Revealing a Correlation between Structure andin vitroactivity of mRNA Lipid Nanoparticles

ABSTRACT mRNA-loaded lipid nanoparticles (mRNA-LNPs) represent a promising platform for disease prevention, cancer immunotherapy, and gene editing, etc. Despite the success of mRNA-LNPs based vaccines, the relationship between their structure and efficacy remains elusive. Here, we generated a series of mRNA-LNPs with varied structural properties and in vitro cellular activities by altering their processing and storage conditions to investigate the structure-activity relationship (SAR). Our findings revealed a moderate anticorrelation between particle size distribution and in vitro activity. Importantly, the intensity of a characteristic peak, as detected by small angle X-ray scattering (SAXS), demonstrated a strong correlation with in vitro activity while the encapsulation efficiency is high. Additionally, the peak width and area were moderately correlated and anticorrelated with activity, respectively. These observations suggest that a more ordered internal structure is likely associated with enhanced in vitro activity of mRNA-LNPs. Further analysis using 31 P nuclear magnetic resonance indicated that lyophilization might induce phase separation of mRNA and lipids within the LNPs, leading to a diminished SAXS peak and reduced in vitro activity. 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.