Multiparametric functional characterization of individual lipid nanoparticles using surface-sensitive light-scattering microscopy

• DOI: 10.1073/pnas.2426601122
• Publication Date: 2025-05-22T15:10:46Z
• Authors: Mattias Sjöberg; Erik Olsén; Mokhtar Mapar; Petteri Parkkila; Simon Niederkofler; Sara Mohammadi; Yujia Jing; Gustav Emilsson; Lennart Lindfors; Björn Agnarsson; Fredrik Höök
• Keywords: Characterization

The most efficient lipid nanoparticles (LNPs) for gene therapeutics rely on specific lipids that protect the oligonucleotide cargo and aid cellular uptake subsequent endosomal escape. Yet, efficacy of current state-of-the-art LNP formulations remains low, a few percent at best. A deeper understanding how cargo, composition, stoichiometry, size, structure, pH-induced conformational changes influence their efficiency is therefore necessary improved design. Given variability these properties, preferred screening methods should offer single-particle-resolved multiparametric characterization. In this work, we employ combined surface-sensitive fluorescence label-free scattering microscopy with single resolution, which when integrated microfluidics liquid exchange between media varying refractive index, enables quantification content. We investigate two that, while similar in size mRNA content, exhibit differences functional delivery. Correlating content Cy5-labeled revealed scaled volume both types LNPs, index varied marginally across size. While fingerprinting alone could not distinguish formulations, use same experimental platform to show difference fusogenicity supporting bilayer under early conditions (drop pH from 7.4 6.0) correlates observed vitro data. This highlights limitation toolbox situ characterization, generally focuses structural properties suspended may adequately capture performance.