Spektro-mikroskopische Charakterisierung von Nano-Bio-Wechselwirkungen in Zellen

In this thesis, the interaction of nanoparticles of different physico-chemical properties with eukaryotic cells (fibroblasts, macrophages, and erythrocytes) was investigated in the context of cellular uptake, processing and cytotoxicity of the nanoparticles. Complementary spectroscopic and microscopic methods were used to study the chemical environment of gold, silver and silica nanoparticles and their distribution within the cellular ultrastructure. The cytotoxic effects were investigated by XTT and live-dead biological tests.
The molecular environment of gold and silver nanoparticles inside the cell during uptake and processing was studied by surface-enhanced Raman scattering (SERS) microscopy. Intrinsic information about the biomolecules in the very proximity of the particle surface or from label molecules, such as molecules that measure intra-endosomal pH, were obtained. This allowed to draw conclusions about the molecular composition of the nanoparticles’ corona in live cells and about molecular changes in the course of endosomal maturation, and to verify transport mechanisms. Multiplexing experiments using different SERS probes prove the capabilities of SERS to differentiate the cellular uptake behavior of different nanoprobes. For the first time, X-ray tomography of cryofixed cells (cryo-XT) was utilized for the visualization of the 3-dimensional particle distribution and arrangement of nanoparticles in the cellular ultrastructure in whole vitrified cells. These experiments illustrate the uptake of the particles by endocytosis and phagocytosis and the encapsulation in vesicles within the cells. A quantitative method based on laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was established and applied to determine the number of different types of internalized nanoparticles at the single-cell level. This involved the development of a new, matrix-matched calibration procedure for particle quantification with LA-ICP-MS.
In order to adopt the SERS capabilities of novel metal nanoparticles to other particle surfaces and to study the properties of other nanomaterials, core-shell particles were designed, which provide spectral information on the silica nano-bio-interaction inside eukaryotic cells (BrightSilica). The metal core also enables quantification of the BrightSilica nanoparticles using LA-ICP-MS micromapping and localization in the 3D cellular ultrastructure by cryo-XT.
Processing of the nanoparticles studied here and their interaction with the cells and with the biomolecules show strong dependence on the chemical nature of the nanoparticle in contact with the cells as well as on their aggregate geometry, formation of a biomolecule corona through adsorption of proteins, particle size and other parameters. With the results of this thesis, methodological progress with respect to the development and combination of analytical methods is demonstrated. Furthermore, new aspects in nanoparticle interaction with complex biological systems are identified.