Deciphering the heterogeneity of mRNA-containing lipid nanoparticles (LNPs) is essential for understanding the relationship between their microscopic properties and therapeutic function. Here, by combining alternating laser excitation (ALEX) with convex lens-induced confinement (CLiC) microscopy, we simultaneously measure size, multicolor fluorescence, mRNA payload, and Förster resonance energy transfer (FRET) of individual suspended LNPs containing labeled lipid and mRNA molecules. By varying formulation parameters, including ionizable lipids, formulation buffers, and molecular ratios, we investigated and correlated key microscopic properties for relevant vaccine formulations. While per-particle lipid fluorescence was lower for empty versus mRNA-loaded particles for all formulations, the relative size of empty versus mRNA-loaded particles depended upon the formulation and intraparticle structure. When comparing CLiC-ALEX to cryogenic transmission electron microscopy measurements (Cryo-TEM), for the LNP formulations with a major subpopulation of bleb-LNPs, the subpopulation of bleb-LNPs appears to overlap with the subpopulation of mRNA-containing LNPs. CLiC-ALEX also enabled quantification of per-particle mRNA fluorescence and FRET signals, thereby revealing heterogeneity in the mRNA copy number and mRNA-LNP structural arrangements; where the results were compared with biophysical estimates based on the LNP formulations. These rigorous biophysical insights are critical to inform our understanding of structure-activity relationships and inform the rational design of nanomedicines.
mRNA Technology