Lipid nanoparticles (LNPs) are critical drug delivery vehicles that enable functional mRNA delivery into cells to produce desired proteins, enabling vaccine and therapeutic applications. The structure and formulation of mRNA-containing LNPs (mRNA-LNPs) critically impact the delivery and protein production. Here, we generated LNPs with various structural lipids to improve the nanoparticle drug product stability and delivery profiles. We incorporated glyceryl monooleate and phytantriol as alternatives to traditional phospholipids (e.g., distearoylphosphatidylcholine). The physicochemical attributes of mRNA-LNPs were assessed by the hydrodynamic particle size, mRNA encapsulation, and mRNA integrity. mRNA-LNP morphology was assessed by synchrotron small-angle X-ray scattering, pulsed gradient stimulated echo proton nuclear magnetic resonance, and cryogenic-transmission electron microscopy.activity, including transfection and cytokine stimulation were evaluated in HeLa cells and whole human blood, respectively, for select mRNA-LNPs with varied morphologies. Alternative structural lipids in mRNA-LNPs produced different mesophases depending on the lipid type and molar ratio. Specific lipid combinations yielded distinct quality attributes and stability profiles, with uniqueactivities. Hexagonal and cubic phase structures enhanced mRNA-LNP stability at refrigerated and room temperatures for over six months and three months, respectively. These novel mRNA-LNPs provided promising benefits by reducing immunostimulatory cytokines, warranting further exploration. Our efforts demonstrate the nuances of LNP composition and its effects on structure, stability, and activity. In vitro in vitro
