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. In this study, we synthesized a DSPE-CpG covalent complex via amide bond conjugation and fabricated a DSPE-CpG-mRNA LNP nano-delivery system using microfluidic technology. The immune activation efficacy of this complex was comprehensively evaluated through a multi-dimensional analytical platform, including cryo-electron microscopy, agarose gel electrophoresis, flow cytometry, fluorescence microscopy, confocal laser scanning microscopy (CLSM), in vivo imaging, and intracellular cytokine staining (ICS). These assessments were performed in mRNA vaccine models encoding the SARS-CoV-2 spike (S) protein and hepatitis B virus (HBV) antigens. The system demonstrates three breakthrough advantages: DSPE-CpG-mRNA LNP can be internalized via the clathrin-mediated pathway, enhancing DC uptake efficiency by 4-fold; its lymph node targeting efficiency surpasses that of free CpG, effectively promoting DC recruitment; in the SARS-CoV-2 model, it induces high levels of IFN-γ+CD8+ T cells, while in the HBV model, it significantly enhances antigen-specific CD107a+ and FasL+ CTLs. Acute toxicity tests reveal no statistically significant difference in ALT/AST levels compared to the saline control group (p >0.05). This study successfully constructed a DSPE-CpG composite system with amphiphilic properties and optimized its performance through three key strategies: covalent conjugation to ensure the linkage between CpG and DSPE, enhancing its hydrophobicity; utilization of the amphiphilic nature of the composite to effectively improve cell membrane penetration, thereby increasing delivery efficiency; and lymph-targeted delivery to promote innate immune pathway activation, further enhancing immune responses. The DSPE-CpG composite provides an innovative strategy for mRNA vaccine adjuvant design, with broad potential for clinical translation.