工程化细菌膜仿生纳米药物递送系统的建立及在脑胶质瘤治疗中的作用研究  

Establishment of an Engineered Bacterial Membrane Biomimetic Nanodrug Delivery System and Its Role in the Treatment of Glioma

作　　者：赵印震 李玉林 李娇 倪明立 王继创 王晓军 程蕾 牛文革 张应福 王云龙 ZHAO Yinzhen;LI Yulin;LI Jiao;NI Mingli;WANG Jichuang;WANG Xiaojun;CHENG Lei;NIU Wenge;ZHANG Yingfu;WANG Yunlong(Luoyang Vocational and Technical College,Luoyang 471099,China;Henan Provincial Biotechnology Research Center,Zhengzhou 450001,China;Henan Provincial Staff Hospital,Zhengzhou 450002,China)

机构地区：[1]洛阳职业技术学院,洛阳471099 [2]河南省生物工程技术研究中心,郑州450001 [3]河南省职工医院,郑州450002

出　　处：《四川大学学报（医学版）》2024年第4期861-871,共11页Journal of Sichuan University(Medical Sciences)

基　　金：中原学者工作站项目(No.234400510005)资助。

摘　　要：目的 制备工程化细菌膜仿生纳米粒Angiopep-2 E. coli membrane (ANG-2 EM)@PDA-PEI-CpG(简称ANG-2 EM@PPC),实现药物高效地靶向输送,治疗脑胶质瘤。方法 利用实验室构建inaX-N-angiopep-2工程菌表达,溶菌酶处理超滤离心获得ANG-2 EM;采用超声法包覆细菌膜制备ANG-2 EM@PPC;Western blot、琼脂糖凝胶电泳、透射电子显微镜(TEM)验证制备情况;检测粒径和Zeta电位以考察ANG-2 EM@PPC的稳定性。细胞水平研究:CCK-8法考察ANG-2 EM@PPC的对中性粒细胞存活率的影响。设计并构建流动腔模型,流式细胞术测定中性粒细胞的摄取效率,以考察在炎症环境下ANG-2 EM@PPC搭中性粒细胞便车的效率。荧光显微镜表征中性粒细胞死亡方式,Western blot和流式细胞术考察中性粒细胞凋亡小体的产生。动物水平研究:颅内注射小鼠胶质瘤-荧光素酶标记(GL261-Luc)细胞,建立原位脑胶质瘤小鼠模型,并验证小鼠肿瘤组织炎性环境。将肿瘤模型小鼠分为3组(均n=3),分别尾静脉注射DiR、ANG-2EM@PDA-PEI-CpG、EM@PDA-PEI-CpG(均10 mg/kg),荧光图像检测3种制剂在体内和脑内的分布情况;肿瘤模型小鼠分为6组(均n=4),分别尾静脉注射PBS、PDA、PC、PPC、EM@PPC、ANG-2 EM@PPC(均10 mg/kg),活体成像观察肿瘤消退状况并检测小鼠生存率、体质量以评价体内药效学,TUNEL染色(脑组织)、HE染色(脑、心、肝、脾、肺、肾组织)评价治疗效果。结果 TEM结果显示成功制备工程化细菌膜仿生纳米粒,PPC具有明显的壳核结构,壳厚约8.2 nm。由于ANG-2EM的包覆,ANG-2 EM@PPC的壳厚度增加到约9.6 nm且其表面有1层边界明显的细菌膜覆盖。放置1周内,稳定性良好。细胞实验中,CCK-8法示,ANG-2 EM@PPC对中性粒细胞的活力没有明显的影响。流式细胞术检测示,ANG-2EM@PPC在激活中性粒细胞中的摄取增强,在静止状态下搭中性粒细胞便车的效率比流动条件下高;与EM@PPC组相比,ANG-2 EM@PPC组搭中性粒细胞Objective To develop engineered bacterial membrane biomimetic nanoparticles,Angiopep-2 E.coli membrane(ANG-2 EM)@PDA-PEI-CpG(ANG-2 EM@PPC),for efficient targeted drug delivery in the treatment of glioma,and to provide theoretical and technical support for targeted glioma therapy.Methods The expression of inaX-N-angiopep-2 engineered bacteria was constructed in the laboratory,and ANG-2 EM was obtained through lysozyme treatment and ultrafiltration centrifugation.ANG-2 EM@PPC was prepared by ultrasonication of bacterial membranes.Western blotting,agarose gel electrophoresis,and transmission electron microscopy(TEM)were used to verify the preparation.Particle size and Zeta potential were measured to investigate the stability of ANG-2 [e-mail] cell experiments,CCK-8 assay was performed to determine the effect of ANG-2 EM@PPC on the survival rate of neutrophils.A flow chamber model was designed and constructed,and the uptake efficiency of neutrophils was measured by flow cytometry to investigate the hitchhiking efficiency of ANG 2 EM@PPC on neutrophils in inflammatory environment.Neutrophil death patterns were characterized by fluorescence microscopy,and flow cytometry and Western blotting were performed to examine neutrophil apoptotic bodies and the proportion of apoptotic bodies produced.Regarding animal experiments,a mouse model of in situ glioma was established and the inflammatory environment of tumor tissue was verified.The tumor model mice were divided into three groups,including DiR group,EM@PPC group,and ANG-2 EM@PPC group(all n=3),which were injected with DiR,ANG-2 EM@PDA-PEI-CpG,and EM@PDA-PEI-CpG via the tail vein,respectively(all at 10 mg/kg).Fluorescence images of organs and the brain were used to examine the distribution of the three formulations in vivo and in the brain.The tumor model mice were further divided into PBS group,PDA group,PC group,PPC group,EM@PPC group,and ANG-2 EM@PPC group(all n=4),which were injected with PBS,PDA,PC,PPC,EM@PPC,and ANG-2 EM@PPC injected via the tail vein,respec

关 键 词：中性粒细胞 脑胶质瘤 炎症 血脑屏障 工程化细菌膜 

分 类 号：R739.41[医药卫生—肿瘤]