Exosomes are nano-size extracellular vesicles secreted by all cells, they are classified as nanoparticle based on size and they transport proteins and genetic information between cells. These exosomes hold promise to become powerful tools for targeted drug-delivery approaches.
But how exosomes can be used as drug delivery systems? Drugs can be produced and load into exosomes (inside the cell), which means that they can be packed with proteins, RNA/miRNAs and lipids. Therefore, they transport that information to the rest of the cells in a non-cytotoxic manner. In this sense, exosomes can be purified previously and loaded with drugs in order to be provided to the patients.
Exosomal particular size opens up the possibility of using exosomes as a nanoparticle drug delivery system. There are studies that have successfully demonstrated that drug-loaded engineered exosomes appear to be greatly well tolerated, as exosomes are present in all biological fluids.
Delivery of exosomes with different cargo has been demonstrated in vivo in multiple clinical diseases models using different proteins and drug playlots and the results are promising.
For this reason, everyday more and more companies are interested to use the vesicles to package small-molecule, protein, and RNA drugs or even use them as therapies themselves.
Exosomes are unique in their molecular composition, regarding proteins, lipids, and nucleic acids, and this molecular content reflects the state of the cell at the time exosome is generated. The main aspects we can underline about exosomes composition are:
► Highly abundant cytosolic proteins, including metabolic enzymes and cytoskeletal proteins, are missing from exosomes, which means that the uptake of components during exosome biogenesis is not random, is a highly regulated process.
► All exosomes have proteins involve in the exosome biogenesis (Alix, TSG101, Syntenin-1), and also proteins associated with the endosome (GTPasa of Rabbit family, annexins, and flotillin), also integrins and tetraspanins (CD63, CD9, CD81, CD82 among others).
► Several classically used exosome markers like major histocompatibility complex (MHC), heat shock proteins are similarly present in all EVs.
► Regarding nucleic acids, it is not clear if it is possible to find double-strand DNA (dsDNA), however we can find extracellular RNA, such as miRNA, although exosomes do not carry the necessary molecular machinery to carry out cell-independent miRNA biogenesis, like Argonaute proteins.
► Finally, tetraspanins, are considered common EV-markers and are used frequently as markers for exosome identification. In this sense
Furthermore, it is worth highlighting the importance of carrying out combined analysis, because both CD63 and CD9 can be present alone in other type of EVs different to exosomes.
Scientists have known about exosomes for decades, but few papers used to refer them until now. Nowadays, according to PubMed more than 8000 hits will appear if you search on their site about exosomes, and during the last year, several high-profile papers have been published.
As a result of the suddenly arising interest for exosome as effective drug delivery systems a large amount of companies are starting to manipulate these extracellular vesicles to solve drug delivery issues for different types of therapies.
As exosomes can so easily transport molecules that spread disease, scientists have started to think that they might also be useful for the transportation of disease-stopping molecules. In this sense, a recent study even implicates exosomes as responsible for the distribution of amyloid-β, the plaque-forming protein that accumulates in the brains of people with Alzheimer’s disease.
Exosomes are exponentially becoming promising items of study when talking about spreading diseases, including cancer, and metabolic conditions like diabetes and obesity. By now, exosome-based cancer drug delivery diagnostics are already available, and multiple exosome therapies could begin testing in clinical trials soon.
Even though laboratories and biotechnological companies keep moving forward with exosomes in order to facilitate the delivery of therapeutics and the use of exosomes as drug delivery targets, they are confronting some technical challenges in this regard.
Exosomes are important in almost every aspect of biology and medicine; right now, scientists use exosomes that are released from cells grown in laboratories and purified, and in this sense, producing enough of these exosomes suppose one of the big challenges.
Additionally, several subtypes of EV, including exosomes, ectosomes, microvesicles, membrane vesicles and apoptotic bodies, have been identified, and there is a lot of disagreement about how to isolate them, in order differentiate and separate them.
Why is important to isolate the exosomes in order to use them and characterize them? Because research must be consistent and reproducible, an experiment could be compromised if different exosomes cargo different amount of drugs because of their size.
Each extracellular vesicle subpopulation can be derived via different biogenesis pathways , and therefore, contain different information. For example, cancer-derived EVs carry molecular information distinct from carried by stem-cell or blood-cell-derived EVs.
It is precisely because of their specific biogenic origin that is especially important to a comprehensive characterization of the vesicles when talking about using this exosomes in drug delivery research.
Even though these characteristics make exosomes challenging for drug delivery systems, they make them also promising from other perspectives as biomarkers for liquid biopsies in several applications.
However, a complete characterization of exosomes and their communication with cells could be the key for their used in drug delivery applications. In this sense, understanding the mechanism of action of EVs can open up new possibilities in drug delivery engineering.
Scientist work in order to successfully cargo exosomes with all kinds of effective treatments for different diseases. However, our body’s system can be one of the biggest obstacles to the development of new therapies, as many types of developed therapies are too fragile or too large to enter cells and are not easy to target specific diseases.
As a way of addressing these challenges, many companies are using exosomes, because it is a successful approach to take advantage of the body’s own natural delivery and messaging system.
The capacity of exosomes to mediate certain physiological and pathological processes is used to design the specific targets delivery of therapeutic agents, reducing toxicity and immunogenicity.
Exosomes works perfectly in this sense, because of their compatibility with the biological system, which makes them the perfect delivery system for a wide range of therapeutics, including genetic material.
In order to use exosomes as drug delivery systems, the therapeutic substances must be loaded into the exosomes, once these drugs have been incorporated into the exosomes, scientist have explored two possible approaches:
► Passive loading/encapsulation: Passive loading can be accomplished by incubation of the drug with exosomes or incubation of the drug with donor cells. In incubation with exosomes, drugs diffuse into the exosomes based on the concentration gradient, while in incubation with donor cells, cells are initially drug-treated, and these cells thereafter release drug-loaded exosomes
► Active loading/encapsulation: On the other hand, the active drug loading method offers higher drug loading efficiency and makes it easier to cargo larger molecules. Active drug loading can be enabled by sonication, extrusion, electroporation, or drug conjugation techniques.
• In the sonication method, the exosome membrane integrity is compromised to allow the drug to diffuse into the exosomes without disturbing the membrane-bound proteins. Likewise, extrusion also interrupts the membrane diffusion enabled by the syringe lipid extruder.
• In the electroporation method, exosomes are suspended in a conductive solution and subjected to an electric field. The electric field disrupts the phospholipid bilayer and introduces small temporary pores through which the drug diffuses into the exosomes.
• In click chemistry, drug molecules bind directly to exosome surfaces by covalent bonds, whereas in antibody binding, highly specific antibodies are used to bind to a particular antigen on the exosome surface.
All of the above-mentioned drug loading approaches result in different cargo capacities and the choices to be employed are dependent on the active principle characteristics, such as hydrophilicity, lipophilicity, and molecular size. Furthermore, apart from the efficiency of loading, the integrity and stability of the exosome membrane are of critical importance for drug delivery.
In conclusion, scientists and engineers have attempted to take advantage of very unique properties of EVs to develop smart drug delivery systems that have significant advantages in targeting and safety compared to other systems.
As the field of targeted drug delivery has grown, nanotechnology has contributed substantially to the development of smart carriers in recent decades. In particular, cell-derived extracellular vesicle (EV)-based transport systems have drawn considerable interest, offering a promising platform for drug encapsulation, which has led to the clinical translation of various formulations.
When compared to traditional delivery methods, EVs have been shown to deliver functional cargo with lower immune clearance when administered systemically to rodents.
However, more assessments in clinically relevant patients and a comprehensive direct and quantitative comparison with other alternatives are needed to fully evaluate the risk-benefit balance.
Furthermore, the successful translation of EVs will depend on the availability of cost-effective large-scale production, isolation, and characterization methods with high sensitivity to estimate batch-to-batch variations, and on the availability of broadly applicable methods for drug loading.
On the other hand, as it was mentioned above, a large scale of production of exosomes is also required in order to progress in exosome drug delivery systems applications, and that is the reason why Immunostep developed the highest quality lyophilized exosome standards isolated from a variety of biological sources, including cell culture supernatant, human plasma, serum,. This way, we are able to purify large amount of exosomes successfully for its use in research and clinical applications.
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