Single-Particle Multiparametric Microscopy Reveals Structural, Size, and Payload Heterogeneity in mRNA-Loaded Lipid Nanoparticles

Dec 19, 2025ACS nano

Detailed microscopy reveals differences in shape, size, and mRNA content of individual lipid nanoparticles

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mRNA Technology on OpenScience ↗PubMed ↗DOI ↗

Abstract

The study reveals that the size of lipid nanoparticles (LNPs) can vary significantly between empty and mRNA-loaded particles, depending on formulation parameters.

Measurements of individual LNPs show that empty particles consistently exhibit lower lipid fluorescence compared to mRNA-loaded ones across all formulations.
The relative size differences between empty and mRNA-loaded LNPs are influenced by specific formulation components and the internal structure of the particles.
Observations from CLiC-ALEX microscopy suggest that a subpopulation of LNPs, identified as bleb-LNPs, may overlap with mRNA-containing LNPs.
Quantification of mRNA fluorescence and energy transfer signals reveals variability in both mRNA copy numbers and the structural arrangements of mRNA within LNPs.
These findings connect microscopic properties of LNPs to their potential therapeutic functions, highlighting the complex relationship between structure and activity.

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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.