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

The use of nanoparticles has increasingly been implemented in biomedical applications including the diagnosis and treatment of disease. When administered to a biological system, nanoparticles spontaneously interact with their surrounding environment, leading to the surface-adsorption of small molecules and biomacromolecules. The protein component of the surface-adsorbed species, is often referred to as the “protein corona”. The composition of the protein corona is governed by nanoparticle properties, incubation media and parameters related to the environment in which nanoparticle incubations are performed. In this study, we investigated the formation of protein corona on polystyrene nanoparticles which have different surface chemistries and the impact of experimental incubation parameters, including centrifugation-resuspension protocols, incubation duration and shear flow rate conditions. The particle characteristics measured include size distribution, zeta potential and total protein content. Our findings show significant differences in nanoparticle size following exposure to media containing proteins across the three different nanoparticle surface chemistries. These findings were also confirmed by total protein concentration measurements performed on nanoparticles recovered from bulk media, and measurements of the composition of surface-adsorbed proteins by gel electrophoresis. We also found that exposure to different shear flow conditions alters both the thickness and the composition of surface-adsorbed protein coronas. In parallel to analysis of nanoparticles isolated using the centrifugation-resuspension protocol, we performed in situ analysis of nanoparticle size in media containing proteins. Results obtained from these measurements highlight that the recovery procedure is disruptive to the protein corona and therefore the need for investigative methods that do not alter the properties of the nanoparticle coronas. Nanomedicines are generally intended for administration via injection, and our findings show that parameters such as shear flow and media composition can significantly alter nanoparticle physicochemical parameters. Overall, we show that the recovery protocol can significantly alter 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 experimental design in the context of biologically-relevant shear flow conditions and media composition because these parameters can significantly alter particle physical parameters and the subsequent conclusions drawn from such studies.