Isolation and Clinical Application of Exosomes

Exosomes Isolation

Because of the heterogeneity between and within exosomes types, and the feature overlap with other EVs, it is difficult to distinguish exosomes from other EVs. Different exosomes separation techniques have been developed based on the physicochemical properties of exosomes as well as carrying surface biomarkers. Non-specific separation based on the physical properties of exosomes includes ultracentrifugation, size-exclusion chromatography (SEC), and polymer precipitation, and specific enrichment based on the biological properties of exosomes includes immunoaffinity.

Ultracentrifugation has been widely used to isolate exosomes from a variety of sources (e.g., cell culture media, serum, saliva, urine, and cerebrospinal fluid) and is considered to be the gold standard for the isolation of exosomes; however, there are limitations to the method, such as cumbersome and costly instrumentation, long and laborious processing, contamination of aggregated proteins and ribonucleoprotein particles, and the need for a large number of samples.

Polymers precipitate by reducing the hydration of exosomes to reduce the solubility and thus cause precipitation, the precipitated products can be easily separated by low centrifugal force. This method has a short extraction time and can meet the needs of different conditions. However, this method is expensive for large-scale use, lack of specificity and low purity. Heterogeneous polymer particles tend to be produced in the process of co-precipitation, high likelihood of remaining extracellular contaminants due to reagents that similarly reduce the solubility of vesicles and proteins, also co-precipitate a variety of water-soluble substances such as nucleic acids, lipoproteins, proteins, and even viruses.

Size-exclusion chromatography (SEC) separates vesicles and other molecules based on their size by gel filtration, where small molecules diffuse into the pores as the sample enters the gel, while larger molecules elute directly. Therefore, the larger molecules leave the column earlier than the small ones, which means that the residence time of a molecule is correlated with its size. The size-exclusion column contains resins with a pore size of approximately 75 nm, allowing larger vesicles (>75 nm) to quickly pass through and elute out of the voids, while proteins and other smaller molecules temporarily diffuse into the pores and cannot pass through. SEC has little damage to exosomes and low demand for sample volume, but the exosomes prepared by SEC column usually show wider size distribution, especially in a smaller size range, which indicates the existence of pollutants with similar size to exosomes, such as protein aggregates and lipoproteins.

Immunoaffinity targets marker proteins on the surface of the exosome membrane and utilizes specific antibodies to capture common markers of exosomes. In theory, any protein highly present on the exosome membrane and the lack of corresponding free components in the extracellular fluid can capture the exosomes based on the principle of immune affinity.

Figure 2. Different techniques used for isolation of exosomes
(Source: Mahgoub EO, et al. 2020)

Exosomes Therapy

The exosomes themselves can be used as drug-carrying vehicles to achieve therapeutic effects. Compared with liposomes, injection of exosome can effectively enter the cells and deliver functional cargo with a minimum immune clearance rate after exogenous drugs are injected into mice. The exosomes are also well tolerated, with no significant side effects from repeated injections. On this basis, researchers have developed engineered exosomes for central nervous system and cancer, which can carry designated miRNA or small interference RNA. These RNAs can effectively bind to target mRNAs and inhibit gene expression in receptor cells, thus achieving therapeutic goals. The RNA in exosomes can be protected from the degradation of ribonuclease in the blood, and the therapeutic exosomes has better systemic retention than liposome and can function at a distance. Studies have shown that there is CD47 on the surface of exosomes, which sends a "don't eat me" signal, protects exosomes from being swallowed, and reduces clearance in blood circulation.

Figure 3. Cellular uptake of therapeutic exosomes
(Source: Kalluri R, et al. 2020)

At present, the biggest challenge of exosomes-based therapy is to solve the heterogeneity of secreted exosomes. Exosomes-based therapy requires a better understanding of its biogenesis, composition, and heterogeneity. Although exosomes extracted from similar cells is considered to have the same composition, the results show that these exosomes may have different molecular composition and targeting moiety. The heterogeneity of exosomes brings additional complexity to exosome design, dose standardization and delivery in clinical applications. Therefore, an in-depth study of the heterogeneity of exosomes is crucial, not only to identify suitable subpopulations for specific therapeutic purposes, but also to prevent side effects associated with heterogeneity. Different isolation methods have an effect on the purity and physicochemical properties of exosomes, so optimizing the isolation method can better preserve the biological activity of exosomes and reduce the associated side effects.

The composition and carrying information of exosomes are closely related to that of the parental cells, so when using exosomes for treatment, we can choose exosomes that are more favorable for treatment based on the characteristics of exosomes isolated from different types of cells. Transplanted myeloid dendritic cells, melanoma and muscle cell-derived exosomes have been shown to have different distribution patterns in the spleen, lung, and liver. In addition, neutrophil-derived exosomes can cross the blood-brain barrier and can be used for drug delivery to the brain and targeting of gliomas.