Covalent conjugation of TLR9 agonists to phospholipids enhances mRNA-LNP delivery efficiency and dendritic cell cross-priming.

Current mRNA-LNP systems face challenges in efficient co-delivery of nucleic acid adjuvants, limiting their capacity to elicit robust cellular immunity. To overcome the dissociation limitations of physically admixed CpG in conventional lipid nanoparticles, we developed a covalent conjugation platform that integrates TLR9 agonists with phospholipids via amide bonding. We synthesized a DSPE-CpG conjugate, which exhibited high loading efficiency (97.1%) onto mRNA-LNPs. The immune activation efficacy was evaluated in models encoding SARS-CoV-2 spike protein and HBV antigens. The DSPE-CpG-mRNA LNP system demonstrated enhanced cellular uptake via clathrin-mediated endocytosis, superior lymph node targeting of the adjuvant, and improved antigen expression in vivo. In the SARS-CoV-2 model, it induced high levels of IFN-γ⁺CD8⁺ T cells and elicited higher titers of specific IgG and neutralizing antibodies. In the HBV model, it significantly enhanced antigen-specific CD107a+ and FasL+ CTLs. Acute toxicity tests revealed no significant adverse effects. This covalent conjugation strategy, by ensuring co-localized delivery of antigen and adjuvant, provides an innovative and translatable platform for enhancing mRNA vaccine efficacy.