Browsing by Author "Koch, Marcus"
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- ItemBacteriomimetic Liposomes Improve Antibiotic Activity of a Novel Energy-Coupling Factor Transporter Inhibitor(Basel : MDPI, 2021) Drost, Menka; Diamanti, Eleonora; Fuhrmann, Kathrin; Goes, Adriely; Shams, Atanaz; Haupenthal, Jörg; Koch, Marcus; Hirsch, Anna K. H.; Fuhrmann, GregorLiposomes have been studied for decades as nanoparticulate drug delivery systems for cytostatics, and more recently, for antibiotics. Such nanoantibiotics show improved antibacterial efficacy compared to the free drug and can be effective despite bacterial recalcitrance. In this work, we present a loading method of bacteriomimetic liposomes for a novel, hydrophobic compound (HIPS5031) inhibiting energy-coupling factor transporters (ECF transporters), an underexplored antimicrobial target. The liposomes were composed of DOPG (18:1 (Δ9-cis) phosphatidylglycerol) and CL (cardiolipin), resembling the cell membrane of Gram-positive Staphylococcus aureus and Streptococcus pneumoniae, and enriched with cholesterol (Chol). The size and polydispersity of the DOPG/CL/± Chol liposomes remained stable over 8 weeks when stored at 4 °C. Loading of the ECF transporter inhibitor was achieved by thin film hydration and led to a high encapsulation efficiency of 33.19% ± 9.5% into the DOPG/CL/Chol liposomes compared to the phosphatidylcholine liposomes (DMPC/DPPC). Bacterial growth inhibition assays on the model organism Bacillus subtilis revealed liposomal HIPS5031 as superior to the free drug, showing a 3.5-fold reduction in CFU/mL at a concentration of 9.64 µM. Liposomal HIPS5031 was also shown to reduce B. subtilis biofilm. Our findings present an explorative basis for bacteriomimetic liposomes as a strategy against drug-resistant pathogens by surpassing the drug-formulation barriers of innovative, yet unfavorably hydrophobic, antibiotics.
- ItemA correlative analysis of gold nanoparticles internalized by A549 cells(Hoboken, NJ : Wiley, 2014) Böse, Katharina; Koch, Marcus; Cavelius, Christian; Kiemer, Alexandra K.; Kraegeloh, AnnetteFluorescently labeled nanoparticles are widely used to investigate nanoparticle cell interactions by fluorescence microscopy. Owing to limited lateral and axial resolution, nanostructures (<100 nm) cannot be resolved by conventional light microscopy techniques. Especially after uptake into cells, a common fate of the fluorescence label and the particle core cannot be taken for granted. In this study, a correlative approach is presented to image fluorescently labeled gold nanoparticles inside whole cells by correlative light and electron microscopy (CLEM). This approach allows for detection of the fluorescently labeled particle shell as well as for the gold core in one sample. In this setup, A549 cells are exposed to 8 nm Atto 647N-labeled gold nanoparticles (3.3 × 109 particles mL−1, 0.02 μg Au mL−1) for 5 h and are subsequently imaged by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). Eight fluorescence signals located at different intracellular positions are further analyzed by TEM. Five of the eight fluorescence spots are correlated with isolated or agglomerated gold nanoparticles. Three fluorescence signals could not be related to the presence of gold, indicating a loss of the particle shell.
- ItemDifferential cell reaction upon Toll-like receptor 4 and 9 activation in human alveolar and lung interstitial macrophages(London : BioMed Central, 2010) Koch, Marcus; Hoppstädter, Jessica; Diesel, Britta; Zarbock, Robert; Breinig, Tanja; Monz, Dominik; Meyerhans, Andreas; Gortner, Ludwig; Lehr, Claus-Michael; Huwer, Hanno; Kiemer, Alexandra K.Background Investigations on pulmonary macrophages (MΦ) mostly focus on alveolar MΦ (AM) as a well-defined cell population. Characteristics of MΦ in the interstitium, referred to as lung interstitial MΦ (IM), are rather ill-defined. In this study we therefore aimed to elucidate differences between AM and IM obtained from human lung tissue. Methods Human AM and IM were isolated from human non-tumor lung tissue from patients undergoing lung resection. Cell morphology was visualized using either light, electron or confocal microscopy. Phagocytic activity was analyzed by flow cytometry as well as confocal microscopy. Surface marker expression was measured by flow cytometry. Toll-like receptor (TLR) expression patterns as well as cytokine expression upon TLR4 or TLR9 stimulation were assessed by real time RT-PCR and cytokine protein production was measured using a fluorescent bead-based immunoassay. Results IM were found to be smaller and morphologically more heterogeneous than AM, whereas phagocytic activity was similar in both cell types. HLA-DR expression was markedly higher in IM compared to AM. Although analysis of TLR expression profiles revealed no differences between the two cell populations, AM and IM clearly varied in cell reaction upon activation. Both MΦ populations were markedly activated by LPS as well as DNA isolated from attenuated mycobacterial strains (M. bovis H37Ra and BCG). Whereas AM expressed higher amounts of inflammatory cytokines upon activation, IM were more efficient in producing immunoregulatory cytokines, such as IL10, IL1ra, and IL6. Conclusion AM appear to be more effective as a non-specific first line of defence against inhaled pathogens, whereas IM show a more pronounced regulatory function. These dissimilarities should be taken into consideration in future studies on the role of human lung MΦ in the inflammatory response.
- ItemDimethylaminoethyl methacrylate copolymer-siRNA nanoparticles for silencing a therapeutically relevant gene in macrophages(Cambridge : Royal Society of Chemistry, 2015) Jain, Ratnesh; Dandekar, Prajakta; Loretz, Brigitta; Koch, Marcus; Lehr, Claus-MichaelTherapeutic gene silencing using small-interfering RNA (siRNA) for treatment of bacterial infections has been neglected in comparison with cancer and viral infections. The aim of our investigation was to formulate siRNA-loaded nanoparticles, using an established cationic polymethacrylate polymer, to enhance the delivery of siRNA into the cytoplasm of macrophages that host many pathogenic bacterial species, including tuberculosis. Nanoparticles of cationic dimethylaminoethyl methacrylate copolymer (Eudragit[registered sign] E 100) were successfully formulated and were found to efficiently bind the siRNA molecules (Cy3-siRNA, Bfl1/A1 siRNA). The efficiency of nanoparticles in overcoming cellular barriers to intracellular siRNA delivery and the precise pathway of endocytosis of nanoparticles were both confirmed using confocal microscopy. Through efficient siRNA release into the cytoplasm, the siRNA-loaded nanoparticles enabled a five-fold enhancement in the knockdown efficiency of the endogenous host gene Bfl1/A1, when the formulation was compared with free siRNA. Persistence of Bfl1/A1 is useful for phagolysosomal survival of tuberculosis bacteria in macrophages, and the nanoparticles offer a promising concept for exploitation as an anti-tuberculosis therapy.
- ItemDistribution of SiO2 nanoparticles in 3D liver microtissues(Macclesfield : Dove Medical Press, 2019) Fleddermann, Jana; Susewind, Julia; Peuschel, Henrike; Koch, Marcus; Tavernaro, Isabella; Kraegeloh, AnnetteIntroduction: Nanoparticles (NPs) are used in numerous products in technical fields and biomedicine; their potential adverse effects have to be considered in order to achieve safe applications. Besides their distribution in tissues, organs, and cellular localization, their impact and penetration during the process of tissue formation occurring in vivo during liver regeneration are critical steps for establishment of safe nanomaterials. Materials and methods: In this study, 3D cell culture of human hepatocarcinoma cells (HepG2) was used to generate cellular spheroids, serving as in vitro liver microtissues. In order to determine their differential distribution and penetration depth in HepG2 spheroids, SiO2 NPs were applied either during or after spheroid formation. The NP penetration was comprehensively studied using confocal laser scanning microscopy and scanning electron microscopy. Results: Spheroids were exposed to 100 µg mL-1 SiO2 NPs either at the beginning of spheroid formation, or during or after formation of spheroids. Microscopy analyses revealed that NP penetration into the spheroid is limited. During and after spheroid formation, SiO2 NPs penetrated about 20 µm into the spheroids, corresponding to about three cell layers. In contrast, because of the addition of SiO2 NPs simultaneously to cell seeding, NP agglomerates were located also in the spheroid center. Application of SiO2 NPs during the process of spheroid formation had no impact on final spheroid size. Conclusion: Understanding the distribution of NPs in tissues is essential for biomedical applications. The obtained results indicate that NPs show only limited penetration into already formed tissue, which is probably caused by the alteration of the tissue structure and cell packing density during the process of spheroid formation.
- ItemEnhanced uptake and siRNA-mediated knockdown of a biologically relevant gene using cyclodextrin polyrotaxane(Cambridge : Royal Society of Chemistry, 2015) Dandekar, P.; Jain, R.; Keil, M.; Loretz, B.; Koch, Marcus; Wenz, G.; Lehr, Claus-MichaelIdeal cationic polymers for siRNA delivery could result in its enhanced cellular internalization, escape from endosomal degradation, and rapid release in cell cytoplasm, to facilitate knockdown of the target gene. In this study, we have investigated the ability of an in-house synthesized cationic polyrotaxane to bind siRNA into nanometric complexes. This polymer, which had earlier shown improved transfection of model siRNA (luciferase), was used to improve the cellular internalization of the siRNA molecule with therapeutic implications. In cellular assays, the polymer enhanced the knockdown of a gene involved in the pathogenesis of tuberculosis, when the nanocomplexes were compared with free siRNA. The efficacy and cellular non-toxicity of this polymer encourage its further exploitation in animal models of tuberculosis and other intracellular bacterial infections.
- ItemEnhancing the Stabilization Potential of Lyophilization for Extracellular Vesicles(Weinheim : Wiley-VCH, 2021) Trenkenschuh, Eduard; Richter, Maximilian; Heinrich, Eilien; Koch, Marcus; Fuhrmann, Gregor; Friess, WolfgangExtracellular vesicles (EV) are an emerging technology as immune therapeutics and drug delivery vehicles. However, EVs are usually stored at −80 °C which limits potential clinical applicability. Freeze-drying of EVs striving for long-term stable formulations is therefore studied. The most appropriate formulation parameters are identified in freeze-thawing studies with two different EV types. After a freeze-drying feasibility study, four lyophilized EV formulations are tested for storage stability for up to 6 months. Freeze-thawing studies revealed improved colloidal EV stability in presence of sucrose or potassium phosphate buffer instead of sodium phosphate buffer or phosphate-buffered saline. Less aggregation and/or vesicle fusion occurred at neutral pH compared to slightly acidic or alkaline pH. EVs colloidal stability can be most effectively preserved by addition of low amounts of poloxamer 188. Polyvinyl pyrrolidone failed to preserve EVs upon freeze-drying. Particle size and concentration of EVs are retained over 6 months at 40 °C in lyophilizates containing 10 mm K- or Na-phosphate buffer, 0.02% poloxamer 188, and 5% sucrose. The biological activity of associated beta-glucuronidase is maintained for 1 month, but decreased after 6 months. Here optimized parameters for lyophilization of EVs that contribute to generate long-term stable EV formulations are presented. © 2021 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH
- ItemFailure mechanism analysis based on laser-based surface treatments for aluminum-polyamide laser joining(Amsterdam [u.a.] : Elsevier, 2021) Elahi, Amne; Koch, Marcus; Bardon, Julien; Addiego, Frédéric; Plapper, PeterThe development of strong metal to polymer assemblies is currently an important research subject thanks to its prominence to develop lightweight structures. Furthermore, laser welding is known to be a fast, reliable, and versatile joining process, and it was demonstrated recently that it can be applied to such metal to polymer systems. To enhance the mechanical properties of the laser-joined aluminum-polyamide (Al-PA) specimens, laser polishing and laser ablation processes have been implemented on the aluminum surface before joining. The polyamide surface was also treated with the laser beam, separately. The surfaces were tested by several characterization techniques before and after each surface treatment. Then aluminum and polyamide samples with different surface treatments have been joined with an identical laser joining process. The mechanical properties of the joints in single lap shear configuration are reported and the failure mechanisms are discussed based on micro-computed x-ray tomography imaging of joined specimens and microscopic analysis before failure. Results show that both surface treatments of aluminum significantly improve the shear load of the joint; however, with different failure mechanisms. Polyamide surface treatment and increasing degree of crystallinity are effective when combined with the laser polishing of the Al surface. This combination is responsible for further enhancement of the shear load of the joint to the limit of base metal strength which is approximately 60 % improvement compared to the untreated samples. Finally, energy dispersive X-ray mapping shows the physicochemical bonding between aluminum oxide and polyamide at the interface.
- ItemFriction force microscopy of tribochemistry and interfacial ageing for the SiOx/Si/Au system(Frankfurt am Main : Beilstein-Institut, 2018) Petzhold, Chritinane; Koch, Marcus; Bennewitz, RolandFriction force microscopy was performed with oxidized or gold-coated silicon tips sliding on Au(111) or oxidized Si(100) surfaces in ultrahigh vacuum. We measured very low friction forces compared to adhesion forces and found a modulation of lateral forces reflecting the atomic structure of the surfaces. Holding the force-microscopy tip stationary for some time did not lead to an increase in static friction, i.e., no contact ageing was observed for these pairs of tip and surface. Passivating layers from tip or surface were removed in order to allow for contact ageing through the development of chemical bonds in the static contact. After removal of the passivating layers, tribochemical reactions resulted in strong friction forces and tip wear. Friction, wear, and the re-passivation by oxides are discussed based on results for the temporal development of friction forces, on images of the scanned area after friction force microscopy experiments, and on electron microscopy of the tips.
- ItemGivinostat-Liposomes: Anti-Tumor Effect on 2D and 3D Glioblastoma Models and Pharmacokinetics(Basel : MDPI, 2022) Taiarol, Lorenzo; Bigogno, Chiara; Sesana, Silvia; Kravicz, Marcelo; Viale, Francesca; Pozzi, Eleonora; Monza, Laura; Carozzi, Valentina Alda; Meregalli, Cristina; Valtorta, Silvia; Moresco, Rosa Maria; Koch, Marcus; Barbugian, Federica; Russo, Laura; Dondio, Giulio; Steinkühler, Christian; Re, FrancescaGlioblastoma is the most common and aggressive brain tumor, associated with poor prognosis and survival, representing a challenging medical issue for neurooncologists. Dysregulation of histone-modifying enzymes (HDACs) is commonly identified in many tumors and has been linked to cancer proliferation, changes in metabolism, and drug resistance. These findings led to the development of HDAC inhibitors, which are limited by their narrow therapeutic index. In this work, we provide the proof of concept for a delivery system that can improve the in vivo half-life and increase the brain delivery of Givinostat, a pan-HDAC inhibitor. Here, 150-nm-sized liposomes composed of cholesterol and sphingomyelin with or without surface decoration with mApoE peptide, inhibited human glioblastoma cell growth in 2D and 3D models by inducing a time-and dose-dependent reduction in cell viability, reduction in the receptors involved in cholesterol metabolism (from −25% to −75% of protein levels), and reduction in HDAC activity (−25% within 30 min). In addition, liposome-Givinostat formulations showed a 2.5-fold increase in the drug half-life in the bloodstream and a 6-fold increase in the amount of drug entering the brain in healthy mice, without any signs of overt toxicity. These features make liposomes loaded with Givinostat valuable as potential candidates for glioblastoma therapy.
- ItemImproving the electrical and structural stability of highly piezoresistive nickel–carbon sensor thin films(Göttingen : Copernicus Publ., 2022) Schultes, Günter; Cerino, Mario; Lellig, Angela; Koch, MarcusThe family of sputter deposited granular metal-based carbon-containing sensor films is known for their high sensitivity transforming force-dependent strain into electrical resistance change. Among them nickel–carbon thin films possess a gauge factor of up to 30, compared to only 2 for traditional sensor films of metal alloys. This high sensitivity is based on disordered interparticle tunneling through barriers of graphite-like carbon walls between metal–carbon particles of columnar shape. Force and pressure sensors would benefit a lot from the elevated piezoresistivity. A disadvantage, however, is a disturbing temporal creep and drift of the resistance under load and temperature. This contribution shows how to stabilize such sensor films. A significant stabilization is achieved by partially replacing nickel with chromium, albeit at the expense of sensitivity. The more chromium used in these NixCr1−x-C layers, the higher the optimum annealing temperature can be selected and the better the electrical stabilization. A good compromise while maintaining sensitivities well above the standard of 2 is identified for films with x=0.5 to 0.9, stabilized by optimized temperature treatments. The stabilizing effect of chromium is revealed by transmission electron microscopy with elemental analysis. The post-annealing drives segregation processes in the layer material. While the interior of the layer is depleted of chromium and carbon, boundary layers are formed. Chromium is enriched near the surface boundary, oxidized in air and forms chromium-rich oxide sub-layers, which are chemically very stable and protect against further reactions and corrosion. As a result, creep and drift errors are greatly reduced, so that the optimized sensor coatings are now suitable for widespread use.
- ItemKinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles(London : Biomed Central, 2018) Yasar, Hanzey; Biehl, Alexander; De Rossi, Chiara; Koch, Marcus; Murgia, Xabi; Loretz, Brigitta; Lehr, Claus-MichaelBackground: Messenger RNA (mRNA) has gained remarkable attention as an alternative to DNA-based therapies in biomedical research. A variety of biodegradable nanoparticles (NPs) has been developed including lipid-based and polymer-based systems for mRNA delivery. However, both systems still lack in achieving an efficient transfection rate and a detailed understanding of the mRNA transgene expression kinetics. Therefore, quantitative analysis of the time-dependent translation behavior would provide a better understanding of mRNA's transient nature and further aid the enhancement of appropriate carriers with the perspective to generate future precision nanomedicines with quick response to treat various diseases. Results: A lipid-polymer hybrid system complexed with mRNA was evaluated regarding its efficiency to transfect dendritic cells (DCs) by simultaneous live cell video imaging of both particle uptake and reporter gene expression. We prepared and optimized NPs consisting of poly (lactid-co-glycolid) (PLGA) coated with the cationic lipid 1, 2-di-O-octadecenyl-3-trimethylammonium propane abbreviated as LPNs. An earlier developed polymer-based delivery system (chitosan-PLGA NPs) served for comparison. Both NPs types were complexed with mRNA-mCherry at various ratios. While cellular uptake and toxicity of either NPs was comparable, LPNs showed a significantly higher transfection efficiency of ~ 80% while chitosan-PLGA NPs revealed only ~ 5%. Further kinetic analysis elicited a start of protein translation after 1 h, with a maximum after 4 h and drop of transgene expression after 48 h post-transfection, in agreement with the transient nature of mRNA. Conclusions: Charge-mediated complexation of mRNA to NPs enables efficient and fast cellular delivery and subsequent protein translation. While cellular uptake of both NP types was comparable, mRNA transgene expression was superior to polymer-based NPs when delivered by lipid-polymer NPs.
- ItemLight emission intensities of luminescent Y2O3:Eu and Gd2O3:Eu particles of various sizes(Basel : MDPI, 2017) Adam, Jens; Metzger, Wilhelm; Koch, Marcus; Rogin, Peter; Coenen, Toon; Atchison, Jennifer S.; König, PeterThere is great technological interest in elucidating the effect of particle size on the luminescence efficiency of doped rare earth oxides. This study demonstrates unambiguously that there is a size effect and that it is not dependent on the calcination temperature. The Y2O3:Eu and Gd2O3:Eu particles used in this study were synthesized using wet chemistry to produce particles ranging in size between 7 nm and 326 nm and a commercially available phosphor. These particles were characterized using three excitation methods: UV light at 250 nm wavelength, electron beam at 10 kV, and X-rays generated at 100 kV. Regardless of the excitation source, it was found that with increasing particle diameter there is an increase in emitted light. Furthermore, dense particles emit more light than porous particles. These results can be explained by considering the larger surface area to volume ratio of the smallest particles and increased internal surface area of the pores found in the large particles. For the small particles, the additional surface area hosts adsorbates that lead to non-radiative recombination, and in the porous particles, the pore walls can quench fluorescence. This trend is valid across calcination temperatures and is evident when comparing particles from the same calcination temperature.
- ItemLipid droplets as a novel cargo of tunnelling nanotubes in endothelial cells(London : Nature Publishing Group, 2015) Astanina, Ksenia; Koch, Marcus; Jüngst, Christian; Zumbusch, Andreas; Kiemer, Alexandra K.Intercellular communication is a fundamental process in the development and functioning of multicellular organisms. Recently, an essentially new type of intercellular communication, based on thin membrane channels between cells, has been reported. These structures, termed intercellular or tunnelling nanotubes (TNTs), permit the direct exchange of various components or signals (e.g., ions, proteins, or organelles) between non-adjacent cells at distances over 100 μm. Our studies revealed the presence of tunnelling nanotubes in microvascular endothelial cells (HMEC-1). The TNTs were studied with live cell imaging, environmental scanning electron microscopy (ESEM), and coherent anti-Stokes Raman scattering spectroscopy (CARS). Tunneling nanotubes showed marked persistence: the TNTs could connect cells over long distances (up to 150 μm) for several hours. Several cellular organelles were present in TNTs, such as lysosomes and mitochondria. Moreover, we could identify lipid droplets as a novel type of cargo in the TNTs. Under angiogenic conditions (VEGF treatment) the number of lipid droplets increased significantly. Arachidonic acid application not only increased the number of lipid droplets but also tripled the extent of TNT formation. Taken together, our results provide the first demonstration of lipid droplets as a cargo of TNTs and thereby open a new field in intercellular communication research.
- ItemLipid–Polymer Hybrid Nanoparticles for mRNA Delivery to Dendritic Cells: Impact of Lipid Composition on Performance in Different Media(Basel : MDPI, 2022) Kliesch, Lena; Delandre, Simon; Gabelmann, Aljoscha; Koch, Marcus; Schulze, Kai; Guzmán, Carlos A.; Loretz, Brigitta; Lehr, Claus-MichaelTo combine the excellent transfection properties of lipids with the high stability of polymeric nanoparticles, we designed a hybrid system with a polymeric core surrounded by a shell of different lipids. The aim is to use this technology for skin vaccination purposes where the transfection of dendritic cells is crucial. Based on a carrier made of PLGA and the positively charged lipid DOTMA, we prepared a panel of nanocarriers with increasing amounts of the zwitterionic phospholipid DOPE in the lipid layer to improve their cell tolerability. We selected a nomenclature accordingly with numbers in brackets to represent the used mol% of DOPE and DOTMA in the lipid layer, respectively. We loaded mRNA onto the surface and assessed the mRNA binding efficacy and the degree of protection against RNases. We investigated the influence of the lipid composition on the toxicity, uptake and transfection in the dendritic cell line DC 2.4 challenging the formulations with different medium supplements like fetal calf serum (FCS) and salts. After selecting the most promising candidate, we performed an immune stimulation assay with primary mouse derived dendritic cells. The experiments showed that all tested lipid–polymer nanoparticles (LPNs) have comparable hydrodynamic parameters with sizes between 200 and 250 nm and are able to bind mRNA electrostatically due to their positive zetapotential (20–40 mV for most formulations). The more of DOPE we add, the more free mRNA we find and the better the cellular uptake reaching approx. 100% for LPN(60/40)–LPN(90/10). This applies for all tested formulations leading to LPN(70/30) with the best performance, in terms of 67% of live cells with protein expression. In that case, the supplements of the medium did not influence the transfection efficacy (56% vs. 67% (suppl. medium) for live cells and 63% vs. 71% in total population). We finally confirmed this finding using mouse derived primary immune cells. We can conclude that a certain amount of DOTMA in the lipid coating of the polymer core is essential for complexation of the mRNA, but the zwitterionic phospholipid DOPE is also important for the particles’ performance in supplemented media.
- ItemMelt Electrowriting of Graded Porous Scaffolds to Mimic the Matrix Structure of the Human Trabecular Meshwork(Washington, DC : ACS Publ., 2022) Włodarczyk-Biegun, Małgorzata K.; Villiou, Maria; Koch, Marcus; Muth, Christina; Wang, Peixi; Ott, Jenna; del Campo, AranzazuThe permeability of the human trabecular meshwork (HTM) regulates eye pressure via a porosity gradient across its thickness modulated by stacked layers of matrix fibrils and cells. Changes in HTM porosity are associated with increases in intraocular pressure and the progress of diseases such as glaucoma. Engineered HTMs could help to understand the structure-function relation in natural tissues and lead to new regenerative solutions. Here, melt electrowriting (MEW) is explored as a biofabrication technique to produce fibrillar, porous scaffolds that mimic the multilayer, gradient structure of native HTM. Poly(caprolactone) constructs with a height of 125-500 μm and fiber diameters of 10-12 μm are printed. Scaffolds with a tensile modulus between 5.6 and 13 MPa and a static compression modulus in the range of 6-360 kPa are obtained by varying the scaffold design, that is, the density and orientation of the fibers and number of stacked layers. Primary HTM cells attach to the scaffolds, proliferate, and form a confluent layer within 8-14 days, depending on the scaffold design. High cell viability and cell morphology close to that in the native tissue are observed. The present work demonstrates the utility of MEW for reconstructing complex morphological features of natural tissues.
- ItemMelt Electrowriting of Scaffolds with a Porosity Gradient to Mimic the Matrix Structure of the Human Trabecular Meshwork(New York : Cold Spring Harbor Laboratory, 2022) Włodarczyk-Biegun, Małgorzata K.; Villiou, Maria; Koch, Marcus; Muth, Christina; Wang, Peixi; Ott, Jenna; del Campo, AranzazuThe permeability of the Human Trabecular Meshwork (HTM) regulates eye pressure via a porosity gradient across its thickness modulated by stacked layers of matrix fibrils and cells. Changes in HTM porosity are associated with increases in intraocular pressure and the progress of diseases like glaucoma. Engineered HTMs could help to understand the structure-function relation in natural tissues, and lead to new regenerative solutions. Here, melt electrowriting (MEW) is explored as a biofabrication technique to produce fibrillar, porous scaffolds that mimic the multilayer, gradient structure of native HTM. Poly(caprolactone) constructs with a height of 125-500 μm and fiber diameters of 10-12 μm are printed. Scaffolds with a tensile modulus between 5.6 and 13 MPa, and a static compression modulus in the range of 6-360 kPa are obtained by varying the scaffolds design, i.e., density and orientation of the fibers and number of stacked layers. Primary HTM cells attach to the scaffolds, proliferate, and form a confluent layer within 8-14 days, depending on the scaffold design. High cell viability and cell morphology close to that in the native tissue are observed. The present work demonstrates the utility of MEW to reconstruct complex morphological features of natural tissues.
- ItemMicro-mechanical response of ultrafine grain and nanocrystalline tantalum(Rio de Janeiro : Elsevier, 2021) Yang, Wen; Ruestes, Carlos J.; Li, Zezhou; Torrents Abad, Oscar; Langdon, Terence G.; Heiland, Birgit; Koch, Marcus; Arzt, Eduard; Meyers, Marc A.In order to investigate the effect of grain boundaries on the mechanical response in the micrometer and submicrometer levels, complementary experiments and molecular dynamics simulations were conducted on a model bcc metal, tantalum. Microscale pillar experiments (diameters of 1 and 2 μm) with a grain size of ~100–200 nm revealed a mechanical response characterized by a yield stress of ~1500 MPa. The hardening of the structure is reflected in the increase in the flow stress to 1700 MPa at a strain of ~0.35. Molecular dynamics simulations were conducted for nanocrystalline tantalum with grain sizes in the range of 20–50 nm and pillar diameters in the same range. The yield stress was approximately 6000 MPa for all specimens and the maximum of the stress–strain curves occurred at a strain of 0.07. Beyond that strain, the material softened because of its inability to store dislocations. The experimental results did not show a significant size dependence of yield stress on pillar diameter (equal to 1 and 2 um), which is attributed to the high ratio between pillar diameter and grain size (~10–20). This behavior is quite different from that in monocrystalline specimens where dislocation ‘starvation’ leads to a significant size dependence of strength. The ultrafine grains exhibit clear ‘pancaking’ upon being plastically deformed, with an increase in dislocation density. The plastic deformation is much more localized for the single crystals than for the nanocrystalline specimens, an observation made in both modeling and experiments. In the molecular dynamics simulations, the ratio of pillar diameter (20–50 nm) to grain size was in the range 0.2–2, and a much greater dependence of yield stress to pillar diameter was observed. A critical result from this work is the demonstration that the important parameter in establishing the overall deformation is the ratio between the grain size and pillar diameter; it governs the deformation mode, as well as surface sources and sinks, which are only important when the grain size is of the same order as the pillar diameter.
- ItemMyxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections(Basel : MDPI, 2020) Goes, Adriely; Lapuhs, Philipp; Kuhn, Thomas; Schulz, Eilien; Richter, Robert; Panter, Fabian; Dahlem, Charlotte; Koch, Marcus; Garcia, Ronald; Kiemer, Alexandra K.; Müller, Rolf; Fuhrmann, GregorIn 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is Staphylococcus aureus, a bacterium that can invade and survive within mammalian cells. S. aureus intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria Cystobacter velatus strain Cbv34 and Cystobacter ferrugineus strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular S. aureus. We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular S. aureus. In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections.
- ItemNano-in-Microparticles for Aerosol Delivery of Antibiotic-Loaded, Fucose-Derivatized, and Macrophage-Targeted Liposomes to Combat Mycobacterial Infections: In Vitro Deposition, Pulmonary Barrier Interactions, and Targeted Delivery(Weinheim : Wiley-VCH, 2022) Huck, Benedikt C.; Thiyagarajan, Durairaj; Bali, Aghiad; Boese, Annette; Besecke, Karen F.W.; Hozsa, Constantin; Gieseler, Robert K.; Furch, Marcus; Carvalho‐Wodarz, Cristiane; Waldow, Franziska; Schwudke, Dominik; Metelkina, Olga; Titz, Alexander; Huwer, Hanno; Schwarzkopf, Konrad; Hoppstädter, Jessica; Kiemer, Alexandra K.; Koch, Marcus; Loretz, Brigitta; Lehr, Claus‐MichaelNontuberculous mycobacterial infections rapidly emerge and demand potent medications to cope with resistance. In this context, targeted loco-regional delivery of aerosol medicines to the lungs is an advantage. However, sufficient antibiotic delivery requires engineered aerosols for optimized deposition. Here, the effect of bedaquiline-encapsulating fucosylated versus nonfucosylated liposomes on cellular uptake and delivery is investigated. Notably, this comparison includes critical parameters for pulmonary delivery, i.e., aerosol deposition and the noncellular barriers of pulmonary surfactant (PS) and mucus. Targeting increases liposomal uptake into THP-1 cells as well as peripheral blood monocyte- and lung-tissue derived macrophages. Aerosol deposition in the presence of PS, however, masks the effect of active targeting. PS alters antibiotic release that depends on the drug's hydrophobicity, while mucus reduces the mobility of nontargeted more than fucosylated liposomes. Dry-powder microparticles of spray-dried bedaquiline-loaded liposomes display a high fine particle fraction of >70%, as well as preserved liposomal integrity and targeting function. The antibiotic effect is maintained when deposited as powder aerosol on cultured Mycobacterium abscessus. When treating M. abscessus infected THP-1 cells, the fucosylated variant enabled enhanced bacterial killing, thus opening up a clear perspective for the improved treatment of nontuberculous mycobacterial infections.