• Three-dimensional hierarchical cultivation of human skin cells on bio-adaptive hybrid fibers.

      Planz, Viktoria; Seif, Salem; Atchison, Jennifer S; Vukosavljevic, Branko; Sparenberg, Lisa; Kroner, Elmar; Windbergs, Maike; Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS),Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2016-07-11)
      The human skin comprises a complex multi-scale layered structure with hierarchical organization of different cells within the extracellular matrix (ECM). This supportive fiber-reinforced structure provides a dynamically changing microenvironment with specific topographical, mechanical and biochemical cell recognition sites to facilitate cell attachment and proliferation. Current advances in developing artificial matrices for cultivation of human cells concentrate on surface functionalizing of biocompatible materials with different biomolecules like growth factors to enhance cell attachment. However, an often neglected aspect for efficient modulation of cell-matrix interactions is posed by the mechanical characteristics of such artificial matrices. To address this issue, we fabricated biocompatible hybrid fibers simulating the complex biomechanical characteristics of native ECM in human skin. Subsequently, we analyzed interactions of such fibers with human skin cells focusing on the identification of key fiber characteristics for optimized cell-matrix interactions. We successfully identified the mediating effect of bio-adaptive elasto-plastic stiffness paired with hydrophilic surface properties as key factors for cell attachment and proliferation, thus elucidating the synergistic role of these parameters to induce cellular responses. Co-cultivation of fibroblasts and keratinocytes on such fiber mats representing the specific cells in dermis and epidermis resulted in a hierarchical organization of dermal and epidermal tissue layers. In addition, terminal differentiation of keratinocytes at the air interface was observed. These findings provide valuable new insights into cell behaviour in three-dimensional structures and cell-material interactions which can be used for rational development of bio-inspired functional materials for advanced biomedical applications.
    • Tobacco Smoke and Inhaled Drugs Alter Expression and Activity of Multidrug Resistance-Associated Protein-1 (MRP1) in Human Distal Lung Epithelial Cells .

      Selo, Mohammed Ali; Delmas, Anne-Sophie; Springer, Lisa; Zoufal, Viktoria; Sake, Johannes A; Clerkin, Caoimhe G; Huwer, Hanno; Schneider-Daum, Nicole; Lehr, Claus-Michael; Nickel, Sabrina; et al. (Frontiers, 2020-09-08)
      Multidrug resistance-associated protein-1 (MRP1/ABCC1) is highly expressed in human lung tissues. Recent studies suggest that it significantly affects the pulmonary disposition of its substrates, both after pulmonary and systemic administration. To better understand the molecular mechanisms involved, we studied the expression, subcellular localization and activity of MRP1 in freshly isolated human alveolar epithelial type 2 (AT2) and type 1-like (AT1-like) cells in primary culture, and in the NCI-H441 cell line. Moreover, the effect of cigarette smoke extract (CSE) and a series of inhaled drugs on MRP1 abundance and activity was investigated in vitro. MRP1 expression levels were measured by q-PCR and immunoblot in AT2 and AT1-like cells from different donors and in several passages of the NCI-H441 cell line. The subcellular localization of the transporter was studied by confocal laser scanning microscopy and cell surface protein biotinylation. MRP1 activity was assessed by bidirectional transport and efflux experiments using the MRP1 substrate, 5(6)-carboxyfluorescein [CF; formed intracellularly from 5(6)-carboxyfluorescein-diacetate (CFDA)] in AT1-like and NCI-H441 cell monolayers. Furthermore, the effect of CSE as well as several bronchodilators and inhaled corticosteroids on MRP1 abundance and CF efflux was investigated. MRP1 protein abundance increased upon differentiation from AT2 to AT1-like phenotype, however, ABCC1 gene levels remained unchanged. MRP1 abundance in NCI-H441 cells were comparable to those found in AT1-like cells. The transporter was detected primarily in basolateral membranes of both cell types which was consistent with net basolateral efflux of CF. Likewise, bidirectional transport studies showed net apical-to-basolateral transport of CF which was sensitive to the MRP1 inhibitor MK-571. Budesonide, beclomethasone dipropionate, salbutamol sulfate, and CSE decreased CF efflux in a concentration-dependent manner. Interestingly, CSE increased MRP1 abundance, whereas budesonide, beclomethasone dipropionate, salbutamol sulfate did not have such effect. CSE and inhaled drugs can reduce MRP1 activity in vitro, which implies the transporter being a potential drug target in the treatment of chronic obstructive pulmonary disease (COPD). Moreover, MRP1 expression level, localization and activity were comparable in human AT1-like and NCI-H441 cells. Therefore, the cell line can be a useful alternative in vitro model to study MRP1 in distal lung epithelium.
    • Tobramycin Liquid Crystal Nanoparticles Eradicate Cystic Fibrosis-Related Pseudomonas aeruginosa Biofilms.

      Thorn, Chelsea R; Carvalho-Wodarz, Cristiane de Souza; Horstmann, Justus C; Lehr, Claus-Michael; Prestidge, Clive A; Thomas, Nicky; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Wiley-VCH, 2021-05-12)
      Pseudomonas aeruginosa biofilms cause persistent and chronic infections, most known clinically in cystic fibrosis (CF). Tobramycin (TOB) is a standard anti-pseudomonal antibiotic; however, in biofilm infections, its efficacy severely decreases due to limited permeability across the biofilm matrix. Herewith, a biomimetic, nanostructured, lipid liquid crystal nanoparticle-(LCNP)-formulation is discovered to significantly enhance the efficacy of TOB and eradicate P. aeruginosa biofilm infections. Using an advanced, biologically-relevant co-culture model of human CF bronchial epithelial cells infected with P. aeruginosa biofilms at an air-liquid interface, nebulized TOB-LCNPs completely eradicated 1 × 109 CFU mL-1 of P. aeruginosa after two doses, a 100-fold improvement over the unformulated antibiotic. The enhanced activity of TOB is not observed with a liposomal formulation of TOB or with ciprofloxacin, an antibiotic that readily penetrates biofilms. It is demonstrated that the unique nanostructure of the LCNPs drives the enhanced penetration of TOB across the biofilm barrier, but not through the healthy lung epithelium barrier, significantly increasing the available antibiotic concentration at the site of infection. The LCNPs are an innovative strategy to improve the performance of TOB as a directed pulmonary therapy, enabling the administration of lower doses, reducing the toxicity, and amplifying the anti-biofilm activity of the anti-pseudomonal antibiotic.
    • Tofacitinib Loaded Squalenyl Nanoparticles for Targeted Follicular Delivery in Inflammatory Skin Diseases.

      Christmann, Rebekka; Ho, Duy-Khiet; Wilzopolski, Jenny; Lee, Sangeun; Koch, Marcus; Loretz, Brigitta; Vogt, Thomas; Bäumer, Wolfgang; Schaefer, Ulrich F; Lehr, Claus-Michael; et al. (MDPI, 2020-11-24)
      Tofacitinib (TFB), a Janus kinase inhibitor, has shown excellent success off-label in treating various dermatological diseases, especially alopecia areata (AA). However, TFB's safe and targeted delivery into hair follicles (HFs) is highly desirable due to its systemic adverse effects. Nanoparticles (NPs) can enhance targeted follicular drug delivery and minimize interfollicular permeation and thereby reduce systemic drug exposure. In this study, we report a facile method to assemble the stable and uniform 240 nm TFB loaded squalenyl derivative (SqD) nanoparticles (TFB SqD NPs) in aqueous solution, which allowed an excellent loading capacity (LC) of 20%. The SqD NPs showed an enhanced TFB delivery into HFs compared to the aqueous formulations of plain drug in an ex vivo pig ear model. Furthermore, the therapeutic efficacy of the TFB SqD NPs was studied in a mouse model of allergic dermatitis by ear swelling reduction and compared to TFB dissolved in a non-aqueous mixture of acetone and DMSO (7:1 v/v). Whereas such formulation would not be acceptable for use in the clinic, the TFB SqD NPs dispersed in water illustrated a better reduction in inflammatory effects than plain TFB's aqueous formulation, implying both encouraging good in vivo efficacy and safety. These findings support the potential of TFB SqD NPs for developing a long-term topical therapy of AA.
    • Towards a Continuous Manufacturing Process of Protein-Loaded Polymeric Nanoparticle Powders.

      Schiller, Stefan; Hanefeld, Andrea; Schneider, Marc; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer, 2020-10-06)
      To develop a scalable and efficient process suitable for the continuous manufacturing of poly(lactic-co-glycolic acid) (PLGA) nanoparticles containing ovalbumin as the model protein. PLGA nanoparticles were prepared using a double emulsification spray-drying method. Emulsions were prepared using a focused ultrasound transducer equipped with a flow cell. Either poly(vinyl alcohol) (PVA) or poloxamer 407 (P-407) was used as a stabilizer. Aliquots of the emulsions were blended with different matrix excipients and spray dried, and the yield and size of the resuspended nanoparticles was determined and compared against solvent displacement. Nanoparticle sizes of spray-dried PLGA/PVA emulsions were independent of the matrix excipient and comparable with sizes from the solvent displacement method. The yield of the resuspended nanoparticles was highest for emulsions containing trehalose and leucine (79%). Spray drying of PLGA/P-407 emulsions led to agglomerated nanoparticles independent of the matrix excipient. PLGA/P-407 nanoparticles pre-formed by solvent displacement could be spray dried with limited agglomeration when PVA was added as an additional stabilizer. A comparably high and economically interesting nanoparticle yield could be achieved with a process suitable for continuous manufacturing. Further studies are needed to understand the robustness of a continuous process at commercial scale.
    • Towards drug quantification in human skin with confocal Raman microscopy.

      Franzen, Lutz; Selzer, Dominik; Fluhr, Joachim W; Schaefer, Ulrich F; Windbergs, Maike; Biopharmaceutics and Pharmaceutical Technology, Saarland University, Saarbruecken, Germany. lutz.franzen@mx.uni-saarland.de (2013-06)
      Understanding the penetration behaviour of drugs into human skin is a prerequisite for the rational development and evaluation of effective dermal drug delivery. The general procedure for the acquisition of quantitative drug penetration profiles in human skin is performed by sequential segmentation and extraction. Unfortunately, this technique is destructive, laborious and lacks spatial resolution. Confocal Raman microscopy bares the potential of a chemically selective, label free and nondestructive analysis. However, the acquisition of quantitative drug depth profiles within skin by Raman microscopy is impeded by imponderable signal attenuation inside the tissue. In this study, we present a chemical semi-solid matrix system simulating the optical properties of human skin. This system serves as a skin surrogate for investigation of Raman signal attenuation under controlled conditions. Caffeine was homogeneously incorporated within the skin surrogate, and Raman intensity depth profiles were acquired. A mathematical algorithm describing the Raman signal attenuation within the surrogate was derived from these profiles. Human skin samples were incubated with caffeine, and Raman intensity depth profiles were similarly acquired. The surrogate algorithm was successfully applied to correct the drug profiles in human skin for signal attenuation. For the first time, a mathematical algorithm was established, which allows correction of Raman signal attenuation in human skin, thus facilitating reliable drug quantification in human skin by confocal Raman spectroscopy.
    • Towards More Predictive, Physiological and Animal-free in vitro Models: Advances in Cell and Tissue Culture 2020 Conference Proceedings.

      Singh, Bhumika; Abdelgawad, Mohamed Essameldin; Ali, Zulfiqur; Bailey, Jarrod; Budyn, Elisa; Civita, Prospero; Clift, Martin J D; Connelly, John T; Constant, Samuel; Hittinger, Marius; et al. (Fund for the Replacement of Animals in Medical Experiments (FRAME), 2021-07-06)
      Experimental systems that faithfully replicate human physiology at cellular, tissue and organ level are crucial to the development of efficacious and safe therapies with high success rates and low cost. The development of such systems is challenging and requires skills, expertise and inputs from a diverse range of experts, such as biologists, physicists, engineers, clinicians and regulatory bodies. Kirkstall Limited, a biotechnology company based in York, UK, organised the annual conference, Advances in Cell and Tissue Culture (ACTC), which brought together people having a variety of expertise and interests, to present and discuss the latest developments in the field of cell and tissue culture and in vitro modelling. The conference has also been influential in engaging animal welfare organisations in the promotion of research, collaborative projects and funding opportunities. This report describes the proceedings of the latest ACTC conference, which was held virtually on 30th September and 1st October 2020, and included sessions on in vitro models in the following areas: advanced skin and respiratory models, neurological disease, cancer research, advanced models including 3-D, fluid flow and co-cultures, diabetes and other age-related disorders, and animal-free research. The roundtable session on the second day was very interactive and drew huge interest, with intriguing discussion taking place among all participants on the theme of replacement of animal models of disease.
    • Towards nanotechnology regulation – Publish the unpublishable

      Hankin, Steve; Boraschi, Diana; Duschl, Albert; Lehr, Claus-Michael; Lichtenbeld, Hera (2012-10-05)
    • Towards the sustainable discovery and development of new antibiotics.

      Miethke, Marcus; Pieroni, Marco; Weber, Tilmann; Brönstrup, Mark; Hammann, Peter; Halby, Ludovic; Arimondo, Paola B; Glaser, Philippe; Aigle, Bertrand; Bode, Helge B; et al. (Springer Nature, 2021-08-19)
      An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.
    • Vibrational spectroscopic imaging and live cell video microscopy for studying differentiation of primary human alveolar epithelial cells.

      Vukosavljevic, Branko; Hittinger, Marius; Hachmeister, Henning; Pilger, Christian; Murgia, Xabier; Gepp, Michael M; Gentile, Luca; Huwer, Hanno; Schneider-Daum, Nicole; Huser, Thomas; et al. (Wiley-VCH, 2019-02-20)
    • Vitamin D Deficiency Does Not Result in a Breach of Host Defense in Murine Models of Pneumonia.

      Niederstrasser, Julia; Herr, Christian; Wolf, Lisa; Lehr, Claus M; Beisswenger, Christoph; Bals, Robert; HIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus 8.1, 66123 Saarbrücken, Germany. (2016-11-01)
      Vitamin D (VitD) has a role in the regulation of calcium and phosphate metabolism and in addition impacts the activity of the immune system. VitD deficiency might be linked to increased susceptibility to respiratory tract infection. The aim of the present study was to characterize the impact of VitD deficiency on the susceptibility to bacterial infection in murine models. C57BL/6N mice were fed a diet with or without VitD for 10 weeks. The VitD-deficient or -sufficient mice were infected with Pseudomonas aeruginosa or Streptococcus pneumoniae The colonization and inflammatory response in the lung were analyzed at defined time points. The serum 25-hydroxy-VitD concentration was significantly lower in mice on the VitD-deficient diet. In infection experiments with Pseudomonas aeruginosa or Streptococcus pneumoniae, no differences could be observed in the numbers of viable bacteria or in differential cell counts in the bronchoalveolar lavage fluids. Measurements of inflammatory cytokines (KC and interleukin-1β [IL-1β]) did not show significant differences between the groups. In conclusion, VitD-deficient animals did not show significantly increased susceptibility to infection or an altered course of infection. The immune systems of humans and mice likely respond differently to VitD. Murine models are likely not appropriate for drawing conclusions on the role of VitD in human pulmonary host defense.