NanoKON – Systematic evaluation of health effects of nanoscale contrast agents
The NanoKon project was dedicated to assessing the health effects of orally administered nanoscale contrast agents. For that specific purpose, nanoparticles were produced and tested together with commercially available reference particles in numerous in vitro, in vivo, and in silico assays.
Within work package 1, the respective nanomaterials were thoroughly characterised and their specific properties e.g., ion release of toxically relevant species (Ba, Gd) in artificial gastric juice analysed. The changing conditions (pH, high ionic strength) were tolerated only insufficiently by most of the (commercially available and self-synthesised) particle systems causing agglomeration for the majority of the tested nanoparticles. In the course of the project different modifications of the nanoparticles’ surface were investigated with the objective of obtaining as stable as possible suspensions that are functional with regards to reproducibility.
Work packages 2 and 3 were dedicated to in vitro analyses of the respective nanoparticles in relevant cell lines and barrier models. The objective was to investigate the contrast agents’ fate and uptake in systems coming into contact with them when administered orally. In addition, the adsorption of surrounding proteins on the surface of the contrast agent during formation of a protein corona was investigated. Concordantly, an uptake of the respective contrast agents (FexOy and FexOy@SiO2) and SiO2-NP (reference surface) was observed in intestinal epithelial cells, endothelial cells and macrophages. In the case of some examples, particle uptake was detected already after 15 to 30 minutes. The tested iron oxide nanoparticles were absorbed by endothelial cells and revealed no cytotoxicity in the relevant concentration range. However, under these conditions, cell-type specific biological effects such as a decrease in the impedance pointing towards a change in the barrier properties of the cells could be observed.
In addition to the above, a complex multicellular in vitro model of the intestinal barrier was established to analyse the effects of nanoparticles (see also work package 3). For this coculture model, the intestinal epithelial cell line Caco-2 was used together with the microvascular endothelial cells ISO-HAS-1. In that system, the used nanoparticles proved to be neither toxic nor inflammatory.
Besides, the studies proved that the formation of the nanoparticle protein corona is a decisive factor for (nano)-medical and (nano)-biotechnological applications. The time-resolved quantitative analysis of the protein corona shows that the protein envelope forms extremely rapidly, is highly complex, and affects the toxicity or intracellular uptake of the nanoparticles.
After uptake the particles used in the different cell types were localized in lysosomes, which indicates uptake via endocytosis or phagocytosis. After uptake, the nanoparticles were found to be located outside the cytosol. They cannot reach the latter before having penetrated the vesicular membrane. There was found to be no uptake of nanoparticles into the nucleus of intestinal epithelial cells.
Within work package 4, the biological effects of nanoscale contrast agents were analysed in vivo in the mouse model. There, none of the tested particle suspensions caused any significant organ toxicity. Besides, all tested serum parameters proved to be inconspicuous. The distribution of the particles after oral administration was analysed by means of magnetic resonance tomography (MRT) and computer tomography (CT). Additionally, element and electron-microscopic analyses were carried out on various organs. Whereas approximately 95 percent of the administered quantity of the respective nanoparticles suspensions was directly excreted, the major part of the remaining 5 percent was removed from the stomach, small intestine, and rectum by rinsing with a buffer solution. For the respective organism, this means that less than 0.5 percent of the applied nanoparticle dose remained in the body.
Work package 5 was dedicated to establishing and developing different image analysis elements to be applied to the results of the microscopy data. By applying tailor-made image analysis methods, the three-dimensional structures of the cell membrane and MT network were reconstructed. The simulation model developed within work package 5 considers different uptake mechanisms at the cell membrane and the dynamics of the nanoparticles in different cell compartments. The analysis of the MT structure suggests an influence of the nanoparticles on the persistence length of the MT network.
The analyses yielded the following basic results:
- Nanoscale contrast agents are taken up by the majority of the tested cell lines (intestinal epithelial cells, endothelial cells, and macrophages)
- As a rule, no organo-toxic, cytotoxic or inflammatory effects were observed for the tested systems neither in vitro nor in vivo. Exceptions to this rule are the activation and amplification of inflammatory signalling pathways in macrophages by silica reference particles as well as the slightly inflammatory potential of Gd-doped BaSO4 nanoparticles in Caco-2 monocultures
- As a function of the particle surface, the presence of protein-containing biological media creates a protein corona influencing the physicochemical and biological behaviour of the nanoparticles
- In vitro and in vivo, Gd-containing BaSO4 suspensions revealed good X-ray contrasting potential which was weaker for the MRT in the in vivo situation
- The correlation between the structure of the close-to-membrane actin network and the particle dynamics was determined.
- It was shown that the absorption of nanoparticles influences the persistence length of the MT filaments.
Grant Number: BMBF - FKZ 03X0100
Duration: 01.10.2010 - 30.09.2013 (extended to 31.12.2013)