In The excretory vesicles known as exosomes are tiny extracellular balls (usually around 30 to 150 nm in diameter) that have become very popular in recent years for their use as delivery devices. These vesicles are produced by nearly all cells, and they are a major source of intercellular signaling, smuggled across cells with a variety of biomolecules from proteins to lipids to RNA. It is this intrinsic capacity to transport bioactive molecules that make exosomes particularly appealing for targeting drugs. Isolation of exosomes from culture media is now one of the major steps in taking advantage of their therapeutic potential for cancer, gene therapy and regenerative medicine. Yet exosome isolation is an elaborate task, and the exosomes must be isolated with a degree of accuracy and efficiency so that they remain quality and pure, as well as ready for use as a drug delivery agent. How to isolate exosomes from culture media where cells grow are critical to producing quality exosome-based drug delivery systems. There are usually multiple steps involved in separating exosomes from cell culture media that all work to isolate them from other contamination (cellular debris, proteins, larger vesicles). Most often used to isolate exosomes is differential centrifugation, a process that cuts particles according to size and density. For this, the cell culture supernatant is passed through multiple centrifugation cycles at varying speeds so that smaller particles will be ground. The oligo-exosome-containing fraction is accessible after the final high-speed spin, usually in the supernatant. This approach is straightforward and popular, but it can take time and do not always produce ultra-pure exosomes. These constrictions are typically overcome by other techniques, such as ultrafiltration, size-exclusion chromatography or immunoaffinity capture. Ultrafiltration size-separates particles via a membrane filter; size-exclusion chromatography sizes exosomes by size and molecular weight. Immunoaffinity capture, meanwhile, employs antibodies that specifically target the proteins on the exosomal surface and is more sensitive in isolation of exosomes. Pureness and yield of exosomes purified from cell culture media are essential when a drug delivery device is to be used. It needs to be made with high-quality exosomes, so therapeutic payloads are loaded quickly, and the exosomes remain as naturally targeted as they were. Density gradient centrifugation (where the gradient is dominated by sucrose or other compounds) can even make isolated exosomes more pure, dissociating them from the rest of the particles in accordance with their buoyancy. Furthermore, the exosome isolation procedure is also increasingly being conducted using high-throughput liquid chromatography (HPLC) and sophisticated filtration techniques with high throughput solutions for large-scale exosome manufacturing in the clinic. These sophisticated techniques also increase exosome preparations' reproducibility and reliability (critical to achieving reproducibility and reliability in drug delivery experiments). Even with all these steps forward, exosome purification from cell culture media is still an obstacle, and there are still pitfalls associated with contamination of the purified exosomes with extracellular vesicles or proteins that can disrupt the therapeutic efficacy of the exosomes. Scientists are now devising protocols for optimal isolation to circumvent these limitations and make exosome preparations more efficient, scalable and more clinically relevant.
Figure 1. Conventional and emerging exosome isolation techniques. (Willis GR, et al.; 2017)
Exosomes are already fast becoming a broad-spectrum drug delivery system, given that they are biocompatible, stable and have the capacity to penetrate biological obstacles such as the blood-brain barrier or the endothelial lining of blood vessels. They can be packed with any number of therapeutic agents, from small molecules, proteins, RNA (including siRNA or mRNA), and even CRISPR-Cas9 elements for gene editing. But successful exosome-based drug delivery is predicated on the isolation. To isolate exosomes from cell culture media is not merely to get a vesicle fraction, it is to get a high-purity, reproducible, and useful purified exosome that can be infused with a drug or nucleic acid and used to target a disease target. Cells automatically identify exosomes, and their membrane proteins (tetraspanins (CD63, CD81, CD9), integrins and lipid molecules decide which ones interact with which cells). So exosome surface features can be altered or designed during isolation to enhance their ability to be targeted. By, for instance, putting targeting ligands or antibodies on the surface of exosomes, scientists could target the vesicles to specific tissues or cell types (like tumor cells or immune cells). This cell-specificity gives the delivery system an edge over other standard drug delivery systems that don't usually have the precision to target cancer cells and weed out healthy ones. This selective targeting is a prime concern in cancer treatment, where the lowest possible side-effects is as critical as the greatest therapeutic success. What's more, exosomes can also float their cargo directly into the cytoplasm of its target cells without endosomal escape, which is a common issue for many nanoparticle-based drug delivery devices. Besides being targeted, exosomes made from cell culture media can be customised to harbour multiple therapeutic molecules, thus being highly flexible drug carriers. Small molecules such as chemotherapeutic drugs can be wrapped around the exosomes or attached to their surface, so that the drug goes straight to the tumor or other cells. Exosomes also contain RNA molecules (siRNA, miRNA or mRNA) that could be used to silencing or therapy genes. The ability to be carriers of RNA-based drugs opens up new possibilities to treat diseases on a genetic level – for example, to silence pathogenic genes or generate therapeutic proteins in target cells. Furthermore, exosomes are relatively stable in the bloodstream, which guards their cargo against breakdown by nucleases or enzymes, prolonging the half-life of the therapeutic molecules and maximising their therapeutic efficacy. While exosome-based drug delivery continues to develop, scientists are also working on ways to incorporate several payloads into a single exosome, so that different aspects of disease could be addressed by a combination therapy — such as chemoresistance in cancer or infection.
Exosome separation from cell culture media promises a lot in the way of drug delivery, but there are still a few obstacles. One of the biggest issues is standardization of isolation protocols. Current exosome isolators, from differential centrifugation to ultrafiltration to size-exclusion chromatography, are less efficient and more variable in their exosomes. Differential centrifugation is popular and relatively straightforward, but it's also slow and won't always discriminate exosomes from other types of extracellular vesicles or protein bundles. Ultrafiltration is another popular method that uses size-exclusion membranes to remove exosomes based on their size, but it loses vesicle integrity and yields. Size-exclusion chromatography is a more precise approach for size- and molecular-weight isolation of exosomes, but it's an equipment-intensive procedure that may not scale to mass production. Immunoaffinity capture – which uses antibodies to bind to the exosome surface proteins – is more specific but is constrained by the lack of antibodies and subsequent optimization. A second big problem is the lack of a consistent test for purity and yield of isolated exosomes. Exosomes are not like any other nanoparticle or drug delivery system – they are different in size and molecular makeup. That variation can impact how well exosomes function as carriers of drugs. Scientists have come up with an array of analyses – nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), electron microscopy – to measure exosome size and concentration, although most provide only rough estimates of exosome purity and functional quality. To be therapeutically effective, exosome preparations have to be consistently good, especially in clinical conditions. Consequently, we have a perpetual need for standardised exosome isolation, purification and quality control protocols that can be used in different research labs and in clinical trials with confidence. Exosome-based drug delivery will continue to advance with novel exosome isolation methods, cargo loading systems and functionalization methods. The use of microfluidic technology, for example, has allowed for ultra-thin, ultra-speed platforms that can separate exosomes from cell culture medium in record time and with high purity. Also possible with the use of CRISPR/Cas9 technology and other gene-editing tools is the possibility of manipulating exosomes to have particular genes or to target specific cells even more precisely. As scientists refine and perfect these processes, exosomes will become a far more important target in targeted drug delivery systems – for cancer, genetic diseases and other ailments. Additionally, as regulators provide more lucid instructions for exosome-based therapies, delivery mechanisms will go from lab to clinic, and the potential for individualized and highly effective therapies relying on exosome's natural properties will be extended.
Alternate Names:
Exosome Extraction
Exosome Purification
Exosome Enrichment
Exosome Separation
Exosome Isolation Protocol
Exosome Concentration
Extracellular Vesicle Isolation
Exosome Harvesting
Exosome Isolation from Cell Culture Supernatant
Nanovesicle Isolation
References:
1. Willis GR, et al.; Toward Exosome-Based Therapeutics: Isolation, Heterogeneity, and Fit-for-Purpose Potency. Front Cardiovasc Med. 2017, 4:63.
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