Application of extracellular vesicles to an enzyme prodrug therapy approach

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, researchers at Imperial College London embedded EVs 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. The researchers show 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.

Characterization of poly(vinyl alcohol) hydrogels containing enzyme-loaded vesicles

 a) Schematic overview of EV- or liposome-encapsulated enzyme (β-glucuronidase) incorporated into PVA hydrogels. Enzyme-functionalized hydrogels are incubated with a glucuronide prodrug to release an active drug in a selective and controlled manner. b) Photographs of PVA hydrogels containing free (nonencapsulated) β-glucuronidase, EV- or liposome-encapsulated β-glucuronidase (EV hydrogel or liposome hydrogel, respectively), or nonloaded control gels. c) Mechanical properties of PVA hydrogels. EV hydrogels and liposome hydrogels were loaded with 1.7 × 10⁸ or 3.5 × 10⁸ vesicles per gel (indicated by number in (c)). Unconfined elastic modulus of compression of hydrogels compressed to 10% strain at 0.5% strain min⁻¹. Values are represented as mean ± SD, n = 5, no differences by one-way ANOVA (p < 0.05). d) Confocal micrographs of EVs fluorescently labeled with PKH67 and incorporated into PVA hydrogels at higher and lower concentration (3.5 × 10⁸ and 1.7 × 10⁸ EVs per gel, respectively). e–j) Scanning electron microscopy imaging of PVA hydrogels containing optically more dense uranyl-labeled EVs (EV-uranyl, 3.5 × 10⁸ EVs per gel, indicated by arrows) or no vesicles (nonloaded control gels). EV-uranyl-hydrogel samples exhibit distinct and localized vesicular structures (arrows) while control nonloaded hydrogels show unspecific sample charging without clear vesicular structures. Images were obtained by e,f) in-lens electron detector and g,h) in backscattered electron mode; and by i,j) density-dependent SEM analysis with the in-lens or secondary electron image assigned to the green channel and the backscattering signal assigned to the red channel.