Nanoparticles (NPs) entering a biological fluid undergo surface modification due to dynamic, physicochemical interactions with biological components, especially proteins. In this work we used complementary bio-physico-chemical approaches to characterize the effects of interactions between CeO(2) NPs, immunoglobulins (IgGs) and bovine serum albumin (BSA) of a similar size on protein structural evolution as well as formation of (hetero-) aggregates. Using circular dichroism we showed that IgGs and BSA underwent significant structural changes after interaction with NPs. The NPs and protein-NPs were observed after size exclusion chromatography, highlighting the fact that few aggregates were stable enough to pass this mild separation step. X-ray absorption spectroscopy suggested that the surface chemistry of NPs was not affected by these proteins, also implying weak interactions. Competitive experiments revealed that, while the serum was more concentrated for BSA, IgG-NP aggregates were more stable. Altogether, our results indicate that, under our experimental conditions, the formation of a "protein corona" is an unstable and reversible mechanism. This indicates that, when NPs and proteins are similar in size, the adsorption concept (i.e. protein corona concept) cannot be applied to model the NP-protein interaction, and a heteroaggregation model is more appropriate.

It has been established that transferrin binds a variety of metals. These include toxic uranyl ions which form rather stable uranyl-transferrin derivatives. We determined the extent to which the iron binding sites might accommodate the peculiar topographic profile of the uranyl ion and the consequences of its binding on protein conformation. Indeed, metal intake via endocytosis of the transferrin/transferrin receptor depends on the adequate coordination of the metal in its site, which controls protein conformation and receptor binding. Using UV-vis and Fourier transform infrared difference spectroscopy coupled to a microdialysis system, we showed that at both metal binding sites two tyrosines are uranyl ligands, while histidine does not participate with its coordination sphere. Analysis by circular dichroism and differential scanning calorimetry (DSC) showed major differences between structural changes associated with interactions of iron or uranyl with apotransferrin. Uranyl coordination reduces the level of protein stabilization compared to iron, but this may be simply related to partial lobe closure. The lack of interaction between uranyl-TF and its receptor was shown by flow cytometry using Alexa 488-labeled holotransferrin. We propose a structural model summarizing our conclusion that the uranyl-TF complex adopts an open conformation that is not appropriate for optimal binding to the transferrin receptor.