Early Stage Soft Protein Corona Identification on Nanoparticles via Extended Size Exclusion Chromatography.

Unlike the hard protein corona (HPC), the soft protein corona (SPC) is a dynamic and complex layer for which limited knowledge exists regarding its structure and composition, primarily due to critical challenges in separating and purifying SPC from nanoparticles (NPs). In this study, we report the identification of SPC on the surface of polystyrene NPs at low time scales (i.e., a few to a hundred seconds). Using extended size exclusion chromatography (SEC) columns (i.e., 200 mm) and relatively large NPs (i.e., 300 nm), we successfully separated SPC-coated NPs from excess plasma. Our results show that SPC-coated NPs elute in early fractions, while excess plasma elutes in later fractions due to the significant size difference between plasma proteins and NPs, as well as the long column length. Additionally, SPC-coated NPs exhibit a relatively thicker shell compared with HPC-coated NPs, likely due to the rapid adsorption of large, abundant proteins such as albumin, as confirmed by both TEM and DLS measurements. Liquid chromatography-mass spectrometry (LC-MS) analysis further revealed that SPC formed at early incubation times (5-120 s) exhibited significantly lower protein diversity (59-93 proteins) compared to the hard protein corona (HPC, 152 proteins). The SPC composition dynamically evolved within seconds, highlighting the rapid competitive adsorption and displacement of abundant plasma proteins at nanoparticle interfaces in the first few seconds. These findings underscore the critical need for time-resolved approaches to accurately characterize nanoparticle-protein interactions at biologically relevant time scales.