氟化超支化聚酰胺-胺作为流感DNA疫苗递送载体的研究  

Fluorinated hyperbranched poly(amido amine)s as carriers of influenza DNA vaccine

作　　者：范雪莲 徐月桦 陈刚 FAN Xue-lian;XU Yue-hua;CHEN Gang(Institute of Comparative Medicine,College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学兽医学院(比较医学研究院),江苏扬州225009 [2]扬州大学,江苏高校动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《药学学报》2020年第6期1282-1287,共6页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(90612002);江苏高校优势学科建设工程资助项目(PAPD)。

摘　　要：本文设计合成了氟化超支化聚酰胺-胺并研究其作为流感DNA疫苗递送载体的能力。首先采用迈克尔加成反应合成超支化聚酰胺-胺聚合物[hyperbranched poly(amido amine)s,HP],并进行氟化修饰(fluorinated HP,F-HP)通过设计特异性引物扩增目的基因,构建流感病毒PR8的核蛋白(nucleoprotein,NP)基因真核表达质粒,作为DNA疫苗,F-HP与NP质粒通过静电作用形成纳米复合物(F-HP/NP)。结果表明:所合成的HP的分子质量为59.7 kDa多分散系数(polydispersity index,PDI)为2.67,F-HP中氟元素的含量为20%(质量比,w/w),聚合物能与NP质粒络合形成球形的纳米粒。聚合物与DNA质量比为5~10时,纳米复合物的粒径分布在100 nm左右,并随质量比的升高而减小。氟化修饰有利于提高纳米复合物的细胞摄取及溶酶体逃逸能力,进一步提高NP的细胞内表达水平。体内免疫实验表明,与HP/NP纳米复合物相比,氟化修饰的F-HP/NP纳米复合物对CD8^+T细胞具有更强的免疫应答诱导效应。以上结果说明氟化修饰的超支化聚酰胺-胺作为DNA疫苗载体具有可行性。动物实验经扬州大学大学实验动物伦理委员会批准。This study aims to synthesize fluorinated hyperbranched poly(amido amine)s for the delivery of influenza DNA vaccine.Hyperbranched poly(amido amine)s(HP)were synthesized by using Michael-type polyaddition and then fluorinated to obtain fluorinated polymers(F-HP).The target gene was amplified by designing specific primers to construct the eukaryotic expression plasmid of influenza viral PR8 nucleoprotein(NP)gene as DNA vaccines.Then the polyplexes(F-HP/NP)were prepared by the electrostatic interactions between polymers and plasmid.The results suggested that the molecular weight of HP was 59.7 kDa and polydispersity index(PDI)was 2.67.The fluorine content in F-HP was found to be 20%(w/w).Transmission electron microscopy(TEM)revealed that the polyplexes had spherical shapes with sizes around 100 nm(w/w 5-10)and decreased with increasing w/w ratios.F-HP polyplexes showed improved cell uptake,lysosomal escape and elevated expression levels of NP in vitro than polyplexes based on HP.Finally,the in-vivo immunization by F-HP/NP polyplexes triggered improved CD8+T cell responses.This study suggests that fluorinated hyperbranched poly(amido amine)s represent one of the effective carriers for DNA vaccine delivery.The animal experiments were approved by the Experimental Animal Ethics Committee of Yangzhou University.

关 键 词：氟化修饰 阳离子聚合物 流感 DNA疫苗 纳米复合物 

分 类 号：R943[医药卫生—药剂学]