Structural Profiling of Lipid Nanoparticles at Sub-10 nm Resolution via AF4 Coupled Online to SAXS and SANS

Precise mapping of structural heterogeneity at the sub-10 nm scale is pivotal for rational nanoparticle design, yet conventional analytical workflows remain inadequate. Here we integrate dilution-controlled asymmetric flow field-flow fractionation (AF4) with small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) to interrogate ellipsoidal solid-liquid lipid nanoparticles (LNPs). The dilution-controlled AF4 mode amplifies scattering contrast, enabling robust, shape-resolved analysis across entire elution profiles. Coupling AF4 to SANS in D₂O further sharpens resolution for the smallest fractions by reducing particle diffusion through increased solvent viscosity. Comparative sizing shows that SAXS/SANS accurately capture primary particles down to ∼5 nm, whereas multi-angle light scattering chiefly detects loosely associated aggregates. Morphology profiling reveals that surfactant identity governs particle shape, polydispersity, and overall architecture. Joint SAXS/SANS modeling uncovers a 2-3 nm polar shell enveloping an internal core-shell morphology. Together, these insights refine our understanding of LNP size, morphology and drug localization and establish dilution-controlled AF4-SAXS/SANS as a high-resolution platform for dissecting complex nanoparticle systems relevant to biomedical applications.