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- 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.
- 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.
- ItemTargeted T1 Magnetic Resonance Imaging Contrast Enhancement with Extraordinarily Small CoFe2O4 Nanoparticles(Washington, DC : American Chemical Society, 2019) Piché, Dominique; Tavernaro, Isabella; Fleddermann, Jana; Lozano, Juan G.; Varambhia, Aakash; Maguire, Mahon L.; Koch, Marcus; Ukai, Tomofumi; Hernández Rodríguez, Armando J.; Jones, Lewys; Dillon, Frank; Reyes Molina, Israel; Mitzutani, Mai; González Dalmau, Evelio R.; Maekawa, Toru; Nellist, Peter D.; Kraegeloh, Annette; Grobert, NicoleExtraordinarily small (2.4 nm) cobalt ferrite nanoparticles (ESCIoNs) were synthesized by a one-pot thermal decomposition approach to study their potential as magnetic resonance imaging (MRI) contrast agents. Fine size control was achieved using oleylamine alone, and annular dark-field scanning transmission electron microscopy revealed highly crystalline cubic spinel particles with atomic resolution. Ligand exchange with dimercaptosuccinic acid rendered the particles stable in physiological conditions with a hydrodynamic diameter of 12 nm. The particles displayed superparamagnetic properties and a low r2/r1 ratio suitable for a T1 contrast agent. The particles were functionalized with bile acid, which improved biocompatibility by significant reduction of reactive oxygen species generation and is a first step toward liver-targeted T1 MRI. Our study demonstrates the potential of ESCIoNs as T1 MRI contrast agents.
- 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.
- 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.
- ItemOptical detection of di- and triphosphate anions with mixed monolayer-protected gold nanoparticles containing zinc(II)–dipicolylamine complexes(Frankfurt, Main : Beilstein-Institut zur Förderung der Chemischen Wissenschaften, 2020) Reinke, Lena; Bartl, Julia; Koch, Marcus; Kubik, StefanGold nanoparticles covered with a mixture of ligands of which one type contains solubilizing triethylene glycol residues and the other peripheral zinc(II)–dipicolylamine (DPA) complexes allowed the optical detection of hydrogenphosphate, diphosphate, and triphosphate anions in water/methanol 1:2 (v/v). These anions caused the bright red solutions of the nanoparticles to change their color because of nanoparticle aggregation followed by precipitation, whereas halides or oxoanions such as sulfate, nitrate, or carbonate produced no effect. The sensitivity of phosphate sensing depended on the nature of the anion, with diphosphate and triphosphate inducing visual changes at significantly lower concentrations than hydrogenphosphate. In addition, the sensing sensitivity was also affected by the ratio of the ligands on the nanoparticle surface, decreasing as the number of immobilized zinc(II)–dipicolylamine groups increased. A nanoparticle containing a 9:1 ratio of the solubilizing and the anion-binding ligand showed a color change at diphosphate and triphosphate concentrations as low as 10 μmol/L, for example, and precipitated at slightly higher concentrations. Hydrogenphosphate induced a nanoparticle precipitation only at a concentration of ca. 400 μmol/L, at which the precipitates formed in the presence of diphosphates and triphosphates redissolved. A nanoparticle containing fewer binding sites was more sensitive, while increasing the relative number of zinc(II)–dipicolylamine complexes beyond 25% had a negative impact on the limit of detection and the optical response. Transmission electron microscopy provided evidence that the changes of the nanoparticle properties observed in the presence of the phosphates were due to a nanoparticle crosslinking, consistent with the preferred binding mode of zinc(II)–dipicolylamine complexes with phosphate anions which involves binding of the anion between two metal centers. This work thus provided information on how the behavior of mixed monolayer-protected gold nanoparticles is affected by multivalent interactions, at the same time introducing a method to assess whether certain biologically relevant anions are present in an aqueous solution within a specific concentration range.