共載超微米氧化鐵奈米粒子與阿黴素之微氣泡於大鼠腦瘤之化療與磁性導向標靶治療的可行性研究

Feasibility study of ultrasmall superparamagnetic iron oxide nanoparticle and doxorubicin encapsulated microbubbles for chemotherapy and magnetic targeting delivery in a xenograft rat glioma model

林函融(Han-Jung Lin)

指導教授 ： 葉秩光

國立清華大學/原子科學院/生醫工程與環境科學系/碩士(2012年)

摘要

神經膠質母細胞瘤是最常見的原發性惡性腦瘤，即使經過適當治療仍無法有效增加病人的平均壽命。除了受限於腦組織中特殊的保護結構—血腦屏障阻礙化療藥物遞送至腦腫瘤區域的劑量之外，化療藥物本身對於身體其他部位的正常組織所造成的毒性也限制了臨床上投藥的劑量。近年來，許多研究致力於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被證實能有效地非侵入性的增加血腦屏障的通透大幅提升藥物累積於腦組織中的劑量。另一方面，改良藥物載體的設計或是將藥物載體修飾上特定分子亦可以增加藥物於目標處的累積量。
本研究的目的是發展共載超微米氧化鐵奈米粒子(ultrasmall superparamagnetic iron oxide nanoparticle, USPIO)與阿黴素(doxorubicin, DOX)的磷脂質微氣泡(簡稱UD-MB)，並評估UD-MB作為神經膠質母細胞瘤多功能治療用藥的可行性。此外，本研究將利用USPIO的趨磁性吸引UD-MB以增加藥物於腫瘤組織中的累積量，更藉著USPIO本身具有超順磁的特性來產生MRI影像對比作為治療中的影像導引以及治療後的預後評估。
本研究藉由電性吸附的方式將阿黴素修飾於微氣泡殼層上，超微米氧化鐵奈米粒子則以疏水作用力與磷脂質殼層相接來製備UD-MB。接著為了評估UD-MB作為超音波或MRI對比劑的能力將進行聲學以及磁學的特性量測。治療成效的評估則以C-6腫瘤細胞劑型細胞毒性測試。最後將以中心頻率為0.4 MHz的聚焦式超音波在特定參數下(328 kPa、1000 cycles、PRF 1Hz、照射90 sec)配合UD-MB來治療經植入C-6腫瘤細胞株的Sprague–Dawley大鼠進行血腦屏障開啟的評估。
實驗結果顯示UD-MB可以承載675± 62 mg/mL DOX以及707± 57 mg/mL USPIO。仿體實驗發現UD-MB能造成超音波對比影像增強約37 dB，MRI影像上每單位莫爾體積分率的UD-MB即可增加14.1 sec-1的橫向遲緩率。由細胞實驗則可確定在尚未以超音波進行釋藥前，UD-MB的細胞毒性與自由狀態下的DOX具有顯著性的差異(p value < 0.05)。並以強力磁鐵置於細胞培養品下方來吸引UD-MB的實驗可觀察到細胞攝取DOX以及USPIO的能力增加。最後，動物實驗的結果顯示UD-MB確實可以用來增加血腦屏障的通透。總結以上實驗結果可以證實自製UD-MB具有針對神經膠質母細胞瘤作為磁力導向的標靶治療藥物的能力。
未來工作希望進一步將腫瘤標的物質修飾上UD-MB，希望藉著標的物質來達到降低藥物於非目標區域的累積量，同時以磁力導向的標靶技術來增強效果。

關鍵字：神經膠質母細胞瘤、血腦屏障、微氣泡、聚焦式超音波、化學治療、超順磁性氧化鐵奈米粒子、標靶治療

關鍵字

神經膠質母細胞瘤 ； 血腦屏障 ； 微氣泡 ； 聚焦式超音波 ； 化學治療 ； 超順磁性氧化鐵奈米粒子 ； 標靶治療

並列摘要

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor. Most GBM patients have poor prognosis even after proper treatment. The major obstacles include less drugs reaching targeted area due to the existence of blood-brain barrier (BBB) and appearance of severe side effects preventing further increase in prescription dosage. Recently many techniques have been proposed to resolve these problems. Among these techniques, focus ultrasound combined with microbubbles provided the potential for elevating amount of drugs delivered into brain tissue. In order to reduce the exposure of normal tissue to drugs, designing a drug carrier or chelating a targeting molecule to drugs is needed.
The aim of this study is to develop the multi-functional phospholipid microbubbles (UD-MB) which loaded with doxorubicin (DOX) and ultrasmall superparamagnetic iron oxide nanoparticle (USPIO). As a drug carrier, UD-MB can reduce DOX toxicity to normal tissue and increase the accumulation of drugs in brain when combined with focus ultrasound insonation. Moreover, loading with USPIO on microbubbles, UD-MB can be transported to the targeting site by magnet and used as MRI contrast agents.
The DOX molecules with positive charge were electrostatically complex to anion phospholipid molecules and surface modified USPIO were trapped onto the inner shell by hydrophobic linkage between phospholipid molecules and USPIO. We also conducted the in-vitro experiments to quantify and qualify the sono-properties and magnetic properties of UD-MB. The cells toxicity of UD-MB was evaluated by using C-6 glioma cells. The degree of BBB opening by UD-MB with focused ultrasound (328 kPa, 1000 cycles, PRF 1 Hz, sonicate for 90 sec) in Sprague–Dawley rats bearing GBM tumors were also evaluated.
The results show the payloads of 675± 62 mg/mL DOX and 707± 57 mg/mL USPIO on the fabricated UD-MB. The ultrasound images can be enhanced by 37 dB and MRI relaxivity was 107.3 s-1mM-1 by using UD-MB. Moreover, intact UD-MB can reduce cells toxicity significantly compared to ionizing DOX. A magnet loacated on the beneath cell culture dish would enhance uptakes of DOX and USPIO by cells. In-vivo experiments demonstrate that UD-MB have the potential to increase the permeability of BBB.
In conclusion, UD-MB can be used as a dual imaging modality contrast agents and a magnetic targeting therapeutic drug for treating GBM. Future works include chelating a targeting molecule on UD-MB and applying the magnet to enhance the accumulation.

Keywords: glioblastoma multiforme (GBM), blood-brain barrier (BBB), microbubbles , focused ultrasound, chemotherapy, superparamagnetic iron oxide nanoparticle (SPIO), targeting therapy

並列關鍵字

glioblastoma multiforme (GBM) ； blood-brain barrier (BBB) ； microbubbles ； focused ultrasound ； chemotherapy ； superparamagnetic iron oxide nanoparticle (SPIO) ； targeting therapy

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