Establishment of methods for high throughput screening of nanoparticle toxicity in mammalian cells
MetadataShow full item record
The use of nanomaterials is getting more extensive, both within the industrial and the medical field. The production and implementation of nanomaterials into consumer market is rapid and at such a rate that they are preceding safety/toxicity assessments. There is an urgent need for consistent and accurate high-throughput in vitro systems for rapid assessment of emerging nanomaterials. This is the motivation for this master thesis. The main goal of this work is to establish HTS-methods for detecting nanotoxicity in representative cell lines and use these methods for detection of cytotoxicity of a range of available nanomaterials. Thereafter the aim is to get a deeper understanding of cellular responses following exposure of selected toxic nanomaterials. In this thesis five cell lines representing vital organs for detoxification or entry routes of nanomaterials were established for in vitro assay in high-throughput screening (HTS) format for detection of nanoparticle toxicity. Four of these cell based HTS assays were used to screen for cytotoxic effect in 52 nanomaterials manufactured for use in medical and industrial applications. Several nanomaterials with cytotoxic effect were detected, some of these were chosen for further studies. A new method for studying cellular uptake of nanomaterials under flow conditions was established for Human vascular endothelial cell line (HUV-EC-C). This assay was used to study uptake of poly(alkylcyanoacrylate) particles in HUV-EC-Cs under flow conditions compared to static incubation. Cellular uptake was evaluated by confocal microscopy and flow cytometry, no significant differences were found between cellular uptake under perfusion compared to static incubation. Effect on cells was also studied by investigating stress and toxicity related cellular pathways. Reporter arrays were transfected into HepG2, human hepatocellular carcinoma, for detection of cellular pathways following nanomaterial exposure. Heat shock response NFκB, oxidative stress, glucocorticoid, and MAPK/JNK was found to be induced in HepG2 after exsposure to poly(alkylcyanoacrylate) particles. The established screening methods resulted in consistent and reproducible data. These methods provide important tools for high-throughput screening of nanoparticle toxicity in mammalian cells. The perfusion assay provide a new method to compare cytotoxicity under flow conditions and static conditions. The reporter assay in HepG2 cells provide opportunities to study cellular pathways in toxicity induced by nanomaterials. This is of importance for future evaluation of new nanomaterials to be used in industrial and medical applications. 5 Use of nanomaterials is increasing. Efficient and established assays for nanotoxicity are a prerequisite for implementation of new nanomaterials in industrial and medical applications. The toolbox established in this thesis including high-throughput methods, perfusion assays and cellular pathway analysis provide important tools for future nanomaterial evaluation in the SINTETF/NTNU environment.