Three-Component Zwitterionic LNPs Enhance Spleen-Targeted mRNA Delivery and Mitigate Inflammatory Responses

During the COVID-19 pandemic, mRNA–lipid nanoparticle (LNP) vaccines by Pfizer-BioNTech and Moderna proved highly effective. However, a small number of recipients developed acute inflammatory symptoms. The cause remains unclear, but ionizable lipids in LNPs are known to activate immune responses, especially when co-delivered with nucleic acids. Moreover, current LNP formulations tend to accumulate in the liver. Cholesterol promotes uptake by hepatocytes, and Poly (ethylene glycol) (PEG)-ylated lipids trigger anti-PEG immunity, limiting their efficacy in cancer vaccines and repeated dosing. To address these challenges, we developed a Three-Component (ThrCo) LNP by replacing cholesterol and PEGylated lipids in Pfizer-BioNTech LNPs with zwitterionic pyridine carboxybetaine (PyCB) ionizable lipids, achieving ~70% lower liver accumulation and a 4.5-fold increase in spleen-specific mRNA translation. PyCB improves hydrophilicity and stabilizes the LNP surface, compensating for cholesterol loss. It also enhances mRNA release by protonating in endosomes. The zwitterionic surface reduces protein adsorption and avoids the accelerated blood clearance (ABC) effect seen with PEGylated lipids. ThrCo LNPs combines the advantages of the safety of Pfizer-BioNTech LNPs and the efficiency of Moderna LNPs, exhibiting lower immunogenicity than Moderna LNPs, higher mRNA translation efficiency than Pfizer-BioNTech LNPs and better spleen targeting than either, offering a promising strategy for cancer vaccines requiring repeated dosing.