<em>Gold Nanoparticles contaminated by Bacterial Endotoxin: biophysical characterization, imaging and nanotoxicology</em>

Abstract

Gold nanoparticles (AuNPs) are nanodevices that can have many uses in biomedical applications but sometime they show nanotoxic effects on biological system. Among these effects, the activation of the innate immune system (inflammatory response) is considered a central issue for assessing health risks of Au NPs. Although the origin of this nanotoxicity is not well known, the cause could be associated to the presence of contaminants on nanoparticles’ surface, such as bacterial endotoxin. Bacterial Endotoxin, also known as Lipopolysaccharide (LPS), is the main component on cell walls of gram-negative bacteria and it is considered one of the major contaminant in the environment. The main goal of this study is to identify and analyse the activation of the inflammatory response associated to AuNPs and/or to the presence of LPS on the nanoparticles’ surface. To this aim, the interaction of AuNPs with LPS is analysed, the presence of LPS molecules on NPs is quantified, and the interaction of AuNPs with human primary macrophages is investigated, in order to distinguish the intrinsic NPs biological effects from those induced by LPS. LPS dose-dependent adsorption on 50 nm AuNPs was studied by DLS and by SERS technique in order to understand the amount of LPS that binds to NPs surface and quantify it. Internalization of bare and LPS coated 50 nm AuNPs was studied in macrophages by TEM and Raman imaging and their inflammatory effect was studied by in vitro stimulation through evaluation of inflammatory cytokine production (TNF-α). DLS results indicate that a uniform LPS corona (8712 molecules) is formed around all NPs (2 µg) when incubated with doses greater than 500 ng, while analysis of SERS signals show a Limit of Detection (LOD) for LPS amount of the order of fg. These promising results show how SERS technique can be a reliable LPS-Sensor, while NPs imaging studies showed that NPs are localized in cytoplasmic vesicles inside macrophages. Moreover, bare NPs do not induce the production of TNF-α cytokine in treated macrophages.