Exosomes are subtype of extracellular vesicles, which are closed lipid bilayer structures derived from cells and secreted by almost all types of cells, including exosomes (30-150 nm), microvesicles ( 150 nm to 1 μm) and apoptotic bodies (1-5 μm). These vesicles have long been thought of as a way of loading metabolic waste, responsible for transporting waste produced by cells. Until the 1980s, when researchers were studying the development of sheep reticulocytes, they initially determined the role of some 30-150 nm vesicles and named them exosomes. Under an electron microscope, the shape of exosomes is generally cup-shaped or spherical, which protect and deliver functional macromolecules between cells, including nucleic acids, proteins, lipids and carbohydrates, and transfer their "cargo" to the recipient cells.
Based on years of research, the industry has recognized the potential of exosomes in a variety of applications. In current clinical trials, exosomes are used as biomarkers, cell-free therapy (exosome therapy), drug delivery systems, and anti-tumor vaccines, among others. Its sources include mesenchymal cells, T cells, and dendritic cells, as well as other engineered cell lines.
Exosomes have irreplaceable advantages as drug delivery vehicles, including low immunogenicity, excellent biocompatibility, and biostability. In addition to using natural exosomes without any genetic/chemical modification, there are two main ways to load payloads into exosomes: In the direct method, exosomes are prepared and purified to load therapeutic drugs (exogenous loading), while in the indirect approach, appropriate cells are genetically engineered or co-cultured with therapeutic drugs to produce engineered exosomes (endogenous loading).
For exogenous loading, the industry has explored various strategies to load drugs into exosomes to maximize their delivery potential, including simple incubation as well as electroporation, sonication, freeze-thaw, etc. There are often some differences between studies attributed to different parental cell biology and reagent properties. Furthermore, exosomes are inherently loaded with native proteins and nucleic acids, which greatly reduces the required loading efficiency. The correct approach to achieve optimal loading, which in turn depends to some extent on the load molecule, must be carefully selected in advance, and loading capacity, drug retention, and potential impact on exosome properties should be considered. Limitations of the direct loading strategy have limited the use of exosome-based therapies in clinical trials.
The creation and use of rationally and purposefully designed engineered exosomes with highly defined and reproducible properties and a known mechanism of action is a compelling alternative to naturally derived exosomes because naturally derived exosomes usually have high heterogeneity and the mechanism of action is not clear, and engineered exosomes are more feasible basis for the development of important new drugs. However, engineering methods need to achieve certain improvements in maintaining the ideal physicochemical properties of exosomes and improving loading efficiency. Yet another challenge is that most methods for exosome engineering struggle to find a balance between stable loading of the desired load and surface modification vs. maintaining exosome biocompatibility.
Another bottleneck in scaling exosome-based therapeutics to industrial-scale production and subsequently to the clinical trial is the production of large-scale clinical-grade exosomes. The production of exosomes is highly dependent on its parent cells, which is limited by the different ability of cells to secrete exosomes and the high difficulty and high cost of large-scale cell culture. For the pharmaceutical exosome industry, scaling up to an industrial level is still in its infancy, and it is of utmost importance to decide early on methods that will be able to produce exosomes in the required quantities and containing therapeutic payloads.
The inefficiency of large-scale exosome isolation methods is another obstacle to the development of clinical-grade exosomes. The number, physicochemical characteristics, and composition of exosomes released by different cell types may vary. Currently, techniques based on different principles have been used for exosome isolation, including differential/ultracentrifugation, filtration, size exclusion chromatography, immunoaffinity-based capture, polymer precipitation, etc. Although some exosome purification methods have been developed and optimized, it is still difficult to find a specific method that solves all related challenges such as low isolation efficiency, sample loss, low exosome recovery and purity, and batch-to-batch difference. Accordingly, comprehensive characterization of exosomes is also critical, especially regarding size, morphology, concentration, presence of exosomal markers/contents, and removal of contaminants.
Commonly used exosome isolation methods and their advantages and disadvantages
Separation technology | Principle | Advantages | Disadvantages |
Differential/ultracentrifugation based techniques | Sequential separation based on density and particle size | Gold standard for exosome isolation, high throughput | Time-consuming, operator-dependent, large volume to start, high equipment cost, and exosomes may be damaged by high-speed centrifugation |
Ultrafiltration | Particle size | Easy to operation, good portability and high scalability | Potential exosome degradation and lysis (thus loss of exosomes) due to shear forces, blockage and capture of exosomes into the filter membrane |
Size exclusion chromatography | Particle size | High-purity exosomes, gravity flow retains the structure, integrity and biological activity of exosomes (not affected by shear stress), with good reproducibility | Time-consuming, moderate cost of equipment, special equipment is required, and difficult to scale up |
Flow field - flow fractionation | Particle size | Gentle separation, which allows for buffer exchange due to the absence of shear forces, is especially important when isolated exosome subpopulations have potential therapeutic applications. | May require additional pre-concentration steps before further studies, low sample volume, difficult to scale up |
Microfluid-based technologies | Immunoaffinity, particle size and density | High efficiency, fast sample processing, high portability, easy automation and integration | Requires large amounts of starting material to increase yield, low sample throughput |
Immunoaffinity capture | Exosome capture based on the use of specific exosome markers | Obtain exosomes with high specificity and purity | High cost of reagents, low yield, limited use |
Precipitation | Solubility and dispersibility | Easy to use, does not require special equipment, large sample processing capacity, and can be scaled. | Lack of specificity, selectivity and low purity (other non-exosomal contaminants such as proteins and polymeric material may co-precipitate) |
While challenges and limitations remain, various pharmaceutical companies and start-ups have paved the way for the development of clinical-grade exosome therapeutics. A growing number of companies are focusing on developing such exosome-based therapeutics to address drug delivery for a variety of therapies, including small molecules, RNA therapeutics, proteins, viral gene therapies, and even CRISPR gene editing tool. Some of these companies are also seeking more innovative exosome engineering methods to design exosome-based therapeutics to increase drug loading and improve targeting capabilities.
Traditional methods of delivering RNA, protein, and chemical drugs have shown some limitations, and exosomes as drug delivery vehicles have great advantages including low immunogenicity, long-term safety, and no cytotoxicity. There are still challenges that must be overcome in terms of clinical translation, large-scale production, stable preparation, storage protocols, and quality control. Further development of cell-derived engineered exosomes and their isolation, purification, and drug loading techniques will help overcome these shortcomings. Engineered exosomes have significant commercial advantages in increasing productivity. Furthermore, by anchoring specific surface molecules to exosomes, the local concentration of exosomes in target cells or disease sites can be increased, thereby reducing toxicity and adverse reactions, and maximizing therapeutic efficacy. In the future, the industry will likely develop new types of multifunctional engineered exosomes to improve healthcare, therefore, further research is needed to explore new strategies for exosome-mediated therapies.
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