Nanoparticle isolation from biological media for protein corona analysis: the impact of incubation and recovery protocols on nanoparticle properties

Nanoparticles are increasingly implemented in biomedical applications, including the diagnosis and treatment of disease. When exposed to complex biological media, nanoparticles spontaneously interact with their surrounding environment, leading to the surface-adsorption of small and bio- macromolecules- termed the “corona”. Corona composition is governed by nanoparticle properties and incubation parameters. While the focus of most studies is on the protein signature of the nanoparticle corona, the impact of experimental protocols on nanoparticle size in the presence of complex biological media, and the impact of nanoparticle recovery from biological media has not yet been reported. Here using a non-degradable robust model, we show how centrifugation-resuspension protocols used for the isolation of nanoparticles from incubation media, incubation duration and shear flow conditions alter nanoparticle parameters including particle size, zeta potential and total protein content. Our results show significant changes in nanoparticle size following exposure to media containing protein under different flow conditions, which also altered the composition of surface-adsorbed proteins profiled by SDS-PAGE. Our in situ analysis of nanoparticle size in media containing protein using particle tracking analysis highlights that centrifugation-resuspension is disruptive to agglomerates that are spontaneously formed in protein containing media, highlighting the need for in situ analytical methods that do not alter the intermediates formed following nanoparticle exposure to biological media. Nanomedicines are mostly intended for parenteral administration, and our findings show that parameters such as shear flow can significantly alter nanoparticle physicochemical parameters. Overall, we show that the centrifugation-resuspension isolation of nanoparticles from media significantly alters particle parameters in addition to the overall protein composition of surface-adsorbed proteins. We recommend that nanoparticle characterization pipelines studying bio-nano interactions during early nanomedicine development consider biologically-relevant shear flow conditions and media composition that can significantly alter particle physical parameters and subsequent conclusions from these studies.