ИСТИНА

It is known that, upon contact with biological fluids, nanoparticles (NPs) get coated with one or more layers of biomolecules, forming a corona. These adsorbed biomolecules subsequently modulate the interaction between cells and the nanomaterial, with significant implications for toxicology, as the amount and type of proteins in the coating have been shown to determine the cellular uptake and trafficking of NPs. While the strongly attached protein corona has been extensively studied the weakly attached proteins are challenging to investigate via ex situ techniques since they exchange rapidly. In this work, we investigate the so called hard and soft coronas that form around polyvinylpyrrolidone (PVP) coated silver nanocubes (~35 nm edge length), upon incubation in media supplemented with foetal bovine serum (FBS). We used the optical properties of the nanocubes related to local surface plasmon resonances (LSPRs) to quantify both the hard and soft coronas and investigate the location of proteins across the particle at different times. Red shifts in the wavelengths of the maximum absorbance of LSPRs specific for our NPs were tracked on a UV-Vis spectrophotometer for several incubation time-points. A two-layer model accounting for strong and weak binding interactions was developed based on finite-difference time-domain simulations and, together with LSPR peak shifts, was used to quantify both slowly and rapidly exchanging proteins around NPs. The corona was tracked over several incubation times, ranging from 1 minute to 24 hours, showing the formation and time-dependant densification of a monolayer composed of strongly-binding biomolecules. By tracking LSPRs localised either at the nanocube facets or edges/corners we showed that, for short time-points, the biomolecule layer binding kinetics at cube edges and corners versus facets are different. Time of Flight Secondary Ion Mass Spectrometry (Tof-SIMS) studies on the nanocube surfaces at various incubation times (0 minutes, 1 minute, 1 hour, 12 hours) suggest that the presence of PVP may be one of the factors responsible for the late onset of the hard corona on certain regions of the geometrically anisotropic particles. Subsequently, quantification of the second layer of weakly-binding, rapidly-exchanging proteins showed it to be relatively constant throughout the incubation. To further investigate this additional layer, we resuspended NPs with preformed hard coronas in media containing various FBS concentrations and measured the absorbance of LSPR signals. We observe an increased red shift of the plasmon signals with higher serum contents, but this shift reaches a plateau and remains constant above 3% serum, probably due to a saturation of the rapidly exchanging biomolecules layer. The soft corona formed at high concentration contains similar amounts of protein to the underlying hard corona. Our results suggest that the soft corona protein content and patterns depend on the serum content in the dispersion medium.