顱內注射能量剝奪的阿托伐醌溫感性水凝膠介導粒線體功能障礙以協同放射治療膠質母細胞瘤

Abstract

高級惡性膠質瘤，尤其是惡性膠質母細胞瘤，是常見且高度侵襲性的成人腦瘤。目前的標準治療包括腫瘤切除、化療和放療。雖然放射治療會引起 DNA 斷裂，但同時腫瘤細胞也會啟動 DNA 修復機制並驅動腫瘤的代謝重編程，促使腫瘤侵襲性增強並對治療產生抗性。粒線體調節生物體的生物合成和代謝反應，在腫瘤代謝中發揮至關重要的作用，促使腫瘤呈現更高的代謝可塑性。Atovaquone 是一種FDA許可的抗瘧疾藥物，可以靶向抑制氧化磷酸化中的電子傳遞鏈的細胞色素 bc1 複合物。我們提出了一種新策略，利用Atovaquone的能量剝奪來開發透明質酸/Pluronic F127複合水凝膠的藥物遞送系統，透過抑制DNA修復與輻射協同作用來根除高級別膠質瘤。由透明質酸和Pluronic F127組成的可注射水凝膠應用於手術後腦瘤移除的空腔。 Atovaquone水凝膠具有溫感性質和緩慢降解兩個特點。透過流變儀測試可知聚合物水凝膠在37℃左右會發生溶膠-凝膠轉變，增加臨床使用者的便利性。由分光光度計進行測試可知水凝膠能夠控制釋放Atovaquone長達兩周，以此達到局部緩慢釋放的目的。使用 WST-1 的細胞活性測驗確定了抑制細胞增殖的有效Atovaquone濃度。粒線體壓力測試顯示Atovaquone不僅可以抑制粒線體產生ATP，同時，阻礙腫瘤整體代謝導向糖解作用。透過免疫螢光染色和電子顯微鏡可以觀察到粒線體自噬的過程。Colony formation實驗證明了Atovaquone與輻射的協同抗癌作用。彗星實驗和蛋白質印跡分析表明經由Atovaquone以及放射治療的組別DNA 損傷增強，並且抑制腫瘤DNA 修復機制，從而導致神經膠質瘤細胞侵襲和遷移減少、增強癌細胞凋亡。在動物實驗中，使用雞胚胎模型模擬局部給藥之狀況並且使用C57BL/6小鼠建立小鼠模型來驗證協同抗癌作用。綜上所述，Atovaquone的能量剝奪特性可介導粒線體代謝，並在腫瘤切除的空腔內進行顱內注射並持續緩慢釋放，與放射治療達到協同作用，從而根除高級別膠質瘤。我們的研究表明發展局部遞送改變腫瘤代謝之藥物增加放射敏感性用於腦瘤治療的潛力。

High-grade gliomas, especially glioblastoma, are common and highly aggressive adult brain tumors. The current standard treatments include tumor excision followed by chemotherapy and radiotherapy. While radiation therapy induces DNA breakage, it concurrently triggers DNA repair mechanisms and drives metabolic reprogramming, culminating in heightened tumor aggressiveness resistance to treatment. Mitochondria, regulating biosynthesis and metabolic reactions of organisms, plays a crucial role in cancer metabolism, thereby endowing tumors with remarkable metabolic plasticity. Atovaquone is an antimicrobial medication, which inhibits the electron transport system at the level of cytochrome bc1 complex. We proposed a novel strategy to utilize energy deprivation of atovaquone to develop a drug delivery system of Hyaluronic acid/Pluronic F127 composites hydrogel synergizing with radiation by inhibiting DNA repair to eradicate high-grade glioma. The injectable hydrogel composed of hyaluronic acid and Pluronic F127 is aim to apply on the surgical cavity intracranially after surgery. There are two characteristics of the Atovaquone hydrogel, thermosensitive and slow degradation. The polymeric hydrogel could undergo sol-gel transition at around 37℃, which is tested by rheometer. The polymeric hydrogel enables 2-week controlled release of atovaquone before radiotherapy, which is tested by UV-VIS spectroscopy. The cell viability using WST-1 identified the effective atovaquone concentration to inhibit cell proliferation. The mitochondria stress test illustrated Atovaquone not only inhibit ATP production by mitochondria but also obstruct metabolism transform to glycolysis. The process of mitochondrial autophagy can be observed through immunofluorescence staining and electron microscopy. The colony formation demonstrated the synergistic anticancer effect of atovaquone synergizing with radiation. The Comet assay and Western blotting analysis of γ-H2AX treated by atovaquone and radiation demonstrated enhancing DNA damage and reducing DNA repairment, which led to a reduction in glioma cell invasion and migration, while also enhancing cancer cell apoptosis. In animal studies, a chorioallantoic Membrane (CAM) simulated local administration scenarios, while a C57BL/6 mouse model was established to validate the synergistic anticancer efficacy. These results suggest that the energy deprivation feature of atovaquone mediates mitochondrial metabolism with sustained-release in the tumor-removed cavity to synergize with radiation for eradicating high-grade glioma, suggesting its potential developing localized delivery of agents that alter tumor metabolism to enhance radiosensitivity for the treatment of brain tumors.