Zhao Yinzhen, Li Yulin, Li Jiao, Ni Mingli, Wang Jichuang, Wang Xiaojun, Cheng Lei, Niu Wenge, Zhang Yingfu, Wang Yunlong
（ 471099） Luoyang Vocational and Technical College, Luoyang 471099, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2024 Jul 20;55(4):861-871. doi: 10.12182/20240760203.
To develop engineered bacterial membrane biomimetic nanoparticles, Angiopep-2 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.
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 EM@PPC. Regarding 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 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 =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 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 =4), which were injected with PBS, PDA, PC, PPC, EM@PPC, and ANG-2 EM@PPC injected via the tail vein, respectively (all at 10 mg/kg). Imaging was performed to observe tumor regression, and the survival rate and body mass of mice were measured to evaluate pharmacodynamics. TUNEL staining (brain tissue) and HE staining (brain, heart, liver, spleen, lung and kidney tissues) were performed to evaluate the therapeutic effect.
The results of TEM showed successful preparation of engineered bacterial membrane biomimetic nanoparticles, with PPC exhibiting a distinct shell-core structure and a shell thickness of about 8.2 nm. Due to the coating of ANG-2 EM, the shell thickness of ANG-2 EM@PPC increased to about 9.6 nm, with a clear bacterial membrane layer on the surface. Stability was maintained for at least one week. ANG-2 EM@PPC had no significant effect on the activity of neutrophils according to the findings from the CCK-8 assay. Flow cytometry showed that ANG-2 EM@PPC uptake is enhanced in activated neutrophils and hitchhiking on neutrophils was more efficient in the stationary state than that in the flowing condition. Compared with the EM@PPC group, the neutrophil hitchhiking ability of the ANG-2 EM@PPC group was enhanced (uptake efficiency 24.9% vs. 31.1%). Fluorescence microscopy showed that ANG-2 EM@PPC changed the death pathway of neutrophils from neutrophil extracellular traps-osis (NETosis) to apoptosis. Western blot confirmed the production of neutrophil apoptotic bodies, and flow cytometry showed that the production rate was as high as 77.7%. Animal experiments showed that there was no significant difference in the distribution of engineered bacterial membrane biomimetic nanoparticles in the organs (heart, liver, spleen, lungs, and kidney) in the DiR group, the EM@PPC gropu, and the ANG-2 EM@PPC group (>0.05), but there was higher distribution in the brain tissue in EM@PPC and ANG-2 EM@PPC groups compared to the DiR group (<0.05). Engineered bacterial membrane biomimetic nanoparticles crossed the blood-brain barrier (BBB), and exhibited high affinity to and internalization by neutrophils located in brain tumors. Compared with PBS, PDA, PC, and PPC groups, the survival rate and body mass of mice in the EM@PPC group were improved, tumor fluorescence intensity was weakened, and apoptotic cells were increased. These trends were even more prominent in the ANG-2 EM@PPC group. No abnormality was found in the HE staining of any group.
An ANG-2 EM@PPC nanodelivery system with inflammation response characteristics was successfully prepared, capable of crossing BBB and targeting the tumor inflammatory microenvironment to improve the anti-glioma efficacy. This study provides a new drug delivery strategy for glioma treatment and offers a new idea for targeted drug delivery in the non-invasive inflammatory microenvironments in other central nervous system diseases.
构建工程化细菌膜仿生纳米粒Angiopep-2膜（ANG-2 EM）@PDA-PEI-CpG（ANG-2 EM@PPC），用于高效靶向递送药物治疗胶质瘤，为胶质瘤靶向治疗提供理论和技术支持。
在实验室构建inaX-N-angiopep-2工程菌表达，经溶菌酶处理和超滤离心获得ANG-2 EM。通过对细菌膜进行超声处理制备ANG-2 EM@PPC。采用蛋白质免疫印迹法、琼脂糖凝胶电泳和透射电子显微镜（TEM）对制备物进行验证。测量粒径和Zeta电位以研究ANG-2 EM@PPC的稳定性。细胞实验方面，采用CCK-8法检测ANG-2 EM@PPC对中性粒细胞存活率的影响。设计并构建流动腔模型，通过流式细胞术测量中性粒细胞的摄取效率，以研究ANG-2 EM@PPC在炎症环境中对中性粒细胞的搭车效率。通过荧光显微镜表征中性粒细胞死亡模式，并进行流式细胞术和蛋白质免疫印迹法检测中性粒细胞凋亡小体及产生的凋亡小体比例。动物实验方面，建立小鼠胶质瘤模型并验证肿瘤组织的炎症环境。将肿瘤模型小鼠分为三组，包括DiR组、EM@PPC组和ANG-2 EM@PPC组（每组均为3只），分别经尾静脉注射DiR、ANG-2 EM@PDA-PEI-CpG和EM@PDA-PEI-CpG（均为10 mg/kg）。利用器官和脑的荧光图像检查三种制剂在体内及脑中的分布。将肿瘤模型小鼠进一步分为PBS组、PDA组、PC组、PPC组、EM@PPC组和ANG-2 EM@PPC组（每组均为4只），分别经尾静脉注射PBS、PDA、PC、PPC、EM@PPC和ANG-2 EM@PPC（均为10 mg/kg）。进行成像观察肿瘤消退情况，并测量小鼠的存活率和体重以评估药效学。进行TUNEL染色（脑组织）和HE染色（脑、心、肝、脾、肺和肾组织）以评估治疗效果。
TEM结果显示成功制备了工程化细菌膜仿生纳米粒，PPC呈现明显的核壳结构，壳厚度约为8.2 nm。由于ANG-2 EM的包裹，ANG-2 EM@PPC的壳厚度增加至约9.6 nm，表面有清晰的细菌膜层。稳定性至少维持一周。CCK-8法检测结果显示ANG-2 EM@PPC对中性粒细胞活性无显著影响。流式细胞术显示，ANG-2 EM@PPC在活化中性粒细胞中的摄取增强，且在静止状态下对中性粒细胞的搭车效率高于流动状态。与EM@PPC组相比，ANG-2 EM@PPC组中性粒细胞的搭车能力增强（摄取效率分别为24.9%和31.1%）。荧光显微镜显示ANG-2 EM@PPC使中性粒细胞的死亡途径从中性粒细胞胞外诱捕网形成（NETosis）转变为凋亡。蛋白质免疫印迹法证实了中性粒细胞凋亡小体的产生，流式细胞术显示产生率高达77.7%。动物实验表明，DiR组、EM@PPC组和ANG-2 EM@PPC组工程化细菌膜仿生纳米粒在器官（心、肝、脾、肺和肾）中的分布无显著差异（>0.05），但EM@PPC组和ANG-2 EM@PPC组在脑组织中的分布高于DiR组（<0.05）。工程化细菌膜仿生纳米粒可穿过血脑屏障（BBB），并对位于脑肿瘤中的中性粒细胞具有高亲和力和内化作用。与PBS、PDA、PC和PPC组相比，EM@PPC组小鼠的存活率和体重提高，肿瘤荧光强度减弱，凋亡细胞增加。这些趋势在ANG-2 EM@PPC组中更为明显。各组HE染色均未发现异常。
成功制备了具有炎症反应特性的ANG-2 EM@PPC纳米递送系统，能够穿过BBB并靶向肿瘤炎症微环境，提高抗胶质瘤疗效。本研究为胶质瘤治疗提供了一种新的药物递送策略，也为其他中枢神经系统疾病非侵入性炎症微环境中的靶向药物递送提供了新思路。
