Frit-inlet asymmetric flow field-flow fractionation for the analysis of lipid nanoparticle-protein interactions

Research into nanoparticle interactions with biomolecules has become increasingly important in nanomedicine. While lipid nanoparticles (LNPs) are widely used as drug delivery systems, there remains a gap in understanding their fate in circulation, which is crucial for selecting appropriate lipids during formulation development. This study is the first to use Asymmetric Flow Field Flow Fractionation (AF4) to compare two types of LNPs: MC3-LNPs and SM-102-LNPs, and their interactions with a model protein, bovine serum albumin (BSA). AF4 offers high-resolution separation, with the ability to simultaneously perform multiparametric inline analysis with multiple detectors. In this study, the impact of LNP size, morphology and PDI on BSA corona formation were examined using inline multiangle light scattering (MALS) and dynamic light scattering (DLS). AF4 separation revealed two subpopulations for MC3-LNPs, while SM102-LNPs exhibited a single population. Analysis of shape factor indicated a shape factor of 0.783 for SM-102-BSA and 0.741 and 0.795 (peak 1 and 2) for MC3-BSA, confirming interaction between LNPs and BSA. Both LNPs exhibited LNP-BSA induced aggregation. Overall, this study demonstrates the effectiveness of AF4, particularly when hyphenated with multidetector systems, for simultaneously separating LNPs from complex biological media and studying LNP-protein interactions.