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Blechinger, Julia (2012): Nanoparticles and human health: development and application of an experimental strategy to estimate cytotoxicity of nanopaticles. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

The potential toxicity of nanoparticles currently raises many discussions in public and scientific life. The question whether nanoparticles are a threat to human health cannot be answered to complete satisfaction at the current state of knowledge. A versatile tool to investigate nanotoxicity are fluorescence microscopy and live-cell imaging as they provide excellent resolution and direct insight into cellular processes. In this work fluorescence based methods are used to investigate the influence of silica nanoparticles on human health, more precisely on the blood vessel system. At first, the synthesis and characterization of the following three different types of perylene labeled amorphous SiO 2 nanoparticle species is described: surface-labeled monodisperse particles, particles with a dye-containing silica core and a non-fluorescent silica shell and a surface-labeled nanoparticle network. The labeling of nanoparticles should not induce artificial cytotoxic effects when they are used for cytotoxicity assessment. This is achieved either by incorporating the dye into the nanoparticle’s structure or by covering only a minor surface fraction by dye molecules. The surface-labeled silica species are used to investigate nanotoxicity throughout this thesis. Another prerequisite for reliable dose-dependent nanotoxicity studies is the knowledge about the number of nanoparticles taken up by an individual cell. We therefore developed the Nano_In_Cell_3D ImageJ macro which is able to quantify nanoparticle uptake into cells. Nano_In_Cell_3D uses the fluorescence image of the cell membrane to segment the cell into an intracellular space, a transition region (e-membrane region) and an extracellular space. The number of present nanoparticles is calculated from the fluorescence intensity of each region. This custom-made method offers the possibility to quantify nanoparticles in the individual cellular regions. Nano_In_Cell_3D was validated by comparing the results to the well established quenching method. By using Nano_In_Cell_3D we could show that the cytotoxic impact of nanoparticles onto different cell lines correlates to their intracellular uptake. Primary human vascular endothelial cells (HUVEC) take up 310 nm silica nanoparticles more efficiently and are more sensitive to this nanoparticle species than cancer cells derived from the cervix carcinoma (HeLa). Upon nanoparticle contact, cellular viability of HUVEC is strongly reduced and membrane permeability increases leading to apoptosis. In contrast, HeLa cells show a considerable lower effect in both cellular viability and membrane permeability and do not show apoptosis. In consistence to these findings, HUVEC take up approximately 20 times more particles than HeLa cells within 4h. Interestingly nanoparticle uptake is clathrin mediated in both cell types. HUVEC grow in the blood vessel system under natural conditions and are therefore exposed to blood flow conditions. The latter can be simulated using a microfluidic system. We chose a microfluidic system based on the surface acoustic wave (SAW) technology which was characterized concerning fluid evaporation behavior, fluid temperature and flow velocities. Based on these results the system can be further improved to allow the assessment of nanotoxicity at blood flow conditions in a next step. The last part of the thesis focuses on interactions between silica nanoparticles and giant unilamellar vesicles (GUV)s. The latter serve as a simple model for the cell membrane. Nanoparticles in contact with the lipid membrane influence the morphological behavior of the vesicles during phase transition. In absence of nanoparticles, vesicles typically show extracellular budding processes. Nanoparticlesin contact with the cell membrane induce intravesicular budding of daughter vesicles, similar to endocytosis observed in living cells. Furthermore exocytosis processes were observed where daughter vesicles crossed the GUV membrane and were transferred from the intravesicular to the extravesicular space. These observations suggest that the fundamental mechanism of endocytosis can partly be explained by simple physical effects. In summary, this theses provides an experimental strategy to investigate the impact of nanoparticles onto human cells using SiO 2 nanoparticles as an example. Starting with the synthesis and characterization of nanoparticles it tackles the question how to quantify nanoparticle uptake into cells. Furthermore we could prove that cytotoxic effects can be correlated to nanoparticle uptake and were able to show that nanoparticles influence artificial membranes which is a first step to understand the basic mechanisms of nano-toxicity. The methodology developed in this thesis is expected to provide insight into cytotoxicity of a broad variety of different nanoparticle types.