ИСТИНА

It is known that, upon contact with biological fluids, nanoparticles (NPs) get coated with biomolecules forming a corona. This corona is thought to modulate the interactions with cells. In the present work we investigate the strongly and weakly binding protein layers around PVP-coated silver nanocubes (AgNCs) in media relevant for nanotoxicology studies. This is done taking advantage of AgNCs specific plasmonic signals. Localised surface plasmon resonance (LSPR) measurements were performed in the UV-Vis region, on NPs incubated in culture medium containing various amounts of foetal bovine serum. Several time-points, from 15 minutes to 24 hours, were studied, showing kinetics of corona formation. Experimental results of shifts in the wavelength of maximum absorbance were coupled with finitedifference time-domain (FDTD) simulations of the optical response. The resulting model shows the formation of a strongly-binding protein monolayer increasing in density up to the limit imposed by random sequential absorbance. Furthermore, it allowed us to estimate the average number of protein molecules bound to a NP at each time-point. The results at 24 h incubation were confirmed by experimentally quantifying total strongly-bound proteins with the bicinchoninic acid assay. Subsequently, weakly-binding protein layers were studied. We showed soft corona patterns rapidly change upon variation in the serum content. Furthermore, taking into account the thickness of the strongly-binding protein layer and the near field decay, we could quantify weakly-interacting proteins. The LSPR spectrum of AgNCs exhibits two different, spectrally resolved resonance modes which FDTD simulations show have different spatial distributions and we explore the possibility to distinguish the different corona-formation patterns at cube facets versus cube rounded corners. The results indicate that LSPR is a sensitive tool for probing the protein corona around nanoparticles in situ in biological media.