Publikationen

Herein an assay toward a rapid and reliable profiling of extracellular matrix of Escherichia coli (E. coli) utilizing a tandem of GC-MS as a tool for definition of the exact chemical nature of low molecular weight compounds and cyclic voltammetry for their high throughput detection is presented. Briefly, during a set of investigations the formation of glycerol in the extracellular matrix (ECM) of E. coli at physiological relevant conditions of cells was revealed. Based on the obtained knowledge, the electrochemical protocol allowing both qualitative and quantitative analyses of glycerol in E. coli ECMs at palladium ink-modified screen printed electrodes with precision values (RSD) <10 % and recovery rates ranged from 98 % to 102 % was proposed. The provided protocol for a rapid electrochemical profiling of the bacterial ECMs can readily be used as a guideline for the controlled electroanalysis of target electroactive signaling analytes in complex biological samples.

In living therapeutic materials (LTMs), organisms genetically programmed to produce and deliver drugs are encapsulated in porous matrices acting as physical barriers between the therapeutic organisms and the host cells. LTMs consisting of engineered E. coli encapsulated in Pluronic F127-based hydrogels have been frequently used in LTM designs but their immunogenicity has not been tested. In this study, we investigate the response of human peripheral blood mononuclear cells (PBMCs) exposed to this bacteria/hydrogel combination. The release of inflammation-related cytokines and cytotoxic proteins and the subsets of natural killer cells and T cells were examined. Encapsulation of the bacteria in hydrogels considerably lowers their immunogenicity. ClearColi, an endotoxin-free variant of E. coli, did not polarize NK cells into the more cytolytic CD16dim subset as E. coli. Our results demonstrate that ClearColi-encapsulated hydrogels generate low immunogenic response and are suitable candidates for the development of LTMs for in vivo testing to assess a potential clinical use. Nevertheless, we observed a stronger immune response (elevated levels of IFNγ, IL-6 and cytotoxic proteins) in pro-inflammatory PBMCs characterized by a high spontaneous release of IL-2. This highlights the need to identify recipients who have a higher likelihood of experiencing undesired immune responses to LTMs with IL-2 serving as a potential predictive marker. Additionally, including anti-inflammatory measures in living therapeutic material designs could be beneficial for such recipients.

Hydrogel precursors that crosslink within minutes are essential for the development of cell encapsulation matrices and their implementation in automated systems. Such timescales allow sufficient mixing of cells and hydrogel precursors under low shear forces and the achievement of homogeneous networks and cell distributions in the 3D cell culture. The previous work showed that the thiol-tetrazole methylsulfone (TzMS) reaction crosslinks star-poly(ethylene glycol) (PEG) hydrogels within minutes at around physiological pH and can be accelerated or slowed down with small pH changes. The resulting hydrogels are cytocompatible and stable in cell culture conditions. Here, the gelation kinetics and mechanical properties of PEG-based hydrogels formed by thiol-TzMS crosslinking as a function of buffer, crosslinker structure and degree of TzMS functionality are reported. Crosslinkers of different architecture, length and chemical nature (PEG versus peptide) are tested, and degree of TzMS functionality is modified by inclusion of RGD cell-adhesive ligand, all at concentration ranges typically used in cell culture. These studies corroborate that thiol/PEG-4TzMS hydrogels show gelation times and stiffnesses that are suitable for 3D cell encapsulation and tunable through changes in hydrogel composition. The results of this study guide formulation of encapsulating hydrogels for manual and automated 3D cell culture.

Microbial biofactories allow the upscaled production of high-value compounds in biotechnological processes. This is particularly advantageous for compounds like flavonoids that promote better health through their antioxidant, antibacterial, anticancer and other beneficial effects but are only produced in small quantities in their natural plant-based hosts. Bacteria like E. coli have been genetically modified with enzyme cascades to produce flavonoids like naringenin and pinocembrin from coumaric or cinnamic acid. Despite advancements in yield optimization, the production of these compounds still involves high costs associated with their biosynthesis, purification, storage and transport. An alternative production strategy could involve the direct delivery of the microbial biofactories to the body. In such a strategy, ensuring biocontainment of the engineered microbes in the body and controlling production rates are major challenges. In this study, these two aspects are addressed by developing engineered living materials (ELMs) consisting of probiotic microbial biofactories encapsulated in biocompatible hydrogels. Engineered probiotic E. coli Nissle 1917 able to efficiently convert cinnamic acid into pinocembrin were encapsulated in poly(vinyl alcohol)-based hydrogels. The biofactories are contained in the hydrogels for a month and remain metabolically active during this time. Control over production levels is achieved by the containment inside the material, which regulates bacteria growth, and by the amount of cinnamic acid in the medium.

Methylsulfone derivatized poly(ethylene) glycol (PEG) macromers can be biofunctionalized with thiolated ligands and cross-linked with thiol-based cross-linkers to obtain bioactive PEG hydrogels for in situ cell encapsulation. Methylsulfonyl-thiol (MS-SH) reactions present several advantages for this purpose when compared to other thiol-based cross-linking systems. They proceed with adequate and tunable kinetics for encapsulation, they reach a high conversion degree with good selectivity, and they generate stable reaction products. Our previous work demonstrated the cytocompatibility of cross-linked PEG-MS/thiol hydrogels in contact with fibroblasts. However, the cytocompatibility of the in situ MS-SH cross-linking reaction itself, which generates methylsulfinic acid as byproduct at the cross-linked site, remains to be evaluated. These studies are necessary to evaluate the potential of these systems for in vivo applications. Here we perform an extensive cytocompatibility study of PEG hydrogels during in situ cross-linking by the methylsulfonyl-thiol reaction. We compare these results with maleimide–thiol cross-linked PEGs which are well established for cell culture and in vivo experiments and do not involve the release of a byproduct. We show that fibroblasts and endothelial cells remain viable after in situ polymerization of methylsulfonyl-thiol gels on the top of the cell layers. Cell viability seems better than after in situ cross-linking hydrogels with maleimide–thiol chemistry. The endothelial cell proinflammatory phenotype is low and similar to the one obtained by the maleimide–thiol reaction. Finally, no activation of monocytes is observed. All in all, these results demonstrate that the methylsulfonyl-thiol chemistry is cytocompatible and does not trigger high pro-inflammatory responses in endothelial cells and monocytes. These results make methylsulfonyl-thiol chemistries eligible for in vivo testing and eventually clinical application in the future.

Coating laser-patterned stainless-steel surfaces with carbon nanotubes (CNT) or carbon onions (CO) forms a tribological system that provides effective solid lubrication. Lubricant retention represents the fundamental mechanism of this system, as storing the particles inside the pattern prevents lubricant depletion in the contact area. In previous works, we used direct laser interference patterning to create line patterns with three different structural depths on AISI 304 stainless-steel platelets. Electrophoretic deposition subsequently coated the patterned surfaces with either CNTs or COs. Ball-on-disc friction tests were conducted to study the effect of structural depth on the solid lubricity of as-described surfaces. The results demonstrated that the shallower the textures, the lower the coefficient of friction, regardless of the applied particle type. This follow-up study examines the carbon nanoparticles’ structural degradation after friction testing on substrates patterned with different structural depths (0.24, 0.36, and 0.77 µm). Raman characterization shows severe degradation of both particle types and is used to classify their degradation state within Ferrari’s three-stage amorphization model. It was further shown that improving CNT lubricity translates into increasing particle defectivity. This is confirmed by electron microscopy, which shows decreasing crystalline domains. Compared to CNTs, CO-derived tribofilms show even more substantial structural degradation.

ABSTRACTWe present an approximate, analytical treatment for the linearly elastic response of a film with arbitrary Poisson's ratio ?, which is indented by a flat cylindrical punch while resting on a rigid foundation. Our approach is based on a simple scaling argument allowing the vast changes of the elastomer?s effective modulus E? with the ratio of film height h and indenter radius a to be described with a compact, analytical expression. This yields exact asymptotics for large and small reduced film heights h/a, whereby it also reproduces the observation that E?(h/a) has a pronounced minimum for ?>0.49 at h/a≈1.6. Using Green?s function molecular dynamics (GFMD), we demonstrate that the predictions for E?(h/a) are reasonably correct and generate accurate reference data for effective modulus and pull-off force. GFMD also reveals that the nature of surface instabilities occurring during stable crack growth as well as the crack initiation itself depend sensitively on the way how continuum mechanics is terminated at small scales, that is, on parameters beyond the two dimensionless numbers h/a and ? defining the continuum problem.

We study how the commonly neglected coupling of normal and in-plane elastic response affects tribological properties when Hertzian or randomly rough indenters slide past an elastic body. Compressibility-induced coupling is found to substantially increase maximum tensile stresses, which cause materials to fail, and to decrease friction such that Amontons’ law is violated macroscopically even when it holds microscopically. Confinement-induced coupling increases friction and enlarges domains of high tension. Moreover, both types of coupling affect the gap topography and thereby leakage. Thus, coupling can be much more than a minor perturbation of a mechanical contact.

Viscoelasticity is well known to cause significant hysteresis of crack closure and opening when an elastomer is brought in and out of contact with a flat, rigid, adhesive counterface. A separate origin of adhesive hysteresis is small-scale, elastic multistability. Here, we study a system in which both mechanisms act concurrently. Specifically, we compare the simulated and experimentally measured time evolution of the interfacial force and the real contact area between a soft elastomer and a rigid, flat punch, to which small-scale, single-sinusoidal roughness is added. To this end, we further the Green’s function molecular dynamics method and extend recently developed imaging techniques to elucidate the rate- and preload-dependence of the pull-off process. Our results reveal that hysteresis is much enhanced when the saddle points of the topography come into contact, which, however, is impeded by viscoelastic forces and may require sufficiently large preloads. A similar coaction of viscous- and multistability effects is expected to occur in macroscopic polymer contacts and to be relevant, e.g., for pressure-sensitive adhesives and modern adhesive gripping devices.

In this study, fundamental aspects that have impact on the electroanalytical detection of hydrazine in phosphate, acetate and yeast fermentation medium in an analytically significant concentration range by several types of palladium (Pd)-modified electrodes, namely, Pd-ink, Pd-sputtered films and palladium nanoparticles (Pd-NPs) were systematically studied. The efficiency of hydrazine electrooxidation is not affected by the composition of multicomponent medium (i), presence of oxygen (ii), morphology or electroactive area (iii), but more likely depends on the purity degree of the electrode surface from residual palladium oxides (iv). In addition, using advanced methods of nanoanalytics and quantum chemistry, the crucial role of hydrazine surface adsorption (v) on oxide-free and oxide-based Pd-electrodes is highlighted. The obtained knowledge will provide future development strategies of electrodes based on nanoparticles of noble metals for tuned and efficient hydrazine electrooxidation in complex fermentation media.