• 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.
    • 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.
    • 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.
    • 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; Ayre, D Craig; Bach, Jean-Marie; Bachurski, Daniel; Baharvand, Hossein; Balaj, Leonora; Baldacchino, Shawn; Bauer, Natalie N; Baxter, Amy A; Bebawy, Mary; Beckham, Carla; Bedina Zavec, Apolonija; Benmoussa, Abderrahim; Berardi, Anna C; Bergese, Paolo; Bielska, Ewa; Blenkiron, Cherie; Bobis-Wozowicz, Sylwia; Boilard, Eric; Boireau, Wilfrid; Bongiovanni, Antonella; Borràs, Francesc E; Bosch, Steffi; Boulanger, Chantal M; Breakefield, Xandra; Breglio, Andrew M; Brennan, Meadhbh Á; Brigstock, David R; Brisson, Alain; Broekman, Marike LD; Bromberg, Jacqueline F; Bryl-Górecka, Paulina; Buch, Shilpa; Buck, Amy H; Burger, Dylan; Busatto, Sara; Buschmann, Dominik; Bussolati, Benedetta; Buzás, Edit I; Byrd, James Bryan; Camussi, Giovanni; Carter, David RF; Caruso, Sarah; Chamley, Lawrence W; Chang, Yu-Ting; Chen, Chihchen; Chen, Shuai; Cheng, Lesley; Chin, Andrew R; Clayton, Aled; Clerici, Stefano P; Cocks, Alex; Cocucci, Emanuele; Coffey, Robert J; Cordeiro-da-Silva, Anabela; Couch, Yvonne; Coumans, Frank AW; Coyle, Beth; Crescitelli, Rossella; Criado, Miria Ferreira; D’Souza-Schorey, Crislyn; Das, Saumya; Datta Chaudhuri, Amrita; de Candia, Paola; De Santana, Eliezer F; De Wever, Olivier; del Portillo, Hernando A; Demaret, Tanguy; Deville, Sarah; Devitt, Andrew; Dhondt, Bert; Di Vizio, Dolores; Dieterich, Lothar C; Dolo, Vincenza; Dominguez Rubio, Ana Paula; Dominici, Massimo; Dourado, Mauricio R; Driedonks, Tom AP; Duarte, Filipe V; Duncan, Heather M; Eichenberger, Ramon M; Ekström, Karin; EL Andaloussi, Samir; Elie-Caille, Celine; Erdbrügger, Uta; Falcón-Pérez, Juan M; Fatima, Farah; Fish, Jason E; Flores-Bellver, Miguel; Försönits, András; Frelet-Barrand, Annie; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany.