During their life cycle, cells are subject to various external cues. In an organism, biochemical signaling via hormones and other messenger molecules is crucial for physiological processes and cell function. An example of a response of cells to a biochemical cue is the epithelial-to-mesenchymal transition (EMT), which plays an important role during in physiological processes like tissue development and wound healing.(Kalluri and Weinberg, 2009) The result of this biochemically triggered event is the transition of cells from an epithelial state into a mesenchymal, more motile phenotype, which encompasses loss of cell-cell contacts as well as cytoskeletal rearrangement. Albeit, EMT can also contribute in a pathophysiological processes like tumor progression. (Thiery, 2002) A key step in the formation of metastasis is the detachment of tumor cells from the primary tumor and invasion into the surrounding tissue, which could be initialized by EMT.(Kumar and Weaver, 2009)
Furthermore, not only soluble molecules or hormones like TGF-β in the cell’s environment influence the behavior of a cell, also chemical composition, topography and the rigidity of the underlying substrate are crucial for cellular behavior. In the last decades it has also been demonstrated that rigidity as well as porosity of the substrate influence migration and proliferation of cells and interestingly, is able to direct the fate of stem cell differentiation.(Clark et al., 1991; Engler et al., 2006; Lo et al., 2000; Teo et al., 2013; Wang et al., 2000) Substrate properties are usually sensed via focal adhesions, which facilitate cell adhesion via heterodimeric transmembrane receptors called intergrins. On intracellular side these receptors are associated with a vast variety of proteins including the actin network.(Geiger et al., 2009) Thereby, the tension generated by the actomyosin cytoskeleton has been shown to be crucial for mechanosensing as well as force generation demonstrating a direct link between substrate properties, cellular shape and mechanics.(Janshoff et al., 2010; Tee et al., 2011; Wolfenson et al., 2011)
From a physics point of view an eukaryotic cell consists of a thin, largely inextensible shell made of surfactant molecules, which is filled with an aqueous, colloidal solution of proteins as well as flexible biopolymer networks or gels that are attached to the outer shell via linkers. (Boal, 2012; Zhou et al., 2009) All together contribute to shape and mechanical resistivity of the cell over a wide range of extensional and compressional stresses.
Interaction between an extracellular particle, i.e. a nanoparticle, and a cell is largely governed by membrane mechanics and the strength of the intermolecular forces between membrane and particle as shown in coarse grain simulations.(Yue et al., 2014) Isotropic particles have been demonstrated to be wrapped by the membrane or form inverse micellar structures within the membrane and are usually taken up by the cell via common endocytotic pathways. (Treuel et al., 2013; Yue et al., 2014) However, in recent years a new subset of nanoparticles emerged. Janus particles or grains, named after the two-faced roman god by soft matter physicist Pierre-Gille de Gennes in his nobel lecture, are anisotropic and possess amphiphilic properties.(De Gennes, 1992; Wurm and Kilbinger, 2009) Coarse grain simulations by Reynwar et al. and Alexeev et al. showed that these particles have effects on the membrane, which differ from the effect of isotropic particles: Janus particles were able to induce large membrane deformations, vesiculation or pore formation. (Alexeev et al., 2008;
Reynwar et al., 2007) On the one hand, these mechanisms could be used in medical applications for drug delivery to circumvent the membrane barrier of cells and increase uptake rates substantially. On the other hand, an unintended uptake of nanomaterials by cells could pose threat to human health. Therefore, profound knowledge of the interaction between cells and these new nanoparticles is necessary and experimental evidence for the proposed types of interactions between particles and membrane has to be provided.
The first part of this thesis will focus on the effects of Janus nanoparticles and their isotropic counterparts on artificial membranes and living cells. Giant unilamellar vesicles (GUVs) serve as a model system for the cellular membrane. The influence of Janus particles on the GUVs is followed using confocal laser scanning microscopy. To quantify the interaction between particles and membrane surface plasmon resonance spectroscopy is employed. Furthermore, uptake of the particles is evaluated by fluorescence microscopy. Finally, cytotoxicity of the particles is measured using electric cell-substrate impedance sensing as well as biochemical assays.
As demonstrated in the first part, cellular mechanics play an important role in the interplay between cells and their environment. Therefore, mechanics of cells exposed to different external stimuli will be examined in the next part of this thesis. For this purpose, a technique introduced by Alcaraz et al. is introduced in the laboratory enabling the measurement of frequency dependent rheological data using the atomic force microscope.(Alcaraz et al., 2003) This technique is used to measure the influence of different chemical drugs and the aforementioned epithelial-to-mesenchymal transition on cellular mechanics. Furthermore, to compare benign and malign cells according to their mechanical properties, rheological properties of eight different cell
Introduction lines with diverging metastatic potential are measured. A difference in the mechanical behavior of malignant and non-malignant cell lines is found. Then, the influence of substrate properties on cellular mechanics and cytoskeletal arrangement is evaluated showing reorganization of the actin cytoskeleton in cells grown on porous substrates, which is accompanied by a softening of cells.
In the final chapter of this thesis, a new technique is presented, which enables measurement of distances between a fluorophore and a metal mirror with nanometer precision by metal-induced energy transfer (MIET) fluorescence lifetime imaging. The method is based on the distance dependent modulation of the fluorescence liftetime of a fluorophore in proximity to a metal a layer up to a fluorophore/metal layer distance of approximately 200 nm. As a first application the distance between the basal membrane of three different cell lines and the substrate is measured. Additionally, the spreading process of cells was followed. However, the method is not restricted to the mapping of the basal membrane of living cells and can be used for applications, which necessitate nanometer resolution in a distance range up to 200 nm.