The interactions between nanoparticles (NPs) and proteins in complex biological application media such as blood serum are capable of inducing aggregate formation which can lead to subsequent changes in biological activity. Here, we correlate surface charge, aggregation-tendency, and surface serum protein adsorption with cellular uptake and biodistribution in mice.Polystyrene-based NPs (80 -170 nm) with different surface functionalizations were synthesized and incubated with human serum. Interaction of NPs with serum proteins and aggregate formation were analyzed by mass spectrometryanalysis and dynamic light-scattering. Influence of surface functionalization on specific cellular uptake and organdistribution was characterized.Localization and organ targeting of intravenously applied NPs preferentially depended on their aggregationbehavior in the presence of serum. Whereas strongly aggregating particles mainly located to liver, non-aggregating particles distributed to all organs. Determination of aggregate formation of NPs in the presence of serum and further analysis of the protein corona allows for pre-selection of NPs for in vivo application. studies were done by dynamic light scattering applying a previously developed pre-in vivo screening method [10]. This enabled us to analyze the effect of surface functionalization on aggregation and protein binding in the presence of human serum under physiologically relevant conditions. To allow analyzing the cellular uptake of NPs in cells of the peripheral blood as well as in professional antigen-presenting cells, the NPs were fluorescently labeled with boron-dipyrromethene (BODIPY) for flow cytometry and immune fluorescence. In addition, the particles were loaded with a near-infrared dye (IR-Dye 780) for in vivo imaging. Using quantitative high performance liquid chromatography coupled with mass spectrometry, we determined the composition of the protein corona. Using these particles, we were able to demonstrate that the pattern of protein binding in the presence of serum correlates with the tendency to form aggregates in vitro and in vivo. In addition, the charge of the particles' surface influenced their uptake in phagocytes and endocytosis as well as their biodistribution after intravenous application in mice.

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