载美罗培南细菌外膜囊泡的制备及其对抗耐药细菌的作用研究  

Preparation of meropenem-loaded bacterial outer membrane vesicles and their anti-drug-resistant bacteria property

作　　者：邱晓涵 徐文杰 胡雄彬[2] 吴军勇[2] 向大雄[2] QIU Xiao-han;XU Wen-jie;HU Xiong-bin;WU Jun-yong;XIANG Da-xiong(The FirstAffiliated Hospital of Hunan Medical Institute,Huaihua Hunan 418000;The Second XiangyaHospital of Central South University,Changsha 410000)

机构地区：[1]湖南医药学院第一附属医院,湖南怀化418000 [2]中南大学湘雅二医院,长沙410000

出　　处：《中南药学》2025年第2期356-361,共6页Central South Pharmacy

基　　金：湖南省教育厅科学研究项目(No.22C1185);湖南省卫健委科研项目(No.W20243128)。

摘　　要：目的以大肠埃希菌来源细菌外膜囊泡(OMVs)为载体,包载美罗培南(MEM),制备载药细菌外膜囊泡(MEM@OMVs)并用于抗耐药细菌作用研究。方法培养大肠埃希菌DH5α,采用超滤和超速离心相结合的方法分离纯化OMVs,利用动态光散射技术(DLS)、纳米粒子跟踪分析(NTA)、透射电镜(TEM)和十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)对OMVs进行粒径分布、电势、粒子数、形态和标志蛋白鉴定。采用孵育法制备MEM@OMVs,并对其进行粒径表征和载药量测定。采用微量肉汤稀释法、结晶紫染色半定量法以及荧光分析法对MEM@OMVs体外抗耐碳青霉烯类的鲍曼不动杆菌(CRAB)、耐碳青霉烯类的铜绿假单胞菌(CRPA)的作用进行研究。结果成功提取分离OMVs,粒径峰值分布在92 nm左右,TEM下呈球形囊泡结构,OMVs标志蛋白OmpA、OmpF与OmpC均有表达。孵育法制备的MEM@OMVs载药量可达2 mg/10^(12)个粒子,且对粒径分布的影响不大。MEM@OMVs在体外能够抑制CRAB、CRPA的生长,且与浓度成正相关,浓度越高,抑制作用越强。MEM@OMVs能够抑制CRAB、CRPA生物膜的形成,清除成熟的生物膜。当MEM@OMVs质量浓度大于16μg·mL^(-1),CRAB的相对生物膜含量在40%以下;CRPA的相对生物膜含量在15%以下。结论制备的MEM@OMVs提高了CRAB、CAPA对MEM的敏感性,抑制了CRAB、CRPA的生长及生物膜的形成,清除成熟生物膜,为抗耐药细菌的研究提供了新的策略。Objective To prepare meropenem(MEM)-loaded Escherichia coli DH5αouter membrane vesicles(MEM@OMVs)and determine the anti-drug-resistant bacterial activity.Methods Escherichia coli DH5αwas cultured and OMVs were extracted and purified by ultrafiltration and ultracentrifugation.The particle size,zeta potential,particle amount,morphology and biomarker proteins of OMVs were characterized and determined by dynamic light scattering(DLS),nanoparticle tracking analysis(NTA),transmission electron microscopy(TEM)and sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).MEM@OMVs were prepared by co-incubating MEM with OMVs.The size distribution was determined and drug loading efficiency was evaluated.Then antibacterial effects of MEM@OMVs against carbapenem-resistant Acinetobacter baumannii(CRAB)and carbapenem-resistant Pseudomonas aeruginosa(CRPA)were assessed by broth microdilution method,crystal violet staining,and fluorescence analysis.Results The successfully extracted OMVs displayed a peak size distribution around 92 nm and a spherical structure under TEM,with the expression of biomarkers including OmpA,OmpF,and OmpC.MEM@OMVs prepared by co-incubation exhibited a drug loading capacity of 2 mg per 10^(12) particles with minimal change on the particle size.MEM@OMVs showed dose-dependent inhibition of CRAB and CRPA growth in vitro.The inhibitory effect was positively correlated with the concentration of MEM,meaning higher concentration of MEM@OMVs leading to better antibacterial effect.MEM@OMVs also prevented biofilm formation and eradicated mature biofilms in both strains.For CRAB,MEM@OMVs above 16μg·mL^(-1)reduced the relative content of biofilm to less than 40%.For CRPA,when the concentration of MEM@OMVs exceeded 16μg·mL^(-1),the relative content of biofilm was below 15%.Conclusion MEM@OMVs are successfully prepared and can enhance CRAB and CRPA sensitivity to MEM,inhibiting the bacteria growth and biofilm formation in vitro,thereby providing a promising strategy for combating drug-resistant bacteri

关 键 词：细菌外膜囊泡 美罗培南 抗感染 药物递送 

分 类 号：R94[医药卫生—药剂学] R96[医药卫生—药学]