Time meets protein corona: the Vroman effect dictates macrophage recognition of PEGylated nanoparticles

The phenomenon of the protein corona is a major contributor to the limited bench-to-bedside translation of nanomedicines, as it confers a new biological identity that can lead to rapid bloodstream clearance. The composition of the protein corona is dynamic and varies over time. This so-called Vroman effect implies that nanoparticles (NPs) should display a time-dependent biological identity that would modulate how they interact with the living environment as time evolves. Incorporating polyethylene glycol (PEG) into nanoformulations is the gold-standard for minimizing this rapid recognition by the immune system. Nonetheless, whether the protein corona and, consequently, the biological identity imparted by this stealth polymer are immutable over time remain unexplored. Here, we provide a facile assay to evaluate how the Vroman effect dictates macrophage recognition of NPs upon incubation with plasma. For that purpose, we have engineered two types of PEGylated mesoporous silica nanoparticles that have been subjected to short-term (5 min) or long-term (1 h) incubation with human plasma to allow for protein corona formation. The physicochemical characterization has demonstrated appreciable changes according to the incubation time (e.g., mean size, amount of protein adsorbed…). The protein coronas have been fully analyzed by means of proteomics, validating the existence of an underlying Vroman effect. The key finding and novelty of our work is that flow cytometry experiments of time-dependent protein coronas suggest the existence of a transient, undesired protein corona that enhances immune recognition before evolving into a less immunogenic state, regardless of the PEGylation degree. Altogether, these findings open the door to more exhaustive analyses of nanomedicines and could contribute to the rational design of in vivo experimentation.