The unique physicochemical properties of two-dimensional (2D) graphene oxide (GO) could greatly benefit the biomedical field; however, recent research demonstrated that GO could induce in vitro and in vivo toxicity. We determined the mechanism of GO induced toxicity, and our in vitro experiments revealed that pristine GO could impair cell membrane integrity and functions including regulation of membrane- and cytoskeleton-associated genes, membrane permeability, fluidity and ion channels. Furthermore, GO induced platelet depletion, pro-inflammatory response and pathological changes of lung and liver in mice. To improve the biocompatibility of pristine GO, we prepared a series of GO derivatives including aminated GO (GO-NH2), poly(acrylamide)-functionalized GO (GO-PAM), poly(acrylic acid)-functionalized GO (GO-PAA) and poly(ethylene glycol)-functionalized GO (GO-PEG), and compared their toxicity with pristine GO in vitro and in vivo. Among these GO derivatives, GO-PEG and GO-PAA induced less toxicity than pristine GO, and GO-PAA was the most biocompatible one in vitro and in vivo. The differences in biocompatibility were due to the differential compositions of protein corona, especially immunoglobulin G (IgG), formed on their surfaces that determine their cell membrane interaction and cellular uptake, the extent of platelet depletion in blood, thrombus formation under short-term exposure and the pro-inflammatory effects under long-term exposure. Overall, our combined data delineated the key molecular mechanisms underlying the in vivo and in vitro biological behaviors and toxicity of pristine GO, and identified a safer GO derivative that could be used for future applications.

The physicochemical properties of a set of 21 different gold nanoparticles (spherical and rod-shaped nanoparticles (NPs) of different diameters with three different surface coatings) were studied. Protein corona formation, in vitro uptake, effect on cell viability and proliferation, and in vivo biodistribution of these NPs were determined. The relation of the results of the different NPs was analyzed by hierarchical cluster analysis, which will tell which NPs have the most similar physicochemical properties and biological effects, without having to specify individual physicochemical parameters. The results show that the physicochemical properties of gold nanoparticles (Au NPs) are mainly accounted for by their hydrodynamic diameter and their zeta-potential. The formation of the protein corona is determined by the pH-dependence of their zeta-potential. While several reports found that in vitro uptake and in vivo biodistribution of NPs are correlated to individual physicochemical parameters, e. g., size, shape, or surface chemistry, such direct dependence in the investigated multidimensional set of NPs was not found in our study. This most likely is due to entanglement of the different parameters, which complicates the prediction of the biological effect of NPs in case multiple physicochemical properties are simultaneously varied. The in vitro uptake and in vivo biodistribution of NPs seem to be not directly driven by the protein corona, but the physicochemical properties determine as well the corona as they influence in vitro/ in vivo behaviors, and thus the effect of the protein corona would be rather indirect.