基于多肽-泊洛沙胺纳米颗粒载体的呼吸道黏膜 mRNA疫苗研究

Development of mucosal vaccine via respiratory tract based on mRNA-peptide-poloxamine nanoparticles

目的设计并制备mRNA-多肽-泊洛沙胺纳米颗粒(mRNA peptidepolymerternary complex,mRNA-PPTC),考察其理化特性、体外和体内呼吸道组织相关细胞递送效率、免疫后小鼠特异性抗体诱导能力。方法通过体外转录分别制备编码萤火虫荧光素酶(fireflyluciferase,F-luc)、增强型绿色荧光蛋白(enhancedgreenfluorescentprotein,EGFP)和新冠病毒受体结合域蛋白(receptorbinding domain,RBD)的mRNA,利用动态光散射法检测mRNA-PPTC的粒径、多分散系数、Zeta电位。通过透射电镜观察纳米颗粒形态。将F-lucmRNA-PPTC及对照组制剂分别转染至人支气管上皮细胞(16 human bronchial epithelial cell,16HBE)、鼠源树突状细胞(dendritic cell2.4,DC2.4)、鼠源巨噬细胞(mouse leukemia cells of monocyte macrophage,RAW264.7),检测荧光素酶报告基因转染效率和细胞活性;将EGFPmRNA-PPTC及对照组制剂转染至16HBE细胞,使用荧光显微镜观察EGFP表达情况。利用活体成像技术考察F-lucmRNA-PPTC在小鼠呼吸道的转染效率。选用编码RBD蛋白的mRNA作为疫苗抗原模型,通过酶联免疫吸附实验检测免疫后小鼠血清中抗原特异性免疫球蛋白G(immunoglobulin G,IgG)和支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中抗原特异性分泌型免疫球蛋白A(secretory immunoglobulin A,sIgA)水平。结果成功制备粒径约100nm近球形且均一稳定的弱阳离子mRNA-PPTC纳米颗粒;mRNA-PPTC能有效转染16HBE、DC2.4、RAW264.7细胞,与对照组比较差异有统计学意义(P<0.01),并且细胞毒性较低;mRNA-PPTC通过滴鼻免疫后,在小鼠鼻部和肺组织中检测到F-luc报告基因的有效表达,与对照组比较差异有统计学意义(P<0.01);在初免后第28天的小鼠血清和BALF样品中,能分别检测到高效价的RBD抗原特异性IgG和sIgA,与对照组比较差异有统计学意义(P<0.01,P<0.05)。结论mRNA-PPTC纳米颗粒有良好的体外mRNA递送能力且细胞毒性较低;滴鼻给药后在小鼠上下呼吸道均能高效表达目的基因;小鼠滴鼻免疫后能产生高水平的抗原特异性血清IgG和支气管肺泡灌洗液sIgA。

Objective To design and prepare mRNA-peptide-poloxamine ternary complex(mRNA-PPTC)nanoparticles and explore their physicochemical properties,in vitro/vivo delivery efficiency,and induction of specific antibody production in immunized mice.Methods The mRNAs coding firefly luciferase(F-luc),enhanced green fluorescent protein(EGFP)and SARS-CoV-2 receptor binding domain(RBD)were prepared through in vitro transcription.The size,polydispersity(PDI)and Zeta potential of the mRNA-PPTC nanoparticles were studied with dynamic light scattering,and their morphology was observed with transmission electron microscopy(TEM).After F-luc reporter gene was loaded in the mRNA-PPTC nanoparticles,the obtained F-luc mRNA-PPTC nanoparticles were used to transfect human bronchial epithelial cell line 16HBE,mouse dendritic cell line DC2.4 and mouse macrophage cell line RAW264.7.The transfection efficiency and cell viability were observed with luminescence reporter assay.Fluorescence microscopy was employed to observe the expression of EGFP in EGFP mRNA-PPTC nanoparticles-transfected 16HBE cells.In vivo imaging system was adopted to investigate the delivery efficiency through the intranasal administration in mice.Then,RBD mRNA was used as antigen for vaccine preparation,the levels of specific IgG in the serum and sIgA in bronchoalveolar lavage fluid(BALF)were detected with ELISA after the mice were immunized with RBD mRNA-PPTC nanoparticles.Results Our mRNA-PPTC nanoparticles were successfully assembled,as weak cationic nanoparticles in sphere shape,in a diameter of about 100 nm,and with low PDI.The obtained mRNA-PPTC could effectively transfect 16HBE,DC2.4 and RAW264.7 cells(vs controls,P<0.01)with low cytotoxicity.The mRNA-PPTC nanoparticles effectively expressed the reporter gene F-luc in the nose and lung of mice immunized though an intranasal route(vs controls,P<0.01).High titers of antigen-specific IgG and sIgA were found in the serum and BALF from the mice in 28 d after intranasal immunization with RBD mRNA-PPTC(vs controls,P<0.01,P<0.05).Conclusion Our prepared mRNA-PPTC have good capacity to delivery mRNA in vitro without significant cytotoxicity and in vivo at respiratory mucosal site,and can induce high levels of specific serum IgG and BALF sIgA by intranasal inoculation.