Structural Characterization of mRNA Lipid Nanoparticles in the Presence of Intrinsic Drug-free Lipid Nanoparticles

Abstract

Lipid nanoparticles (LNPs) have emerged as a versatile platform for mRNA delivery across a range of applications, including disease prevention, cancer immunotherapy, and gene editing. Structural models of mRNA-containing lipid nanoparticles (mRNA-LNPs) have also been proposed based on characterization of samples by using various advanced techniques. Among these, small angle neutron scattering (SANS) has proven essential for elucidating the lipid distribution within mRNA-LNPs, a factor crucial to both their preparation and efficacy. However, recent findings suggest that the mRNA-LNP samples prepared via commercial microfluidic techniques may contain a substantial fraction of drug-free LNPs, casting doubt on the validity of earlier structural models. In this study, we employed contrast variation SANS to characterize both drug-free LNPs and our mRNA-LNP sample, and quantified the proportion of drug-free LNPs present to be [~]30% in our mRNA-LNP sample using nano flow cytometry. By removing the contributions of drug-free LNPs from the SANS data of our mRNA-LNP sample, we were able to precisely characterize the structure of mRNA-LNPs. Consequently, we proposed structural models for both drug-free LNPs and mRNA-LNPs. Notably, our analysis revealed similar lipid distributions and shell thicknesses between the two particle types, while the solvent content in mRNA-LNPs was significantly higher, leading to a larger core size. This work not only offers a method for accurately characterizing the structure of mRNA-LNPs, but also establishes criteria for selecting appropriate analytical techniques based on the structural parameters of interest. Therefore, our findings hold significant implications for the mechanistic understanding and quality control of mRNA-based vaccines. SignificancePrecise structural determination of mRNA-containing lipid nanoparticles (mRNA-LNPs) is vital for mechanistic insights into their preparation, delivery, immunogenicity, and storage, which are critical to the development of mRNA-based vaccines. However, most previous studies overlooked the substantial presence of drug-free LNPs within these samples. Here, we identified that approximately 30% of the nanoparticles in our mRNA-LNP sample were drug-free. By integrating contrast variation small angle neutron scattering (SANS) data from both drug-free LNPs and mRNA-LNPs, we developed structural models for both particle types, and provided a guidance for characterization technique selection based on concerned structural features. Beyond mechanistic insight on structure, our approach offers a robust method for quality assessment and process monitoring in mRNA-based vaccine production.