Going hi-res in bulk: flowless multiangle dynamic light scattering for detection on asymmetric flow field flow...

Nanomedicine is an emerging field of research, demanding new analytical methods to respond to stringent regulatory and safety requirements. Particle size and size distributions are critical quality attributes that require accurate and affordable measurements suitable for both R&D activities and routine quality control in production pipelines. Here we propose a new approach combining multidetector asymmetric flow field flow fractionation (MD-AF4) with multiangle dynamic light scattering (MADLS) for determination of particle size on bioinspired colloids and on polymeric particles, without the need for calibration materials. We benchmarked our results against established analytical methods, using a set of model liposome formulations to cover a wide range of particle sizes. The liposomes, prepared by the traditional lipid thin-film extrusion method, were characterized by cryogenic transmission electron microscopy (cryoTEM) and MADLS, showing high monodispersity and long-term stability. These model samples allowed us to evaluate the effect of key variables on the performance of AF4 coupled to in-flow dynamic light scattering, identifying the influence of sample concentration on analytical bias for size determination. We extended the analysis to other types of particles (i.e. polystyrene and polymethyl methacrylate), which are relevant for environmental and drug delivery applications. Our results demonstrate that the analytical bias for in-flow DLS measurements follows a universal law, nearly independent of the chemical nature of the nanoparticles investigated. Subsequently, we combined MADLS and AF4 techniques, exploiting a programmable fraction collection system, enabling the accurate determination of particle size and number concentration in the absence of flow, and providing higher resolution for the size distributions. Our approach could be extended to any kind of colloid suitable for light scattering methods and paves the way for the revisitation of AF4-DLS instrumentation that may become widely available in the future, expanding the set of analytical tools for nanomedicine applications.