靶向脑胶质瘤的Ang外泌体递送系统的构建

Construction of Ang exosome delivery system targeting glioblastoma

目的构建可以精准靶向胶质母细胞瘤(glioblastoma,GBM)的外泌体。方法构建靶向GBM的Ang-Lamp2b-HA质粒,将Ang-Lamp2b-HA质粒和对照pcDNA3.1载体质粒分别转染入HEK293T细胞后提取工程化外泌体Ang-exo和对照未修饰外泌体Unmod-exo,使用蛋白免疫印迹技术观察两组外泌体标志蛋白表达情况,使用扫描电镜和纳米颗粒分析仪观察两组外泌体形态学表征。建立基于Transwell系统的bEnd.3/LN229体外血脑屏障模型,上下腔室分别接种bEnd.3细胞和LN229细胞,生成完整屏障后,分别在上腔室中加入已染色的Ang-exo和Unmod-exo,通过共聚焦激光扫描显微镜观察两组外泌体在下腔室中胶质瘤细胞荧光强度,验证Ang-exo穿透血脑屏障能力和靶向的能力。已染色的Ang-exo和Unmod-exo外泌体分别通过尾静脉注射入荷瘤小鼠模型,通过活体动物成像和离体器官成像观察两组外泌体在荷瘤小鼠体内荧光分布变化,验证其穿透血脑屏障能力和靶向效果。将Ang-exo与生理盐水分别尾静脉注射入健康小鼠体内,观察比较器官变化情况,对比血液生物化学和炎性因子指标差异。结果转染Ang-Lamp2b-HA质粒和对照pcDNA3.1载体质粒的HEK293T细胞分别产生分泌Ang-exo和Unmod-exo,两组外泌体外形和粒径大小无明显差异。经过体外血脑屏障模型功能鉴定,与Unmod-exo组相比,Ang-exo组穿透血脑屏障的数目明显较高(P<0.05)。与Unmod-exo组相比,Ang-exo组脑部肿瘤区域的荧光强度较高(P<0.05)。与生理盐水组比较,Ang-exo组小鼠血液学指标天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、碱性磷酸酶(ALP)、血尿素氮(BUN)和肌酐(Cr)等肝肾损伤标志物水平,γ干扰素(IFN-γ)、肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)等血清炎性细胞因子水平,脑、心、肺、肝、脾和肾等器官均无明显变化(P>0.05)。结论Ang-exo可以精准靶向GBM,是一种可靠的药物运载平台,为GBM治疗药物的递送提供了新的策略。

Objective To construct exosomes that can accurately target glioblastoma(GBM).Methods The Ang-Lamp2b-HA plasmid targeting GBM was constructed,and the Ang-Lamp2b-HA plasmid and the control pcDNA3.1 vector plasmid were transfected into HEK293T cells,respectively.Then the engineered exosome Ang-exo and the control unmodified exosome Unmod-exo were extracted.The expression of exosome marker proteins in the two groups was observed by Western blotting,and the morphological characterization of exosomes was observed by scanning electron microscopy and nanoparticle analyzer.A bEnd.3/LN229 blood-brain barrier model based on Transwell system was established in vitro.The bEnd.3 cells and LN229 cells were inoculated in the upper and lower chambers,and stained Ang-exo and Unmod-exo were added to the upper chambers after the complete barrier was formed.The fluorescence intensity of exosomes in glioma cells in the inferior compartment was observed in the two groups by confocal laser scanning microscopy to verify the ability of Ang-exo penetrating the blood-brain barrier and its targeting ability.The stained Ang-exo and Unmod-exo exosomes were injected into the tumor-bearing mouse model via the tail vein,respectively.The fluorescence distribution changes of exosomes in tumor-bearing mice were observed by in vivo imaging and in vitro organ imaging,and the ability to penetrate the blood-brain barrier(BBB)and the targeting effect were verified.Ang-exo and saline were respectively injected into the tail vein of healthy mice to observe the changes of organs,blood biochemistry and inflammatory factors.Results The HEK293T cells transfected with Ang-Lamp2b-HA plasmid and control pcDNA3.1 vector plasmid secreted Ang-exo and Unmod-exo,respectively.Morphological analysis of exosomes showed no significant differences in shape and particle size between the two groups.After functional identification of BBB model in vitro,the cell number of penetrating BBB in Ang-exo group was significantly higher than that in Unmod-exo group(P<0.05).Compared with Unmod-exo group,the fluorescence intensity of brain tumor areas in Ang-exo group was increased(P<0.05).There was no significant difference in the levels of hematologic indexes such as aspartic acid aminotransferase(AST),alanine aminotransferase(ALT),alkaline phosphatase(ALP),blood urea nitrogen(BUN)and creatinine(Cr),the levels of serum inflammatory cytokines such as interferonγ(IFN-γ),tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β),and the changes of brain,heart,lung,liver,spleen and kidney between normal saline group and Ang-exo group(P>0.05).Conclusion Ang-exo can accurately target GBM and is a reliable drug delivery platform,which provides a new idea for improving the therapeutic effect of GBM.