AbstractEndothelial cells lining the lumen of blood vessels serve as a physiological barrier controlling nanoparticle movement from the vasculature into the tissue. For exploring the effect of polymer hydrophilicity on nanoparticle interactions with human umbilical vein endothelial cells (HUVECs) in vitro, a series of monomodal poly[acrylonitrile-co-(N-vinylpyrrolidone)] model nanoparticles with increasing hydrophilicity as related to their increasing content (0–30 mol.%) of N-vinylpyrrolidone (NVP) were synthesized by miniemulsion polymerization. Nanoparticles with a low NVP content were rapidly endocytized into all cells independent from the particle dose with toxic effects only observed at high particle concentrations, while only 10–30% of the cells incorporated particles with ⩾20 mol.% NVP. Since pathologies are often related to inflammation, an inflammatory HUVEC culture condition with IL-1β stimulation has been introduced and suggested to be widely applied for studying nanocarriers, since cellular uptake in this assay was clearly increased for NVP contents ⩾20 mol.%. Importantly, the secretion of functional biological mediators by HUVECs was not relevantly influenced by the nanoparticles for both homeostatic and inflammatory conditions. These findings may motivate concepts for nanocarriers specifically targeted to pathologic regions. Additionally, rapidly endocytized Rhodamin B loaded particles with low NVP content may be explored for cell labeling and tracking.