• Decoding (patho-)physiology of the lung by advanced in vitro models for developing novel anti-infectives therapies.

      Montefusco-Pereira, Carlos Victor; Carvalho-Wodarz, Cristiane de Souza; Seeger, Johanna; Kloft, Charlotte; Michelet, Robin; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Elsevier, 2020-11-21)
      Advanced lung cell culture models provide physiologically-relevant and complex data for mathematical models to exploit host-pathogen responses during anti-infective drug testing.
    • Development and evaluation of a quality control system based on transdermal electrical resistance for skin barrier function in vitro.

      Knoth, Katharina; Zäh, Ralf-Kilian; Veldung, Barbara; Burgio, Dominic; Wiegand, Birgit; Smola, Hans; Bock, Udo; Lehr, Claus-Michael; Hittinger, Marius; Groß, Henrik; et al. (Wiley & Sons, 2021-01-06)
      Background: In vitro skin permeation experiments are highly relevant for pharmaceutical, cosmetic, agricultural developments, and regulatory evaluation. A key requirement is the skin barrier integrity, that is accompanied by an intact stratum corneum (SC) which implements high skin quality. A variety of integrity tests are currently available, for example, measurement of transepidermal water loss, monitoring the permeation of tritiated water and the measurement of transdermal electrical resistance (TER). Materials and methods: We aimed for a non-destructive examination of barrier integrity as quality control system, based on TER. Therefore, the in-house developed instrument SkinTER measures electrical resistance on excised human skin samples in a non-invasive and easy-to-use pattern. In this proof of concept study, we compared three human in vitro skin models with focus on their TER and permeation properties. The skin integrity was impaired to mimic conditions of skin during age, lifestyle (eg, shaving) or diseases (eg, obesity, psoriasis, and atopic dermatitis). The OECD permeation marker caffeine was correlated to the corresponding TER value. Results: A correlation between both was obtained by having a Pearson coefficient of -0.830. Hereby, a minimum TER value for intact skin samples of ~1.77 kΩ*cm2 was suggested. Intact samples are significantly different (α = ≤0.05) to their impaired counterparts in flux and TER values. Conclusion: The new SkinTER instrument gives a quick and non-invasive feedback on skin quality before a permeation experiment.
    • Development of artemether-loaded nanostructured lipid carrier (NLC) formulation for topical application.

      Nnamani, Petra O; Hansen, Steffi; Windbergs, Maike; Lehr, Claus-Michael (2014-12-30)
      NLC topical formulation as an alternative to oral and parenteral (IM) delivery of artemether (ART), a poorly water-soluble drug was designed. A Phospholipon 85G-modified Gelucire 43/01 based NLC formulation containing 75% Transcutol was chosen from DSC studies and loaded with gradient concentration of ART (100-750mg). ART-loaded NLCs were stable (-22 to -40mV), polydispersed (0.4-0.7) with d90 size distribution range of 247-530nm without microparticles up to one month of storage. The encapsulation efficiency (EE%) for ART in the NLC was concentration independent as 250mg of ART loading achieved ∼61%. DSC confirmed molecular dispersion of ART due to low matrix crystallinity (0.028J/g). Ex vivo study showed detectable ART amounts after 20h which gradually increased over 48h achieving ∼26% cumulative amount permeated irrespective of the applied dose. This proves that ART permeates excised human epidermis, where the current formulation served as a reservoir to gradually control drug release over an extended period of time. Full thickness skin study therefore may confirm if this is a positive signal to hope for a topical delivery system of ART.
    • 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.
    • Dimethylaminoethyl methacrylate copolymer-siRNA nanoparticles for silencing a therapeutically relevant gene in macrophages

      Jain, Ratnesh; Dandekar, Prajakta; Loretz, Brigitta; Koch, Marcus; Lehr, Claus-Michael; elmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Campus A4 1, Saarland University, Saarbrücken, 66123 Germany. (2015)
    • Dissolution techniques for in vitro testing of dry powders for inhalation.

      May, Sabine; Jensen, Birte; Wolkenhauer, Markus; Schneider, Marc; Lehr, Claus Michael; PharmBioTec GmbH, Saarbrücken, Germany. (2012-08)
      To evaluate different dissolution testing methods and subsequently develop a simple to perform but reproducible and discriminating dissolution technique for inhalative powders.
    • Diverse Applications of Nanomedicine.

      Pelaz, Beatriz; Alexiou, Christoph; Alvarez-Puebla, Ramon A; Alves, Frauke; Andrews, Anne M; Ashraf, Sumaira; Balogh, Lajos P; Ballerini, Laura; Bestetti, Alessandra; Brendel, Cornelia; et al. (2017-03-28)
      The design and use of materials in the nanoscale size range for addressing medical and health-related issues continues to receive increasing interest. Research in nanomedicine spans a multitude of areas, including drug delivery, vaccine development, antibacterial, diagnosis and imaging tools, wearable devices, implants, high-throughput screening platforms, etc. using biological, nonbiological, biomimetic, or hybrid materials. Many of these developments are starting to be translated into viable clinical products. Here, we provide an overview of recent developments in nanomedicine and highlight the current challenges and upcoming opportunities for the field and translation to the clinic.
    • Drug delivery for fighting infectious diseases: a global perspective.

      Loretz, Brigitta; Oh, Yu-Kyoung; Hudson, Sarah; Gu, Zhen; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (SPringer, 2021-06-09)
      [No abstract available]
    • 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)
    • Editorial EJPB - Biobarriers 2018.

      Fuhrmann, Gregor; Loretz, Brigitta; Schneider-Daum, Nicole; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Elsevier, 2020-11-02)
      [No abstract available]
    • 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)
    • Exploring the permeation of fluoroquinolone metalloantibiotics across outer membrane porins by combining molecular dynamics simulations and a porin-mimetic in vitro model.

      Sousa, Carla F; Coimbra, João T S; Richter, Robert; Morais-Cabral, João H; Ramos, Maria J; Lehr, Claus-Michael; Fernandes, Pedro A; Gameiro, Paula; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Elsevier, 2021-12-08)
      The misuse and overuse of fluoroquinolones in recent years have triggered alarming levels of resistance to these antibiotics. Porin channels are crucial for the permeation of fluoroquinolones across the outer membrane of Gram-negative bacteria and modifications in porin expression are an important mechanism of bacterial resistance. One possible strategy to overcome this problem is the development of ternary copper complexes with fluoroquinolones. Compared to fluoroquinolones, these metalloantibiotics present a larger partition to the lipid bilayer and a more favorable permeation, by passive diffusion, across bacteriomimetic phospholipid-based model membranes. To rule out the porin-dependent pathway for the metalloantibiotics, we explored the permeation through OmpF (one of the most abundant porins present in the outer membrane of Gram-negative bacteria) using a multi-component approach. X-ray studies of OmpF porin crystals soaked with a ciprofloxacin ternary copper complex did not show a well-defined binding site for the compound. Molecular dynamics simulations showed that the translocation of the metalloantibiotic through this porin is less favorable than that of free fluoroquinolone, as it presented a much larger free energy barrier to cross the narrow constriction region of the pore. Lastly, permeability studies of different fluoroquinolones and their respective copper complexes using a porin-mimetic in vitro model corroborated the lower rate of permeation for the metalloantibiotics relative to the free antibiotics. Our results support a porin-independent mechanism for the influx of the metalloantibiotics into the bacterial cell. This finding brings additional support to the potential application of these metalloantibiotics in the fight against resistant infections and as an alternative to fluoroquinolones.
    • Expression and Activity of Breast Cancer Resistance Protein (BCRP/ABCG2) in Human Distal Lung Epithelial Cells In Vitro.

      Nickel, Sabrina; Selo, Mohammed Ali; Fallack, Juliane; Clerkin, Caoimhe G; Huwer, Hanno; Schneider-Daum, Nicole; Lehr, Claus Michael; Ehrhardt, Carsten; Helmholtz Institut für Pharmaceutischr Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-05-03)
      Breast cancer resistance protein (BCRP/ABCG2) has previously been identified with high expression levels in human lung. The subcellular localisation and functional activity of the transporter in lung epithelia, however, remains poorly investigated. The aim of this project was to study BCRP expression and activity in freshly isolated human alveolar epithelial type 2 (AT2) and type 1-like (AT1-like) cells in primary culture, and to compare these findings with data obtained from the NCI-H441 cell line.
    • 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.
    • Extracellular vesicles as antigen carriers for novel vaccination avenues.

      Mehanny, Mina; Lehr, Claus-Michael; Fuhrmann, Gregor; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Elsevier, 2021-03-26)
      Antigen delivery has always been a challenge in scientific practice of vaccine formulation. Yet, mammalian extracellular vesicles (EVs) or bacterial membrane vesicles (MVs) provide an innovative avenue for safe and effective delivery of antigenic material. They include intrinsically loaded antigens from EV-secreting cells or extrinsically loaded antigens onto pre-formed vesicles. Interestingly, many studies shed light on potential novel anti-cancer vaccination immunotherapy for therapeutic applications from mammalian cell host-derived EVs, as well as conventional vaccination for prophylactic applications using bacterial cell-derived MVs against infectious diseases. Here, we discuss the rationale, status quo and potential for both vaccine applications using EVs.
    • Extracellular vesicles as novel assay tools to study cellular interactions of anti-infective compounds - A perspective.

      Richter, Robert; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Elsevier, 2021-04-20)
      Sudden outbreaks of novel infectious diseases and the persistent evolution of antimicrobial resistant pathogens make it necessary to develop specific tools to quickly understand pathogen-cell interactions and to study appropriate drug delivery strategies. Extracellular vesicles (EVs) are cell-specific biogenic transport systems, which are gaining more and more popularity as either diagnostic markers or drug delivery systems. Apart from that, there are emerging possibilities for EVs as tools to study drug penetration, drug-membrane interactions as well as pathogen-membrane interactions. However, it appears that the potential of EVs for such applications has not been fully exploited yet. Considering the vast variety of cells that can be involved in an infection, vesicle-based analytical methods are just emerging and the number of reported applications is still relatively small. Aim of this review is to discuss the current state of the art of EV-based assays, especially in the context of antimicrobial research and therapy, and to present some new perspectives for a more exhaustive and creative exploration in the future.
    • Extracellular vesicles protect glucuronidase model enzymes during freeze-drying.

      Frank, Julia; Richter, Maximilian; de Rossi, Chiara; Lehr, Claus-Michael; Fuhrmann, Kathrin; Fuhrmann, Gregor; HIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus 8.1, 66123 Saarbrücken, Germany. (2018-08-17)
      Extracellular vesicles (EVs) are natural nanoparticles that play important roles in intercellular communication and are increasingly studied for biosignalling, pathogenesis and therapy. Nevertheless, little is known about optimal conditions for their transfer and storage, and the potential impact on preserving EV-loaded cargoes. We present the first comprehensive stability assessment of different widely available types of EVs during various storage conditions including -80 °C, 4 °C, room temperature, and freeze-drying (lyophilisation). Lyophilisation of EVs would allow easy handling at room temperature and thus significantly enhance their expanded investigation. A model enzyme, β-glucuronidase, was loaded into different types of EVs derived from mesenchymal stem cells, endothelial cells and cancer cells. Using asymmetric flow field-flow fractionation we proved that the model enzyme is indeed stably encapsulated into EVs. When assessing enzyme activity as indicator for EV stability, and in comparison to liposomes, we show that EVs are intrinsically stable during lyophilisation, an effect further enhanced by cryoprotectants. Our findings provide new insight for exploring lyophilisation as a novel storage modality and we create an important basis for standardised and advanced EV applications in biomedical research.
    • 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.
    • Formulation and evaluation of transdermal nanogel for delivery of artemether.

      Nnamani, Petra O; Ugwu, Agatha A; Nnadi, Ogechukwu H; Kenechukwu, Franklin C; Ofokansi, Kenneth C; Attama, Anthony A; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer, 2021-03-19)
      rtemether (ART) is second to artesunate in being the most widely used derivatives of artemisinin in combination therapy of malaria. Nanostructured lipid carrier (NLC) formulations were prepared following our previous report using optimized ART concentration of 0.25 g dissolved in 5% w/v mixture of solid (Gelucire 43/01 and Phospholipon 85G) and liquid (Transcutol) lipids at 90 °C. An aqueous surfactant phase at 90 °C was added (dropwise) under magnetic stirring (1000 rpm) for 5 min. The pre-emulsion was speedily homogenized at 28,000 rpm for 15 min and further probe sonicated at 60% amplitude (15 min). Resultant sample was cooled at room temperature and frozen at - 80 °C prior to lyophilization. The freeze-dried sample was used for solid-state characterization as well as in the formulation of transdermal nanogels using three polymers (Carbopol 971P, Poloxamer 407, and Prosopis africana peel powder) to embed the ART-NLC, using ethanol as a penetration enhancer. Transdermal ART-nanogels were characterized accordingly (physical examination, pH, drug content, rheology, spreadability, stability, particle size and morphology, skin irritation, in vitro and ex vivo skin permeation, and analysis of permeation data), P < 0.05. Results indicated that ART nanogels showed good encapsulation, drug release, pH-dependent swelling, stability, and tolerability. Overall, ART nanogels prepared from Poloxamer 407 showed the most desirable drug permeation, pH, swellability, spreadability, viscosity, and transdermal antiplasmodial properties superior to PAPP-ANG > C971P-ANG. A two-patch/week concurrent application of the studied nanogels could offer 100% cure of malaria as a lower-dose (50 mg ART) patient-friendly regimen devoid of the drug's many side effects.
    • Freeze-drying as a preserving preparation technique for in vitro testing of human skin.

      Franzen, Lutz; Vidlářová, Lucie; Kostka, Karl-Heinz; Schaefer, Ulrich F; Windbergs, Maike (2013-01)
      In vitro testing of drugs with excised human skin is a valuable prerequisite for clinical studies. However, the analysis of excised human skin presents several obstacles. Ongoing drug diffusion, microbial growth and changes in hydration state influence the results of drug penetration studies. In this work, we evaluate freeze-drying as a preserving preparation method for skin samples to overcome these obstacles. We analyse excised human skin before and after freeze-drying and compare these results with human skin in vivo. Based on comprehensive thermal and spectroscopic analysis, we demonstrate comparability to in vivo conditions and exclude significant changes within the skin samples due to freeze-drying. Furthermore, we show that freeze-drying after skin incubation with drugs prevents growth of drug crystals on the skin surface due to drying effects. In conclusion, we introduce freeze-drying as a preserving preparation technique for in vitro testing of human skin.