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

Background: 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. Methods: 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. Results: 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). Conclusion: 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.

Background:

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.

Methods:

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.

Results:

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).

Data Summary:

PEI/mRNA/PAN2H nanoparticles achieved approximately twofold higher expression of GFP- and Luc-mRNA in vitro compared with PEI-mediated transfection. In vivo, vaccination with PEI/mRNA/PAN2H elicited significantly stronger antibody responses and cellular immune activation than the PEI control. The nanoparticles also exhibited low cytotoxicity.

Conclusions:

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.

Practical Significance:

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). This approach offers a promising chitosan-derived platform for developing next-generation mRNA vaccines.