As part of normal homeostasis, cells release different types of small-sized membrane vesicles to traffic analytes across membranes, communicate with other cells and remove unwanted cell content. These vesicles can range in size from 30nm to 1,000nm and are collectively known as Extracellular Vesicles (EVs).

EVs can be found in circulating blood, serum (1) and in most biological fluids. In recent years, it has been shown that EVs are able to interfere with biological functions, stimulating the growth of EV research particularly in their characterisation as a novel class of therapeutics, with the limelight on drug delivery and diagnostics (2).

EVs are ideal candidates for developing and testing new drug delivery methods. In this piece, we explore the growth of the EV-mediated drug delivery sector and the tools available to exhaustively characterise EVs and their cargoes. Recent advances in EV research are technology-driven, with a particular focus on high sensitivity imaging methods, such as single-molecule localisation microscopy. These technologies support engineering of EVs and enable the better understanding of EV-mediated drug delivery.

Different subtypes of extracellular vesicles

EVs are highly stable membrane-enclosed particles that act as carriers of active molecules like RNA, DNA, proteins, metabolites and lipids, which can either be membrane-bound or enclosed within vesicles. Once secreted, EVs can mediate intercellular signalling, modulate stroma tissue or even regulate inflammatory and immune responses by trafficking to local or distant targets and governing various biological functions.

Circulating EVs are considered small long-distance signalling units travelling around the human body with the ability to cross biological barriers. They have different characteristics according to the cell type releasing them, and cargoes are reflective of the producer cell. Released EVs also have distinctive composition in pathological conditions. This makes EVs appealing in early disease diagnostics and biomarker development tools and their use in targeted drug delivery to tackle diseases, including cancer or neuronal disorders.

For many years, EVs have been classified according to their size and origin as exosomes (nanoscalesized, of endocytic origin), microvesicles/microparticles (various sizes, shed from the plasma membrane), apoptotic bodies and Golgi vesicles. However, after the 2019 International Society for Extracellular Vesicles (ISEV) annual conference in Kyoto, Japan, a statement issued by the society endorsed the use of the generic term extracellular vesicles (EV) for particles naturally released from the cell, delimited by a lipid bilayer and without a functional replicating nucleus (3).

It was highlighted that unless specific methods can be used to reliably establish the origin of EVs, scientists should stay clear of using popular terms such as exosomes or microvesicles, as these can present contradictory or inaccurate definitions. These guidelines aim to unify the denomination of EV subtypes according to their physical characteristics, including: density or size (small EVs <200nm, or medium/large EVs), their biochemical composition (tetraspanins CD63+/CD81+- EVs, Annexin A5-stained EVs, etc) and cell of origin (eg podocyte EVs, hypoxic EVs, large oncosomes, apoptotic bodies, multivesicular bodies).

EVs as a novel class of biotherapeutics

EVs can cross biological barriers, such as the blood-brain barrier, and get internalised into cells with a high degree of specificity. A growing body of evidence highlights their critical role in cell-tocell communication pathways (2). The natural properties of EVs enable their forward engineering and further refinement to make EVs carrying specific drug candidates to target specific molecular pathways, cells and tissues. Additionally, EVs are believed to evade the immune system, which opens new possibilities when approaching therapy development for a broad range of diseases.

In recent years the number of EV-related publications has increased, with a study reporting an approximate increase of 733-fold in the past nine years4 – a trajectory analogous to that of fields such as T-cell research and circulating tumour cells. EV-related patents (UTSPO’s database) have also increased over the past decade, with a total of 524 US patents citing exosome-related terms between 2000-16. In the same period, a total of 948 NIH grants cited exosome(s) and/or microvesicle(s), with a 201% increase in 2016 (4).

Advances in EV research and technologies are driving the increased interactions between the pharmaceutical industry and academic scientists working in neuroscience, cell and gene therapy, molecular biology and imaging; accelerating the effective translation of bench discoveries to clinical pipelines. Both start-up and more established companies are successfully focusing on engineering EVs, mainly using smaller so-called exosomes, to facilitate drug delivery to target organs, paving the way towards drug delivery to challenging tissues, such as the central nervous system.

Development of next-generation therapeutic platforms involving EVs spans across various tissues, including those hard to reach. CAP-2003 from Capricor Therapeutics (USA) is currently undergoing pre-clinical studies, highlighting the drug’s potential as a treatment for diseases caused by inflammation and fibrosis through the modulation of the immune system to restore cell damage. The therapeutic agent consists of exosomes isolated from the company’s proprietary cardiospherederived cells (CDCs). These exosomes also have the capacity to reduce scarring.

Another US-based start-up company, Kimera Labs, specialises in the production of exosomes isolated from Mesenchymal Stem Cells (MSC). Its highest-purity MSC exosomes are optimised to stimulate healing processes and tissue regeneration. XoGlo® is a sterile, cell-free isolate of MSC exosomes containing growth factors involved in skin repair, beautification and regeneration.

The proprietary pipeline of Evox Therapeutics, based in Oxford (UK), encompasses seven exosome- based therapeutic products, ranging from therapeutic discovery stage to FDA Investigational New Drug assessment stage. These drugs have the potential to address lysosomal storage disorders (LSDs), with a combined incidence of around one in every 5,000 births. Another area of focus for Evox are genetic diseases described as Inborn Errors of Metabolism (IEMs), where toxic substances accumulate impacting normal metabolism and resulting in impaired muscle functioning. Evox’s technology revolves around the loading of therapeutics and tissue targeting moieties on the surfaces of exosomes, which facilitates highly-specific delivery of therapeutic proteins to the target organ.

A broad spectrum of serious diseases is also addressed by Codiak (USA), whose engEx™ Platform aims at engineering exosomes targeting immune-based diseases, metabolic and fibrotic disorders, neurodegenerative disorders, cancer and rare diseases. The current product pipelines encompass eight candidates in various therapeutic areas.

A new way to tackle cancer and control the tumor microenvironment is through exosome depletion, potentially as an adjuvant therapy. EVs generated by cancer cells can lead to the inhibition of immune responses and stimulation of new blood vessels. Depletion of cancer cell-generated exosomes may lead to inhibition of tumour growth and increased efficiency of anticancer agents. USbased Aethlon Medical has devised a therapeutic hemofiltration technology to capture circulating viruses and cancer-promoting exosomes through affinity attachment. At present, the Hemopurifier® is being advanced under an FDA-approved clinical study. Aethlon is also the majority owner of Exosome Sciences, Inc, a company focused on the discovery of exosomal biomarkers to diagnose and monitor life-threatening diseases.

The growth of exosomes diagnostics and therapeutics

Altogether, the global market for exosome diagnostics and therapeutic market is expected to grow from $34.7 million in 2018 to $186.2 million in 2023, with a substantial five-year compound growth of 39.9%5. Major factors that are contributing to the growth of the market include the increased demand for the use of exosomes as biomarkers in cancer diagnosis, new approaches to develop EVs in the liquid biopsy market (a growing sector in cancer diagnostics), the potential of using EVs for targeted delivery vehicles of therapeutic molecules to target cells, as well as the increased prevalence of cancer in a global ageing population.

In the US and Europe, the commercialisation of EV-mediated targeted therapeutics is financially more rewarding than companion diagnostics, which is boosting the commercialisation of EV pipelines with product launches in the UK, the US, Italy and Korea. North America accounted for more than half of the market share in 2016. Emerging therapeutics in countries with ‘fasttrack’” EV regulatory authorities including Korea, Italy and China, will also contribute to the growth, even if the FDA takes a longer time for its decision to approve EV therapeutics.

Furthermore, EVs have the great potential to impact and accelerate diagnostics and discovery of therapeutics in a vast range of markets, such as the biomarker and diagnostic industry, the microRNA market, the liquid biopsy market, the stem cell technologies market, the prenatal diagnostic market, the agriculture and food industry, microfluidics and nanotechnology and the skincare industry (5,6).

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 distribution, heterogeneity and concentration of the purified EV population, as well as the cellular uptake and the mechanism of action of the EVmediated therapy.

Characterising EVs at the molecular scale

Despite increasing interest in using EVs as a new class of biological medicines, designing and engineering 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 therapeutic release requires close monitoring of the efficiencies 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 preparation 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 specialised knowledge and reagents, and is a multistep procedure that can be time-consuming and can prove difficult for multiplexed imaging. While electron 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 flowbased 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 tracking- 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 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 understanding of the role of EVs in all aspects of cellular transmission; from packaging of signalling molecules and nucleic acids during vesicle biogenesis, to characterising the cargo loading efficiency and tracking their uptake and fate after internalisation within selected target cells or tissues. Advances in this field (see section below), combined with other techniques, are making EV visualisation an essential tool for researchers in the field.

An indirect, albeit powerful, method complementing EV visualisation at a single molecule level is Next-Generation Sequencing (NGS). This rapidly- growing field enables analysis of nucleic acid sequences (both RNA and DNA) in small sample volumes in a fast and relatively inexpensive manner. The rapid improvement in labelling and imaging sensitivity, as well as sequencing, allows researchers to monitor EV production for therapeutic 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-effective, 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 identifying different sub-populations of vesicles. In the last decade, several techniques have emerged known as super-resolution microscopy, which surpass 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 localisation 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 simultaneously 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 composition of EV membranes can be reconstructed with SMLM and used to identify the specific biomolecules involved in EV signalling and targeting; revealing crucial information on the mechanism 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 characterisation techniques. Multimodal SMLM has the ability to combine imaging and quantitative characterisation 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 accumulation analysis, cluster formation and distribution measurements, to understand the efficiency of EV cargo loading and evaluate heterogeneity of EV populations – both when engineering and upscaling production. Having a successful outcome therapy will depend on engineering functional, heterogenous EVs with specific cargoes and developing 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 candidates 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 development of EV-mediated therapies is a growing market segment, with a number of start-up and more established companies moving forward in this field. Most of the recent advances in EV research are technology-driven and particularly linked to high sensitivity imaging capabilities, such as single-molecule localisation microscopy. This enables quantification of EV cargoes and subtypes, and tracking of EV dynamics with unprecedented precision, helping to better understand their composition and uptake mechanisms by living cells.

Overall, the combination of established methods and emerging disruptive technologies is progressively allowing the more precise characterisation of EV therapies and understanding of their mechanism of action, helping to modulate drug dosage and delivery and bringing to light more efficient treatments for hard-to-treat conditions such as cancer and neurodegenerative diseases. DDW

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This article originally featured in the DDW Winter 2019/20 issue

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Dr Anna Caballe has a PhD in Molecular and Cell Biology and applied super-resolution microscopy during her postdoctoral research at the University of Oxford. Anna joined ONI as a Grant Writer in March 2019, working across Business Development and Marketing, driving grant proposals for the development of new products and applications using ONI’s technology and expanding her role into Product Management.

Dr Katarzyna Ginda-Mäkelä trained in super-resolution microscopy at the Biochemistry Department, University of Oxford, before joining ONI as Application Scientist. Currently her role is focused on product marketing management and content generation for various marketing channels.

References

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3 Théry, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles (2018) 23; 7(1):1535750.

4 Roy, S, Hochberg, FH and Jones, PS. Extracellular vesicles: the growth as diagnostics and therapeutics; a survey. J Extracell Vesicles (2018) 7(1): 1438720.

5 Exosome Diagnostics and Therapeutics Market 2019 Global Analysis, Opportunities And Forecast To 2024 (2019). Medgadget: https://www. medgadget.com/2019/09/exoso me-diagnostics-andtherapeutics- market-2019- global-analysis-opportunitiesand- forecast-to-2024.html.

6 Exosome Diagnostic and Therapeutic Market Overview, Global Opportunity Analysis and Industry Forecasts, 2014- 2022 (2016) Allied Market Research: https://www. alliedmarketresearch.com/exos ome-diagnostic-andtherapeutic- market