血腦屏障是哺乳類動物腦部具有的特異化結構，其存在能使腦組織免於有害物質侵入，但同時也增加了腦部疾病治療的困難，近年來已經發現多種方法能提升血腦屏障的通透性，其中聚焦式超音波配合微氣泡的方法，在藉由傳統磷脂質殼層微氣泡於超音波聲場下產生振動及破裂的現象，已被證實能夠局部且非侵入性地開啟血腦屏障，提升血管通透性及藥物輸送效率；然而磷脂質微氣泡破裂時所伴隨產生的慣性穴蝕效應在活體腦組織內可能造成不欲見之出血和傷害。因此，本研究發展出一種新型微氣泡(磷脂質包覆之聚合微氣泡)，藉由在磷脂質單層膜內之疏水單體及交聯劑的自由基聚合反應，於磷脂質殼層內部形成二維網狀聚合物。
製備上，先將磷脂質、聚合單體及交聯劑等材料經由強烈震盪形成微氣泡，再加入起始劑及加速劑在室溫環境下聚合3小時完成。接著我們測試磷脂質包覆之聚合微氣泡的物化及聲學性質，包括共振頻率、非線性共振能力、慣性穴蝕效應強度及擊破閥值。此外，為了避免高頻超音波(10-MHz)在組織內的衰減效應，我們另外製作了大粒徑的磷脂質包覆之聚合微氣泡並配合低頻超音波(2-MHz)來開啟血腦屏障，同時評估其開啟效率及出血情形。
低溫穿透式電子顯微鏡清楚驗證了此二維網狀聚合結構確實形成於磷脂質單層膜內的疏水相。小粒徑微氣泡方面，在低聲壓時磷脂質包覆之聚合微氣泡與未聚合微氣泡除了擁有相同的共振頻率(12-MHz)外，也在5-MHz處表現出相似的次諧波訊號強度；然而，在高聲壓時磷脂質包覆之聚合微氣泡的慣性穴蝕效應強度卻低了未聚合微氣泡1.3倍，且磷脂質包覆之聚合微氣泡的擊破閥值也低了未聚合微氣泡約800 KPa，此結果顯示網狀聚合物殼層不但不會影響磷脂質殼層的震動能力，還能降低其慣性穴蝕效應。另一方面，大粒徑磷脂質包覆之聚合微氣泡的聲學性質大部分皆與小粒徑的相似，除了其慣性穴蝕效應強度只有在超音波脈衝重複頻率高於20 Hz時才會較未聚合微氣泡來的低。最後，我們於活體實驗中使用此磷脂質包覆之聚合微氣泡配合2-MHz的超音波來開啟血腦屏障，結果顯示磷脂質包覆之聚合微氣泡造成了比未聚合微氣泡更多的依文氏藍染劑滲漏，亦即能較有效率地提升血腦屏障通透性，但卻能產生較少的出血傷害。
此研究首先發展了一種新型的微氣泡，其能夠在配合聚焦式超音波開啟血腦屏障時提供更安全的應用；然而，仍然有許多參數及材料可以更進一步調整和嘗試，例如優化磷脂質、聚合單體及交聯劑的材料組成比例，或是使用硬性材料像是熱固性高分子以形成更堅固的聚合物殼層而達到更好的應用效果。

Blood-brain barrier (BBB) is an unique structure in mammals’ brain. Although BBB can prevent harmful substances from entering brain tissues, it makes treatments of brain diseases difficult. Recently, a lot of methods have been discovered to enhance the permeability of BBB, among which traditional phospholipid-shelled microbubbles (MBs) combined with focused ultrasound (FUS) have been approved to induce non-invasive and local BBB disruption by the oscillation and destruction of MBs under insonification of acoustic field, facilitating vascular permeability and drug delivery efficiency. However, the destruction effects of phospholipid MBs may also accompany inertial cavitation (IC) that cause unwanted hemorrhage and damage in vivo. Therefore, in this study, a novel MBs (phospholipid-coated polymer MBs, PP-MBs) was developed by polymerization of hydrophobic monomer and cross-linker inside the phospholipid monolayer of MBs using a radical mechanism, forming a two-dimensional (2D) polymer network inside phospholipid shell.
During the fabrication of PP-MBs, phospholipids, monomer and cross-linker were intensively shook, followed by polymerization via adding initiator and accelerator for 3 hours at room temperature. Then, physicochemical and acoustic properties of PP-MBs, including resonance frequency, nonlinear resonance capacity, inertial cavitation (IC) dose and destruction threshold were measured. Beside, in order to avoid the attenuation effect of high frequency ultrasound (10-MHz) in tissues, PP-MBs with larger size were fabricated for BBB disruption with low frequency ultrasound (2-MHz), and the BBB disruption efficiency and hemorrhage levels were assessed.
Cryo-transmission electron microscopy provided clear evidence that the 2D polymer network structure was indeed incorporated inside the hydrophobic phase of phospholipid monolayer. In the aspects of PP-MBs with smaller size, PP-MBs and unpolymeried MBs behaved not only the same ultrasound resonance frequencies (measured as 12-MHz), but also similar subharmonic intensity characterized at 5-MHz under low acoustic pressure. However, under high acoustic pressure, the IC dose of PP-MBs was 1.3-fold lower than unpolymerized MBs. In addition, the destruction threshold of PP-MBs was about 800 KPa lower than unpolymerized MBs. These results suggested that the polymer network shell of PP-MBs could reduce the inertial cavitation with no influence on oscillation properties of phospholipid shell. On the other hand, PP-MBs with larger size showed similar acoustic properties with the smaller-sized PP-MBs, except for their lower IC dose under ultrasound pulse repetition frequency of >20 Hz than that of smaller-sized PP-MBs. Finally, PP-MBs were applied to disrupt BBB in vivo with 2-MHz FUS. PP-MBs were demonstrated to exhibit more Evans blue leakage (i.e. enhance the permeability of BBB more effectively) but less hemorrhage damage than unpolymerized MBs did.
In this study, we first developed a novel MBs which had great potential to provide safer application for BBB disruption in the combination with FUS. However, there were still some parameters and different materials to adjust and attempt. For example, optimizing the composition of phospholipid, monomer and cross-linker, or using stiff materials such as thermosetting polymers to form more rigid polymer shell to achieve better effectiveness.

第一章 緒論 1
1.1聚焦式超音波(Focused ultrasound, FUS) 1
1.1.1超音波和生物組織的相互效應 2
1.1.1.1熱效應 2
1.1.1.2機械效應 2
1.1.1.3穴蝕效應 2
1.2微氣泡超音波對比劑(Ultrasound contrast agents, UCAs) 3
1.2.1脂質微氣泡(Lipid microbubbles) 5
1.2.2高分子聚合微氣泡(Polymer microbubbles) 6
1.3血腦屏障(Blood-Brain Barrier, BBB) 7
1.3.1提升血腦屏障通透性之方法 8
1.3.2利用聚焦式超音波與微氣泡提升血腦屏障通透性 9
1.4研究概念與動機 10
1.4.1磷脂質包覆之聚合微氣泡(Phospholipid-coated polymer microbubbles, PP-MBs) 10
1.4.2研究動機與目的 12
第二章 實驗材料與方法 13
2.1概論 13
2.2磷脂質包覆之聚合微氣泡製備 13
2.2.1網狀交聯聚合殼層 16
2.2.2粒徑分析 16
2.2.3光學顯微鏡 17
2.2.4穿透式電子顯微鏡(Transmission electron microscope, TEM) 17
2.2.5體外穩定性 17
2.3體外聲學實驗 18
2.3.1共振頻率 18
2.3.1.1系統架構 18
2.3.1.2實驗步驟 20
2.3.1.3訊號分析處理 20
2.3.2微氣泡的非線性共振及慣性穴蝕效應強度 21
2.3.2.1仿體製備 21
2.3.2.2系統架構 21
2.3.2.3實驗步驟 23
2.3.2.4訊號分析處理 24
2.3.3微氣泡的擊破閥值 25
2.3.3.1仿體製備 25
2.3.3.2系統架構 26
2.3.3.3實驗步驟 27
2.3.3.4訊號分析處理 28
2.4活體動物實驗 29
2.4.1活體內穩定性 30
2.4.1.1實驗架構 30
2.4.1.2實驗步驟 31
2.4.1.3結果分析 32
2.4.2血腦屏障開啟及傷害性評估 33
2.4.2.1實驗架構 33
2.4.2.2實驗步驟 33
2.4.2.3 Evans blue染劑定量 35
2.4.2.4腦組織冷凍切片與H & E染色法 35
2.4.2.5血腦屏障開啟之傷害性定量 37
2.4.2.6血管收縮測試 37
第三章 結果與討論 39
3.1小粒徑微氣泡 39
3.1.1 PP-MB的物化性質 39
3.1.1.1粒徑分析 39
3.1.1.2顯微鏡光學影像 40
3.1.1.3穿透式電子顯微鏡 41
3.1.1.4體外穩定性 43
3.1.2體外聲學實驗 44
3.1.2.1微氣泡共振頻率 44
3.1.2.2微氣泡的非線性共振 45
3.1.2.3微氣泡的慣性穴蝕效應強度 47
3.1.2.4微氣泡的擊破閥值 51
3.2大粒徑微氣泡 53
3.2.1 PP-MB的物化性質 53
3.2.1.1粒徑分析 53
3.2.1.2顯微鏡光學影像 54
3.2.1.3體外穩定性 55
3.2.2體外聲學實驗 56
3.2.2.1微氣泡的共振頻率 56
3.2.2.2微氣泡的非線性共振 57
3.2.2.3微氣泡的慣性穴蝕效應強度 58
3.2.2.4改變脈衝長度 61
3.2.2.5改變脈衝重複頻率 62
3.2.2.6微氣泡的擊破閥值 64
3.2.3活體動物實驗 66
3.2.3.1活體內穩定性 66
3.2.3.2以PP-MB增加血腦屏障通透性及Evans blue染劑定量 67
3.2.3.3血管收縮效應測試 70
3.2.3.4以PP-MB開啟血腦屏障的傷害性評估 72
第四章 結論與未來工作 77
4.1結論 77
4.2未來工作 78
4.2.1改變配方濃度及比例 79
4.2.2使用硬性的聚合材料 79
參考文獻 80

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