**5. Conclusions and future research**

Overall the binding of plasma proteins to nanoparticles, based on our findings, seems to vary with origin, surface properties and size of the particles. A large portion of the interacting proteins we identified by 2-DE/MS are proteins involved in the immune defense and reverse cholesterol transport to the liver, but we also identified proteins mediating brain uptake. Most likely these protein patterns of the nanoparticles represent a mixture of particle-protein and protein-protein interactions. Extensive research in this field is therefore needed before conclusions could be drawn regarding potential health effects of nanoparticles and their associated protein "corona". One major difficulty to overcome is how to characterize the particles used in different studies in such a way that comparisons and generalized conclusions are allowed. Most types of nanoparticles seem to form aggregates, especially so in water suspensions and the "corona" seen might be heavily influenced by the size/diameter of these aggregates rather than by other particle characteristics. Thus, characterization of particles and the aggregates they form prior to exposure of plasma proteins, cells or other biological systems is therefore extremely important. One way of doing that, as we have showed herein, is by DLS. These analyses gives valuable information about the trends in size distribution connected to sample preparation methods and choice of solvent. Sample preparation methods are indeed very important as well as choice of solvent and care should be taken when choosing fitting model and the model-inbuilt parameters. Thus, information obtained from DLS is important for everybody that is doing research on nanoparticles in liquids. The numbers given as product information i.e. the size and size distribution are often relevant for the core-size of the nanocrystals within the material. However the nanoparticles are most often not soluble to that extent. Consequently, nanoparticles obtained in dry state and then dispersed in liquid usually form aggregates as shown in this study.

Given ample attention to the characterization of the NPs used, future studies of the NP– protein complex behavior in different biological systems are needed. Questions that need to be addressed are which properties of the NPs that govern the protein "corona" formed around the NPs in biological fluids and how these complexes interact with endothelial cells, platelets, cells of the immune system etc. One interesting finding in this study is Amyloid β A4, not previously identified in plasma, which was only associated to ZnO particles. This protein may act as a chelator forming metal-amyloid aggregates and needs further attention in toxicological studies.

In summary, the improved 2-DE/MS protocols shown herein underline this proteomic approach as a powerful tool in human nano-particle toxicology. Furthermore, thorough characterisation of the particles studied, e.g. with DLS, is crucial to evaluate the results.
