The development and evaluation of extracellular vesicles as a biocompatible anti-infective drug delivery system

Approximately 700,000 people worldwide die from infections with antimicrobial-resistant bacteria annually. Extracellular vesicles (EVs) can help to delay or even prevent the development of this resistance by releasing high concentrations of antimicrobial agents specifically at the site of infection. In this context, vesicles derived from immune cells and myxobacteria were tested for the use as anti-infective drug delivery systems. Five main research objects were investigated: i) characterization of the vesicles, ii) storage stability iii) biocompatibility, iv) uptake in bacteria and cells and v) their antimicrobial activity against bacterial pathogens. The vesicles exhibited sufficient stability and showed exceptional biocompatibility with low endotoxin levels, minor cytokine release by primary immune cells and no toxicity in zebrafish larvae. Vesicles were taken up into bacteria, cells and the epithelial layer of a 3D gastrointestinal co-culture model. An inherent antibacterial effect against Escherichia coli was observed with vesicles derived from Cystobacter velatus. Non-inherently active vesicles were successfully loaded with a broad-spectrum antibiotic, which thus inhibited the growth of Shigella flexneri. The findings in this work demonstrate the exceptional properties of EVs to treat infections and provide the foundation for a successful translation towards clinical application.Weltweit sterben jährlich ca. 700.000 Menschen an einer Infektion mit antimikrobiell resistenten Bakterien. Extrazelluläre Vesikel (EVs) können dazu beitragen, die Entwicklung der Resistenzen zu verzögern oder gar zu verhindern, indem sie gezielt am Ort der Infektion hohe Konzentrationen an antimikrobiellen Wirkstoffen freisetzen. In diesem Zusammenhang wurden Vesikel, von Immunzellen und Myxobakterien, auf ihre Verwendung als antiinfektive Wirkstofftransportsysteme getestet. Fünf Hauptforschungspunkte wurden untersucht: i) Charakterisierung der Vesikel, ii) Stabilität iii) Biokompatibilität, iv) Aufnahme in Bakterien und Zellen und v) ihre antimikrobielle Aktivität gegenüber Pathogenen. Die Vesikel wiesen eine ausreichende Stabilität auf und zeigten hohe Biokompatibilität mit niedrigen Endotoxinwerten, geringer Zytokinfreisetzung durch primäre Immunzellen und keine Toxizität in Zebrafischlarven. Die Vesikel wurden in Bakterien, Zellen und in die Epithelschicht eines 3D-Gastrointestinalen Kokulturmodells aufgenommen. Eine inhärente antibakterielle Wirkung gegen Escherichia coli wurde mit Vesikeln von Cystobacter velatus beobachtet. Nicht inhärent aktive Vesikel wurden erfolgreich mit einem Breitspektrum-Antibiotikum beladen, welche so das Wachstum von Shigella flexneri hemmten. Die Ergebnisse dieser Arbeit weisen auf die außergewöhnlichen Eigenschaften von EVs zur Behandlung von Infektionen und bilden die Grundlage für eine erfolgreiche Translation in die klinische Anwendung

Enhancing the Stabilization Potential of Lyophilization for Extracellular Vesicles

Extracellular vesicles (EV) are an emerging technology as immune therapeutics and drug delivery vehicles. However, EVs are usually stored at −80 °C which limits potential clinical applicability. Freeze-drying of EVs striving for long-term stable formulations is therefore studied. The most appropriate formulation parameters are identified in freeze-thawing studies with two different EV types. After a freeze-drying feasibility study, four lyophilized EV formulations are tested for storage stability for up to 6 months. Freeze-thawing studies revealed improved colloidal EV stability in presence of sucrose or potassium phosphate buffer instead of sodium phosphate buffer or phosphate-buffered saline. Less aggregation and/or vesicle fusion occurred at neutral pH compared to slightly acidic or alkaline pH. EVs colloidal stability can be most effectively preserved by addition of low amounts of poloxamer 188. Polyvinyl pyrrolidone failed to preserve EVs upon freeze-drying. Particle size and concentration of EVs are retained over 6 months at 40 °C in lyophilizates containing 10 mm K- or Na-phosphate buffer, 0.02% poloxamer 188, and 5% sucrose. The biological activity of associated beta-glucuronidase is maintained for 1 month, but decreased after 6 months. Here optimized parameters for lyophilization of EVs that contribute to generate long-term stable EV formulations are presented. © 2021 The Authors. Advanced Healthcare Materials published by Wiley-VCH Gmb

Cell-Derived Vesicles for Antibiotic Delivery-Understanding the Challenges of a Biogenic Carrier System

Recently, extracellular vesicles (EVs) sparked substantial therapeutic interest, particularly due to their ability to mediate targeted transport between tissues and cells. Yet, EVs’ technological translation as therapeutics strongly depends on better biocompatibility assessments in more complex models and elementary in vitro–in vivo correlation, and comparison of mammalian versus bacterial vesicles. With this in mind, two new types of EVs derived from human B-lymphoid cells with low immunogenicity and from non-pathogenic myxobacteria SBSr073 are introduced here. A large-scale isolation protocol to reduce plastic waste and cultivation space toward sustainable EV research is established. The biocompatibility of mammalian and bacterial EVs is comprehensively evaluated using cytokine release and endotoxin assays in vitro, and an in vivo zebrafish larvae model is applied. A complex three-dimensional human cell culture model is used to understand the spatial distribution of vesicles in epithelial and immune cells and again used zebrafish larvae to study the biodistribution in vivo. Finally, vesicles are successfully loaded with the fluoroquinolone ciprofloxacin (CPX) and showed lower toxicity in zebrafish larvae than free CPX. The loaded vesicles are then tested effectively on enteropathogenic Shigella, whose infections are currently showing increasing resistance against available antibiotics