• The bacterial cell envelope as delimiter of anti-infective bioavailability - An in vitro permeation model of the Gram-negative bacterial inner membrane.

      Graef, Florian; Vukosavljevic, Branko; Michel, Jean-Philippe; Wirth, Marius; Ries, Oliver; De Rossi, Chiara; Windbergs, Maike; Rosilio, Véronique; Ducho, Christian; Gordon, Sarah; et al. (2016)
      Gram-negative bacteria possess a unique and complex cell envelope, composed of an inner and outer membrane separated by an intermediate cell wall-containing periplasm. This tripartite structure acts intrinsically as a significant biological barrier, often limiting the permeation of anti-infectives, and so preventing such drugs from reaching their target. Furthermore, identification of the specific permeation-limiting envelope component proves difficult in the case of many anti-infectives, due to the challenges associated with isolation of individual cell envelope structures in bacterial culture. The development of an in vitro permeation model of the Gram-negative inner membrane, prepared by repeated coating of physiologically-relevant phospholipids on Transwell®filter inserts, is therefore reported, as a first step in the development of an overall cell envelope model. Characterization and permeability investigations of model compounds as well as anti-infectives confirmed the suitability of the model for quantitative and kinetically-resolved permeability assessment, and additionally confirmed the importance of employing bacteria-specific base materials for more accurate mimicking of the inner membrane lipid composition - both advantages compared to the majority of existing in vitro approaches. Additional incorporation of further elements of the Gram-negative bacterial cell envelope could ultimately facilitate model application as a screening tool in anti-infective drug discovery or formulation development.
    • 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.
    • Barriers and motivations for non-invasive drug delivery.

      Loretz, Brigitta; Schneider-Daum, Nicole; Windbergs, Maike; Schaefer, Ulrich; Schneider, Marc; Lehr, Claus Michael; HIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus E8.1,66123 Saarbrücken, Germany. (2017-09)
    • Biodegradable starch derivatives with tunable charge density-synthesis, characterization, and transfection efficiency.

      Thiele, Carolin; Loretz, Brigitta; Lehr, Claus Michael; Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS),Saarland Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2016-10-03)
      Regioselective oxidation of water-soluble starch and conversion with alkyl diamines resulted in defined cationic starch derivatives. Those were assessed in their potential for polyplex formation, biocompatibility, and transfection efficacy. The new polymers have the advantage of being biodegradable, being not cytotoxic at rather high concentrations (LC50 > 400 μg/ml) for C2 substitution, and reach transfection efficiencies comparable to commercial transfection reagents. The polymer with the highest transfection efficacy is a C12 substituted polymer (degree of substitution = 30 %) at N/P 3. The LC50 value of that highly modified polymer is still one order of magnitude lower than that of PEI 25 kDa.
    • Bioinspired Liposomes for Oral Delivery of Colistin to Combat Intracellular Infections by Salmonella enterica.

      Menina, Sara; Eisenbeis, Janina; Kamal, Mohamed Ashraf M; Koch, Marcus; Bischoff, Markus; Gordon, Sarah; Loretz, Brigitta; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Wiley-VCH, 2019-07-22)
      Bacterial invasion into eukaryotic cells and the establishment of intracellular infection has proven to be an effective means of resisting antibiotic action, as anti-infective agents commonly exhibit a poor permeability across the host cell membrane. Encapsulation of anti-infectives into nanoscaled delivery systems, such as liposomes, is shown to result in an enhancement of intracellular delivery. The aim of the current work is, therefore, to formulate colistin, a poorly permeable anti-infective, into liposomes suitable for oral delivery, and to functionalize these carriers with a bacteria-derived invasive moiety to enhance their intracellular delivery. Different combinations of phospholipids and cholesterol are explored to optimize liposomal drug encapsulation and stability in biorelevant media. These liposomes are then surface-functionalized with extracellular adherence protein (Eap), derived from Staphylococcus aureus. Treatment of HEp-2 and Caco-2 cells infected with Salmonella enterica using colistin-containing, Eap-functionalized liposomes resulted in a significant reduction of intracellular bacteria, in comparison to treatment with nonfunctionalized liposomes as well as colistin alone. This indicates that such bio-invasive carriers are able to facilitate intracellular delivery of colistin, as necessary for intracellular anti-infective activity. The developed Eap-functionalized liposomes, therefore, present a promising strategy for improving the therapy of intracellular infections.
    • 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)
    • Calcifediol-loaded liposomes for local treatment of pulmonary bacterial infections.

      Castoldi, Arianna; Herr, Christian; Niederstraßer, Julia; Labouta, Hagar Ibrahim; Melero, Ana; Gordon, Sarah; Schneider-Daum, Nicole; Bals, Robert; Lehr, Claus Michael; HIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus 8.1, 66123 Saarbrücken, Germany. (2017-09)
      The influence of vitamin D3 and its metabolites calcifediol (25(OH)D) and calcitriol on immune regulation and inflammation is well described, and raises the question of potential benefit against bacterial infections. In the current study, 25(OH)D was encapsulated in liposomes to enable aerosolisation, and tested for the ability to prevent pulmonary infection by Pseudomonas aeruginosa. Prepared 25(OH)D-loaded liposomes were nanosized and monodisperse, with a negative surface charge and a 25(OH)D entrapment efficiency of approximately 23%. Jet nebulisation of liposomes was seen to yield an aerosol suitable for tracheo-bronchial deposition. Interestingly, 25(OH)D in either liposomes or ethanolic solution had no effect on the release of the proinflammatory cytokine KC from Pseudomonas-infected murine epithelial cells (LA-4); treatment of infected, human bronchial 16-HBE cells with 25(OH)D liposomes however resulted in a significant reduction in bacterial survival. Together with the importance of selecting an application-appropriate in vitro model, the current study illustrates the feasibility and practicality of employing liposomes as a means to achieve 25(OH)D lung deposition. 25(OH)D-loaded liposomes further demonstrated promising effects regarding prevention of Pseudomonas infection in human bronchial epithelial cells.
    • Calcium Phosphate System for Gene Delivery: Historical Background and Emerging Opportunities.

      Mostaghaci, Babak; Loretz, Brigitta; Lehr, Claus-Michael; Helmholtz Institut f?r Pharmazeutische Forschung Saarland, Universit?tscampus E8.1, 66123 Saarbr?cken, Germany. (2016)
      Calcium phosphate system has been used widely in in vitro gene delivery for almost four decades. Excellent biocompatibility and simple application have motivated the researchers to always consider this system in their transfection experiments. However, there was a major drawback regarding the low transfection efficiency of calcium phosphate. Hence, there have been many efforts in order to increase the gene delivery potential of this system. In this paper, the application of calcium phosphate in gene delivery is introduced. Moreover, the recent progresses in the application of calcium phosphate in the delivery of (oligo)nucleotides and different approaches to improve the properties of this system are reviewed.
    • Capturing the Onset of Bacterial Pulmonary Infection in Acini-On-Chips

      Artzy-Schnirman, Arbel; Zidan, Hikaia; Elias-Kirma, Shani; Ben-Porat, Lee; Tenenbaum-Katan, Janna; Carius, Patrick; Fishler, Ramy; Schneider-Daum, Nicole; Lehr, Claus Michael; Sznitman, Josué (Wiley-VCH, 2019-09-01)
    • Cellular delivery of polynucleotides by cationic cyclodextrin polyrotaxanes.

      Dandekar, Prajakta; Jain, Ratnesh; Keil, Manuel; Loretz, Brigitta; Muijs, Leon; Schneider, Marc; Auerbach, Dagmar; Jung, Gregor; Lehr, Claus-Michael; Wenz, Gerhard; et al. (2012-12-28)
      Cationic polyrotaxanes, obtained by temperature activated threading of cationic cyclodextrin derivatives onto water-soluble cationic polymers (ionenes), form metastable nanometric polyplexes with pDNA and combinations of siRNA with pDNA. Because of their low toxicity, the polyrotaxane polyplexes constitute a very interesting system for the transfection of polynucleotides into mammalian cells. The complexation of Cy3-labeled siRNA within the polyplexes was demonstrated by fluorescence correlation spectroscopy. The uptake of the polyplexes (red) was imaged by confocal fluorescence microscopy using the A549 cell line as a model (blue: nuclei, green: membranes). The results prove the potential of polyrotaxanes for further investigations involving knocking down genes of therapeutic interest.
    • Challenges and Strategies in Drug Delivery Systems for Treatment of Pulmonary Infections.

      Ho, Duy-Khiet; Nichols, Brittany L B; Edgar, Kevin J; Murgia, Xabier; Loretz, Brigitta; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Elsevier, 2019-09-04)
      Inhalation therapy has been reported as the most effective treatment for respiratory bacterial infections due to the increasing relevance of drug bioavailability. Drug delivery systems (DDS) have the capacity to overcome pulmonary biological barriers limiting the bioavailability of inhaled anti-infectives. This is important to eradicate bacterial infections and to prevent the development of bacterial resistance. Despite substantial efforts in the field, the current state-of-the-art often fails to achieve those goals, and we still observe increasing bacterial resistance. We give a brief insight on benefits and challenges in pulmonary delivery of anti-infectives. In the context of drug delivery development for pulmonary infections, particularly focusing on Pseudomonas aeruginosa (PA) infections, this mini review will critically discuss the main requirements, as well as the recent strategies of drug delivery system synthesis and preparation. Finally, interaction of DDS with crucial pulmonary biological barriers will be of great importance for the success of future applications of the developed DDS.
    • Characterization and evaluation of β-glucan formulations as injectable implants for protein and peptide delivery.

      Jacobs, Simone; Bunt, Craig R; Wu, Zimei; Lehr, Claus-Michael; Rupenthal, Ilva D; Biopharmaceutics and Pharmaceutical Technology, Saarland University, Saarbrücken, Germany. (2012-11)
      Injectable implants are biodegradable, syringeable formulations that are injected as liquids, but form a gel inside the body due to a change in pH, ions or temperature.
    • 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.
    • 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.