Structural characterization of mRNA lipid nanoparticles (LNPs) in the presence of mRNA-free LNPs

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 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 mRNA-free LNPs, casting doubt on the validity of earlier structural models. In this study, we employed contrast variation SANS to characterize both mRNA-free LNPs and our mRNA-LNP sample, and quantified the proportion of mRNA-free LNPs present to be ∼30 % in our mRNA-LNP sample using nano flow cytometry. By removing the contributions of mRNA-free LNPs from the SANS data of our mRNA-LNP sample, we were able to precisely characterize the structure of mRNA-loaded LNPs. Consequently, we proposed structural models for both mRNA-free and mRNA-loaded LNPs. Notably, our analysis revealed similar lipid distributions and shell thicknesses between the two particle types, while the solvent content in mRNA-loaded LNPs was significantly higher, leading to a larger core size. This work not only offers a method for accurately characterizing the structure of mRNA-loaded 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.