• Different macro- and micro-rheological properties of native porcine respiratory and intestinal mucus.

      Bokkasam, Harish; Ernst, Matthias; Guenther, Marco; Wagner, Christian; Schaefer, Ulrich F; Lehr, Claus-Michael; Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken. (2016-06-13)
      Aim of this study was to investigate the similarities and differences at macro- and microscale in the viscoelastic properties of mucus that covers the epithelia of the intestinal and respiratory tract. Natural mucus was collected from pulmonary and intestinal regions of healthy pigs. Macro-rheological investigations were carried out through conventional plate-plate rheometry. Microrheology was investigated using optical tweezers. Our data revealed significant differences both in macro- and micro-rheological properties between respiratory and intestinal mucus.
    • Impact of PEG and PEG-b-PAGE modified PLGA on nanoparticle formation, protein loading and release.

      Rietscher, René; Czaplewska, Justyna A; Majdanski, Tobias C; Gottschaldt, Michael; Schubert, Ulrich S; Schneider, Marc; Lehr, Claus-Michael (2016-03-16)
      The effect of modifying the well-established pharmaceutical polymer PLGA by different PEG-containing block-copolymers on the preparation of ovalbumin (OVA) loaded PLGA nanoparticles (NPs) was studied. The used polymers contained poly(d,l-lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG) and poly(allyl glycidyl ether) (PAGE) as building blocks. The double emulsion technique yielded spherical NPs in the size range from 170 to 220nm (PDI<0.15) for all the differently modified polymers, allowing to directly compare protein loading of and release. PEGylation is usually believed to increase the hydrophilic character of produced particles, favoring encapsulation of hydrophilic substances. However, in this study simple PEGylation of PLGA had only a slight effect on protein release. In contrast, incorporating a PAGE block between the PEG and PLGA units, also eventually enabling active targeting introducing a reactive group, led to a significantly higher loading (+25%) and release rate (+100%), compared to PLGA and PEG-b-PLGA NPs.
    • Characterization of Microvesicles Released from Human Red Blood Cells.

      Nguyen, Duc Bach; Thuy Ly, Thi Bich; Wesseling, Mauro Carlos; Hittinger, Marius; Torge, Afra; Devitt, Andrew; Perrie, Yvonne; Bernhardt, Ingolf; Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS),Saarland 9 University, 66123 Saarbrücken, Germany. (2016)
      Extracellular vesicles (EVs) are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs) and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs) under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C.
    • Human alveolar epithelial cells expressing tight junctions to model the air-blood barrier.

      Kuehn, Anna; Kletting, Stephanie; de Souza Carvalho-Wodarz, Cristiane; Repnik, Urska; Griffiths, Gareth; Fischer, Ulrike; Meese, Eckart; Huwer, Hanno; Wirth, Dagmar; May, Tobias; et al. (2016-03-17)
      This paper describes a new human alveolar epithelial cell line (hAELVi - human Alveolar Epithelial Lentivirus immortalized) with type I-like characteristics and functional tight junctions, suitable to model the air-blood barrier of the peripheral lung. Primary human alveolar epithelial cells were immortalized by a novel regimen, grown as monolayers on permeable filter supports and characterized morphologically, biochemically and biophysically. hAELVi cells maintain the capacity to form tight intercellular junctions, with high trans-epithelial electrical resistance (> 1000 Ω*cm²). The cells could be kept in culture over several days, up to passage 75, under liquid-liquid as well as air-liquid conditions. Ultrastructural analysis and real time PCR revealed type I-like cell properties, such as the presence of caveolae, expression of caveolin-1, and absence of surfactant protein C. Accounting for the barrier properties, inter-digitations sealed with tight junctions and desmosomes were also observed. Low permeability of the hydrophilic marker sodium fluorescein confirmed the suitability of hAELVi cells for in vitro transport studies across the alveolar epithelium. These results suggest that hAELVi cells reflect the essential features of the air-blood barrier, as needed for an alternative to animal testing to study absorption and toxicity of inhaled drugs, chemicals and nanomaterials.
    • Enhanced uptake and siRNA-mediated knockdown of a biologically relevant gene using cyclodextrin polyrotaxane

      Dandekar, P.; Jain, R.; Keil, M.; Loretz, B.; Koch, M.; Wenz, G.; Lehr, C.-M.; Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS),Saarland 9 University, 66123 Saarbrücken, Germany. (2015)
    • Nanocarriers for optimizing the balance between interfollicular permeation and follicular uptake of topically applied clobetasol to minimize adverse effects.

      Mathes, C; Melero, A; Conrad, P; Vogt, T; Rigo, L; Selzer, D; Prado, W A; De Rossi, C; Garrigues, T M; Hansen, S; et al. (2016-02-10)
      The treatment of various hair disorders has become a central focus of good dermatologic patient care as it affects men and women all over the world. For many inflammatory-based scalp diseases, glucocorticoids are an essential part of treatment, even though they are known to cause systemic as well as local adverse effects when applied topically. Therefore, efficient targeting and avoidance of these side effects are of utmost importance. Optimizing the balance between drug release, interfollicular permeation, and follicular uptake may allow minimizing these adverse events and simultaneously improve drug delivery, given that one succeeds in targeting a sustained release formulation to the hair follicle. To test this hypothesis, three types of polymeric nanocarriers (nanospheres, nanocapsules, lipid-core nanocapsules) for the potent glucocorticoid clobetasol propionate (CP) were prepared. They all exhibited a sustained release of drug, as was desired. The particles were formulated as a dispersion and hydrogel and (partially) labeled with Rhodamin B for quantification purposes. Follicular uptake was investigated using the Differential Stripping method and was found highest for nanocapsules in dispersion after application of massage. Moreover, the active ingredient (CP) as well as the nanocarrier (Rhodamin B labeled polymer) recovered in the hair follicle were measured simultaneously, revealing an equivalent uptake of both. In contrast, only negligible amounts of CP could be detected in the hair follicle when applied as free drug in solution or hydrogel, regardless of any massage. Skin permeation experiments using heat-separated human epidermis mounted in Franz Diffusion cells revealed equivalent reduced transdermal permeability for all nanocarriers in comparison to application of the free drug. Combining these results, nanocapsules formulated as an aqueous dispersion and applied by massage appeare to be a good candidate to maximize follicular targeting and minimize drug penetration into the interfollicular epidermis. We conclude that such nanotechnology-based formulations provide a viable strategy for more efficient drug delivery to the hair follicle. Moreover, they present a way to minimize adverse effects of potent glucocorticoids by releasing the drug in a controlled manner and simultaneously decreasing interfollicular permeation, offering an advantage over conventional formulations for inflammatory-based skin/scalp diseases.
    • Surface-modified yeast cells: A novel eukaryotic carrier for oral application.

      Kenngott, Elisabeth E; Kiefer, Ruth; Schneider-Daum, Nicole; Hamann, Alf; Schneider, Marc; Schmitt, Manfred J; Breinig, Frank; Helmholtz Institute for Pharmaceutical Research Saarland (HIPS);Saarland University, Building A4.1, 66123 Saarbruecken, Germany. (2016-02-28)
      The effective targeting and subsequent binding of particulate carriers to M cells in Peyer's patches of the gut is a prerequisite for the development of oral delivery systems. We have established a novel carrier system based on cell surface expression of the β1-integrin binding domain of invasins derived from Yersinia enterocolitica and Yersinia pseudotuberculosis on the yeast Saccharomyces cerevisiae. All invasin derivatives were shown to be effectively expressed on the cell surface and recombinant yeast cells showed improved binding to both human HEp-2 cells and M-like cells in vitro. Among the different derivatives tested, the integrin-binding domain of Y. enterocolitica invasin proved to be the most effective and was able to target Peyer's patches in vivo. In conclusion, cell surface-modified yeasts might provide a novel bioadhesive, eukaryotic carrier system for efficient and targeted delivery of either antigens or drugs via the oral route.
    • A strategy for in-silico prediction of skin absorption in man.

      Selzer, Dominik; Neumann, Dirk; Neumann, Heike; Kostka, Karl-Heinz; Lehr, Claus-Michael; Schaefer, Ulrich F; Helmholtz Institute for Pharmaceutical Research Saarland (HIPS);Saarland University, Building A4.1, 66123 Saarbruecken, Germany. (2015-09)
      For some time, in-silico models to address substance transport into and through the skin are gaining more and more importance in different fields of science and industry. In particular, the mathematical prediction of in-vivo skin absorption is of great interest to overcome ethical and economical issues. The presented work outlines a strategy to address this problem and in particular, investigates in-vitro and in-vivo skin penetration experiments of the model compound flufenamic acid solved in an ointment by means of a mathematical model. Experimental stratum corneum concentration-depth profiles (SC-CDP) for various time intervals using two different in-vitro systems (Franz diffusion cell, Saarbruecken penetration model) were examined and simulated with the help of a highly optimized three compartment numerical diffusion model and compared to the findings of SC-CDPs of the in-vivo scenario. Fitted model input parameters (diffusion coefficient and partition coefficient with respect to the stratum corneum) for the in-vitro infinite dose case could be used to predict the in-use conditions in-vitro. Despite apparent differences in calculated partition coefficients between in-vivo and in-vitro studies, prediction of in-vivo scenarios from input parameters calculated from the in-vitro case yielded reasonable results.
    • Solid Phase Extraction as an Innovative Separation Method for Measuring Free and Entrapped Drug in Lipid Nanoparticles.

      Guillot, Alexis; Couffin, Anne-Claude; Sejean, Xavier; Navarro, Fabrice; Limberger, Markus; Lehr, Claus-Michael; Helmholtz Institute for Pharmaceutical Research Saarland (HIPS);Saarland University, Building A4.1, 66123 Saarbruecken, Germany. (2015-12)
      Contrary to physical characterization techniques for nanopharmaceuticals (shape, size and zeta-potential), the techniques to quantify the free and the entrapped drug remain very few and difficult to transpose in routine analytical laboratories. The application of Solid Phase Extraction (SPE) technique was investigated to overcome this challenge.
    • Bacteriomimetic invasin-functionalized nanocarriers for intracellular delivery.

      Labouta, Hagar Ibrahim; Menina, Sara; Kochut, Annika; Gordon, Sarah; Geyer, Rebecca; Dersch, Petra; Lehr, Claus-Michael; Helmholtz Institute for Pharmaceutical Research Saarland (HIPS);Saarland University, Building A4.1, 66123 Saarbruecken, Germany. (2015-12-28)
      Intracellular bacteria invade mammalian cells to establish an infectious niche. The current work models adhesion and subsequent internalization strategy of pathogenic bacteria into mammalian cells to design a bacteriomimetic bioinvasive delivery system. We report on the surface functionalization of liposomes with a C-terminal fragment of invasin (InvA497), an invasion factor in the outer membrane of Yersinia pseudotuberculosis. InvA497-functionalized liposomes adhere to mammalian epithelial HEp-2 cell line at different infection stages with a significantly higher efficiency than liposomes functionalized with bovine serum albumin. Covalent attachment of InvA497 results in higher cellular adhesion than liposomes with physically adsorbed InvA497 with non-specific surface protein alignment. Uptake studies in HEp-2 cells indicate active internalization of InvA497-functionalized liposomes via β1-integrin receptor-mediated uptake mechanism mimicking the natural invasion strategy of Y. pseudotuberculosis. Uptake studies in Caco-2 cells at different polarization states demonstrate specific targeting of the InvA497-functionalized liposomes to less polarized cells reflecting the status of inflamed cells. Moreover, when loaded with the anti-infective agent gentamicin and applied to HEp-2 cells infected with Y. pseudotuberculosis, InvA497-functionalized liposomes are able to significantly reduce the infection load relative to non-functionalized drug-loaded liposomes. This indicates a promising application of such a bacteriomimetic system for drug delivery to intracellular compartments.
    • Dual flow bioreactor with ultrathin microporous TEER sensing membrane for evaluation of nanoparticle toxicity

      Sbrana, Tommaso; Ucciferri, Nadia; Favrè, Mèlanie; Ahmed, Sher; Collnot, Eva-Maria; Lehr, Claus-Michael; Ahluwalia, Arti; Liley, Martha; Helmholtz Institute for Pharmaceutical Research Saarland (HIPS);Saarland University, Building A4.1, 66123 Saarbruecken, Germany. (2016-02)
    • Inhalable Clarithromycin Microparticles for Treatment of Respiratory Infections.

      Dimer, Frantiescoli; de Souza Carvalho-Wodarz, Cristiane; Haupenthal, Jörg; Hartmann, Rolf; Lehr, Claus-Michael; Helmholtz-Institute for Pharmaceutical 8 Research Saarland (HIPS),Saarland 9 University, 66123 Saarbrücken, Germany. (2015-12)
      The aim of this work was to develop clarithromycin microparticles (CLARI-MP) and evaluate their aerodynamic behavior, safety in bronchial cells and anti-bacterial efficacy.
    • Expression and function of the epithelial sodium channel δ-subunit in human respiratory epithelial cells in vitro.

      Schwagerus, Elena; Sladek, Svenja; Buckley, Stephen T; Armas-Capote, Natalia; Alvarez de la Rosa, Diego; Harvey, Brian J; Fischer, Horst; Illek, Beate; Huwer, Hanno; Schneider-Daum, Nicole; et al. (2015-11)
      Using human airway epithelial cell lines (i.e. NCI-H441 and Calu-3) as well as human alveolar epithelial type I-like (ATI) cells in primary culture, we studied the contribution of the epithelial sodium channel δ-subunit (δ-ENaC) to transepithelial sodium transport in human lung in vitro. Endogenous δ-ENaC protein was present in all three cell types tested; however, protein abundance was low, and no expression was detected in the apical cell membrane of these cells. Similarly, known modulators of δ-ENaC activity, such as capsazepine and icilin (activators) and Evans blue (inhibitor), did not show effects on short-circuit current (I SC), suggesting that δ-ENaC is not involved in the modulation of transcellular sodium absorption in NCI-H441 cell monolayers. Over-expression of δ-ENaC in NCI-H441 cells resulted in detectable protein expression in the apical cell membrane, as well as capsazepine and icilin-stimulated increases in I SC that were effectively blocked by Evans blue and that were consistent with δ-ENaC activation and inhibition, respectively. Consequently, these observations suggest that δ-ENaC expression is low in NCI-H441, Calu-3, and ATI cells and does not contribute to transepithelial sodium absorption.
    • Macrophage uptake of cylindrical microparticles investigated with correlative microscopy.

      Tscheka, Clemens; Hittinger, Marius; Lehr, Claus-Michael; Schneider-Daum, Nicole; Schneider, Marc; Helmholtz Institute for Pharmaceutical Research Saarland (HIPS);Saarland University, Building A4.1, 66123 Saarbruecken, Germany. (2015-09)
      Cylindrical particles offer the opportunity to develop controlled and sustained release systems for the respiratory tract. One reason is that macrophages can phagocyte such particles only from either of the two ends. We investigated the uptake behaviour of murine alveolar macrophages incubated with elongated submicron-structured particles. For that purpose, fluorescent model silica nanoparticles were interconnected with the biocompatible polysaccharide agarose, building up cylindrical particles within the pores of track-etched membranes. In contrast to common approaches we determined the uptake at different time points with scanning electron microscopy, fluorescence microscopy, and the combination of both techniques - correlative microscopy (CLEM). As a consequence, we could securely identify uptake events and observe in detail the engulfment of particles and confirm, that phagocytosis could only be observed from the tips of the cylinders. CLEM allowed a comparison of the uptake measured with different techniques at identical macrophages. Qualitative and quantitative evaluation of this cylindrical particle uptake showed substantial differences between fluorescence microscopy, electron microscopy and the combination of both (CLEM) within 24h.
    • Biological barriers - Advanced drug delivery, in vitro modelling, and their implications for infection research.

      Schneider, Marc; Loretz, Brigitta; Windbergs, Maike; Schneider-Daum, Nicole; Schaefer, Ulrich F; Lehr, Claus-Michael; Helmholtz Institute for Pharmaceutical Research Saarland (HIPS);Saarland University, Building A4.1, 66123 Saarbruecken, Germany. (2015-09)
    • Organic cation transporter function in different in vitro models of human lung epithelium.

      Salomon, Johanna J; Gausterer, Julia C; Yahara, Tohru; Hosoya, Ken-Ichi; Huwer, Hanno; Hittinger, Marius; Schneider-Daum, Nicole; Lehr, Claus-Michael; Ehrhardt, Carsten; Helmholtz Institute for Pharmaceutical Research Saarland (HIPS);Saarland University, Building A4.1, 66123 Saarbruecken, Germany. (2015-12-01)
      Organic cation transporters (OCT) encoded by members of the solute carrier (SLC) 22 family of genes are involved in the disposition of physiological substrates and xenobiotics, including drugs used in the treatment of chronic obstructive lung diseases and asthma. The aim of this work was to identify continuously growing epithelial cell lines that closely mimic the organic cation transport of freshly isolated human alveolar type I-like epithelial cells (ATI) in primary culture, and which consequently, can be utilised as in vitro models for the study of organic cation transport at the air-blood barrier. OCT activity was investigated by measuring [(14)C]-tetraethylammonium (TEA) uptake into monolayers of Calu-3, NCI-H441 and A549 lung epithelial cell lines in comparison to ATI-like cell monolayers in primary culture. Levels of time-dependent TEA uptake were highest in A549 and ATI-like cells. In A549 cells, TEA uptake had a saturable and a non-saturable component with Km=528.5±373.1μM, Vmax=0.3±0.1nmol/min/mg protein and Kd=0.02μl/min/mg protein. TEA uptake into Calu-3 and NCI-H441 cells did not reach saturation within the concentration range studied. RNAi experiments in A549 cells confirmed that TEA uptake was mainly facilitated by OCT1 and OCT2. Co-incubation studies using pharmacological OCT modulators suggested that organic cation uptake pathways share several similarities between ATI-like primary cells and the NCI-H441 cell line, whereas more pronounced differences exist between primary cells and the A549 and Calu-3 cell lines.
    • Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

      Gordon, Sarah; Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C23, D-66123 Saarbrücken, Germany. (2015)
    • Focused Ultrasound as a Scalable and Contact-Free Method to Manufacture Protein-Loaded PLGA Nanoparticles.

      Schiller, Stefan; Hanefeld, Andrea; Schneider, Marc; Lehr, Claus-Michael; Helmholtz Institute for Pharmaceutical Research Saarland,Saarbru¨ cken, Saarland 66123, Germany. (2015-09)
      Although nanomaterials are under investigation for a very broad range of medical applications, only a small fraction of these are already commercialized or in clinical development. A major challenge for the translation of nanomedicines into the clinic is the missing scalability of the available lab scale preparation methods and, ultimately, non-identical samples during early and late research.
    • Semi-automated nanoprecipitation-system--an option for operator independent, scalable and size adjustable nanoparticle synthesis.

      Rietscher, René; Thum, Carolin; Lehr, Claus-Michael; Schneider, Marc; Helmholtz Institute for Pharmaceutical Research Saarland (HIPS);Saarland University, Building A4.1, 66123 Saarbruecken, Germany. (2015-06)
      The preparation of nano-sized carrier systems increasingly moved into focus of pharmaceutical research and industry in the past decades. Besides the drug load and properties of the selected polymer/lipid, the size of such particles is one of the most important parameters regarding their use as efficient drug delivery systems. However, the preparation of nanoparticles with different sizes in a controlled manner is challenging, especially in terms of reproducibility and scale-up possibility. To overcome these hurdles we developed a system relying on nanoprecipitation, which meets all these requirements of an operator independent, scalable and size-adjustable nanoparticle synthesis-the Semi-Automated Nanoprecipitation-System. This system enables the adaption of the particle size to specific needs based on the process parameters-injection rate, flow rate and polymer concentration-identified within this study. The basic set-up is composed of a syringe pump and a gear pump for a precise control of the flow and injection speed of the system. Furthermore, a home-made tube-straightener guarantees a curvature-free injection point. Thus it could be shown that the production of poly(lactide-co-glycolide) nanoparticles from 150 to 600 nm with a narrow size distribution in a controlled semi-automatic manner is possible.
    • pH-triggered drug release from biodegradable microwells for oral drug delivery.

      Nielsen, Line Hagner; Nagstrup, Johan; Gordon, Sarah; Keller, Stephan Sylvest; Østergaard, Jesper; Rades, Thomas; Müllertz, Anette; Boisen, Anja (2015-06)
      Microwells fabricated from poly-L-lactic acid (PLLA) were evaluated for their application as an oral drug delivery system using the amorphous sodium salt of furosemide (ASSF) as a model drug. Hot embossing of PLLA resulted in fabrication of microwells with an inner diameter of 240 μm and a height of 100 μm. The microwells were filled with ASSF using a modified screen printing technique, followed by coating of the microwell cavities with a gastro-resistant lid of Eudragit® L100. The release behavior of ASSF from the coated microwells was investigated using a μ-Diss profiler and a UV imaging system, and under conditions simulating the changing environment of the gastrointestinal tract. Biorelevant gastric medium (pH 1.6) was employed, after which a change to biorelevant intestinal release medium (pH 6.5) was carried out. Both μ-Diss profiler and UV imaging release experiments showed that sealing of microwell cavities with an Eudragit® layer prevented drug release in biorelevant gastric medium. An immediate release of the ASSF from coated microwells was observed in the intestinal medium. This pH-triggered release behavior demonstrates the future potential of PLLA microwells as a site-specific oral drug delivery system.