Preparation of the Protein Corona: How Washing Shapes the Proteome and Influences Cellular Uptake of Nanocarriers

Abstract A protein coat, termed the protein corona, assembles around the nanocarriers´ surface once it gets in contact with a biological environment. We show that the media used for the washing of protein corona can be crucial. This is true for the downstream analysis as well as for the precoating used in in vitro or in vivo. This has been widely overlooked so far. In this paper we focus on eight different washing media and analyze how they influence the composition of the hard protein corona of several nanocarriers incubated with human blood plasma and serum. SDS-PAGE and LC-MS analysis showed major differences in protein corona profiles when using diverse washing media. While plasma and serum proteins already have different complexities, each washing media changes the composition of proteins detected by downstream methods with different key proteins bound to the nanocarriers´ surface. Furthermore, the protein structure of the most abundant blood proteins incubated in the different media was analyzed with nanoDSF. This also emphasized the importance of the washing media, which had a significant influence on the protein adsorption stability. Lastly, cell uptake experiments for HeLa and RAW 264.7 macrophages also indicated an influence of the washing media. In conclusion, picking a specific washing media is on the one hand an important factor for downstream detection of protein compositions and may on the other hand be used to deliberately tune the protein corona for pre-adsorbed proteins from complex protein compositions. This might further support a guided delivery of the nanocarrier to a desired location within a physiological environment.