[關(guān)鍵詞]
[摘要]
目的 制備肝癌細胞Hep1-6外泌體并對化療藥物阿霉素(DOX)進行包載,以期實現(xiàn)對腫瘤細胞更高的靶向活性與殺傷作用。方法 采用梯度離心法對腫瘤細胞Hep1-6來源的外泌體進行制備分離;采用透射電鏡技術(shù)、表面標記蛋白表征以及納米顆粒追蹤分析技術(shù)對外泌體的形態(tài)、特征蛋白、粒徑分布和濃度進行表征;采用電穿孔方法實現(xiàn)外泌體對DOX的有效包載,制備包載阿霉素外泌體(EXODOX)。采用CCK-8法檢測EXODOX與DOX(0.5、1、2、3、5、10 μg·mL-1)體外對 Hep1-6細胞增殖的影響,采用激光共聚焦顯微鏡觀察體外 Hep1-6細胞對 EXODOX與 DOX(1 μg·mL-1)的靶向攝取作用。結(jié)果 所制備的外泌體具有形態(tài)良好 、 粒度均一的特性且具備外泌體特征膜蛋白 CD63、 CD81、 腫瘤易感基因101(TSG101)的表達;在電穿孔條件為150 V和75 μF下外泌體對DOX具備良好的包載特性;相比于單獨給藥DOX,在同等質(zhì)量濃度下 EXODOX對 Hep1-6細胞增殖抑制作用顯著增強(P<0.05、0.01),同時腫瘤細胞對 EXODOX的攝取更具靶向性。結(jié)論 制備的EXODOX較DOX具有更強的體外細胞毒活性,EXODOX表現(xiàn)出對腫瘤細胞更高的靶向特性。
[Key word]
[Abstract]
Objective To extract exosomes from hepatoma cells Hep1-6 and carry out effective inclusion of doxorubicin (DOX) as a chemotherapy drug to achieve higher targeting activity and killing effect on tumor cells.Methods Gradient centrifugation was used to prepare and isolate exosomes from tumor cells. The morphology, characteristic marker protein, particle size distribution and concentration of exosomes were characterized by transmission electron microscopy, Western blotting and nanoparticle tracking analysis (NTA). Exosomes were effectively loaded with doxorubicin by electroporation to prepare exosomes-doxorubicin (EXODOX). The tumor killing activity and targeted uptake of EXODOX and DOX (0.5, 1, 2, 3, 5, 10 μg·mL-1)were evaluated by CCK-8 and laser confocal assay.Results EXODOX with good morphology and uniform particle size were prepared with the expression of the characteristic membrane proteins of exosomes, such as CD63, CD81, and TSG101. The exosomes have good encapsulation characteristics for DOX under 150 V and 75 μF electroporation condition. Compared with DOX alone, EXODOX achieve efficient targeted uptake of tumor cells at the same dose, and further improve the killing effect on tumor cells.Conclusions EXODOX achieve higher targeting characteristics and stronger cytotoxic activity against tumor cells.
[中圖分類號]
R945;R965
[基金項目]
北京市自然科學(xué)基金面上項目(7212174)