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Different types of natural and synthetic polymeric nanocarriers are being tested for diverse biomedical applications ranging from drug/ gene delivery vehicles to imaging probes. The development of such innovative nanoparticulate systems (NPs) should include in the very beginning of their conception a comprehensive evaluation of the nano-bio interactions. Specifically, intrinsic physicochemical properties as size, surface charge and shape may have an impact on cellular uptake, intracellular trafficking, exocytosis and cyto- or genocompatibility. Those properties can be tuned for effectiveness purposes such as targeting intracellular organelles, but at the same time inducing unforeseen adverse nanotoxicological effects. Further, those properties may change due to the adsorption of biological components (e.g. proteins) with a tremendous impact on the cellular response. The evaluation of these NPs is highly challenging and has produced some controversial results. Future research work should focus on the standardization of analytical or computational methodologies, aiming the identification of toxicity trends and the generation of a useful meta-analysis database on polymeric nanocarriers.