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Therapeutics


The use of extracellular vesicles as therapeutics


is rapidly growing. In order to reach their full potential, EV-mediated therapies must be precisely characterised at multiple stages as it is crucial to understand the cargo loading efficiency and distri- bution, heterogeneity and concentration of the purified EV population, as well as the cellular uptake and the mechanism of action of the EV- mediated therapy.


Characterising EVs at the molecular scale Despite increasing interest in using EVs as a new class of biological medicines, designing and engi- neering EVs carrying specific cargoes and targeting desired cells poses significant challenges. The array of tools available to determine EV size often do not agree, and seeing EVs at the single vesicle level remains difficult. Successful commercial therapeu- tic release requires close monitoring of the efficien- cies of production and purification at an industrial scale. In order to ensure the purity and consistency of production lines, exosomes should ideally be characterised using a multimodal approach. For decades, the gold standard for visualising


nanoscale details of EVs has been electron microscopy (EM). However, EM sample prepara- tion can substantially affect EV morphology and composition, yielding images of deformed EVs, diminishing the consistency and quality of the obtained results. Additionally, EM relies on spe- cialised knowledge and reagents, and is a multistep procedure that can be time-consuming and can prove difficult for multiplexed imaging. While elec- tron microscopy techniques have the capability to resolve individual EVs, they do not easily allow detection of multiple markers at the same time, and are limited to fixed cells. One of the most popular methods for studying


EV populations is nano-flow cytometry, a flow- based analysis of particles that enables robust nanoparticle size distribution and concentration measurements. However, it is not always capable of detecting the lower end of the EV size spectrum (30-150nm) due to its low spatial resolution. Nanoparticle Tracking Analysis (NTA) is a track- ing-based analysis of single particles in solution. NTA calculates the rate of particle movement and estimates the size of the nanoparticles. As NTA analysis detects single particles, it is also useful in measuring the concentration of particles, however, in population-based techniques it can produce false positives or unspecific binding of EVs to the beads, resulting in data misinterpretation. Currently, the most precise and accessible


Drug Discovery World Winter 2019/20


Figure 2: Imaging and characterisation of EVs using single-molecule localisation microscopy (SMLM). Sizing and distribution of an EV isolated from human keratinocyte culture media, observed by dSTORM. A: Schematic of the immunostaining of an EV. B: The image reveals the spatial organisation of tetraspanins, CD63 (blue) and CD81 (yellow) at a molecular level, relative to the vesicle membrane surface (WGA, magenta). C: Particle distribution along the line depicted in panel (B); image adapted from: www.oni.bio/ev


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