Amphiphilic Chitosan-PEI Hybrid Nanocarrier Enhances Delivery Efficiency and Immunogenicity of PEDV mRNA Vaccines

mRNA vaccines require efficient and safe delivery systems to achieve robust antigen expression and immune activation. Chitosan is widely recognized for its biocompatibility and adjuvant properties; however, its application in mRNA delivery remains underexplored. This study aimed to develop a chitosan-derived nanoparticle system capable of enhancing mRNA transfection efficiency and vaccine immunogenicity.

A novel chitosan derivative, PAN2H, was synthesized by grafting palmitic acid (PA) onto N-2-hydroxypropyl trimethyl ammonium chloride chitosan (N-2-HACC). mRNA-loaded nanoparticles (PEI/mRNA/PAN2H) were constructed by first complexing mRNA with polyethyleneimine (PEI) and subsequently electrostatically binding the complex to PAN2H. Transfection efficiency was evaluated using GFP- and Luc-mRNA. Porcine epidemic diarrhea virus (PEDV) S1- and RBD-mRNA were selected as vaccine antigens for in vivo immunization to assess cellular and humoral immune responses.

Compared with PEI-mediated transfection, PEI/mRNA/PAN2H nanoparticles achieved approximately twofold higher expression of GFP- and Luc-mRNA in vitro, indicating that PAN2H incorporation markedly enhances mRNA transfection. In vivo, PEI/mRNA/PAN2H vaccination elicited significantly stronger antibody responses and cellular immune activation than the PEI control, supporting improved antigen expression and immune stimulation. Importantly, the PAN2H-PEI hybrid strategy provides a practical way to enhance PEI-based delivery by combining electrostatic condensation (PEI) with amphiphilicity-driven assembly and chitosan-associated biocompatibility/adjuvanticity (PAN2H).

The cationic PEI/mRNA/PAN2H nanoparticles exhibit low cytotoxicity and markedly improved mRNA delivery capabilities. These findings highlight PAN2H as a promising chitosan-derived platform for developing next-generation mRNA vaccines.