Compositional variation insights into nanoprecipitation through Asymmetric flow field-flow fractionation (AF4): Impact of lipid composition and surfactant placement on particle formation

Lipid nanoparticles (LNPs) are attractive drug delivery platforms, yet a fundamental limitation of conventional batch characterizations is their inability to resolve compositional heterogeneity across polydisperse particle size distributions under nanoprecipitation. Here, the impact of two key formulation parameters, surfactant placement and solid/liquid lipid composition, are systematically investigated on particle formation, with particular emphasis on the size distribution and partitioning of physical encapsulation within nanoparticles. The model system, LNPs incorporating a Förster resonance energy transfer (FRET) donor-acceptor fluorophore pair (FLNPs), are used to probe nanoscale payload distribution. Integration of batch characterization (dynamic light scattering (DLS), transmission electron microscopy (TEM), and photophysical analysis), flow-induced dispersion analysis (FIDA) with asymmetrical flow field-flow fractionation (AF4) coupled to multi-angle light scattering (MALS), DLS, UV–vis and fluorescence detection, reveal that surfactant placement is the dominant parameter: when dissolved in the organic phase, the surfactant produces smaller particles with varied shapes and single-donor FLNPs, whereas when the surfactant is dissolved in the aqueous phase it results in larger, more spherical and co-localization particles. The addition of liquid lipid shifts size distributions towards greater size uniformity. These findings link nanoprecipitation parameters to size distribution and component localization, highlight the power of AF4 in resolving heterogeneity, and provide design principles for controlling LNPs size and payload distribution.