甘露糖修饰的壳聚糖聚乳酸-羟基乙酸共聚物纳米微球作为口蹄疫病毒核酸疫苗递送载体的特性评价

doi:10.11843/j.issn.0366-6964.2021.012.022

摘要/Abstract

摘要： 旨在对制备的甘露糖修饰的壳聚糖聚乳酸-羟基乙酸共聚物[poly （D，L-lactide-co-glycolide），PLGA]纳米微球作为口蹄疫病毒（foot-and-mouth disease virus，FMDV）核酸疫苗递送载体进行评价。采用西佛碱反应和元素分析制备具有一定取代度的甘露糖修饰的壳聚糖衍生物（mannose modified chitosan，MCS），然后，经双重乳化挥发法制备得到甘露糖修饰的壳聚糖PLGA纳米微球（MCS-PLGA-NPs）。采用纳米粒径仪检测MCS-PLGA-NPs粒径分布和表面电势（zeta）、扫描电镜考察其形态、琼脂糖凝胶电泳观察其对质粒的吸附和吸附质粒后抵抗核酸酶降解能力、CCK-8法检测MCS-PLGA-NPs的细胞毒性、激光共聚焦观察巨噬细胞对MCS-PLGA-NPs-质粒DNA复合物的摄取、荧光显微镜和Western blot验证MCS-PLGA-NPs加载质粒DNA在细胞中的表达。元素分析结果表明，成功制备了取代度为5%~10%的MCS。纳米粒径测定和扫描电镜结果表明，MCS-PLGA-NPs的zeta为正值、粒径分布均匀且形态规则呈球形。琼脂糖凝胶电泳结果显示，MCS-PLGA-NPs吸附质粒的能力随着其质量的增加而增强并且可以在一定程度上抵抗核酸酶降解质粒DNA。在细胞毒性试验中，不同浓度的MCS-PLGA-NPs与RAW264.7细胞共孵育24 h后，细胞存活率仍在85%以上。在细胞摄取试验中，用激光共聚焦显微镜可以明显观察到质粒DNA结合到纳米微球表面被RAW264.7细胞摄取。荧光显微镜和Western blot试验证明MCS-PLGA-NPs加载质粒DNA可以在细胞中进行表达。综上表明，本研究成功制备了MCS以及具有递送核酸疫苗能力的MCS-PLGA-NPs，为FMDV核酸疫苗的递送研究提供了新的方向和见解，也为该递送载体携带特定抗原靶向抗原递呈细胞表面甘露糖受体以及应用于动物免疫的研究奠定基础。

关键词: 口蹄疫病毒, 甘露糖修饰的壳聚糖, 壳聚糖接枝聚乳酸-羟基乙酸共聚物, 纳米微球, 递送载体, 核酸疫苗

Abstract: This study aimed to evaluate the characteristics of mannosylated chitosan polylactic acid-glycolide copolymer[poly(D,L-lactide-co-glycolide), PLGA] nanospheres as a delivery system of FMDV nucleic acid vaccine. A certain degree of substitution of mannose modified chitosan (MCS) was prepared by schiff base reaction and elemental analysis. Then, Mannose modified chitosan PLGA nanospheres (MCS-PLGA-NPs) were prepared by double emulsification and volatilization. The particle size distribution and surface potential (zeta) of MCS-PLGA-NPS were measured by nanometer particle size analyzer; The morphology of MCS-PLGA-NPS was investigated by scanning electron microscopy; The ability of plasmids adsorbed on MCS-PLGA-NPs to resist nuclease degradation was observed by agarose gel electrophoresis; The cytotoxicity of MCS-PLGA-NPs was detected by CCK-8 method; The MCS-PLGA-NPs-DNA complex uptake by macrophages was observed by confocal laser scanning; The expression of MCS-PLGA-NPs loaded plasmid DNA in cells was verified by fluorescence microscope and Western blot. The results of elemental analysis showed that MCS with a substitution degree of 5% to 10% was successfully prepared; The results of nanometer particle size measurement and scanning electron microscopy showed that the zeta of MCS-PLGA-NPs was positive, the particle size distribution was uniform and the morphology was spherical; The results of agarose gel electrophoresis showed that the ability of MCS-PLGA-NPs to adsorb plasmids increased with the increase of their quality and could resist nuclease degradation of plasmid DNA to a certain extent; In the cytotoxicity test, the survival rate of RAW264.7 cells was still more than 85% after 24 h co-incubation with different concentrations of MCS-PLGA-NPs; In the cell uptake experiment, confocal laser microscopy could clearly observe the plasmid DNA binding to the surface of the nanomicrosphere and uptake by RAW264.7 cells; Fluorescence microscopy and Western blot experiments showed that MCS-PLGA-NPS-loaded plasmid DNA could be expressed in cells. In summary, MCS and MCS-PLGA-NPs with the ability to deliver DNA vaccine of FMDV were successfully prepared, which provided new directions and insights for the delivery of FMDV nucleic acid vaccine, and also laid a foundation for further research on the carrier carrying specific antigen targeting mannose receptor on APCs surface and its application in animal immunity.

Key words: FMDV, mannose modified chitosan, chitosan grafted PLGA, nanospheres, delivery system, DNA vaccine

中图分类号:

Q933
S855.3

李显, 张中旺, 张富东, 吕建亮, 李嘉豪, 潘丽. 甘露糖修饰的壳聚糖聚乳酸-羟基乙酸共聚物纳米微球作为口蹄疫病毒核酸疫苗递送载体的特性评价[J]. 畜牧兽医学报, 2021, 52(12): 3557-3568.

LI Xian, ZHANG Zhongwang, ZHANG Fudong, Lü Jianliang, LI Jiahao, PAN Li. Evaluation of the Characteristics of Mannosylated Chitosan PLGA Nanospheres as a Delivery System for DNA Vaccine of A/FMDV[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(12): 3557-3568.

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