Single particle charge detection mass spectrometry enables molecular characterization of lipid nanoparticles and mRNA packaging

Lipid nanoparticles (LNPs) are effective delivery systems for RNA therapeutics, yet their intrinsic heterogeneity in size and composition make them challenging to characterize. Charge detection mass spectrometry (CDMS) was used to rapidly weigh thousands of individual LNPs. Diameter distributions of empty LNPs from CDMS and cryo-TEM measurements are in excellent agreement demonstrating that these particles are sufficiently stable in the high vacuum environment of the mass spectrometer for accurate mass analysis. A similarly prepared mRNA-packaged LNP sample has a peak mass at ∼70 MDa, 31 MDa higher than that of the empty LNP sample. Four freeze-thaw (FT) cycles of the mRNA-LNPs results in a peak mass at ∼26.5 MDa, indicating significantly degraded LNPs. The degraded LNPs are about 28 % of the population of the mRNA-LNP sample after the first FT cycle. A non-linear least squares fitting routine was developed to convolve the mass distribution of the LNP core with a function that describes the packaging distribution to fit the mRNA-LNP data. Two models of the lipid core mass distribution were used to obtain the distribution of mRNA in the packaged LNPs. These two models provide a lower and upper limit to the average mRNA packaging of 43 and 107 mRNA copies, consistent with a rough estimate of an average of 62 mRNA copies obtained from cryo-TEM images. These results demonstrate the potential for label-free, rapid characterization of mass, diameter, packaging, and stability of LNPs with CDMS.