Bacterial endotoxin binds to the surface of gold nanoparticles and triggers inflammation.

Due to the increasing development of nanomaterials for different applications, safety aspects are becoming of key importance for ensuring their safe use and for sustaining their industrial development. Nanosafety studies need however to reliably report the effects of nanoparticles (NP) on living organisms, so as to understand the possible impact on human and environmental health. The modulation of immune responses is considered a central element for assessing health risks. Reliability of the safety assays, both in vitro and in vivo, implies the accurate characterisation of the nanomaterial under test, which should be devoid not only of chemical impurities but also of biologically active contaminants. In this context, the presence of bacterial endotoxin (lipopolysaccharide, LPS), a potent immune/inflammatory activator and toxic molecule, needs to be assessed in engineered NP preparations, in order to be able to correctly attribute to NPs the inflammatory/toxic effects that may be observed. In this study, we have evaluated the ability of LPS to associate to gold NPs. After synthesis in endotoxin-free conditions, gold NPs (diameter 10 nm, Z-Potential -45 mV, sodium citrate coated) were tested to confirm lack of endotoxin contamination, then exposed to different concentrations of E. coli LPS for different times at different temperatures. The increase in NP size and the decrease in Z-potential indicated a dose-dependent binding of LPS to the NP surface, which increased with time to reach maximal binding after about 60 min at room temperature. At other temperatures (4°C, 37°C) binding occurred with an essentially similar kinetics. Interestingly, binding of LPS did stabilise gold NPs, which did not aggregate after washing, at variance with LPSfree NPs. The ability of LPS-treated gold NPs to induce an inflammatory response was tested in human primary monocytes in vitro. Preliminary results showed that LPS-treated NPs could induce the expression of the IL1B gene and the production of the mature IL-1? protein, suggesting that LPS-carrying NPs not only upregulated the IL1B gene but could also activate the inflammasome-dependent IL-1? maturation. LPS-free NPs were unable to induce either gene expression or protein production. These data suggest that unintentional adsorption of ubiquitous LPS to engineered NP surface, if going undetected, could induce inflammatory/toxic effects that may be erroneously attributed to NPs. Distinguishing the true NP effects from those caused by biologically active contaminants such as LPS is key to a correct and reliable nanosafety evaluation.