Synthesis and characterization of fluorescent iron oxide nanoparticles to study uptake and intracellular trafficking of nanoparticles in neural cells
|Other Titles:||Synthese und Charakterisierung von fluoreszierenden Eisenoxidnanopartikeln zur Untersuchung der Aufnahme und des intrazellulären Transports von Nanopartikeln in neuronalen Zellen||Authors:||Willmann, Wiebke||Supervisor:||Dringen, RalfDr.||1. Expert:||Hofmann, Heinrich||2. Expert:||Colombi Ciacchi, Lucio||Abstract:||
Iron oxide nanoparticles (IONPs) have promising features for biomedical applications and are already used for some therapeutic and diagnostic approaches. As IONPs can reach the brain it is important to study the potential consequences of an exposure to IONPs on brain cells. In the presented thesis, fluorescent IONPs were synthesized by functionalizing the coating material dimercaptosuccinate (DMSA) of the IONPs with either the green dye Oregon Green (OG) or the red dye tetramethylrhodamine (TMR). Comparison to previously used BODIPY-labeled DMSA-coated IONPs, OG- and TMR-IONPs revealed higher fluorescence signal intensities and improved stability, while these fluorescent IONPs had almost identical physicochemical properties and colloidal stability as the corresponding non-fluorescent DMSA-coated IONPs. To investigate the accumulation of IONPs in brain cells, C6 glioma cells were used as model system. IONPs exposure studies revealed that these cells accumulate fluorescent and non-fluorescent IONPs in a time-, concentration- and temperature-dependent manner. Due to the strong fluorescence observed in cells that had been exposed to OG- or TMR-IONPs and due to the slow bleaching of cellular fluorescence, these fluorescent IONPs were considered as suitable tools for further studies of cellular uptake and intracellular trafficking of internalized IONPs. To monitor the intracellular trafficking of fluorescent nanoparticles with improved temporal and spatial resolution, single and double nanoparticle pulse-chase experiments were established for OG- and TMR-IONPs. As IONPs efficiently adsorb to the cell membrane but are not internalized at 4AdegreeC, the fluorescent IONPs were bound to the cells by a 10 min pulse at 4AdegreeC. Subsequently, unbound nanoparticles were removed by washing before an increase of the incubation temperature to 37AdegreeC started a synchronized internalization of the IONPs by the cells. Double nanoparticle pulse-chase experiment with the two types of fluorescent IONPs allowed to even monitor the sequential uptake of OG- and TMR-IONPs. The usage of nanoparticle pulse-chase experiments in the presence of inhibitors of the cytoskeleton integrity revealed an actin-dependent formation of IONPs-containing vesicles and a microtubules-dependent transport of these vesicles to the perinuclear area. Additionally, the separation of the fluorescent DMSA coat and the iron oxide core during the intracellular trafficking was observed in nanoparticle pulse-chase experiments. Finally, limitations, requirements and special challenges of studies on the exposure of in cultured neural cells with fluorescent IONPs were investigated, and are described and discussed. In conclusion, the data presented in this thesis revealed that the synthesized fluorescent IONPs are suitable tools to study the uptake and intracellular fate of DMSA-coated IONPs by microscopical approaches and that the established nanoparticles pulse-chase setup allows to study internalization and mechanisms involved in intracellular IONPs trafficking with improved resolution.
|Keywords:||IONPs, iron oxide nanoparticle, fluorescent||Issue Date:||25-May-2018||URN:||urn:nbn:de:gbv:46-00106576-16||Institution:||Universität Bremen||Faculty:||FB2 Biologie/Chemie|
|Appears in Collections:||Dissertationen|
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