Exosomes and ectosomes, which are derived from endosomes and the plasma membrane, respectively, are the two subtypes of lipid-bound EVs secreted into the extracellular environment. These EVs do not possess a nucleus structure; hence, they cannot replicate. According to the latest position statement indicated in the Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018) report, exosome and ectosome terminologies should only be used if an experimental research design can establish the subcellular origin of the EV subtype being studied. Otherwise, operational terminologies for the subtypes of EVs should be adopted, which are based on (i) particle size (small EVs for particles smaller than 200 nm, and medium and large EVs for particles larger than 200 nm), (ii) surface markers expression or biochemical recognition (such as CD81+, CD63+ or annexin A5-stained), and (iii) the origin or condition of cells where the EVs are isolated (such as hypoxic EVs, apoptotic bodies, and podocyte EVs) [76].

Exosomes can be either native or bioengineered, depending on whether they have been artificially manipulated or not. Both animals and plants can produce native exosomes, hence giving rise to animal-based exosomes or plant-based exosomes. Among the animal-based exosomes, they can be further classified depending on the type of cells that are producing the exosomes—either exosomes produced by normal and healthy cells or those that are synthesized from tumor cells. Amongst the variety of normal cells that can produce exosomes, MSC-derived exosomes have been given considerable attention due to the capability of MSCs to self-renew and undergo multilineage differentiation. Therefore, MSC-derived exosomes are being actively investigated for their tissue regeneration potential [77].

Exosomes, the smallest form of EVs, with diameters ranging from 30–150 nm [78], are usually synthesized through an endosomal-sorting complex required for transport (ESCRT)-dependent or ESCRT-independent pathways [79,80,81,82]. The ESCRT-dependent pathway for exosome biogenesis usually involves a series of budding processes that are facilitated by complex protein machinery, which is primarily made up of four distinct ESCRT proteins (0 to III). This pathway is triggered when ubiquitinated proteins are recognized by ESCRT-0 and are sequestered into specific endosomal membrane domains. After further interacting with ESCRTs I/II and forming a total complex with ESCRT III, plasma membrane infolding begins, resulting in the formation of a cup-shaped early endosome that contains cell-surface proteins and soluble bioactive substances that are accumulated from the extracellular environment. As this early endosome buds inward, it matures into a late endosome that subsequently undergoes a secondary endosomal membrane invagination to form a multivesicular body containing intraluminal vesicles [80,83,84,85,86]. These intraluminal vesicles have similar membrane positioning to the cell surface, where extracellular domains of transmembrane protein face the extracellular environment while enclosing the cytosolic entities [87]. As the multivesicular body moves to the cell surface and fuses with it, the intraluminal vesicles are released into the extracellular space, giving rise to exosomes [77]. Other than ESCRT proteins, numerous other accessory proteins have been shown to be involved in the production of exosomes through the ESCRT-dependent pathway, including programmed cell death 6-interacting protein (ALIX); tumor susceptibility gene 101 protein (TSG101); heat shock cognate protein 70 (HSC70); vacuolar protein sorting-associated protein 4 (VPS4); heat shock protein 90 (HSP90); soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNAREs); cluster of differentiation proteins 9, 81 and 63; and syndecan-1, synthenin-1, and tetraspanins [79,85,87,88]. In the alternative ESCRT-independent pathway, ceramides play an important role as their cone-shaped feature is speculated to promote microdomain-induced budding of the endosomal membrane. These ceramides are usually produced through the removal of the phosphocholine moiety by sphingomyelinases that are abundantly present in those microdomains. The presence of ceramides will also induce the lateral isolation of cargo within the endosomal membrane that leads to the formation of intraluminal vesicles within the multivesicular body, releasing exosomes once it fuses with the plasma membrane [89,90].

Upon secretion into the extracellular space, exosomes will migrate and deliver their contents to their target cells, resulting in the alteration of gene expression as well as the modification of physiological and biological functions. The functional modification relies on the type of exosomal contents present within the exosomes [91]. Invariably, most exosomes are rich in proteins and lipids. To date, about 8000 proteins and 194 lipids have been found to be related to exosomes [92]. Along with these, exosomes are known to carry nucleic acid cargos, including mRNAs and miRNAs, for intercellular communication [93]. Since exosomes are secreted by numerous cell types and the exosomal content is heavily associated with the cell of origin, each exosome may have different roles in intercellular communication for varying physiological effects. For example, platelets secrete exosomes containing prostaglandins that modulate inflammatory activities [94]. Meanwhile, antigen-presenting cells, such as dendritic cells, release exosomes that carry functional major histocompatibility complexes class I and II (MHC I and MHC II) to attenuate or stimulate antigen-specific B-cell or T-cell responses [95]. Similarly, many other studies have also demonstrated the involvement of exosomes in various biological processes, including coagulation, blood vessel formation, red blood cell maturation, and removal of unwanted RNAs and proteins [96].

In view of exosomes’ capability to carry their cargo to the target cells for participation in both normal and pathobiological mechanisms through intercellular communication, extensive investigations have been performed to exploit their therapeutic purposes to restore the diseased tissue to its normal phenotype. One of the approaches involves the isolation of these endogenous exosomes from body fluids so that they can be artificially enriched with therapeutic miRNAs before these bioengineered exosomes are reintroduced back into the patient’s body [93,96]. Exosomes have increasing popularity as a preferred drug delivery vehicle as they are non-immunogenic. In addition to that, other advantages of exosomes over MSCs are their homing ability to the site of injury and passing through tight junctions such as the blood–brain barrier. Since exosomes are very heterogeneous, they can also carry different proteins on their surface that can be delivered into the cell through receptor-mediated endocytosis upon interacting with the target cells [77]. Therefore, the exosome-assisted delivery approach has been utilized by various research groups to deliver a variety of therapeutic compounds to their target cells, including short interfering-RNA (siRNA), recombinant proteins, miRNA, and antagomirs, as well as anti-inflammatory and chemotherapeutic drugs [89]. For instance, exosomes equipped with siRNAs against the mRNA of Huntington’s disease have been successfully introduced into the cortical neurons of mice, resulting in an improvement of the murine Huntington’s disease condition [97]. Through the use of exogenous siRNAs, the MAPK gene in target lymphocytes and monocytes could be silenced. Hence, this proves the feasibility of bio-engineering isolated exosomes for the targeted delivery of therapeutic materials to a specific tissue to exert its function [98]. Alternatively, donor cells can be bio-engineered to contain the therapeutic materials, which will then be synthesized and encapsulated in the exosomes produced. These enriched exosomes can then be used for the treatment of specific diseases. An example would be a study done by Shimbo et al. that introduced miR-143 into MSCs, where this miRNA was then secreted and packaged into the exosomes. Upon delivery to osteosarcoma cells, the miR-143 present within the exosomes were able to significantly inhibit the migration of the cancerous cells [99]. In view of these and many other successful examples in utilizing exosomes as a delivery vehicle for therapeutic purposes, exosomes have been accepted as drug carriers for the treatment of various diseases in clinical trials [79].