Project 13 (PLUS)
Project 13: Identification of specific parameters which influence the impact of nanoparticles on human cells
Fellow: Dr. Matthew Boyles (ER2), 24 months
Tutors: Prof. Dr. Albert Duschl (PLUS)
In direct comparison to stably transfected cell lines (see project 1), primary human cells will be used to study more closely mechanistic aspects of the interaction between human cells and selected NP. As a primary lung epithelium model, normal human bronchial epithelial (NHBE) cells will be used (to be compared with stable A549 cells) and peripheral blood-derived mononuclear cells (PBMC) will be used as model for immune cells present in the blood (to be compared to the stable T cell line Jurkat). Since we have shown in previous studies that a pre- existing activation of the immune system can both increase and decrease responses to specific pollutants, the primary cells will be treated with bacterial LPS or the pro-inflammatory cytokine TNF- ELISA and intracellular protein staining will be applied for these investigations. Special focus will be on the correlation of NP parameters (surface charge, solubility, protein corona) with the results from the in vitro cell systems. NP parameters will be controlled through conjugating different molecules on the surface of the NP and also by using different cell culture media compositions. The role of size and shape of the NP tested will be further examined. Ag NP, which can be produced in many different forms, will be used as paradigmatic example. Moreover, different exposure conditions (flow chamber, inserts) and different incubation times will be analyzed. The uptake of Ag NP with different shapes, sizes and coatings into primary cells will be followed using TEM and molecular mechanisms involved in the induction of apoptosis by toxic particles will be analyzed. The oxidative dissolution of Ag NP will be quantified and correlated with size, shape and coating, as well as with biological effects.
An important result of our research within FP6 DIPNA was that bystander effects were often dominating effects directly induced by NP. Hence, we will investigate the impact of relevant single compounds (stabilizers, coating molecules, residues of chemicals used in the synthesis, etc.) to improve our cell-based methods for nanotoxicology studies. These experiments will be carried out early on and will provide a guideline for all other projects. The project will employ one ER, who will acquire skills in molecular and cell based assays, in the validation of assays regarding bystander effects as well as in the correlation of NP parameters and biological effects. Secondments to partners 4 (validation of tests in ongoing screening projects), 9 (inter-laboratory standardization of test conditions), 11 (optical NP-cell interaction), 12 (characterization of NP under relevant conditions).
In WP1 (tasks 1.1 and 1.5), WP2 (tasks 2.1, 2.4 and 2.5), WP3 (task 3.2) and WP4 (tasks 4.1, 4.2, 4.3 and 4.5).