Revealing a Correlation between Structure andin vitroactivity of mRNA Lipid Nanoparticles

Abstract

AbstractMessenger RNA-containing lipid nanoparticles (mRNA-LNPs) represent a promising platform for disease prevention, cancer immunotherapy, and gene editing, etc. Despite the clinical success of mRNA-LNPs based vaccines in preventing infectious diseases, the relationship between their structure and efficacy remains poorly understood, primarily due to their complex chemical composition. In this study, we generated a series of mRNA-LNPs with varied structural properties andin vitrocellular activities by altering their processing and storage conditions to investigate the structure-activity relationship (SAR) of these nanoparticles. Our findings revealed a moderate anticorrelation between particle size distribution andin vitroactivity, while nanoparticle size and morphology exhibited only weak correlations with efficacy. Notably, the intensity of a characteristic peak, as detected by small angle X-ray scattering (SAXS), demonstrated a strong correlation within vitroactivity. Additionally, the peak width and area were moderately correlated and anticorrelated with activity, respectively. These observations suggest that a more ordered internal structure, as identified by SAXS, is likely associated with enhancedin vitroactivity of mRNA-LNPs. Further analysis using31P nuclear magnetic resonance indicated that lyophilization may induce phase separation of mRNA and lipids within the LNPs, leading to a diminished SAXS peak and reducedin vitroactivity. Overall, our study establishes an SAR for mRNA-LNPs, highlighting that a more ordered internal structure correlates with higher efficacy, which could be instrumental in high-throughput screening of LNP libraries for mRNA loading and monitoring efficacy during storage.SignificanceIn addition to the two mRNA-based vaccines developed for COVID-19, the FDA approved a third mRNA-based vaccine for respiratory syncytial virus in 2024. These vaccines rely on mRNA-containing lipid nanoparticles (mRNA-LNPs), which are also being explored for cancer treatment, gene editing, and antibody production. Despite their promise, the structure-activity relationship of mRNA-LNPs remains inadequately understood, limiting the optimization of lipid formulations, long-term storage, and mechanistic insights. Our study suggests that an ordered internal structure correlates with enhancedin vitroefficacy, consistent with previous findings. We identified the intensity of a peak at ∼0.13 Å-1in small-angle X-ray scattering (SAXS) as a strong indicator of the efficacy. Furthermore, we demonstrated that this correlation aroused from phase separation between mRNA and lipids inside LNPs in the lyophilization process. These insights offer a straightforward method to predictin vitroactivity of mRNA-LNPs with certain formulation and shed light on the mechanisms underlying the loss of mRNA-LNPs’ activity during lyophilization.Table of Content