group leader: Dr. Fuhrmann

Recent Submissions

  • Biogenic and Biomimetic Carriers as Versatile Transporters To Treat Infections.

    Goes, Adriely; Fuhrmann, Gregor; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (American Chemical Society, 2018-03-29)
    Biogenic and biomimetic therapeutics are a relatively new class of systems that are of physiological origin and/or take advantage of natural pathways or aim at mimicking these to improve selective interaction with target tissue. The number of biogenic and bioengineered avenues for drug therapy and diagnostics has multiplied over the past years for many applications, indicating the high expectations associated with this biological route. Nevertheless, the use of "bio"-related approaches for treating or diagnosing infectious diseases is still rare. Given that infectious diseases, in particular bacterial resistances, are seriously on the rise, there is an urgent need to take advantage of biogenic and bioengineered systems to target these challenges. In this manuscript, we first give a definition of the various "bio" terms, including biogenic, biomimetic, bioinspired, and bioengineered and we highlight them using tangible applications in the field of infectious diseases. Our examples cover cell-derived systems, including bioengineered bacteria, virus-like particles, and different cell-mimetics. Moreover, we discuss natural and bioengineered particles such as extracellular vesicles from mammalian and bacterial sources and liposomes. A concluding section outlines the potential for biomaterial-related avenues to overcome challenges associated with difficult-to-treat infections. We critically discuss benefits and risks for these applications and give an outlook on the future of biogenic engineering.
  • Liver-derived extracellular vesicles: A cell by cell overview to isolation and characterization practices.

    Zivko, Cristina; Fuhrmann, Gregor; Luciani, Paola; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Elsevier, 2020-02-19)
    BACKGROUND: Extracellular vesicles (EVs) are a diverse group of membrane-bound nanovesicles potentially released by every cell. With the liver's unique ensemble of cells and its fundamental physiological tasks, elucidating the role of EV-mediated hepatic cellular crosstalk and their role in different pathologies has been gaining the attention of many scientists. SCOPE OF REVIEW: The present review shifts the perspective into practice: we aim to critically discuss the methods used to purify and to biochemically analyse EVs from specific liver resident cells, including hepatocytes, hepatic stellate cells, cholangiocytes, liver sinusoidal endothelial cells, Kupffer cells, liver stem cells. The review offers a reference guide to current approaches. MAJOR CONCLUSIONS: Strategies for EV isolation and characterization are as varied as the research groups performing them. We present main advantages and disadvantages for the methods, highlighting common causes for concern, such as FBS handling, reporting of cell viability, EV yield and storage, differences in differential centrifugations, suboptimal method descriptions, and method transferability. We both looked at how adaptable the research between human and rodent cells in vitro is, and also assessed how well either of them translates to ex vivo settings. GENERAL SIGNIFICANCE: We reviewed methodological practices for the isolation and analysis of liver-derived EVs, making a cell type specific user guide that shows where to start, what has worked so far and to what extent. We critically discussed room for improvement, placing a particular focus on working towards a potential standardization of methods.
  • Streptococcal Extracellular Membrane Vesicles Are Rapidly Internalized by Immune Cells and Alter Their Cytokine Release.

    Mehanny, Mina; Koch, Marcus; Lehr, Claus-Michael; Fuhrmann, Gregor; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Frontiers, 2020-02-14)
    Extracellular vesicles are membranous structures shed by almost every living cell. Bacterial gram-negative outer membrane vesicles (OMVs) and gram-positive membrane vesicles (MVs) play important roles in adaptation to the surrounding environment, cellular components' exchange, transfer of antigens and virulence factors, and infection propagation. Streptococcus pneumoniae is considered one of the priority pathogens, with a global health impact due to the increase in infection burden and growing antibiotic resistance. We isolated MVs produced from the S. pneumoniae reference strain (R6) and purified them via size exclusion chromatography (SEC) to remove soluble protein impurities. We characterized the isolated MVs by nanoparticle tracking analysis (NTA) and measured their particle size distribution and concentration. Isolated MVs showed a mean particle size range of 130-160 nm and a particle yield of around 1012 particles per milliliter. Cryogenic transmission electron microscopy (cryo-TEM) images revealed a very heterogeneous nature of isolated MVs with a broad size range and various morphologies, arrangements, and contents. We incubated streptococcal MVs with several mammalian somatic cells, namely, human lung epithelial A549 and human keratinocytes HaCaT cell lines, and immune cells including differentiated macrophage-like dTHP-1 and murine dendritic DC2.4 cell lines. All cell lines displayed excellent viability profile and negligible cytotoxicity after 24-h incubation with MVs at concentrations reaching 106 MVs per cell (somatic cells) and 105 MVs per cell (immune cells). We evaluated the uptake of fluorescently labeled MVs into these four cell lines, using flow cytometry and confocal microscopy. Dendritic cells demonstrated prompt uptake after 30-min incubation, whereas other cell lines showed increasing uptake after 2-h incubation and almost complete colocalization/internalization of MVs after only 4-h incubation. We assessed the influence of streptococcal MVs on antigen-presenting cells, e.g., dendritic cells, using enzyme-linked immunosorbent assay (ELISA) and observed enhanced release of tumor necrosis factor (TNF)-α, a slight increase of interleukin (IL)-10 secretion, and no detectable effect on IL-12. Our study provides a better understanding of gram-positive streptococcal MVs and shows their potential to elicit a protective immune response. Therefore, they could offer an innovative avenue for safe and effective cell-free vaccination against pneumococcal infections.
  • Diffusion and transport of extracellular vesicles.

    Fuhrmann, Gregor; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer Nature, 2020-02-17)
    Cell-derived extracellular vesicles are important intercellular communicators involved in many biological processes and diseases, including cancer and cardiovascular diseases, but, thus far, how they navigate within complex extracellular matrices has been poorly understood.
  • 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.
  • Myxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections.

    Goes, Adriely; Lapuhs, Philipp; Kuhn, Thomas; Schulz, Eilien; Richter, Robert; Panter, Fabian; Dahlem, Charlotte; Koch, Marcus; Garcia, Ronald; Kiemer, Alexandra K; et al. (MDPI, 2020-01-12)
    In 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is Staphylococcus aureus, a bacterium that can invade and survive within mammalian cells. S. aureus intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria Cystobacter velatus strain Cbv34 and Cystobacter ferrugineus strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular S. aureus. We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular S. aureus. In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections.
  • Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide.

    Hoppstädter, Jessica; Dembek, Anna; Linnenberger, Rebecca; Dahlem, Charlotte; Barghash, Ahmad; Fecher-Trost, Claudia; Fuhrmann, Gregor; Koch, Marcus; Kraegeloh, Annette; Huwer, Hanno; et al. (Frontiers, 2019-01-01)
    Glucocorticoids (GCs) are widely prescribed therapeutics for the treatment of inflammatory diseases, and endogenous GCs play a key role in immune regulation. Toll-like receptors (TLRs) enable innate immune cells, such as macrophages, to recognize a wide variety of microbial ligands, thereby promoting inflammation. The interaction of GCs with macrophages in the immunosuppressive resolution phase upon prolonged TLR activation is widely unknown. Treatment of human alveolar macrophages (AMs) with the synthetic GC dexamethasone (Dex) did not alter the expression of TLRs -1, -4, and -6. In contrast, TLR2 was upregulated in a GC receptor-dependent manner, as shown by Western blot and qPCR. Furthermore, long-term lipopolysaccharide (LPS) exposure mimicking immunosuppression in the resolution phase of inflammation synergistically increased Dex-mediated TLR2 upregulation. Analyses of publicly available datasets suggested that TLR2 is induced during the resolution phase of inflammatory diseases, i.e., under conditions associated with high endogenous GC production. TLR2 induction did not enhance TLR2 signaling, as indicated by reduced cytokine production after treatment with TLR2 ligands in Dex- and/or LPS-primed AMs. Thus, we hypothesized that the upregulated membrane-bound TLR2 might serve as a precursor for soluble TLR2 (sTLR2), known to antagonize TLR2-dependent cell actions. Supernatants of LPS/Dex-primed macrophages contained sTLR2, as demonstrated by Western blot analysis. Activation of metalloproteinases resulted in enhanced sTLR2 shedding. Additionally, we detected full-length TLR2 and assumed that this might be due to the production of TLR2-containing extracellular vesicles (EVs). EVs from macrophage supernatants were isolated by sequential centrifugation. Both untreated and LPS/Dex-treated cells produced vesicles of various sizes and shapes, as shown by cryo-transmission electron microscopy. These vesicles were identified as the source of full-length TLR2 in macrophage supernatants by Western blot and mass spectrometry. Flow cytometric analysis indicated that TLR2-containing EVs were able to bind the TLR2 ligand Pam3CSK4. In addition, the presence of EVs reduced inflammatory responses in Pam3CSK4-treated endothelial cells and HEK Dual reporter cells, demonstrating that TLR2-EVs can act as decoy receptors. In summary, our data show that sTLR2 and full-length TLR2 are released by macrophages under anti-inflammatory conditions, which may contribute to GC-induced immunosuppression.
  • Hot EVs - how temperature affects extracellular vesicles.

    Schulz, Eilien; Karagianni, Anna; Koch, Marcus; Fuhrmann, Gregor; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Elsevier, 2019-12-02)
    In recent years, extracellular vesicles (EVs) and outer membrane vesicles (OMVs) have become an extensive and diverse field of research. They hold potential as diagnostic markers, therapeutics and for fundamental biological understanding. Despite ongoing studies, numerous information regarding function, content and stability of EVs remains unclear. If EVs and OMVs ought to be used as therapeutics and in clinical environments, their stability is one of the most important factors to be considered. Especially for formulation development, EVs and OMVs need to be stable at higher temperatures. To the best of our knowledge, very little work has been published regarding heat stability of neither EVs nor OMVs. In the present study, we investigated B lymphoblastoid cell-derived EVs and OMVs derived from myxobacterial species Sorangiineae as model vesicles. We exposed the vesicles to 37 °C, 50 °C, 70 °C and 100 °C for 1 h, 6 h and 24 h, and also autoclaved them. Interestingly, physico-chemical analyses such as size, particle concentration and protein concentration showed minor alterations, particularly at 37 °C. Flow cytometry analysis emphasised these results suggesting that after heat impact, EVs and OMVs were still able to be taken up by macrophage-like dTHP-1 cells. These data indicate that both mammalian and bacterial vesicles show intrinsic stability at physiological temperature. Our findings are important to consider for vesicle formulation and for advanced bioengineering approaches.
  • Extracellular Vesicles-Connecting Kingdoms.

    Woith, Eric; Fuhrmann, Gregor; Melzig, Matthias F; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2019-11-14)
    It is known that extracellular vesicles (EVs) are shed from cells of almost every type of cell or organism, showing their ubiquity in all empires of life. EVs are defined as naturally released particles from cells, delimited by a lipid bilayer, and cannot replicate. These nano- to micrometer scaled spheres shuttle a set of bioactive molecules. EVs are of great interest as vehicles for drug targeting and in fundamental biological research, but in vitro culture of animal cells usually achieves only small yields. The exploration of other biological kingdoms promises comprehensive knowledge on EVs broadening the opportunities for basic understanding and therapeutic use. Thus, plants might be sustainable biofactories producing nontoxic and highly specific nanovectors, whereas bacterial and fungal EVs are promising vaccines for the prevention of infectious diseases. Importantly, EVs from different eukaryotic and prokaryotic kingdoms are involved in many processes including host-pathogen interactions, spreading of resistances, and plant diseases. More extensive knowledge of inter-species and interkingdom regulation could provide advantages for preventing and treating pests and pathogens. In this review, we present a comprehensive overview of EVs derived from eukaryota and prokaryota and we discuss how better understanding of their intercommunication role provides opportunities for both fundamental and applied biology.
  • Appraisal on the wound healing potential of Melaleuca alternifolia and Rosmarinus officinalis L. essential oil-loaded chitosan topical preparations.

    Labib, Rola M; Ayoub, Iriny M; Michel, Haidy E; Mehanny, Mina; Kamil, Verena; Hany, Meryl; Magdy, Mirette; Moataz, Aya; Maged, Boula; Mohamed, Ahmed; et al. (PLOS, 2019-01-01)
    The present study investigates the wound healing potential of three chitosan-based topical preparations loaded with either tea tree essential oil, rosemary essential oil or a mixture of both oils in vivo. Essential oils of M. alternifolia and R. officinalis were analyzed using GC/MS. Essential oil-loaded chitosan topical preparations were formulated. Wound healing potential was evaluated in vivo using an excision wound model in rats. GC/MS analysis of M. alternifolia and R. officinalis essential oils revealed richness in oxygenated monoterpenes, representing 51.06% and 69.61% of the total oil composition, respectively. Topical application of chitosan-based formulation loaded with a mixture of tea tree and rosemary oils resulted in a significant increase in wound contraction percentage compared to either group treated with individual essential oils and the untreated group. Histopathological examination revealed that topical application of tea tree and rosemary oil combination demonstrated complete re-epithelialization associated with activated hair follicles. The high percentage of oxygenated monoterpenes in both essential oils play an important role in the antioxidant and wound healing potential observed herein. Incorporation of tea tree and rosemary essential oils in chitosan-based preparations in appropriate combination could efficiently promote different stages of wound healing.
  • Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

    Théry, Clotilde; Witwer, Kenneth W; Aikawa, Elena; Alcaraz, Maria Jose; Anderson, Johnathon D; Andriantsitohaina, Ramaroson; Antoniou, Anna; Arab, Tanina; Archer, Fabienne; Atkin-Smith, Georgia K; et al.
  • Biocompatible bacteria-derived vesicles show inherent antimicrobial activity.

    Schulz, Eilien; Goes, Adriely; Garcia, Ronald; Panter, Fabian; Koch, Marcus; Müller, Rolf; Fuhrmann, Kathrin; Fuhrmann, Gregor; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Elsevier, 2018-11-28)
    Up to 25,000 people die each year from resistant infections in Europe alone, with increasing incidence. It is estimated that a continued rise in bacterial resistance by 2050 would lead up to 10 million annual deaths worldwide, exceeding the incidence of cancer deaths. Although the design of new antibiotics is still one way to tackle the problem, pharmaceutical companies investigate far less into new drugs than 30 years ago. Incorporation of antibiotics into nanoparticle drug carriers ("nanoantibiotics") is currently investigated as a promising strategy to make existing antibiotics regain antimicrobial strength and overcome certain types of microbial drug resistance. Many of these synthetic systems enhance the antimicrobial effect of drugs by protecting antibiotics from degradation and reducing their side effects. Nevertheless, they often cannot selectively target pathogenic bacteria and - due to their synthetic origin - may induce side-effects themselves. In this work, we present the characterisation of naturally derived outer membrane vesicles (OMVs) as biocompatible and inherently antibiotic drug carriers. We isolated OMVs from two representative strains of myxobacteria, Cystobacter velatus Cbv34 and Sorangiineae species strain SBSr073, a bacterial order with the ability of lysing other bacterial strains and currently investigated as sources of new secondary metabolites. We investigated the myxobacterias' inherent antibacterial properties after isolation by differential centrifugation and purification by size-exclusion chromatography. OMVs have an average size range of 145-194 nm. We characterised their morphology by electron cryomicroscopy and found that OMVs are biocompatible with epithelial cells and differentiated macrophages. They showed a low endotoxin activity comparable to those of control samples, indicating a low acute inflammatory potential. In addition, OMVs showed inherent stability under different storage conditions, including 4 °C, -20 °C, -80 °C and freeze-drying. OMV uptake in Gram-negative model bacterium Escherichia coli (E. coli) showed similar to better incorporation than liposome controls, indicating the OMVs may interact with model bacteria via membrane fusion. Bacterial uptake correlated with antimicrobial activity of OMVs as measured by growth inhibition of E. coli. OMVs from Cbv34 inhibited growth of E. coli to a comparable extent as the clinically established antibiotic gentamicin. Liquid-chromatography coupled mass spectrometry analyses revealed the presence of cystobactamids in OMVs, inhibitors of bacterial topoisomerase currently studied to treat different Gram-negative and Gram-positive pathogens. This work, may serve as an important basis for further evaluation of OMVs derived from myxobacteria as novel therapeutic delivery systems against bacterial infections.
  • 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.
  • Engineering Extracellular Vesicles with the Tools of Enzyme Prodrug Therapy.

    Fuhrmann, Gregor; Chandrawati, Rona; Parmar, Paresh A; Keane, Timothy J; Maynard, Stephanie A; Bertazzo, Sergio; Stevens, Molly M; HIPS, Helmholtz-Institute für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2018-02-23)
    Extracellular vesicles (EVs) have recently gained significant attention as important mediators of intercellular communication, potential drug carriers, and disease biomarkers. These natural cell-derived nanoparticles are postulated to be biocompatible, stable under physiological conditions, and to show reduced immunogenicity as compared to other synthetic nanoparticles. Although initial clinical trials are ongoing, the use of EVs for therapeutic applications may be limited due to undesired off-target activity and potential "dilution effects" upon systemic administration which may affect their ability to reach their target tissues. To fully exploit their therapeutic potential, EVs are embedded into implantable biomaterials designed to achieve local delivery of therapeutics taking advantage of enzyme prodrug therapy (EPT). In this first application of EVs for an EPT approach, EVs are used as smart carriers for stabilizing enzymes in a hydrogel for local controlled conversion of benign prodrugs to active antiinflammatory compounds. It is shown that the natural EVs' antiinflammatory potential is comparable or superior to synthetic carriers, in particular upon repeated long-term incubations and in different macrophage models of inflammation. Moreover, density-dependent color scanning electron microscopy imaging of EVs in a hydrogel is presented herein, an impactful tool for further understanding EVs in biological settings.
  • Extracellular vesicles - A promising avenue for the detection and treatment of infectious diseases?

    Fuhrmann, Gregor; Neuer, Anna Lena; Herrmann, Inge K; Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-04-07)
    Extracellular vesicles (EVs) have gained increasing attention as novel disease biomarkers and as promising therapeutic agents. These cell-derived, phospholipid-based particles are present in many - if not all - physiological fluids. They have been shown to govern several physiological processes, such as cell-cell communication, but also to be involved in pathological conditions, for example tumour progression. In infectious diseases, EVs have been shown to induce host immune responses and to mediate transfer of virulence or resistance factors. Here, we discuss recent developments in using EVs as diagnostic tools for infectious diseases, the development of EV-based vaccines and the use of EVs as potential anti-infective entity. We illustrate how EV-based strategies could open a viable new avenue to tackle current challenges in the field of infections, including barrier penetration and growing resistance to antimicrobials.