Exosomes were first discovered in vitro in the supernatant of sheep reticulocytes by R.M. Johnstone and others in 1983. However, at that time, the academic community considered exosomes to be nothing more than "metabolic waste" of red blood cells. Therefore, for a long time, exosomes remained a relatively obscure form of “cellular trash”.

Until 2013, when scientists James E. Rothman, Randy W. Schekman, and Thomas C. Südhof discovered the transport regulation mechanism of cell vesicles (including exosomes), they were awarded the Nobel Prize in Physiology or Medicine. This helped the world to rediscover exosomes as small cargo handlers that shuttle freely between cells.

Exosomes are vesicles secreted by cells into the extracellular space (Figure 1), with a diameter of 40-150 nm. They can be released from almost all cells, whether prokaryotic or eukaryotic, and are widely present in various biological fluids such as blood, urine, semen, saliva, breast milk, cerebrospinal fluid, and bile.

Exosomes carry various cellular components, including proteins, lipids, nucleic acids, sugars, and organelles. They shuttle frequently between cells, transporting cargo molecules to the correct destination, thereby facilitating intercellular signaling. Moreover, exosomes vary in size and composition, exhibiting diverse biological activities.

For friends who are new to exosomes, is it difficult to understand the relationship between exosomes and various vesicles? Are you confused by the various abbreviations for vesicles? In order to facilitate everyone's better understanding of this article, we have compiled a list of differentiated vesicles related to exosomes (Table 1). Please save and use it~

Currently, the mainstream view holds that the process of exosome biogenesis involves the inward budding of the cell membrane to form endosomes, which then mature into intracellular multivesicular bodies (MVBs) through secondary inward budding. Finally, MVBs fuse with the plasma membrane and release the intraluminal vesicles (ILVs) of MVBs into the extracellular space by exocytosis, forming exosomes (Figure 2).

The biogenesis of exosomes

1) The formation of ESE and LSE

The extracellular components such as proteins, lipids, metabolites, small molecules, and ions (Figure 2) can enter the cell through endocytosis and membrane invagination along with cell surface proteins. These components can bud on the cytosolic side of the cell to form early sorting endosomes (ESE) or fuse with budding structures composed of endoplasmic reticulum, trans-Golgi network (TGN), and mitochondria to form ESE (ESE can also fuse with ER and TGN). ESE can further develop into late sorting endosomes (LSE).

2) The formation of ILVs and MVBs

LSE is filled with various contents (proteins, nucleic acids, lipids, etc.), so when the LSE membrane invaginates, it encapsulates the contents in a mixed and random manner, forming multiple intraluminal vesicles (ILVs). That is to say, depending on the invagination volume, this process generates ILVs with different contents and sizes. The remaining membrane of the LSE membrane invagination serves as the outer membrane, concentrating the formed ILVs within the LSE lumen. In other words, LSE further forms multivesicular bodies (MVBs). In simple terms, ILVs are formed within MVBs.

3) The formation of exosomes

MVBs can fuse with autophagosomes, and eventually their contents can be degraded in lysosomes (MVBs can also directly fuse with lysosomes for degradation), and the degradation products can be recycled by the cell. In addition, MVBs can also be transported to the plasma membrane through the cellular cytoskeleton and microtubule network, and with the help of docking proteins on MVBs, fuse with the plasma membrane. Through exocytosis, the ILVs of MVBs are secreted into the extracellular space to become exosomes.

Exosomes exhibit heterogeneity

As mentioned earlier, LSE generates ILVs with varying contents through inward membrane invagination, which are secreted by MVBs into the extracellular space to become exosomes. However, the uneven inward invagination of the MVB outer membrane can lead to inconsistency in exosomal contents. Additionally, MVBs can also fuse with other ILVs and organelles, resulting in diversity in exosomal composition. Furthermore, exosomal contents can vary depending on the source cell. In summary, exosomes exhibit heterogeneity.

The heterogeneity of exosomes can be conceptualized (Figure 3) based on their size, content (cargo), functional impact on recipient cells, and cell of origin. Exosomes formed from different combinations of these features exhibit distinct biological activities within the organism.

Currently, exosomes have shown promising research potential in three aspects: disease diagnosis, treatment, and drug delivery (Figure 4).

Exosomes: disease diagnostic markers

Exosomes are small vesicles released by cells, including cancer cells, into the surrounding biological fluids. These exosomes contain materials originating from tumors, such as DNA, RNA, proteins, lipids, glycan structures, and metabolites. Therefore, exosomes generated in pathological microenvironments can capture the complex intracellular molecular signatures specific to particular disease stages or injuries, making them a highly promising repository of biomarkers.

The key point is that proteins overexpressed in tumors isolated from blood or other biological fluids do not necessarily exhibit cancer specificity. Similar biomarkers can also be produced by non-cancerous tissues and may vary in quantity within the normal human body. By enriching exosomes with tissue-specific (or cancer-specific) biomarkers, higher sensitivity and/or specificity can be achieved.

Advantages of exosomes as diagnostic biomarkers for diseases:

1) They are widely present in various biological fluids and are highly stable.
2) They can diagnose diseases in the early stages (exosomes are actively released from cells at various stages of tumor formation).
3) They can achieve the enrichment of cancer-specific biomarkers (Figure 5).
4) Compared to tissue biopsy, liquid biopsy is less traumatic, cost-effective, and provides real-time insights into tumor status.
5) Compared to tissue biopsy, it reduces patient discomfort during sampling.
6) They are easy to store: freezing, freeze-drying, or spray drying.
7) Compared to traditional tissue biopsy, liquid biopsy based on exosomes has higher application value.
Currently, the first prostate cancer detection method based on exosomal RNA has also been developed^[7].

Exosomes: Promising Potential for Disease Treatment

Exosomes extracted from various cells can interact with target cells through various pathways, including endocytosis, direct binding, phagocytosis, and direct fusion, thereby producing specific therapeutic effects. For example, Jeongyeon Heo et al. found that material exchange between different cell types (including endothelial cells, vascular smooth muscle cells, and macrophages) through exosomes can improve atherosclerosis^[9].
Furthermore, exosomes also exhibit biological activity in diseases such as cancer, neurodegenerative disorders, metabolic diseases, as well as immune and inflammatory conditions.

Exosomes: Natural Drug Delivery Vehicles

Exosomes can be modified to target specific cells, carry various types of drugs, and possess superior ability to penetrate various tissue barriers (such as the blood-brain barrier), making them excellent natural drug delivery vehicles. In comparison, exosomes exhibit lower immunogenicity and significantly reduced toxicity compared to lipid nanoparticles (LNPs) (Table 2). Exosomes also demonstrate better drug encapsulation, controlled release, and in vivo biodistribution capabilities. Additionally, exosomes show 10 times higher adhesion and internalization within tumor cells compared to liposomes of the same size, indicating their higher targeting specificity for cancer.

The potential of exosomes as drug delivery vehicles has been extensively researched. Preclinical studies demonstrate that engineered exosomes can efficiently and precisely deliver anti-tumor drugs to the tumor sites in mice, reducing treatment-related adverse reactions (Figure 6).

As shown in Figure 6, after intravenous injection, the engineered exosomes carrying drug molecules are guided to the tumor site by various targeting molecules. Subsequently, chemotherapeutic drugs (such as PTX), ncRNA (such as miR-551-3p), and immune molecules (such as siPDL1) are released into the tumor microenvironment (TME). Ultimately, the internalization of engineered exosomes leads to the death of tumor cells.