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

Abstract

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.

Keywords

• nanoparticles
• nucleic acid delivery
• mrna vaccine
• mucosal vaccine