Lipid nanoparticles (LNPs) have emerged as promising delivery vehicles for RNA therapeutics, but assumptions of uniformity in particle population and mRNA loading often obscure critical heterogeneity that can impact therapeutic efficacy. In this study, we present a single-molecule nanoscopy platform that enables high-resolution analysis of individual LNPs in terms of size, morphology, and encapsulated mRNA content. Using super-resolution imaging with Nile red, we confirm that production-line LNPs appear as spherical particles with a narrow size distribution around 90 nm. By optimizing permeabilization with Triton X-100 and employing specific fluorescent probes, we achieve direct access to and quantification of internalized mRNA while preserving nanoparticle integrity. Quantitative single-particle imaging with Ribogreen dye and sequence-specific fluorescent probes, combined with total internal reflection fluorescence microscopy (TIRFM), reveals a broad distribution of mRNA copies per LNP, with a typical peak at about two molecules per particle and a notable fraction of sparsely loaded or empty nanoparticles—variability hidden by bulk methods. Dynamic probe binding further enables sensitive detection and accurate identification of internalized mRNA cargo. This approach establishes a direct correlation between LNP structural features and mRNA payload at the single-particle level, providing foundational insights into how variations in lipid components influence payload distribution. Understanding these heterogeneities is crucial for optimizing biodistribution, mRNA expression kinetics, and ensuring batch-to-batch consistency. Our methodology delivers a robust quality control framework, paving the way for safer, more effective nucleic acid therapeutics.
Banerjee et al. (Sun,) studied this question.