• 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)
    • 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.
    • Fucosylated lipid nanocarriers loaded with antibiotics efficiently inhibit mycobacterial propagation in human myeloid cells.

      Durán, Verónica; Grabski, Elena; Hozsa, Constantin; Becker, Jennifer; Yasar, Hanzey; Monteiro, João T; Costa, Bibiana; Koller, Nicole; Lueder, Yvonne; Wiegmann, Bettina; et al. (Elsevier, 2021-04-16)
      Antibiotic treatment of tuberculosis (TB) is complex, lengthy, and can be associated with various adverse effects. As a result, patient compliance often is poor, thus further enhancing the risk of selecting multi-drug resistant bacteria. Macrophage mannose receptor (MMR)-positive alveolar macrophages (AM) constitute a niche in which Mycobacterium tuberculosis replicates and survives. Therefore, we encapsulated levofloxacin in lipid nanocarriers functionalized with fucosyl residues that interact with the MMR. Indeed, such nanocarriers preferentially targeted MMR-positive myeloid cells, and in particular, AM. Intracellularly, fucosylated lipid nanocarriers favorably delivered their payload into endosomal compartments, where mycobacteria reside. In an in vitro setting using infected human primary macrophages as well as dendritic cells, the encapsulated antibiotic cleared the pathogen more efficiently than free levofloxacin. In conclusion, our results point towards carbohydrate-functionalized nanocarriers as a promising tool for improving TB treatment by targeted delivery of antibiotics.
    • High-throughput phenotyping by applying digital morphometrics and fluorescence induction curves in seeds to identifying variations: A case study of Annona (Annonaceae) species

      Pontes, Montcharles S.; Montefusco-Pereira, Carlos V.; Misra, Biswapriya B.; Ribeiro-Junior, Howard L.; Graciano, Daniela E.; Santos, Jaqueline S.; Nobrega, Michele A.S.; Fernandes, Shaline S.L.; Caires, Anderson R.L.; Santiago, Etenaldo F.; et al.
    • Human airway mucus alters susceptibility of Pseudomonas aeruginosa biofilms to tobramycin, but not colistin.

      Müller, Laura; Murgia, Xabier; Siebenbürger, Lorenz; Börger, Carsten; Schwarzkopf, Konrad; Sewald, Katherina; Häussler, Susanne; Braun, Armin; Lehr, Claus-Michael; Hittinger, Marius; et al.
      Objectives: In the context of cystic fibrosis, Pseudomonas aeruginosa biofilms often develop in the vicinity of airway mucus, which acts as a protective physical barrier to inhaled matter. However, mucus can also adsorb small drug molecules administered as aerosols, including antibiotics, thereby reducing their bioavailability. The efficacy of antibiotics is typically assessed by determining the MIC using in vitro assays. This widespread technique, however, does not consider either bacterial biofilm formation or the influence of mucus, both of which may act as diffusion barriers, potentially limiting antibiotic efficacy. Methods: We grew P. aeruginosa biofilms in the presence or absence of human tracheal mucus and tested their susceptibility to tobramycin and colistin. Results: A significant reduction of tobramycin efficacy was observed when P. aeruginosa biofilms were grown in the presence of mucus compared with those grown in the absence of mucus. Diffusion of tobramycin through mucus was reduced; however, this reduction was more pronounced in biofilm/mucus mixtures, suggesting that biofilms in the presence of mucus respond differently to antibiotic treatment. In contrast, the influence of mucus on colistin efficacy was almost negligible and no differences in mucus permeability were observed. Conclusions: These findings underline the important role of mucus in the efficacy of anti-infective drugs.
    • 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.
    • A hydrogel-based assay for the fast prediction of antibiotic accumulation in Gram-negative bacteria.

      Richter, Robert; Kamal, Mohamed A M; García-Rivera, Mariel A; Kaspar, Jerome; Junk, Maximilian; Elgaher, Walid A M; Srikakulam, Sanjay Kumar; Gress, Alexander; Beckmann, Anja; Grißmer, Alexander; et al. (Elsevier, 2020-11-02)
      The pipeline of antibiotics has been for decades on an alarmingly low level. Considering the steadily emerging antibiotic resistance, novel tools are needed for early and easy identification of effective anti-infective compounds. In Gram-negative bacteria, the uptake of anti-infectives is especially limited. We here present a surprisingly simple in vitro model of the Gram-negative bacterial envelope, based on 20% (w/v) potato starch gel, printed on polycarbonate 96-well filter membranes. Rapid permeability measurements across this polysaccharide hydrogel allowed to correctly predict either high or low accumulation for all 16 tested anti-infectives in living Escherichia coli. Freeze-fracture TEM supports that the macromolecular network structure of the starch hydrogel may represent a useful surrogate of the Gram-negative bacterial envelope. A random forest analysis of in vitro data revealed molecular mass, minimum projection area, and rigidity as the most critical physicochemical parameters for hydrogel permeability, in agreement with reported structural features needed for uptake into Gram-negative bacteria. Correlating our dataset of 27 antibiotics from different structural classes to reported MIC values of nine clinically relevant pathogens allowed to distinguish active from nonactive compounds based on their low in vitro permeability specifically for Gram-negatives. The model may help to identify poorly permeable antimicrobial candidates before testing them on living bacteria.
    • 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.
    • Improved input parameters for diffusion models of skin absorption.

      Hansen, Steffi; Lehr, Claus-Michael; Schaefer, Ulrich F; Drug Delivery, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz-Center for Infection Research (HZI), Saarbruecken, Germany. Steffi.hansen@helmholtz-hzi.de (2013-02)
      To use a diffusion model for predicting skin absorption requires accurate estimates of input parameters on model geometry, affinity and transport characteristics. This review summarizes methods to obtain input parameters for diffusion models of skin absorption focusing on partition and diffusion coefficients. These include experimental methods, extrapolation approaches, and correlations that relate partition and diffusion coefficients to tabulated physico-chemical solute properties. Exhaustive databases on lipid-water and corneocyte protein-water partition coefficients are presented and analyzed to provide improved approximations to estimate lipid-water and corneocyte protein-water partition coefficients. The most commonly used estimates of lipid and corneocyte diffusion coefficients are also reviewed. In order to improve modeling of skin absorption in the future diffusion models should include the vertical stratum corneum heterogeneity, slow equilibration processes, the absorption from complex non-aqueous formulations, and an improved representation of dermal absorption processes. This will require input parameters for which no suitable estimates are yet available.
    • In vitro and in vivo comparison between poractant alfa and the new generation synthetic surfactant CHF5633.

      Ricci, Francesca; Murgia, Xabier; Razzetti, Roberta; Pelizzi, Nicola; Salomone, Fabrizio; Helmholtz Institut für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-02)
      CHF5633 is a new generation synthetic surfactant containing both SP-B and SP-C analogues developed for the treatment of respiratory distress syndrome. Here, the optimal dose and its performance in comparison to the animal-derived surfactant poractant alfa were investigated.
    • In vitro characterization and in vivo comparison of the pulmonary outcomes of Poractant alfa and Calsurf in ventilated preterm rabbits.

      Guo, Xiaojing; Luo, Siwei; Amidani, Davide; Rivetti, Claudio; Pieraccini, Giuseppe; Pioselli, Barbara; Catinella, Silvia; Murgia, Xabi; Salomone, Fabrizio; Xu, Yaling; et al. (PLOS, 2020-03-13)
      Poractant alfa and Calsurf are two natural surfactants widely used in China for the treatment of neonatal respiratory distress syndrome, which are extracted from porcine and calf lungs, respectively. The purpose of this experimental study was to compare their in vitro characteristics and in vivo effects in the improvement of pulmonary function and protection of lung injury. The biophysical properties, ultrastructure, and lipid composition of both surfactant preparations were respectively analysed in vitro by means of Langmuir-Blodgett trough (LBT), atomic force microscopy (AFM), and liquid-chromatography mass-spectrometry (LC-MS). Then, as core pharmacological activity, both head-to-head (100 and 200 mg/kg for both surfactants) and licensed dose comparisons (70 mg/kg Calsurf vs. 200 mg/kg Poractant alfa) between the two surfactants were conducted as prophylaxis in preterm rabbits with primary surfactant deficiency, assessing survival time and rate and dynamic compliance of the respiratory system (Cdyn). Intrapulmonary surfactant pools, morphometric volume density as alveolar expansion (Vv), and lung injury scores were determined post mortem. AFM and LC-MS analysis revealed qualitative differences in the ultrastructure as well as in the lipid composition of both preparations. Calsurf showed a longer plateau region of the LBT isotherm and lower film compressibility. In vivo, both surfactant preparations improved Cdyn at any dose, although maximum benefits in terms of Vv and intrapulmonary surfactant pools were seen with the 200 mg/kg dose in both surfactants. The group of animals treated with 200 mg/kg of Poractant alfa showed a prolonged survival time and rate compared to untreated but ventilated controls, and significantly ameliorated lung injury compared to Calsurf at any dose, including 200 mg/kg. The overall outcomes suggest the pulmonary effects to be dose dependent for both preparations. The group of animals treated with 200 mg/kg of Poractant alfa showed a significant reduction of mortality. Compared to Calsurf, Poractant alfa exerted better effects if licensed doses were compared, which requires further investigation.
    • In vitro surfactant and perfluorocarbon aerosol deposition in a neonatal physical model of the upper conducting airways.

      Goikoetxea, Estibalitz; Murgia, Xabier; Serna-Grande, Pablo; Valls-i-Soler, Adolf; Rey-Santano, Carmen; Rivas, Alejandro; Antón, Raúl; Basterretxea, Francisco J; Miñambres, Lorena; Méndez, Estíbaliz; et al. (2014)
      Aerosol delivery holds potential to release surfactant or perfluorocarbon (PFC) to the lungs of neonates with respiratory distress syndrome with minimal airway manipulation. Nevertheless, lung deposition in neonates tends to be very low due to extremely low lung volumes, narrow airways and high respiratory rates. In the present study, the feasibility of enhancing lung deposition by intracorporeal delivery of aerosols was investigated using a physical model of neonatal conducting airways.