Impact of tail unsaturation in ionizable lipids on mRNA delivery efficiency and immunogenicity of lipid nanoparticles.

Ionizable lipids are critical for determining the potency of mRNA lipid nanoparticles (LNPs). Various mRNA therapies require LNPs that achieve efficient delivery while maintaining appropriate immunogenicity in vivo. While research has largely focused on screening the amine headgroups, linkers and hydrophobic tails of ionizable lipids, the role of tail unsaturation in influencing mRNA delivery and immunogenicity of LNPs has received less attention. Here, we utilized a four-component Ugi reaction (Ugi-4CR) system to synthesize a small library of 17 ionizable lipids with identical backbones but varying in degrees, positions, and types of unsaturation in hydrophobic tails. We systemically investigated structure-activity relationships and elucidated the influence of tail unsaturation on physicochemical properties, endosomal escape, mRNA delivery efficiency and immunogenicity of LNPs. The findings indicate that optimization of tail unsaturation could serve as a promising strategy to improve mRNA encapsulation and delivery efficacy while modulating LNP immunogenicity in vivo. Using interleukin-27 (IL-27)-encoded mRNA as a model, we demonstrated the potential of selected LNPs for protein supplementation therapies. These insights provide a foundation for the rational design of ionizable lipids, advancing the development of more efficient and safer mRNA delivery systems.