• 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.
    • Chemical imaging of drug delivery systems with structured surfaces-a combined analytical approach of confocal raman microscopy and optical profilometry.

      Kann, Birthe; Windbergs, Maike; Department of Biopharmaceutics and Pharmaceutical Technology, Saarland University, Campus A4.1, 66123 Saarbruecken, Germany. (2013-04)
      Confocal Raman microscopy is an analytical technique with a steadily increasing impact in the field of pharmaceutics as the instrumental setup allows for nondestructive visualization of component distribution within drug delivery systems. Here, the attention is mainly focused on classic solid carrier systems like tablets, pellets, or extrudates. Due to the opacity of these systems, Raman analysis is restricted either to exterior surfaces or cross sections. As Raman spectra are only recorded from one focal plane at a time, the sample is usually altered to create a smooth and even surface. However, this manipulation can lead to misinterpretation of the analytical results. Here, we present a trendsetting approach to overcome these analytical pitfalls with a combination of confocal Raman microscopy and optical profilometry. By acquiring a topography profile of the sample area of interest prior to Raman spectroscopy, the profile height information allowed to level the focal plane to the sample surface for each spectrum acquisition. We first demonstrated the basic principle of this complementary approach in a case study using a tilted silica wafer. In a second step, we successfully adapted the two techniques to investigate an extrudate and a lyophilisate as two exemplary solid drug carrier systems. Component distribution analysis with the novel analytical approach was neither hampered by the curvature of the cylindrical extrudate nor the highly structured surface of the lyophilisate. Therefore, the combined analytical approach bears a great potential to be implemented in diversified fields of pharmaceutical sciences.
    • Chemically modified hCFTR mRNAs recuperate lung function in a mouse model of cystic fibrosis.

      Haque, A K M Ashiqul; Dewerth, Alexander; Antony, Justin S; Riethmüller, Joachim; Schweizer, Georg R; Weinmann, Petra; Latifi, Ngadhnjim; Yasar, Hanzey; Pedemonte, Nicoletta; Sondo, Elvira; et al. (Nature publishing group, 2018-11-13)
      Gene therapy has always been a promising therapeutic approach for Cystic Fibrosis (CF). However, numerous trials using DNA or viral vectors encoding the correct protein resulted in a general low efficacy. In the last years, chemically modified messenger RNA (cmRNA) has been proven to be a highly potent, pulmonary drug. Consequently, we first explored the expression, function and immunogenicity of human (h)CFTR encoded by cmRNA
    • Ciprofloxacin-loaded PLGA nanoparticles against Cystic Fibrosis P. aeruginosa Lung Infections.

      Günday Türeli, Nazende; Torge, Afra; Juntke, Jenny; Schwarz, Bianca C; Schneider-Daum, Nicole; Türeli, Akif Emre; Lehr, Claus-Michael; Schneider, Marc; Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-05-02)
      Current pulmonary treatments against Pseudomonasaeruginosa infections in cystic fibrosis (CF) lung suffer from deactivation of the drug and immobilization in thick and viscous biofilm/mucus blend, along with the general antibiotic resistance. Administration of nanoparticles (NPs) with high antibiotic load capable of penetrating the tight mesh of biofilm/mucus can be an advent to overcome the treatment bottlenecks. Biodegradable and biocompatible polymer nanoparticles efficiently loaded with ciprofloxacin complex offer a solution for emerging treatment strategies. NPs were prepared under controlled conditions by utilizing MicroJet Reactor (MJR) to yield a particle size of 190.4±28.6 nm with 0.089 PDI. Encapsulation efficiency of the drug was 79% resulting in a loading of 14%. Release was determined to be controlled and medium-independent in PBS, PBS+0.2% Tween 80 and simulated lung fluid. Cytotoxicity assays with Calu3 cells and CF bronchial epithelial cells (CFBE41o(-)) indicated that complex loaded PLGA NPs were non-toxic at concentrations >MICcipro against lab strains of the bacteria. Antibacterial activity tests revealed enhanced activity when applied as nanoparticles. NPs' colloidal stability in mucus was proven. Notably, a decrease in mucus turbidity was observed upon incubation with NPs. Herewith, ciprofloxacin complex loaded PLGA NPs are introduced as promising pulmonary nano drug delivery systems against P.aeruginosa infections in CF lung.
    • Circulating Lipoproteins: A Trojan Horse Guiding Squalenoylated Drugs to LDL-Accumulating Cancer Cells.

      Sobot, Dunja; Mura, Simona; Rouquette, Marie; Vukosavljevic, Branko; Cayre, Fanny; Buchy, Eric; Pieters, Grégory; Garcia-Argote, Sébastien; Windbergs, Maike; Desmaële, Didier; et al. (2017-07-05)
      Selective delivery of anticancer drugs to rapidly growing cancercells can be achieved by taking advantage of their high receptor-mediated uptake of low-density lipoproteins (LDLs). Indeed, wehave recently discovered that nanoparticles made of the squa-lene derivative of the anticancer agent gemcitabine (SQGem)strongly interacted with the LDLs in the human blood. In thepresent study, we showed both in vitro and in vivo that suchinteraction led to the preferential accumulation of SQGem incancer cells (MDA-MB-231) with high LDL receptor expression.As a result, an improved pharmacological activity has beenobserved in MDA-MB-231 tumor-bearing mice, an experi-mental model with a low sensitivity to gemcitabine. Accord-ingly, we proved that the use of squalene moieties not onlyinduced the gemcitabine insertion into lipoproteins, but thatit could also be exploited to indirectly target cancer cells in vivo.
    • Co-culture of human alveolar epithelial (hAELVi) and macrophage (THP-1) cell lines.

      Kletting, Stephanie; Barthold, Sarah; Repnik, Urska; Griffiths, Gareth; Loretz, Brigitta; Schneider-Daum, Nicole; de Souza Carvalho-Wodarz, Cristiane; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer Nature, 2018-01-01)
      The air-blood barrier is mainly composed of alveolar epithelial cells and macrophages. Whereas the epithelium acts as a diffusional barrier, macrophages represent an immunological barrier, in particular for larger molecules and nanoparticles. This paper describes a new co-culture of human cell lines representing both cell types. Acquiring, culturing and maintaining primary alveolar epithelial cells presents significant logistical and technical difficulties. The recently established human alveolar epithelial lentivirus immortalized cell line, hAELVi, when grown on permeable filters, forms monolayers with high functional and morphological resemblance to alveolar type I cells. To model alveolar macrophages, the human cell line THP-1 was seeded on pre-formed hAELVi monolayers. The co-culture was characterized regarding cellular morphology, viability and barrier function. Macrophages were homogenously distributed on the epithelium and could be kept in co-culture for up to 7 days. Transmission electron microscopy showed loose contact between THP-1 and hAELVi cells. When grown at air liquid interface, both cells were covered with extracellular matrix-like structure, which was absent in THP-1 mono-culture. In co-culture with macrophages, hAELVi cells displayed similar, sometimes even higher, transepithelial electrical resistance than in mono-cultures. When exposed to silver and starch nanoparticles, hAELVi mono-cultures were more tolerant to the particles than THP-1 mono-cultures. Viability in the co-culture was similar to that of hAELVi mono-cultures. Transport studies with sodium fluorescein in the presence/absence of EDTA proved that the co-culture acts as functional diffusion barrier. These data demonstrate that hAELVi-/THP-1 co-cultures represent a promising model for safety and permeability studies of inhaled chemicals, drugs and nanoparticles.
    • Co-Delivery of mRNA and pDNA Using Thermally Stabilized Coacervate-Based Core-Shell Nanosystems.

      Nasr, Sarah S; Lee, Sangeun; Thiyagarajan, Durairaj; Boese, Annette; Loretz, Brigitta; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2021-11-13)
      Co-delivery of different species of protein-encoding polynucleotides, e.g., messenger RNA (mRNA) and plasmid DNA (pDNA), using the same nanocarrier is an interesting topic that remains scarcely researched in the field of nucleic acid delivery. The current study hence aims to explore the possibility of the simultaneous delivery of mRNA (mCherry) and pDNA (pAmCyan) using a single nanocarrier. The latter is based on gelatin type A, a biocompatible, and biodegradable biopolymer of broad pharmaceutical application. A core-shell nanostructure is designed with a thermally stabilized gelatin-pDNA coacervate in its center. Thermal stabilization enhances the core's colloidal stability and pDNA shielding effect against nucleases as confirmed by nanoparticle tracking analysis and gel electrophoresis, respectively. The stabilized, pDNA-loaded core is coated with the cationic peptide protamine sulfate to enable additional surface-loading with mRNA. The dual-loaded core-shell system transfects murine dendritic cell line DC2.4 with both fluorescent reporter mRNA and pDNA simultaneously, showing a transfection efficiency of 61.4 ± 21.6% for mRNA and 37.6 ± 19.45% for pDNA, 48 h post-treatment, whereas established commercial, experimental, and clinical transfection reagents fail. Hence, the unique co-transfectional capacity and the negligible cytotoxicity of the reported system may hold prospects for vaccination among other downstream applications.
    • Combining MucilAir™ and Vitrocell Powder Chamber for the In Vitro Evaluation of Nasal Ointments in the Context of Aerosolized Pollen.

      Metz, Julia; Knoth, Katharina; Groß, Henrik; Lehr, Claus-Michael; Stäbler, Carolin; Bock, Udo; Hittinger, Marius; HIPS, Helmholtz-Institute für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2018-05-10)
      Hay fever is notoriously triggered when nasal mucosa is exposed to allergenic pollen. One possibility to overcome this pollen exposure may be the application of an ointment with physical protective effects. In this context, we have investigated Bepanthen Eye and Nose Ointment and the ointment basis petrolatum as reference while using contemporary in vitro techniques. Pollen from false ragweed () was used as an allergy-causing model deposited as aerosol using the Vitrocell Powder Chamber (VPC) on Transwell inserts, while being coated with either Bepanthen Eye and Nose Ointment and petrolatum. No pollen penetration into ointments was observed upon confocal scanning laser microscopy during an incubation period of 2 h at 37 °C. The cellular response was further investigated by integrating the MucilAir™ cell system in the VPC and by applying pollen to Bepanthen Eye and Nose Ointment covered cell cultures. For comparison, MucilAir™ were stimulated by lipopolysaccharides (LPS). No increased cytokine release of IL-6, TNF-α, or IL-8 was found after 4 h of pollen exposure, which demonstrates the safety of such ointments. Since nasal ointments act as a physical barrier against pollen, such preparations might support the prevention and management of hay fever.
    • Controlling Supramolecular Structures of Drugs by Light.

      Wiest, Johannes; Kehrein, Josef; Saedtler, Marco; Schilling, Klaus; Cataldi, Eleonora; Sotriffer, Christoph A; Holzgrabe, Ulrike; Rasmussen, Tim; Böttcher, Bettina; Cronin-Golomb, Mark; et al. (American Chemical Society (ACS), 2020-10-29)
      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.
    • Coupling quaternary ammonium surfactants to the surface of liposomes improves both antibacterial efficacy and host cell biocompatibility

      Montefusco-Pereira, Carlos V.; Formicola, Beatrice; Goes, Adriely; Re, Francesca; Marrano, Claudia A.; Mantegazza, Francesco; Carvalho-Wodarz, Cristiane; Fuhrmann, Gregor; Caneva, Enrico; Nicotra, Francesco; et al. (Elsevier BV, 2020-04)
      By functionalizing the surface of PEG-liposomes with linkers bearing quaternary ammonium compounds (QACs), we generated novel bacteria disruptors with anti-adhesive properties and reduced cytotoxicity compared to free QACs. Furthermore, QAC-functionalized liposomes are a promising platform for future drug encapsulation. The QAC (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide (MTAB) was attached to maleimide-functionalized liposomes (DSPE-PEG) via thiol linker. The MTAB-functionalized liposomes were physicochemically characterized and their biological activity, in terms of anti-adherence activity and biofilm prevention in Escherichia coli were assessed. The results showed that MTAB-functionalized liposomes inhibit bacterial adherence and biofilm formation while reducing MTAB toxicity.
    • Crossing biological barriers for advanced drug delivery.

      Schneider, Marc; Windbergs, Maike; Daum, Nicole; Loretz, Brigitta; Collnot, Eva-Maria; Hansen, Steffi; Schaefer, Ulrich F; Lehr, Claus-Michael (2013-06)
      This special issue compiles invited and contributed papers of the 9th International Conference and Workshop "Biological Barriers", 29 February-9 March 2012 at Saarland University, Saarbrücken Germany.
    • A Custom-Made Device for Reproducibly Depositing Pre-metered Doses of Nebulized Drugs on Pulmonary Cells .

      Horstmann, Justus C; Thorn, Chelsea R; Carius, Patrick; Graef, Florian; Murgia, Xabier; de Souza Carvalho-Wodarz, Cristiane; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Frontiers, 2021-04-21)
      The deposition of pre-metered doses (i.e., defined before and not after exposition) at the air-liquid interface of viable pulmonary epithelial cells remains an important but challenging task for developing aerosol medicines. While some devices allow quantification of the deposited dose after or during the experiment, e.g., gravimetrically, there is still no generally accepted way to deposit small pre-metered doses of aerosolized drugs or pharmaceutical formulations, e.g., nanomedicines. Here, we describe a straightforward custom-made device, allowing connection to commercially available nebulizers with standard cell culture plates. Designed to tightly fit into the approximately 12-mm opening of either a 12-well Transwell® insert or a single 24-well plate, a defined dose of an aerosolized liquid can be directly deposited precisely and reproducibly (4.8% deviation) at the air-liquid interface (ALI) of pulmonary cell cultures. The deposited dose can be controlled by the volume of the nebulized solution, which may vary in a range from 20 to 200 μl. The entire nebulization-deposition maneuver is completed after 30 s and is spatially homogenous. After phosphate-buffered saline (PBS) deposition, the viability and barrier properties transepithelial electrical resistance (TEER) of human bronchial epithelial Calu-3 cells were not negatively affected. Straightforward in manufacture and use, the device enables reproducible deposition of metered doses of aerosolized drugs to study the interactions with pulmonary cell cultures grown at ALI conditions.
    • 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]