Examining Uptake of Nanomaterials by Eukaryotic Cells with Digital Image Cytometry
Due to their small size and related interesting properties, artificial nanoma-terials are utilized for a great number of biological and medical applications. Cell entry routes, intracellular trafficking and processing of nanoparticles, which determine their fate, efficiency, and toxicity, are depend...
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|Zusammenfassung:||Due to their small size and related interesting properties, artificial nanoma-terials are utilized for a great number of biological and medical applications. Cell entry routes, intracellular trafficking and processing of nanoparticles, which determine their fate, efficiency, and toxicity, are depending on various parameters of the specific nanomaterial, such as size, surface charge, surface chemistry and elasticity. Nanoparticle-cell interactions are typically elucidated by means of fluorescence microscopy. Cell functions can be observed by a multiplicity of commercially available probes. For the quantification of cell features from images (image cytometry), computer-based algorithms are favoured to avoid bias introduced by the subjective perception of the observer. By applying high throughput microscopy in combination with digital image cytometry the screening of high numbers of cells is made possible. With the large quantity of obtained data, cell populations can be identified and, in general, results that are statistically meaningful are obtained. In the first part of this work this method is applied in order to examine the cellular responses upon exposure to plasmonic poly(methacrylic acid)-coated gold nanoparticles (Au NPs) with respect to morphology and viability of human endothelial and epithelial cells (HUVECs and HeLa cells). Au NPs of 4-5 nm size were chosen which had been thoroughly characterized in terms of their physico-chemical parameters. These particles bear interesting properties for biomedical applications and, for several years, have been in the focus of research. In this work significant impacts on mitochondrial and lysosomal morphology upon exposure to the Au NPs are reported. The alteration of the structure of the cytoskeleton and a dramatically reduced proliferation are described. Interestingly, the smallest dose inducing the described cellular responses was of one or two magnitudes lower than those, where acute cytotoxicity and an increase in the production of reactive oxygen species (ROS) were observed. In the second part the process of endocytosis of polymer capsules is examined. These systems are seen as a promising tool for intracellular cargo delivery and release. After lipid raft-mediated phagocytosis, the capsules are transferred from the neutral extracellular medium to increasingly acidic intracellular vesicles. By embedding a pH-sensitive fluorescent dye into the cavity of the capsule the uptake process and the associated acidification can be monitored time-dependently. It is demonstrated that the kinetic of the acidification process strongly depends on the stiffness of the capsules. Soft particles with minor stiffness are transported faster into lysosomal structures than stiffer ones. Additionally, these sensor particles are used to confirm the importance of the V1G1-subunit of the vacuolar ATPase being responsible for vesicle acidification.|