The interplay of Pseudomonas aeruginosa and Staphylococcus aureus in dual-species biofilms impacts development, antibiotic resistance and virulence of biofilms in in vitro wound infection models.

Due to high tolerance to antibiotics and pronounced virulence, bacterial biofilms are considered a key factor and major clinical challenge in persistent wound infections. They are typically composed of multiple species, whose interactions determine the biofilm's structural development, functional properties and thus the progression of wound infections. However, most attempts to study bacterial biofilms in vitro solely rely on mono-species populations, since cultivating multi-species biofilms, especially for prolonged periods of time, poses significant challenges. To address this, the present study examined the influence of bacterial composition on structural biofilm development, morphology and spatial organization, as well as antibiotic tolerance and virulence on human skin cells in the context of persistent wound infections. By creating a wound-mimetic microenvironment, the successful cultivation of dual-species biofilms of two of the most prevalent wound pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, was realized over a period of 72 h. Combining quantitative analysis with electron microscopy and label-free imaging enabled a comprehensive evaluation of the dynamics of biofilm formation and matrix secretion, revealing a twofold increased maturation of dual-species biofilms. Antibiotic tolerance was comparable for both mono-species cultures, however, dual-species communities showed a 50% increase in tolerance, mediated by a significantly reduced penetration of the applied antibiotic into the biofilm matrix. Further synergistic effects were observed, where dual-species biofilms exacerbated wound healing beyond the effects observed from either Pseudomonas or Staphylococcus. Consequently, predicting biofilm development, antimicrobial tolerance and virulence for multi-species biofilms based solely on the results from mono-species biofilms is unreliable. This study underscores the substantial impact of a multi-species composition on biofilm functional properties and emphasizes the need to tailor future studies reflecting the bacterial composition of the respective in vivo situation, leading to a more comprehensive understanding of microbial communities in the context of basic microbiology and the development of effective treatments.

Unraveling Molecular Composition in Biological Samples-Systematic Evaluation of Statistical Methods for the Analysis of Hyperspectral Raman Data.

Recently, confocal Raman microscopy has gained popularity in biomedical research for studying tissues in healthy and diseased state due to its ability to acquire chemically selective data in a noninvasive approach. However, biological samples, such as brain tissue, are inherently difficult to analyze due to the superposition of molecules in the Raman spectra and low variation of spectral features within the sample. The analysis is further impeded by pathological hallmarks, for example beta-amyloid (Aß) plaques in Alzheimer's disease, which are often solely characterized by subtle shifts in the respective Raman peaks. To unravel the underlying molecular information, convoluted statistical procedures are inevitable. Unfortunately, such statistical methods are often inadequately described, and most natural scientists lack knowledge of their appropriate use, causing unreproducible results and stagnation in the application of hyperspectral Raman imaging. Therefore, we have set out to provide a comprehensive guide to address these challenges with the example of a complex hyperspectral data set of brain tissue samples with Aß plaques. Our study encompasses established as well as novel statistical methods, including univariate analysis, principal component analysis, cluster analysis, spectral unmixing, and 2D correlation spectroscopy, and critically compares the outcomes of each analysis. Moreover, we transparently demonstrate the effect of preprocessing decisions like denoising and scaling techniques, providing valuable insights into implications of spectral quality for data evaluation. Thereby, this study provides a comprehensive evaluation of analysis approaches for complex hyperspectral Raman data, laying out a blueprint for elucidating meaningful information from biological samples in chemical imaging.

Influence of drug molecular weight on self-assembly and intestinal permeation of polymer-based nanocarriers.

For oral delivery, the physicochemical properties of nanocarriers are decisive factors for permeation through the intestinal epithelium. These properties are determined by the composition of the nanocarriers as well as by the process parameters during their self-assembly. For macromolecular drugs, there is still little understanding of the drug-polymer interactions during nanocarrier self-assembly and the effects on carrier properties. In this study, the effect of drug molecular weight on nanocarrier self-assembly, physicochemical properties of nanocarriers as well as their permeation across the intestinal epithelium was investigated. Our results show that the drug molecular weight impacts the physicochemical properties of nanocarriers. Further, the physicochemical properties of the nanocarriers, governed by the molecular weight of the drug, determine their permeation properties across the intestinal epithelium. Comparative in vitro and ex vivo studies revealed that intestinal absorption is dependent on both, the properties of the tissue as well as properties of the carrier system. In conclusion, the molecular weight of drug payload is a key factor determining the physiochemical properties of polymeric nanocarriers and is closely linked to their oral absorption. Using different preclinical models to evaluate intestinal permeation of nanocarriers allows for novel insights into key formulation properties governing oral bioavailability.

Bacteriophage-loaded functional nanofibers for treatment of P. aeruginosa and S. aureus wound infections.

The increasing incidence of infected skin wounds poses a major challenge in clinical practice, especially when conventional antibiotic therapy fails. In this context, bacteriophages emerged as promising alternatives for the treatment of antibiotic-resistant bacteria. However, clinical implementation remains hampered by the lack of efficient delivery approaches to infected wound tissue. In this study, bacteriophage-loaded electrospun fiber mats were successfully developed as next-generation wound dressings for the treatment of infected wounds. We employed a coaxial electrospinning approach, creating fibers with a protective polymer shell, enveloping bacteriophages in the core while maintaining their antimicrobial activity. The novel fibers exhibited a reproducible fiber diameter range and morphology, while the mechanical fiber properties were ideal for application onto wounds. Further, immediate release kinetics for the phages were confirmed as well as the biocompatibility of the fibers with human skin cells. Antimicrobial activity was demonstrated against Staphylococcus aureus and Pseudomonas aeruginosa and the core/shell formulation maintained the bacteriophage activity for 4 weeks when stored at - 20 °C. Based on these promising characteristics, our approach holds great potential as a platform technology for the encapsulation of bioactive bacteriophages to enable the translation of phage therapy into clinical application.

Imitating the microenvironment of native biofilms using nanofibrous scaffolds to emulate chronic wound infections.

Three-dimensional scaffolds of electrospun fibers are widely investigated for in vitro human tissue engineering, but to date, their application in the cultivation of bacterial biofilms has been neglected. In contrast, in a clinical setting, biofilms have received increasing recognition as major determinants of severe and chronic tissue infections, illustrating their immense threat to global public health. Their complex three-dimensional structure enables their persistence in harsh infection environments, tight attachment to human tissue and reduced susceptibility to antimicrobials. For the investigation of biofilm formation and persistence and for the development of novel infection therapies, mimicking the complex biofilm architecture with adequate in vitro models is essential. In this study, electrospun nanofibers were designed to simulate the matrix of native biofilms to serve as scaffolds for a novel biofilm model, which provides an in vivo-like growth environment and comprises biofilm-tissue interfaces. The three-dimensional scaffolds closely imitate the composition and structure of the matrix, while providing high mechanical support. The specific material properties of the developed scaffolds promote bacterial adhesion, infiltration, and homogenous distribution throughout the fiber network. Furthermore, matrix production and increased tolerance against antibiotics were proven, verifying adequate biofilm formation and maturation. In combination with human ex vivo wound models, the chronic state of infected wounds could be emulated allowing for investigation of biofilm-tissue interfaces and biofilm-host interactions. The here-described biofilm model based on nanofibers represents a valuable tool for simulating biofilm-associated infections in a pathophysiologically relevant manner paving new grounds for a multitude of possible applications beyond infection research.

Electrospun fibers loaded with antimicrobial peptides for treatment of wound infections.

The widespread resistance of clinically relevant bacteria against established antibiotics emphasizes the urgent need for novel therapeutics. In this context, wound infections constitute a specific challenge, as most systemically applied antibiotics are insufficiently available at the site of infection. Therefore, the local treatment of infected wounds poses a particular challenge regarding the appropriate release kinetics of actives and their residence time in the wound bed. Consequently, design and development of novel, drug-loaded wound dressings constitute a major research focus for the effective treatment of wound infections. In this study, we employed electrospinning to design drug-loaded wound dressings, incorporating the therapeutically promising antimicrobial peptide tyrothricin. By parallel electrospinning, we combined different ratios of water-soluble polyvinylpyrrolidone and water-insoluble methacrylate copolymer (EudragitE), in order to take advantage of their specific mechanical stability and dissolution properties. We fabricated fiber mats constituting mechanically stable wound dressings with a controlled drug release profile, combining an initial burst release above the minimal inhibitory concentration of known wound pathogens and a subsequent prolonged antimicrobial effect of the active ingredient. Antimicrobial activity against Staphylococcusaureus and Staphylococcusepidermidis was successfully proven, thereby introducing our tyrothricin-loaded fiber mats as a promising prospective therapy against typical wound-associated pathogens.

Discovery of Inhibitors of DNA Methyltransferase 2, an Epitranscriptomic Modulator and Potential Target for Cancer Treatment.

Selective manipulation of the epitranscriptome could be beneficial for the treatment of cancer and also broaden the understanding of epigenetic inheritance. Inhibitors of the tRNA methyltransferase DNMT2, the enzyme catalyzing the S-adenosylmethionine-dependent methylation of cytidine 38 to 5-methylcytidine, were designed, synthesized, and analyzed for their enzyme-binding and -inhibiting properties. For rapid screening of potential DNMT2 binders, a microscale thermophoresis assay was established. Besides the natural inhibitors S-adenosyl-l-homocysteine (SAH) and sinefungin (SFG), we identified new synthetic inhibitors based on the structure of N-adenosyl-2,4-diaminobutyric acid (Dab). Structure-activity relationship studies revealed the amino acid side chain and a Y-shaped substitution pattern at the 4-position of Dab as crucial for DNMT2 inhibition. The most potent inhibitors are alkyne-substituted derivatives, exhibiting similar binding and inhibitory potencies as the natural compounds SAH and SFG. CaCo-2 assays revealed that poor membrane permeabilities of the acids and rapid hydrolysis of an ethylester prodrug might be the reasons for the insufficient activity in cellulo.

Electrospun Scaffolds as Cell Culture Substrates for the Cultivation of an In Vitro Blood-Brain Barrier Model Using Human Induced Pluripotent Stem Cells.

The human blood-brain barrier (BBB) represents the interface of microvasculature and the central nervous system, regulating the transport of nutrients and protecting the brain from external threats. To gain a deeper understanding of (patho)physiological processes affecting the BBB, sophisticated models mimicking the in vivo situation are required. Currently, most in vitro models are cultivated on stiff, semipermeable, and non-biodegradable Transwell® membrane inserts, not adequately mimicking the complexity of the extracellular environment of the native human BBB. To overcome these disadvantages, we developed three-dimensional electrospun scaffolds resembling the natural structure of the human extracellular matrix. The polymer fibers of the scaffold imitate collagen fibrils of the human basement membrane, exhibiting excellent wettability and biomechanical properties, thus facilitating cell adhesion, proliferation, and migration. Cultivation of human induced pluripotent stem cells (hiPSCs) on these scaffolds enabled the development of a physiological BBB phenotype monitored via the formation of tight junctions and validated by the paracellular permeability of sodium fluorescein, further accentuating the non-linearity of TEER and barrier permeability. The novel in vitro model of the BBB forms a tight endothelial barrier, offering a platform to study barrier functions in a (patho)physiologically relevant context.

Application of Confocal Raman Microscopy for the Characterization of Topical Semisolid Formulations and their Penetration into Human Skin Ex Vivo.

PURPOSE: The quality testing and approval procedure for most pharmaceutical products is a streamlined process with standardized procedures for the determination of critical quality attributes. However, the evaluation of semisolid dosage forms for topical drug delivery remains a challenging task. The work presented here highlights confocal Raman microscopy (CRM) as a valuable tool for the characterization of such products. METHODS: CRM, a laser-based method, combining chemically-selective analysis and high resolution imaging, is used for the evaluation of different commercially available topical acyclovir creams. RESULTS: We show that CRM enables the spatially resolved analysis of microstructural features of semisolid products and provides insights into drug distribution and polymorphic state as well as the composition and arrangement of excipients. Further, we explore how CRM can be used to monitor phase separation and to study skin penetration and the interaction with fresh and cryopreserved excised human skin tissue. CONCLUSION: This study presents a comprehensive overview and illustration of how CRM can facilitate several types of key analyses of semisolid topical formulations and of their interaction with their biological target site, illustrating that CRM is a useful tool for research, development as well as for quality testing in the pharmaceutical industry.

Non-invasive analysis of pancreas organoids in synthetic hydrogels defines material-cell interactions and luminal composition.

The cultivation of cells forming three-dimensional structures like organoids holds great potential in different fields of life sciences and is gaining increasing interest with regards to clinical applications and personalised medicine. However, conventional hydrogels used as cell cultivation matrices (e.g. Matrigel®) contain animal-derived components in varying quantities, implicating low reproducibility of experiments and limited applicability for clinical use. Based on the strong need for developing novel, well defined, and animal-free hydrogels for 3D cell cultures, this study presents a comprehensive analysis of pancreas organoid cultivation in two synthetic hydrogels. Besides established visualisation techniques to monitor organoid formation and growth, confocal Raman microscopy was used for the first time to evaluate the gel matrices and organoid formation within the gels. The approach revealed so far not accessible information about material-cell interactions and the composition of the organoid lumen in a non-invasive and label-free manner. Confocal Raman microscopy thereby enabled a systematic characterisation of different hydrogels with respect to cell culture compatibility and allowed for the rational selection of a hydrogel formulation to serve as a synthetic and fully defined alternative to animal-derived cultivation matrices.

Controlling Supramolecular Structures of Drugs by Light.

Controlling physicochemical properties of light-unresponsive drugs, by light, prima facie, a paradox approach. We expanded light control by ion pairing light-unresponsive salicylate or ibuprofen to photoswitchable azobenzene counterions, thereby reversibly controlling supramolecular structures, hence the drugs' physicochemical and kinetic properties. The resulting ion pairs photoliquefied into room-temperature ionic liquids under ultraviolet light. Aqueous solutions showed trans-cis-dependent supramolecular structures under a light with wormlike aggregates decomposing into small micelles and vice versa. Light control allowed for permeation through membranes of cis-ibuprofen ion pairs within 12 h in contrast to the trans ion pairs requiring 72 h. In conclusion, azobenzene ion-pairing expands light control of physicochemical and kinetic properties to otherwise light-unresponsive drugs.

Proline-Based Allosteric Inhibitors of Zika and Dengue Virus NS2B/NS3 Proteases.

The NS2B/NS3 serine proteases of the Zika and Dengue flaviviruses are attractive targets for the development of antiviral drugs. We report the synthesis and evaluation of a new, proline-based compound class that displays allosteric inhibition of both proteases. The structural features relevant for protease binding and inhibition were determined to establish them as new lead compounds for flaviviral inhibitors. Based on our structure-activity relationship studies, the molecules were further optimized, leading to inhibitors with submicromolar IC50 values and improved lipophilic ligand efficiency. The allosteric binding site in the proteases was probed using mutagenesis and covalent modification of the obtained cysteine mutants with maleimides, followed by computational elucidation of the possible binding modes. In infected cells, antiviral activity against Dengue virus serotype 2 using prodrugs of the inhibitors was observed. In summary, a novel inhibitor scaffold targeting an allosteric site shared between flaviviral NS2B/NS3 proteases is presented whose efficacy is demonstrated in vitro and in cellulo.

Formulation development of medicated chewing gum tablets by direct compression using the SeDeM-Diagram-Expert-System.

Medicated chewing gums represent an orally administered dosage form with promising potential for local and systemic drug delivery. However, compared to other solid oral dosage forms, formulation development and release mechanism of medicated chewing gums are extremely complex, and thus only few products reached the approval for the market so far. Therefore, Quality by Design (QbD) approaches for rational formulation development of medicated chewing gums are needed to utilize their full potential. For chewing gum tablets, which are manufactured by direct compression, QbD approaches derived from tableting processes might be exerted. In this context, the SeDeM-Diagram-Expert-System implements the QbD approach while indicating whether a blend is suitable for direct compression and comprises powder properties, which need to be improved to facilitate the formulation development. Here, we present the successful application of the SeDeM-Diagram-Expert-System to the formulation development of medicated chewing gum tablets manufactured by direct compression. Furthermore, limitations of the SeDeM-System for medicated chewing gum tablets are evaluated and potential modifications of the system are suggested and discussed for future use.