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標題: | 探討聚焦超音波開啟血腦障蔽及熱療作用以增強腦部藥物之傳輸 Investigating focused ultrasound-induced blood-brain barrier opening and low-dose hyperthermia to enhance drug delivery to the brain |
作者: | Yu-Hone Hsu 許育弘 |
指導教授: | 林文澧 |
關鍵字: | 血腦障蔽,腦瘤,腦藥物傳輸,聚焦超音波,黏多醣症,奈米藥,超音波熱療, Blood-brain barrier,Brain drug delivery,Brain tumor,Evans blue dye,Focused ultrasound,Mucopolysaccharidosis type I,Nanodrug,Ultrasound hyperthermia,Recombinant human alpha-L-iduronidase., |
出版年 : | 2017 |
學位: | 博士 |
摘要: | 聚焦超音波結合微氣泡使用可開啟血腦障蔽。但關於超音波施打參數對血腦障蔽開啟的影響、超音波開啟血腦障蔽後藥物傳輸的「時間-反應」關係及「劑量-反應」關係、血腦障蔽開啟的持續時間、不同超音波施打流程對血腦障蔽開啟的效果等問題,過去的相關研究並不多。於實驗一我們探討以上問題。我們用弱聚焦超音波來開啟小鼠 (C57/B6 mice) 的血腦障蔽,並以伊凡式藍 (Evans blue) 做為追蹤分子以監測血腦障蔽的開啟。以聲壓0.56 MPa及脈衝重覆頻率1 Hz為固定參數,探討突發時長 (burst length) ,微氣泡劑量及超音波施打時間對於血腦障蔽開啟的影響。我們於超音波開啟血腦障蔽後的1、4、24小時測量腦內伊凡式藍的沉積量,以探討超音波對於腦藥物傳輸的「時間-反應」關係;我們施打不同劑量的伊凡式藍並以超音波開啟血腦障蔽,以探討血腦障蔽開啟後對於腦藥物傳輸的「劑量-反應」關係。我們於超音波開啟血腦障蔽後的不同時間點注射伊凡式藍以探討血腦障蔽開啟的持續時間。我們以超音波施打一次及兩次來開啟血腦障蔽,探討不同的超音波施打流程對於血腦障蔽開啟的效果。實驗結果顯示弱聚焦超音波可廣泛的開啟血腦障蔽。突發時長越長及微氣泡劑量越大,血腦障蔽開啟的效果就越大;超音波施打時間超過60秒並不會增加血腦障蔽開啟的程度;施打超音波後的1小時,伊凡式藍在腦的沉積量最高,24小時後伊凡式藍在腦的沉積量仍保有尖峰量的81%;在血腦障蔽已開啟的狀況下,伊凡式藍的注射量與腦內沉積量呈現高度正相關;超音波施打後,隨著時間過去,血腦障蔽開啟的程度會下降,開啟的效果可維持6小時。超音波施打兩次比施打一次多了74.8%的伊凡式藍腦沉積量。組織切片顯示弱聚焦超音波對腦部造成的傷害輕微 。伊凡式藍在血漿中會與白蛋白結合形成一個83 kDa的聚合體,這個實驗的結果可模擬一個83 kDa的巨分子藥物藉由超音波開啟血腦障蔽後傳輸到腦組織的動力學。
黏多醣症第一型是致殘率極高的遺傳性疾病,主要病因是自身無法製造alpha-L-iduronidase (IDUA),導致身體無法代謝糖胺聚醣 (glycosaminoglycans)。糖胺聚醣在身體每個細胞不當累積,造成嚴重心智障礙及各個器官的功能障礙。目前唯一有效的治療方式是酵素替代療法,也就是定期施打recombinant human alpha-L-iduronidase (rhIDU)。酵素替代療法有效的改善了許多器官的功能但是卻無法改善腦功能,因為血腦障蔽阻絕了rhIDU進入腦組織。於實驗二,我們利用弱聚焦超音波搭配微氣泡使用以開啟黏多醣症第一型小鼠的血腦障蔽,將注射入血液的rhIDU送入腦部。我們將黏多醣症第一型小鼠分成四組:1) 對照組;2) rhIDU酵素替代治療組;3) rhIDU酵素替代治療+聚焦超音波施打一次;4) rhIDU酵素替代治療+聚焦超音波施打二次。所有的聚焦超音波都施打於左腦。比較這四組左右腦部rhIDU的活性量,並利用伊凡式藍模擬rhIDU在腦部的分佈。實驗結果顯示,以聚焦超音波搭配微氣泡使用開啟左腦血腦障蔽,左腦的rhIDU活性量高達右腦的7.81倍,達到正常值的75.84%。伊凡式藍模擬顯示藥物廣泛分佈於腦內。超音波施打二次的藥物輸送效率高於超音波施打一次。這個方式未來有機會成為黏多醣症的治療方式之一。 在腦瘤的治療上,因BBB阻擋大分子進入腦部,使得化療對於腦瘤的治療效果不如其他部位的腫瘤。於實驗三,我們探討低強度聚焦超音波熱療 (low-dose focused ultrasound hyperthermia, UH)是否能夠增強奈米化療藥物pegylated liposomal doxorubicin (PLD)在腦腫瘤的傳輸。我們將小鼠乳癌細胞4T1-luc2植入小鼠腦內以建立腦轉移瘤的動物模型,並以活體影像系統 (IVIS) 觀察腦轉移瘤的生長,於腫瘤細胞植入後6天進行治療。我們將治療小鼠分為五組:1) 對照組 (control);2) Pulsed-wave focused ultrasound hyperthermia組(pUH);3) PLD注射組 (PLD);4) PLD + continuous-wave focused ultrasound hyperthermia組 (PLD+cUH);5) PLD + pulsed-wave focused ultrasound hyperthermia組 (PLD+pUH)。其中pUH及cUH使用相同的acoustic power (2.2-Watt)及sonication duration (10分鐘)。我們利用fluorometry測量doxorubicin在腦瘤組織及正常腦組織的沉積量,以及利用免疫螢光染色法來觀察PLD在腦組織的分佈及細胞凋亡 (apoptosis) 現象。結果顯示pUH可有效的強化PLD在腦瘤組織的沉積量,pUH+PLD有效抑制腦瘤的生長,並且不會對正常腦組織造成傷害。此結果顯示低強度聚焦超音波熱療可選擇性有效增強奈米藥在腦瘤組織的傳輸,並改善腦瘤的治療成效。 Pulsed ultrasound can disrupt blood-brain barrier (BBB) temporarily, but it is not clear about the effectiveness of different ultrasound parameters on BBB disruption, time-response and dose-response relationship on brain drug delivery after BBB disruption, dynamics of BBB disruption, and the effectiveness of different sonication schemes on BBB disruption. In the first portion, we investigated sonication parameter and sonication scheme effectiveness on BBB disruption. We used pulsed weakly focused ultrasound to open the BBB of C57/B6 mice. Evans blue dye (EBD) was used to determine the degree of BBB disruption. Acoustic pressure 0.56 MPa, pulse repetitive frequency 1 Hz, burst length 10 to 50 ms, microbubble dose 100 to 300 μl/kg and sonication time 60 to 150 s were used to open BBB for parameter study; brain EBD accumulation was measured at 1, 4, 24 hours after sonication for time-response relationship study; EBD 100 to 200 mg/kg was administered for dose-response relationship study; EBD injection 0 to 6 hours after sonication was performed for BBB disruption dynamic study; brain EBD accumulation induced by one-spot and two-spot sonication was investigated to study the effectiveness on BBB disruption; histology study was performed for brain tissue damage evaluation. The results showed that pulsed weakly focused ultrasound opened BBB extensively. Longer burst length and larger microbubble dose resulted in higher degree of BBB disruption; sonication time longer than 60 s did not increase BBB disruption; brain EBD accumulation peaked at 1 hour after sonication and remained 81% of peak level at 24 hours after sonication; EBD dose administered correlated to brain EBD accumulation; BBB disruption decreased as time went on after sonication and lasted for 6 hours at least; brain EBD accumulation induced by two-spot sonication increased 74.8% of that induced by one-spot sonication. There was limited adverse effect associated with sonication including petechial hemorrhages and mild neuronal degeneration. We conclude that BBB can be opened extensively and reversibly by pulsed weakly focused ultrasound with limited brain tissue damage. Since EBD combines with albumin in plasma to form a conjugate of 83 kDa, these results may represent ultrasound induced brain delivery of therapeutic molecules of this size scale. Mucopolysaccharidosis type I (MPS I) is a debilitating hereditary disease characterized by alpha-L-iduronidase (IDUA) deficiency and consequent inability to degrade glycosaminoglycans. The pathological accumulation of glycosaminoglycans systemically results in severe mental retardation and multiple organ dysfunction. Enzyme replacement therapy with recombinant human alpha-L-iduronidase (rhIDU) improves the function of some organs but not neurological deficits owing to its exclusion from the brain by the blood-brain barrier (BBB). In the second portion, we tried to deliver rhIDU to MPS I mice brain by FUS induced BBB opening technique. We divided MPS I mice into control group, enzyme replacement group with rhIDU 2.9 mg/kg injection, enzyme replacement with one-spot ultrasound treatment group, and enzyme replacement with two-spot ultrasound treatment group, and compared treatment effectiveness among the groups. All ultrasound treatments were applied on left side brain. Evans blue was used to simulate the distribution of rhIDU in the brain. The result showed that transcranial pulsed weakly focused ultrasound combined with microbubbles facilitated brain rhIDU delivery in MPS I mice receiving systemic enzyme replacement therapy. With intravenously injected rhIDU 2.9 mg/kg, the IDUA enzyme activity on the ultrasound treated side of the cerebral hemisphere raised to 7.81-fold that on the untreated side and to 75.84% of its normal value. Evans blue simulation showed the distribution of the delivered drug was extensive, involving a large volume of the treated cerebral hemisphere. Two-spot ultrasound treatment scheme was more efficient for brain rhIDU delivery than one-spot ultrasound treatment scheme. The clinical application of chemotherapeutics for brain tumors remains a challenge due to limitation of blood-brain barrier/blood-tumor barrier (BBB/BTB). In the third portion, we investigated the effects of low-dose focused ultrasound hyperthermia (UH) on the delivery and therapeutic efficacy of pegylated liposomal doxorubicin (PLD) for brain metastasis of breast cancer. Murine breast cancer cells (4T1-luc2) expressing firefly luciferase were implanted into mouse striatum as a brain tumor model. The mice were intravenously injected with PLD with/without transcranial pulsed-wave/continuous-wave UH (pUH/cUH) treatment on day-6 after tumor implantation. pUH was conducted under equal acoustic power and sonication duration as cUH. The amounts of doxorubicin accumulated in the normal brain and tumor tissues were measured with fluorometry. The tumor growth of the control, pUH, PLD, PLD+cUH, and PLD+pUH groups were evaluated with IVIS. The PLD distribution and cell apoptosis were assessed with immunofluorescence staining. The results showed that pUH significantly enhanced the PLD delivery into brain tumors and the tumor growth was further inhibited by PLD+pUH without damaging the sonicated normal brain tissues. This indicates that low-dose transcranial pUH is a promising method to selectively enhance nanodrug delivery and improve the brain tumor treatment |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67792 |
DOI: | 10.6342/NTU201701917 |
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顯示於系所單位: | 醫學工程學研究所 |
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