Extracellular vesicles (EVs) are nanolipid spheres used by cells throughout the body to transfer molecules such as lipids, proteins, and nucleic acids. They act as important mediators in many physiological and pathological processes, facilitating intercellular communication by transferring biological molecules between cells throughout the body. In order to achieve this movement around the body, EVs must avoid being eliminated by organs and immune cells.
EVs are a heterogeneous population comprising submicron-size microparticles and nanometer-size exosomes, and ongoing research has only served to reinforce our understanding of their complexity. That very diversity presents a wide array of opportunities to use EVs, ranging from drug nanocarriers to therapeutics. This fascinating technology has been proposed to help treat various afflictions, including genetic conditions, cancer, cardiovascular disease, and infectious diseases.
Illustration of EV-mediated cell cross-talk, clearance mechanisms and immune responses.
Source: Nature Nanotechnology
New era, new biologic:
With the evolution of the field of targeted drug delivery, nanotechnology has attracted great interest as a way to develop smart drug carriers. When drugs are transported via such a delivery system, the clearance and distribution profiles of tissues are determined by the properties of the carrier rather than the physicochemical properties of the drug molecule. The ideal approach would have to protect the molecule, overcome barriers in the body, and be safe enough to be used in patients, perhaps even repeatedly. However, synthetic nanoparticles have failed to deliver on this promise due to issues with efficacy and toxicity.
Importantly, extracellular vesicles have low immunogenicity and toxicity, which is usually a major obstacle with conventional synthetic nanoparticles. Some types of EVs also have tissue tropism, so their formulations can be targeted to specific cell types or the migration of inflamed tissue.
EVs may exhibit specific biological activity when released by various cell types, with their function depending on their protein markers and cargo. When EVs are isolated from immune cells such as macrophages or neutrophils, they show pathogenic and therapeutic function. On the other hand, EVs derived from diseased cells may elicit a profile of immune system evasion mechanism. However, tumor cell–derived EVs express specific immune markers, such as MHC-I and II, and may promote oncogenic activity.
EVs are being explored as natural delivery vectors and can be used for various strategies, including small molecules and drugs with suboptimal pharmaceutical properties. It is possible to deliver proteins and RNAs, such as iRNA and microRNA. The development of EVs can facilitate the delivery of drugs to target organs, tissues, and the central nervous system. The amazing properties of EVs offer far-reaching potential and allow the evaluation of exciting options that can be used in the development of new therapies.
Advances in EV research and technology are leading to increased interaction between the pharmaceutical industry and academic scientists in neuroscience, gene therapy, molecular biology, and imaging, accelerating the translation of research findings into the clinic. EV-based therapies have exploited the different aspects of EVs from three different angles: as drug targets, as drugs, and as carriers.
Extracellular vesicles as drug carriers to treat cancer:
Recent studies have provided evidence of the efficacy of extracellular vesicles as a potential tool to deliver synthetic molecules and small interfering RNAs (siRNAs) for therapeutic purposes, including for cancer. EVs derived from immune cells are capable of inducing immunostimulation to recipient cells in tumor microenvironments because of the presence of several stimulatory molecules and tumor antigens on their surface. The effective delivery of genes and proteins represents a huge potential of EVs to serve as cell-derived liposome-like nanoplatforms to treat various diseases, such as cancer.
This excellent efficacy in delivering the therapeutic charge has been researched in various tumor models, including hepatocarcinoma, lymphocytic leukemia, and prostate cancer. The results observed in these studies suggest that EVs are effective and can suppress cancer tumors by delivering chemotherapeutic agents.
In addition to their function as drug carriers, EVs can also help reduce drug toxicity. One interesting study has shown that the chemotherapeutic drug doxorubicin could be delivered to breast cancer cells by intravenous injection of exosomes isolated from peptide-expressing immature dendritic cells (DCs) of mice, resulting in suppression of tumor growth. Doxorubicin is a common antitumor drug used to treat many cancers, but it usually results in severe cytotoxicity or cardiotoxicity. When carried in EVs (Exo-Dox), however, it inhibits breast cancer cell proliferation while significantly reducing cardiotoxicity.
A number of clinical trials have already been conducted demonstrating the use of EVs. A phase I trial conducted in the United States (NCT03608631) evaluated mesenchymal stromal cell–derived EVs with KrasG12DsiRNA against a specific pancreatic cancer, while a phase II trial completed in France in 2018 (NCT01159288) investigated the potential of vaccination with tumor antigen–loaded exosomes derived from dendritic cells against lung cancer.
Potential of extracellular vesicles for infectious diseases:
During infection, intercellular communication is important in regulating the immune system, a process that is mediated by extracellular vesicles. These vesicles can transport pathogen molecules that serve as antigens or agonists of innate immune receptors to induce host defense, or they can act as regulators of immune defense or immune evasion. The involvement of EVs in the induction of immunity is widespread in many pathogens, while their involvement in mechanisms of immune evasion can induce chronic infection and cause acute infection.
By modulating the immune response, EVs can be used as potential therapeutics. In some studies, EVs have been used as vaccines against pathogens. Schnitzer and colleagues demonstrated that DC-derived exosomes containing Leishmania antigens elicit protective immune responses against cutaneous leishmaniasis, while Colino and colleagues found that exosomes stimulated an IgG response specific for diphtheria toxin.
Extracellular vesicles gain traction in science and in the clinic:
As studies show that exosomes can carry RNA biomarkers, the idea of using exosomes as diagnostic biomarkers is of growing interest. The startup ExosomeDx, which was founded in 2008 to help diagnose prostate cancer and decide whether to biopsy it, has developed tests based on EVs that show potential for screening and monitoring patients.
Some studies have shown the potential of extracellular vesicles as biomarkers, ranging from diagnosis of cardiovascular disease to monitoring progression of Chagas disease by measuring EVs isolated from patients’ monocytes. EVs may contain information about the original tissue, the pathophysiological context, and the severity of the disease. Importantly, EVs appear to be a major step toward the highly anticipated liquid biopsies.
Another platform has been developed by Evox Therapeutics. This startup is developing exosomes to deliver protein and nucleic acid–based therapeutics to treat life-threatening rare diseases. Studying platforms to be applied in oncology and neurology, Codiak is developing a pipeline of therapeutic candidates that can impact patients’ lives. The goal of this startup is to explore engineered exosomes to treat some cancers and neurological diseases. There are candidates that are in phase I and others that are still being explored.
Extracellular vesicles’ unfinished puzzle: the challenges
Despite the promising approach of extracellular vesicles and the obvious medical interest, working with these small particles presents many technical challenges. Other uncertainties exist in terms of standardization of protocols and definition of pre-analytical and analytical variables that directly affect the results. The major difficulty in working with EVs is the lack of standardized isolation and purification methods. In clinical applications, there is no uniform standard for the separation of EVs, and extraction and separation from different sources remain challenging.
A second major challenge is to efficiently load EVs with therapeutic targets. The problem is that EVs contain part of the contents of their mother cells during formation, which limits the space available for EVs to hold exogenous drugs. The loading capacity of EVs depends on the loading method and the hydrophobicity of the drug, and optimization is still not completely efficient.
The last important challenge is production. Implementing these EVs in clinics is a major challenge when it comes to production to ensure quality and quantity. There is no modern method that meets the ideal criteria for large-scale production, including scalability, reproducibility, safety, potency, size distribution, surface charge, and purity.
Knowing these difficulties, the International Society of Extracellular Vesicles (ISEV) proposed the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines in 2014. In 2018, these guidelines were updated, including tables and outlines of suggested protocols and steps to follow, representing a step in the right direction for standardization and accountability to aid researchers in overcoming the known technical barriers.
Small size, big potential:
Extracellular vesicles are emerging as a high-potential therapeutic agent, tapping into the therapeutic potential of innate immune properties, as a diagnostic biomarker for specific diseases, and as a “natural” drug delivery system with reduced toxicity. These small molecules provide a link between drug delivery, biologics, and cell therapy. EVs present many possibilities in therapy, such as mimicking the effects of cell therapies, and are safer and present more biocompatibility than artificial vesicles because they are already circulating through the body.
For these reasons, EVs can be considered as an “all-in-one approach” that has great potential in the field of personalized medicine. They allow the detection and monitoring of disease at early stages, as well as the targeted delivery of drugs at the site of the disease. It seems likely that extracellular vesicles will become a platform for highly potent, versatile biopharmaceuticals in the future.
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