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.

The inherent antibiotic activity of myxobacteria-derived autofluorescent outer membrane vesicles is switched on and off by light stimulation.

Outer membrane vesicles are small, lipid-based vesicles shed from the outer membrane of Gram-negative bacteria. They are becoming increasingly recognised as important factors for resistance gene transfer, bacterial virulence factors and host cell modulation. The presence of pathogenic factors and antimicrobial compounds in bacterial vesicles has been proven in recent years, but it remains unclear, if and how environmental factors, such as light specifically regulate the vesicle composition. We report the first example of autofluorescent vesicles derived from non-pathogenic soil-living myxobacteria. These vesicles additionally showed inherent antibiotic activity, a property that is specifically regulated by light stimulation of the producing bacteria. Our data provide a central basis for better understanding the environmental impact on bacteria-derived vesicles, and design of future therapeutic options.

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.