In vitro evaluation of gold nanoparticles toxicity towards human endothelial cells and comparison between static and flow conditions

In the last twenty years fabrication and use of nanoparticles has seen an escalation in every field of science, thanks to their innovative and unique chemical and physical properties. In particular, metal nanoparticles are used as new multi-functional platform for diagnostic and drug delivery (Papasani et al., 2012). At the same time, awareness towards their impact on human and environmental health requires studies that characterize their properties and potential cytotoxic effects (Dykman ann Khlebtsov, 2012). In this work we investigate uptake and toxicity of gold nanoparticles (Au NPs), currently under investigation in many laboratories as diagnostic and therapeutic agents to be administered intravenously. In particular we explore the effects towards cells of the human vascular system (Human Umbilical Vein Endothelial Cells, HUVEC), because these are the first one to get in contact with NPs. The characterization of Au NPs in water and in culture medium is done by standard methods and by Two-Photon Fluorescence Correlation Spectroscopy. Au NPs uptake is investigated through transmission electron microscopy (TEM), fluorescence microscopy and ICP-AES, and NPs toxicity is measured through standard bio-analytical tests (Trypan Blue exclusion test and Live/Dead Cell Viability Assay). We have explored the cytotoxicity of Au NPs by evaluation of gene expression of inflammatory markers and by the production of reactive oxygen species (ROS) after incubation with Au NPs. In parallel, we propose a new in vitro model system, which consists in a linear microfluidic device (MFD) realized in polydimethylsiloxane (PDMS). In this model we add shear stress associated with a flowing medium and a continuous infusion of Au NPs solution in the device, since these phenomena are normally present when NPs formulations are intravenously administrated (Mahto et al., 2010). The tests performed in multiwells, where no flow is present, are also run in the MFD, so as to compare the two model systems. The comparison between static and flow conditions shows that administration of equal concentrations of Au NPs under flow conditions results in reduced sedimentation of NPs on the cells and in reduced uptake, in parallel to a lower cytotoxicity with respect to experiments run in ordinary static conditions: Au NPs concentrations higher than 5×1010 NP/ml display under flow a toxicity approximately 20% lower than that measured in multiwells. These results provide new useful information to evaluate the toxicity and therapeutic efficacy of drugs intravenously administrated by means of nanoparticle-based formulations. References [1] Dykman and Khlebtsov (2012) Gold nanoparticles in biomedical applications: recent advances and perspectives. Chem Soc Rev 41: 2256-2282. [2] Mahto et al. (2010) A new perspective on in vitro assessment method for eva-luating quantum dot toxicity by using microfluidics technology. Biomicrofluidics 4: 034111. [3] Papasani et al. (2012) Gold nanoparticles: the importance of physiological principles to devise strategies for targeted drug delivery. Nanomed: Nanotech, Biol, Med 8: 804-814.