Development of a messenger RNA vaccine using pH-responsive dipeptide-conjugated lipids exhibiting reduced inflammatory properties.

Lipid nanoparticles (LNPs) are used to encapsulate messenger ribonucleic acids (mRNAs) and enhance mRNA vaccine efficacy by producing inflammatory mediators. However, the overproduction of inflammatory mediators via LNP injection causes severe side effects, presenting a potential limitation. To resolve this issue, we developed pH-responsive dipeptide-conjugated lipid (DPL)-based LNPs (DPL-LNPs) for efficient small interfering RNA delivery with excellent biocompatibility. In detail, we optimized the dipeptide sequence and lipid-tail length of DPL, the helper-lipid compositions, and the molecular weight and lipid-tail length of the polyethylene glycol (PEG)-lipid to achieve highly efficient and safe mRNA delivery. Our results revealed that the LNPs prepared using glutamic acid (E)- and arginine (R)-conjugated DPL (DPL-ER) displayed higher protein-expression efficacy than DPL-threonine-R- and DPL-aspartic acid-R-based LNPs. Additionally, the lipid-tail length of the C22-bearing DPL-ER (DPL-ER-C22)-based LNPs displayed higher protein-expression efficacies than their C18 (DPL-ER-C18)- and C24 (DPL-ER-C24)-based LNPs. Moreover, the DPL-ER-C22-based LNPs incorporating low-lipid-tail-length phospholipids and PEG-lipids exhibited efficient protein expression. Most importantly, the injection of optimized DPL-LNPs exhibited comparable antigen-specific antibody production levels, with significantly lower inflammatory-mediator production compared with those of the commercially available LNPs. These results indicate that DPL-based LNPs (DPL-LNPs) can be deployed as highly efficient, safe carriers for mRNA delivery for developing mRNA vaccine formulations.