Lipid nanoparticles (LNPs) containing mRNA can deliver genetic material to cells for use as vaccines or protein replacement therapies. We characterized the effect of solution pH on cationic LNPs containing green fluorescent protein (EGFP) mRNA and their transfection efficiency. We compared the structural and colloidal properties of mRNA LNPs with LNPs not containing mRNA and mRNA free in solution. We used a combination of biophysical technique to build a picture of the structure of the lipids and mRNA across pH and temperature in the form of an empirical phase diagram (EPD). A combination of Fourier-transform infrared (FTIR) spectroscopy and differential scanning calorimetry was used to investigate lipid phase behavior. The mRNA-LNPs transition from an inverse hexagonal phase at pH values below the pKof the cationic lipid to a lamellar phase above the pK. At higher temperatures the mRNA-LNPs also transitioned from an inverse hexagonal phase to a lamellar phase indicating the inverse hexagonal phase is more thermodynamically favorable. Based on circular dichroism, the mRNA within the LNP has more A form structure at pH values below the lipid pKthan above it. Optical density, zeta potential and dynamic light scattering measurements were used to probe the colloidal stability of the mRNA-LNPs. The particles were larger and more prone to aggregation below the pK. A stability study was performed to relate the biophysical characteristics to the storage of the particles in solution at 4 and 25 °C. mRNA-LNPs had the highest transfection efficiency and stability at pH values below the pK. However, there was a trade-off between the stability and aggregation propensity since at very low pH the particles were most prone to aggregation. We performed kinetic experiments to show that the time scale of the pH-dependent phase behavior is slow (6 hour transition) and the transition from lamellar to inverse hexagonal phases is irreversible. This suggests that the lamellar phase is less stable and kinetically trapped. Our findings deepen our structural understanding of mRNA-LNPs and will aid the development of related formulations. a a a a a
