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J Zhejiang Univ (Med Sci)  2017, Vol. 46 Issue (2): 118-126    DOI: 10.3785/j.issn.1008-9292.2017.04.02
    
Bacterial outer membrane vesicles as nano carriers to study immunological activities
CHEN Qi(),WU Min,Bai Hongzhen,Guo Zeling,ZHOU Jun,WANG Qingqing,TANG Guping*()
Department of Chemistry, Zhejiang University, Hangzhou 310028, China
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Abstract  

Objective: To prepare a nano-carrier based on combining bacterial outer membrane vesicles (OMV) with three block polymer pluronic F127 (PEO100-PPO65-PEO100) (OMV-F127) and to investigate its immunological activity. Methods: Attenuated salmonella (sal) was cultivated. OMV were separated by centrifugal ultrafiltration or ultrasonication, and OMV-F127 was prepared by mechanical extrudation method. The protein contents and compositions were tested with BCA and SDS-PAGE; the morphology of OMV, F127 and OMV-F127 were observed with FM and TEM; the particle sizes and their zeta potential were determined with DLS. Mouse macrophage RAW246.7 cells were treated with OMV-F127 (50 μg/mL, 100 μg/mL) in vitro, and the concentrations of IL-12, TNF-α and IFN-γ in culture supernatant were measured with ELISA kits. Results: The contents of protein in separated OMV by centrifugal ultrafiltration and ultrasonication were 2.8 mg/mL and 2.7 mg/mL, respectively. SDS-PAGE showed the marker protein OmpF/C in OMV. Under the FM and TEM, ball-like structure of F127 and OMV-F127 was observed. Size analysis revealed that the diameters of OMV, F127 and OMV-F127 were 72±2 nm, 90±3 nm and 92±2 nm, respectively. ELISA tests revealed that OMV-F127 significantly stimulated the secretion of IL-12, TNF-α and IFN-γ in RAW246.7 cells. Conclusion: A nano-carrier based on bacterial outer membrane vesicles has been prepared, which can stimulate the secretion of cytokines and may have immunomodulatory effects.



Key wordsSalmonella vaccines      Adjuvants, immunologic      Bacterial outer membrane proteins      Nanostructures      Polymers/pharmacology      Macrophages      Cytokines      Cells, cultured     
Received: 02 November 2016      Published: 31 October 2017
Corresponding Authors: TANG Guping     E-mail: cq-chem@zju.edu.cn;tangguping@zju.edu.cn
Cite this article:

CHEN Qi,WU Min,Bai Hongzhen,Guo Zeling,ZHOU Jun,WANG Qingqing,TANG Guping. Bacterial outer membrane vesicles as nano carriers to study immunological activities. J Zhejiang Univ (Med Sci), 2017, 46(2): 118-126.

URL:

http://www.zjujournals.com/xueshu/med/10.3785/j.issn.1008-9292.2017.04.02     OR     http://www.zjujournals.com/xueshu/med/Y2017/V46/I2/118


细菌外膜囊泡纳米载体的制备及其免疫调节作用

目的:以细菌外膜囊泡(OMV)结合三嵌段聚合物普朗尼克F127(PEO100-PPO65-PEO100),构建能够有效促进巨噬细胞分泌抗肿瘤细胞因子的纳米载药体系OMV-F127。方法:培养减毒沙门菌,用超滤离心法与超声破碎法提取OMV,机械挤压法制得OMV-F127;蛋白质定量试剂盒和SDS-PAGE法检测OMV的蛋白含量和成分;荧光显微镜、透射电镜、动态光学散射法检测OMV-F127形态学特征、粒径、电位及其稳定性;以小鼠巨噬细胞RAW246.7为细胞模型,ELISA检测OMV-F127对抗肿瘤细胞因子的分泌作用。结果:两种方法成功提取到减毒沙门菌的OMV,其蛋白质总量分别为2.8 mg/mL和2.7 mg/mL。成功制备OMV-F127且含有OMV的标志蛋白OmpF/C。OMV为纳米级囊泡结构,F127以及OMV-F127为球形纳米颗粒。OMV、F127、OMV-F127颗粒平均直径分别为(72±2)nm、(90±3)nm、(92±2)nm。OMV-F127能促进抗肿瘤细胞因子IF-12、α-TNF、γ干扰素的分泌。结论:基于细菌OMV的纳米载体OMV-F127能够有效促进巨噬细胞分泌抗肿瘤细胞因子,具有免疫调节作用。


关键词: 沙门氏菌菌苗,  佐剂,免疫,  细菌外膜蛋白质类,  纳米结构,  聚合物/药理学,  巨噬细胞,  细胞因子类,  细胞,培养的 
Fig 1 Establish a nano carrier based on bacterial outer membrane vesicles and uptake of OMV -F127 in mice macrophage
Fig 2 The growth curve of attenuated salmonella during 12 h
Fig 3 Protein standard curve
Fig 4 Result of SDS-PAGE
Fig 5 The fluorescence microscope image of OMV
Fig 6 TEM images of OMV-F127
($\bar x \pm s$)
种类 粒径(nm) 电位(mV)
 OMV 72.3±1.8 -40.9±0.9
 F127 89.2±3.1 -5.3±0.7
 OMV-F127 92.0±2.1 -16.8±3.2
Tab 1 Particle size and zeta potential of OMV、F127、OMV-F127
Fig 7 The compare in stability of OMV and OMV-F127
Fig 8 OMV-F127's effect of motivating the secrete of γ-IFN, α-TNF, IL-12 in mice macrophage cells RAW246.7
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