Multiparametric Characterization of Individual Suspended Nanoparticles Using Confocal Fluorescence and Interferometric Scattering Microscopy with Microfluidic Confinement

Detailed characterization of the size, mass, payload,
and structure
of suspended mRNA–lipid nanoparticles (LNPs) is necessary to
improve our understanding of how these heterogeneous properties influence
therapeutic efficacy and potency. Methods currently in use face limitations
in reporting ensemble-average particle properties or requiring dedicated
home-built microscopes that are beyond the reach of nanoparticle developers.
In this work, we overcome these limitations by combining a commercially
available confocal microscope and a convex lens-induced confinement
(CLiC) instrument to achieve simultaneous characterization and correlation
of the size, mass, refractive index, and nucleic acid payload of individual
LNPs. We established the accuracy and precision of our method using
nanosized beads and used it to investigate the size, payload, and
water content of LNPs in different solvent pH. By employing readily
available microscopy tools, we open the door to widespread adoption
of our quantitative, in-solution nanoparticle characterization method.