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- ItemA New PqsR Inverse Agonist Potentiates Tobramycin Efficacy to Eradicate Pseudomonas aeruginosa Biofilms(2021) Schütz, Christian; Ho, Duy-Khiet; Hamed, Mostafa Mohamed; Abdelsamie, Ahmed Saad; Röhrig, Teresa; Herr, Christian; Kany, Andreas Martin; Rox, Katharina; Schmelz, Stefan; Siebenbürger, Lorenz; Wirth, Marius; Börger, Carsten; Yahiaoui, Samir; Bals, Robert; Scrima, Andrea; Blankenfeldt, Wulf; Horstmann, Justus Constantin; Christmann, Rebekka; Murgia, Xabier; Koch, Marcus; Berwanger, Aylin; Loretz, Brigitta; Hirsch, Anna Katharina Herta; Hartmann, Rolf Wolfgang; Lehr, Claus-Michael; Empting, MartinPseudomonas aeruginosa (PA) infections can be notoriously difficult to treat and are often accompanied by the development of antimicrobial resistance (AMR). Quorum sensing inhibitors (QSI) acting on PqsR (MvfR) – a crucial transcriptional regulator serving major functions in PA virulence – can enhance antibiotic efficacy and eventually prevent the AMR. An integrated drug discovery campaign including design, medicinal chemistry-driven hit-to-lead optimization and in-depth biological profiling of a new QSI generation is reported. The QSI possess excellent activity in inhibiting pyocyanin production and PqsR reporter-gene with IC50 values as low as 200 and 11 × 10−9 m, respectively. Drug metabolism and pharmacokinetics (DMPK) as well as safety pharmacology studies especially highlight the promising translational properties of the lead QSI for pulmonary applications. Moreover, target engagement of the lead QSI is shown in a PA mucoid lung infection mouse model. Beyond that, a significant synergistic effect of a QSI-tobramycin (Tob) combination against PA biofilms using a tailor-made squalene-derived nanoparticle (NP) formulation, which enhance the minimum biofilm eradicating concentration (MBEC) of Tob more than 32-fold is demonstrated. The novel lead QSI and the accompanying NP formulation highlight the potential of adjunctive pathoblocker-mediated therapy against PA infections opening up avenues for preclinical development.
- 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
- ItemYields and Immunomodulatory Effects of Pneumococcal Membrane Vesicles Differ with the Bacterial Growth Phase(Weinheim : Wiley-VCH, 2021) Mehanny, Mina; Kroniger, Tobias; Koch, Marcus; Hoppstädter, Jessica; Becher, Dörte; Kiemer, Alexandra K.; Lehr, Claus-Michael; Fuhrmann, GregorStreptococcus pneumoniae infections are a leading cause of death worldwide. Bacterial membrane vesicles (MVs) are promising vaccine candidates because of the antigenic components of their parent microorganisms. Pneumococcal MVs exhibit low toxicity towards several cell lines, but their clinical translation requires a high yield and strong immunogenic effects without compromising immune cell viability. MVs are isolated during either the stationary phase (24 h) or death phase (48 h), and their yields, immunogenicity and cytotoxicity in human primary macrophages and dendritic cells have been investigated. Death-phase vesicles showed higher yields than stationary-phase vesicles. Both vesicle types displayed acceptable compatibility with primary immune cells and several cell lines. Both vesicle types showed comparable uptake and enhanced release of the inflammatory cytokines, tumor necrosis factor and interleukin-6, from human primary immune cells. Proteomic analysis revealed similarities in vesicular immunogenic proteins such as pneumolysin, pneumococcal surface protein A, and IgA1 protease in both vesicle types, but stationary-phase MVs showed significantly lower autolysin levels than death-phase MVs. Although death-phase vesicles produced higher yields, they lacked superiority to stationary-phase vesicles as vaccine candidates owing to their similar antigenic protein cargo and comparable uptake into primary human immune cells.
- ItemAn Outer Membrane Vesicle-Based Permeation Assay (OMPA) for Assessing Bacterial Bioavailability(Weinheim : Wiley-VCH, 2021) Richter, Robert; Kamal, Mohamed A.M.; Koch, Marcus; Niebuur, Bart-Jan; Huber, Anna-Lena; Goes, Adriely; Volz, Carsten; Vergalli, Julia; Kraus, Tobias; Müller, Rolf; Schneider-Daum, Nicole; Fuhrmann, Gregor; Pagès, Jean-Marie; Lehr, Claus-MichaelWhen searching for new antibiotics against Gram-negative bacterial infections, a better understanding of the permeability across the cell envelope and tools to discriminate high from low bacterial bioavailability compounds are urgently needed. Inspired by the phospholipid vesicle-based permeation assay (PVPA), which is designed to predict non-facilitated permeation across phospholipid membranes, outer membrane vesicles (OMVs) of Escherichia coli either enriched or deficient of porins are employed to coat filter supports for predicting drug uptake across the complex cell envelope. OMVs and the obtained in vitro model are structurally and functionally characterized using cryo-TEM, SEM, CLSM, SAXS, and light scattering techniques. In vitro permeability, obtained from the membrane model for a set of nine antibiotics, correlates with reported in bacterio accumulation data and allows to discriminate high from low accumulating antibiotics. In contrast, the correlation of the same data set generated by liposome-based comparator membranes is poor. This better correlation of the OMV-derived membranes points to the importance of hydrophilic membrane components, such as lipopolysaccharides and porins, since those features are lacking in liposomal comparator membranes. This approach can offer in the future a high throughput screening tool with high predictive capacity or can help to identify compound- and bacteria-specific passive uptake pathways.
- ItemOne-Pot Synthesis of Copper Iodide-Polypyrrole Nanocomposites(Basel : MDPI, 2021) Konakov, Artem O.; Dremova, Nadejda N.; Khodos, Igor I.; Koch, Marcus; Zolotukhina, Ekaterina V.; Silina, YuliyaA novel one-pot chemical synthesis of functional copper iodide-polypyrrole composites, CuI-PPy, has been proposed. The fabrication process allows the formation of nanodimensional metal salt/polymer hybrid structures in a fully controlled time- and concentration-dependent manner. The impact of certain experimental conditions, viz., duration of synthesis, sequence of component addition and concentrations of the intact reagents on the structure, dimensionality and yield of the end-product was evaluated in detail. More specifically, the amount of marshite CuI within the hybrid composite can be ranged from 60 to 90 wt.%, depending on synthetic conditions (type and concentration of components, process duration). In addition, the conditions allowing the synthesis of nano-sized CuI distributed inside the polypyrrole matrix were found. A high morphological stability and reproducibility of the synthesized nanodimensional metal-polymer hybrid materials were approved. Finally, the electrochemical activity of the formed composites was verified by cyclic voltammetry studies. The stability of CuI-PPy composite deposited on the electrodes was strongly affected by the applied anodic limit. The proposed one-pot synthesis of the hybrid nanodimensional copper iodide-polypyrrole composites is highly innovative, meets the requirements of Green Chemistry and is potentially useful for future biosensor development. In addition, this study is expected to generally contribute to the knowledge on the hybrid nano-based composites with tailored properties.
- ItemSpray-dried lactose-leucine microparticles for pulmonary delivery of antimycobacterial nanopharmaceuticals(New York, NY [u.a.] : Springer, 2021) Thiyagarajan, Durairaj; Huck, Benedikt; Nothdurft, Birgit; Koch, Marcus; Rudolph, David; Rutschmann, Mark; Feldmann, Claus; Hozsa, Constantin; Furch, Marcus; Besecke, Karen F. W.; Gieseler, Robert K.; Loretz, Brigitta; Lehr, Claus-MichaelPulmonary delivery of nanocarriers for novel antimycobacterial compounds is challenging because the aerodynamic properties of nanomaterials are sub-optimal for such purposes. Here, we report the development of dry powder formulations for nanocarriers containing benzothiazinone 043 (BTZ) or levofloxacin (LVX), respectively. The intricacy is to generate dry powder aerosols with adequate aerodynamic properties while maintaining both nanostructural integrity and compound activity until reaching the deeper lung compartments. Microparticles (MPs) were prepared using vibrating mesh spray drying with lactose and leucine as approved excipients for oral inhalation drug products. MP morphologies and sizes were measured using various biophysical techniques including determination of geometric and aerodynamic mean sizes, X-ray diffraction, and confocal and focused ion beam scanning electron microscopy. Differences in the nanocarriers’ characteristics influenced the MPs’ sizes and shapes, their aerodynamic properties, and, hence, also the fraction available for lung deposition. Spay-dried powders of a BTZ nanosuspension, BTZ-loaded silica nanoparticles (NPs), and LVX-loaded liposomes showed promising respirable fractions, in contrast to zirconyl hydrogen phosphate nanocontainers. While the colloidal stability of silica NPs was improved after spray drying, MPs encapsulating either BTZ nanosuspensions or LVX-loaded liposomes showed the highest respirable fractions and active pharmaceutical ingredient loads. Importantly, for the BTZ nanosuspension, biocompatibility and in vitro uptake by a macrophage model cell line were improved even further after spray drying.
- 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.
- ItemTargeting extracellular lectins of Pseudomonas aeruginosa with glycomimetic liposomes(London [u.a.] : RSC, 2021) Metelkina, Olga; Huck, Benedikt; O'Connor, Jonathan S.; Koch, Marcus; Manz, Andreas; Lehr, Claus-Michael; Titz, AlexanderThe antimicrobial resistance crisis requires novel approaches for the therapy of infections especially with Gram-negative pathogens. Pseudomonas aeruginosa is defined as priority 1 pathogen by the WHO and thus of particular interest. Its drug resistance is primarily associated with biofilm formation and essential constituents of its extracellular biofilm matrix are the two lectins, LecA and LecB. Here, we report microbial lectin-specific targeted nanovehicles based on liposomes. LecA- and LecB-targeted phospholipids were synthesized and used for the preparation of liposomes. These liposomes with varying surface ligand density were then analyzed for their competitive and direct lectin binding activity. We have further developed a microfluidic device that allowed the optical detection of the targeting process to the bacterial lectins. Our data showed that the targeted liposomes are specifically binding to their respective lectin and remain firmly attached to surfaces containing these lectins. This synthetic and biophysical study provides the basis for future application in targeted antibiotic delivery to overcome antimicrobial resistance.
- ItemScanning electron microscopy preparation of the cellular actin cortex: A quantitative comparison between critical point drying and hexamethyldisilazane drying(San Francisco, California, US : PLOS, 2021) Schu, Moritz; Terriac, Emmanuel; Koch, Marcus; Paschke, Stephan; Lautenschläger, Franziska; Flormann, Daniel A.D.The cellular cortex is an approximately 200-nm-thick actin network that lies just beneath the cell membrane. It is responsible for the mechanical properties of cells, and as such, it is involved in many cellular processes, including cell migration and cellular interactions with the environment. To develop a clear view of this dense structure, high-resolution imaging is essential. As one such technique, electron microscopy, involves complex sample preparation procedures. The final drying of these samples has significant influence on potential artifacts, like cell shrinkage and the formation of artifactual holes in the actin cortex. In this study, we compared the three most used final sample drying procedures: critical-point drying (CPD), CPD with lens tissue (CPD-LT), and hexamethyldisilazane drying. We show that both hexamethyldisilazane and CPD-LT lead to fewer artifactual mesh holes within the actin cortex than CPD. Moreover, CPD-LT leads to significant reduction in cell height compared to hexamethyldisilazane and CPD. We conclude that the final drying procedure should be chosen according to the reduction in cell height, and so CPD-LT, or according to the spatial separation of the single layers of the actin cortex, and so hexamethyldisilazane.
- 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.