包覆薑黃素的chitosan-PLGA奈米粒子負載anti-aldehyde dehydrogenase及sialic acid對人類惡性膠質母細胞瘤的標靶傳遞

Chitosan-PLGA Nanoparticles Grafted with Anti-aldehyde Dehydrogenase and Sialic Acid for Targeting Delivery of Curcumin to Human Glioblastoma Cells

王儷蓉(Li-Rung Wang)

指導教授 ： 郭勇志

國立中正大學/工學院/化學工程學系/碩士(2014年)

摘要

為改善薑黃素(curcumin)在生物體內利用度低的特性，利用chitosan-poly(D,L lactide–co–glycolide)奈米粒子作為藥物載體，以anti-aldehyde dehydrogenase (anti-ALDH)和sialic acid (SA)修飾於粒子表面，使薑黃素能夠穿透血腦屏障並標靶至人類惡性膠質母細胞瘤(malignant glioblastoma)。混合使用兩種界面活性劑polysorbate 80 (T80)和Pluronic F-127 (F-127)以微乳化溶劑揮發法製備奈米粒子，再利用人類腦微血管內皮細胞、人類星型細胞及人類周細胞建立體外血腦屏障模型(in vitro blood–brain barrier model)探討薑黃素的穿透率和藥物載體對人類惡性膠質母細胞瘤U87MG cells及腦癌幹細胞的治療，並藉由免疫螢光染色觀察奈米粒子被人類腦微血管內皮細胞、U87MG cells及腦癌幹細胞攝入的結果。由實驗結果顯示，奈米粒子的粒徑會隨著chitosan濃度增加而增加，chitosan-PLGA奈米粒子在界面活性劑濃度為0.1% (w/v)、chitosan濃度為0.02% (w/v)時為最佳粒徑，大約148 nm。由體外血腦屏障模型所測得的跨內皮細胞電阻(transendothelial electrical resistance, TEER)值為371.33 ± 11.09 Ωcm2，適合作為體外實驗的模型。而各組態的粒子穿透率為SA-ACPNPs > ACPNPs > CPNPs，三標靶的奈米粒子SA-ACPNPs的穿透率最高，且對腦癌進行治療的奈米粒子，以SA-ACPNPs最為有效。藉由免疫螢光結果顯示SA改質能提高腦微血管內皮細胞對粒子的攝入，且anti-ALDH可確實標靶U87MG cells和腦癌幹細胞。由上述結果顯示薑黃素經由SA和anti-ALDH表面修飾的chitosan-PLGA nanoparticles包覆後，可有效提升血腦屏障的穿透率，且可抑制U87MG cells和腦癌幹細胞，因此，以SA和anti-ALDH改質chitosan-PLGA nanoparticles未來可期待有效應用於腦癌的治療。

關鍵字

腦癌幹細胞 ； 腦癌 ； 幾丁聚醣-聚乳酸聚甘醇酸奈米粒子 ； 薑黃素 ； 血腦屏障

並列摘要

In order to improve the poor bioavailability of curcumin, chitosan-poly(D,L lactide–co–glycolide) nanoparticles (CS-PLGA NPs) surface modified with anti-aldehyde dehydrogenase (anti-ALDH) and sialic acid (SA) as a drug carrier. The drug carrier can across the blood–brain barrier (BBB) and target to U87MG cells and human brain cancerstem cells (HBCSCs). The CS-PLGA NPs was prepared using emulsion-solvent evaporation techniques in presence of polysorbate 80 (T80) and Pluronic F-127 (F-127). Furthermore, the cell co-culture model was established by consisting human brain-microvascular endothelial cells (HBMECs), human astrocytes (HAs) and human brain vascular pericytes (HPs) to investigate curcumin permeability of the in vitro BBB model and triple-targeting effect for U87MG cells, then observing the cellular uptake of HBMECs, U87MG cells, and HBCSCs by immunofluorescence. The results indicated that increase in the concentration of chitosan made significant increase in the average diameter. The optimum diameter was about 148 nm in 0.02% (w/v) of chitosan and 0.1% (w/v) of surfactant. TEER value of the in vitro BBB model was 371.33±11.09 Ωcm2. It was optimum for the in vitro BBB model. The order of transport across the BBB was SA-ACPNPs > ACPNPs > CPNPs, and the most effective nanoparticles cured barin tumor was SA-ACPNPs. The results of immunofluorescence demonstrated that SA could enhance cellular uptake and anti-ALDH could target U87MG cells and HBCSCs. These evidences suggested that curcumin encapsulated into SA and anti-ALDH grafted chitosan-PLGA nanoparticles could enhance the permeability against BBB. And the nanoparticles could be significantly against U87MG cells and HBCSCs. Therefore, it may be concluded that SA/anti-ALDH chitosan-PLGA nanoparticles has the potential to be applied as a targeted delivery for anticancer drug in the treatment of brain cancer.

並列關鍵字

brain cencer stem cells ； brain cancer ； chitosan-PLGA nanoparticles ； curcumin ； blood–brain barrier

參考文獻

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