible membrane permeability change. The high-speed camera data showed during ADV, the droplet vaporized into bubbles (ADV-bubbles) and the radius of ADV-bubbles rapidly reached maximum and then shirked to stable. When the droplet-to-cell distance larger than “Maximum radius”, whereas Type A occurred. When the distance between Maximum radius and Stable radius, Type B occurred. When the distance smaller than Stable radius, Type C occurred. There were two possible mechanisms of ADV-bubble induced cellular death: (i) the contact between ADV-bubble and cellular membrane; (ii) inertial cavitation. The inertial cavitation also probably increased the cellular membrane permeability. In terms of drug release, shorter droplet-to-cell distance caused higher drug delivery efficiency, but the best release efficiency happened at 8 MPa pressure with 1.5 μm radius droplet, and the drug transferred from droplets to cells only occurred during ADV (<12 μs).

第一章 緒論 11
1.1 癌症 11
1.1.1 癌症治療方法 12
1.1.2 癌症治療瓶頸 12
1.3 超音波結合微氣泡 13
1.3.1 微氣泡 13
1.3.1.1傳統超音波對比劑的發展 13
1.3.1.2 穴蝕效應 13
1.3.2 微氣泡用於生物效應 14
1.3.2.1 細胞膜通透性 14
1.3.2.2 細胞活性 16
1.3.3 微氣泡的缺點 19
1.4 超音波結合相變液滴 19
1.4.1 相變液滴特性 19
1.4.1.1 材質 19
1.4.1.2 相變液滴汽化機制 19
1.4.2 相變液滴應用 20
1.4.2.1 相變液滴應用於物理治療 20
1.4.2.2 觀察汽化機制 20
1.4.2.3 相變液滴應用於化學治療 23
1.4.2.4 觀察釋藥機制 23
1.4.2.5 相變液滴應用於細胞生物效應與藥物釋放之機制探討限制 26
1.5研究目的及論文架構 27
第二章 實驗材料與方法 29
2.1 概論 29
2.2 相變液滴製備 29
2.2.1 濃度與粒徑分佈之量測 30
2.3 細胞培養 31
2.3.1 細胞株-大鼠腦神經膠質瘤細胞 31
2.3.2 大鼠腦腫瘤神經膠質瘤細胞之繼代 31
2.4 高速螢光顯微系統實驗 32
2.4.1 實驗硬體系統架構 32
2.4.2 細胞製備 33
2.4.3 儀器同步時序 33
2.4.4 相變液滴釋放分析 34
2.5多螢光套件顯微系統實驗 35
2.5.1 硬體架構 35
2.5.2 基因轉殖細胞製備 36
2.5.3 染劑介紹 36
2.5.3.1 碘化丙啶 36
2.5.3.2 鈣黃綠素藍-乙酰氧基甲酯 37
2.5.3.3 Lyn-Yellow fluorescent protein 37
2.5.3.4 YFP-FBKT-β-Gal 37
2.5.4 資料分析 38
2.6 統計分析 38
第三章 實驗結果 39
3.1 相變液滴光學影像與粒徑分布 39
3.1.1 相變液滴的光學定性 39
3.1.2相變液滴的粒徑分布 39
3.1.3 定義相變液滴汽化閥值 40
3.2 相變液滴汽化於細胞產生之生物效應 41
3.2.1 細胞種類 42
3.2.1.1 控制組 42
3.2.1.2 Type A 42
3.2.1.3 Type B 43
3.2.1.4 Type C 45
3.2.2 計算細胞螢光強度隨時間變化之情形 45
3.2.3 定量螢光強度的變化量 47
3.2.4 距離影響相變液滴汽化之生物效應 49
3.3 相變液滴膨脹半徑 53
3.4 相變液滴殺死細胞的機制 57
3.4.1 相黏導致細胞死亡 59
3.4.1.2 細胞質內蛋白流出 61
3.4.2 穴蝕效應導致細胞死亡 63
3.5 生物效應的結論 64
3.5.1 距離參數影響 64
3.5.2 聲壓參數影響 64
3.5.3 粒徑參數影響 65
3.6 模擬藥物釋放 65
3.6.1 高速螢光的釋藥影像 65
3.6.2 囊泡特性 66
3.6.3 釋放藥物量定量 67
3.7 藥物釋放於細胞模型 71
3.7.1 藥物釋放量定量 73
3.7.2 釋藥量估算 74
第四章 結論與未來展望 76
4.1 結論 76
4.2未來工作 77
4.3 參考文獻 78

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