The Critical Role of Buffer Composition on the Performance of mRNA-Lipid Nanoparticles

The emergence of mRNA therapeutics has revolutionized our prevention and treatment strategies in infectious diseases, cancers, and inherited diseases. Lipid nanoparticles (LNPs) play a pivotal role in realizing the biomedical applications of RNA-based therapies, demonstrated recently with the safe and efficacious messenger RNA (mRNA) vaccines against COVID-19. Despite this success, mRNA-LNPs are inefficient, with only a small percent (1-10%) that properly escape the endosomes and reach the cytoplasm. Recent studies show that internal crystal phases of LNPs have more favorable curvature for fusion and escape from the endosome, but there is still limited understanding of this relationship. In this study, we quantified the impact of buffer conditions on mRNA-LNP morphology, physiological behavior, and success of mRNA delivery. We found a significant impact of buffer type and molarity on the size, morphology, internal structure, and long-term stability of mRNA-LNPs. Small-angle X-ray scattering (SAXS) and cryo- transmission electron microscopy (cryo-TEM) analyses revealed the coexistence of two distinct ordered phases along with aqueous pockets (“bleb” regions) under some conditions. The presence and extent of these phases was largely dependent on buffer and slightly influenced by salt and cryo protectants. Upon acidification to endosomal conditions all samples exhibited a converging trend toward an ordered structure, driven by ionizable lipid protonation and electrostatic repulsion. Furthermore, stability studies identified pH and buffer molarity as critical factors in preserving the structural integrity of mRNA-LNPs. We find the combination of key morphological features and particle stability, driven by buffer conditions, are correlated to the functional performance of mRNA delivery in vitro. Buffers leading to limited stability and a reduced capacity to modulate mRNA-LNP structure in response to environmental changes show more limited transfection efficiency. Developing such an understanding of the impact of buffer on mRNA LNP structure, stability, and performance will be key in overcoming the critical bottleneck of inefficient delivery.