阳离子PLGA纳米颗粒作为核酸疫苗递送载体的能力及其特性评价

目的对阳离子聚乳酸-羟基乙酸共聚物poly(D,L-lactide-co-glycolide),PLGA]纳米颗粒作为核酸疫苗载体的能力及其特性进行评价。方法采用双重乳化法制备阳离子PLGA纳米颗粒,检测其粒径分布和表面电势,获得合成的最适方案。通过考察其形貌、吸附效率、抵抗核酸酶降解能力、细胞毒性、细胞摄取及转染真核表达质粒的能力,分析其作为核酸疫苗载体的适用性及可行性。结果由两种试剂修饰PLGA制备的纳米颗粒粒径分布、表面电势及分散性最佳;透射电镜及扫描电镜下观察粒径均一且呈规则球形;对质粒DNA的吸附效率可达65%;能够有效保护吸附于纳米颗粒的质粒DNA免受核酸酶降解;两种细胞系中的细胞毒性试验均显示细胞存活率高于80%;共聚焦显微镜下可观察到其能被细胞内化;间接免疫荧光试验结果表明,其能有效转染真核表达质粒并使其正确表达。结论成功制备了具有递送核酸疫苗能力的阳离子PLGA纳米颗粒,为核酸疫苗载体的发展奠定了理论基础。

Ability and characteristics of cationic PLGA nanoparticles as a delivery system of DNA vaccine

Objective To evaluate the ability and characteristics of cationic poly(D,L-lactide-co-glycolide)(PLGA)nanoparticles as a delivery system of DNA vaccine. Methods Cationic PLGA nanoparticles were prepared by a modified double emulsion procedure,of which the size and the zeta potential were characterized to achieve the optimal synthesis.The morphology,adsorption efficiency,resistance to DNaseⅠdegradation,cytotoxicity and the ability of cell uptake and transfecting eukaryotic expression plasmids were investigated to evaluate the applicability and feasibility as a vector for DNA vaccine delivery. Results PLGA nanoparticles modified by two reagents were optimal in size distribution,zeta potential and dispersivity,of which the size was uniform and in regularly spherical shape under scanning and transmission electron microscopes. The adsorption efficiency of DNA reached 65%,and the DNA adsorbed was protected from the degradation by DNaseⅠ. Cytotoxicity test showed that the survival rates of BHK-21 and PK-15 cells were more than 80%.Confocal microscopy demonstrated that cationic PLGA nanoparticles were internalized by BHK-21 cells. Indirect immunofluorescence assay(IFA) eukaryotic expression plasmid was effectively transfected,and the recombinant protein was expressed correctly. Conclusion Cationic PLGA nanoparticles with ability of DNA vaccine delivery were prepared successfully,which provided a theoretical basis for the development of delivery system of DNA vaccine.