Low reactogenicity and high tumour antigen expression from mRNA-LNPs with membrane-destabilizing zwitterionic lipids

Two key challenges in translating messenger RNA-based lipid nanoparticle (mRNA-LNP) cancer vaccines to clinical use are limited mRNA expression and unavoidable inflammatory responses. Here we develop a membrane-destabilizing zwitterionic ionizable lipid that enhances mRNA expression by promoting endosomal escape while reducing inflammatory reactogenicity. This lipid features a pyridine-based carboxybetaine (PyCB) zwitterionic headgroup, biodegradable multitailed alkyl chains and a tertiary amine linker. The PyCB headgroup forms a zwitterionic PyCB-water complex that protonates to a positively charged state below pH 6.8. This allows for good biocompatibility at physiological pH and strong protonation in endosomes, enabling earlier and more efficient mRNA release when synergistic with the tertiary amine and the tail moiety. Incorporating membrane-destabilizing zwitterionic lipids into the LNP formulation used in a commercially available mRNA vaccine significantly boosts mRNA expression in antigen-presenting cells within lymph nodes, enhancing cytotoxic T cell activation. In addition, these membrane-destabilizing zwitterionic lipid-containing nanoparticles show reduced inflammation and neutrophil infiltration at the injection site due to their zwitterionic property. These lipids are also compatible with existing targeted nanoparticle formulations, further improving mRNA delivery.