approach for EV characterisation is visualisation, which is being increasingly adopted by the EV research community. Fluorescent microscopy is an essential and robust technique for the understand- ing of the role of EVs in all aspects of cellular transmission; from packaging of signalling molecules and nucleic acids during vesicle biogene- sis, to characterising the cargo loading efficiency and tracking their uptake and fate after internalisa- tion within selected target cells or tissues. Advances in this field (see section below), com- bined with other techniques, are making EV visu- alisation an essential tool for researchers in the field. An indirect, albeit powerful, method comple-

menting EV visualisation at a single molecule level is Next-Generation Sequencing (NGS). This rapid- ly-growing field enables analysis of nucleic acid sequences (both RNA and DNA) in small sample volumes in a fast and relatively inexpensive man- ner. The rapid improvement in labelling and imag- ing sensitivity, as well as sequencing, allows researchers to monitor EV production for thera- peutic purposes, including EV engineering and cargo loading, all the way through to production scaling. This enables monitoring of therapeutic efficiency to be performed in labs in a cost-effec- tive, more accessible manner than before.

Novel imaging approaches for EV detection: super-resolution microscopy Due to the small size of some EVs, many of them can fall below the resolution limit of light microscopy (200nm), restricting the usefulness of conventional light microscopy techniques in identi- fying different sub-populations of vesicles. In the last decade, several techniques have emerged known as super-resolution microscopy, which sur- pass the resolution barrier of conventional light microscopes, increasing sensitivity and resolution to the nanoscale level. These techniques allow researchers to see inside living cells in non-invasive ways, with a level of resolution similar to classical electron microscopy. Among these techniques, the best solutions for

EV imaging and tracking are those that allow imaging of EVs or exosomes of a small size (30- 150nm), which rely on SMLM (Single-Molecule Localisation Microscopy) approaches, such as PALM (PhotoActivated Localisation Microscopy) and STORM (STochastic Optical Reconstruction Microscopy). STORM is a single-molecule locali- sation technique that has the power to resolve structures with 20nm precision in fixed samples. PALM uses a similar principle but utilises different


fluorophores, which are better suited for live cell imaging and particle tracking studies. SMLM techniques bring high value to EV

research with the ability to identify and visualise EV sub-populations, quantify single proteins, nucleic acids and multiple biomarkers simultane- ously at the sub-vesicular level (Figure 2). dSTORM can be used to directly infer the size of vesicles on a glass surface by imaging them, in the same way that electron microscopy has been used in the past. Additionally, the structural composi- tion of EV membranes can be reconstructed with SMLM and used to identify the specific biomolecules involved in EV signalling and target- ing; revealing crucial information on the mecha- nism of action of therapeutic EVs. For live cell imaging, SMLM imaging is capable

of detecting single EVs in real-time, tracking their interaction and uptake by target cells, as well as visualising their movements post-internalisation and obtaining dynamic intracellular data. In the future, one of the most robust ways of studying EVs, and perhaps the most important one, will be combining multiple complementary characterisa- tion techniques. Multimodal SMLM has the ability to combine imaging and quantitative characterisa- tion of EVs in solution, live and fixed cells. Measurements on EVs at the molecular scale

should be complemented with quick and efficient quantification methods, including biomarker accu- mulation analysis, cluster formation and distribu- tion measurements, to understand the efficiency of EV cargo loading and evaluate heterogeneity of EV populations – both when engineering and upscal- ing production. Having a successful outcome ther- apy will depend on engineering functional, het- erogenous EVs with specific cargoes and develop- ing a scalable manufacturing process for validating the therapy.

Closing remarks Extracellular Vesicles (EVs) are tiny signalling machines involved in a wide variety of cellular functions. They have incredible properties that allow researchers to engineer EVs as carriers for delivery of drug candidates and optimally target tissues of interest, while evading or modulating immune responses. This makes EVs ideal candi- dates for testing novel drug delivery methods, improving therapy efficiency and helping predict patient response to treatment, with the additional emerging role of EVs as biomarkers for disease diagnosis and prognosis. While their substantial diagnostic value appears to dominate part of the market, drug delivery and

Drug Discovery World Winter 2019/20

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