**2. Biogenesis, contents and functions of exosomes**

Exosomes are generated by the endocytic pathway from late endosomes (LE) [11, 12]. LEs are formed by inward budding of the multivesicular body (MVB) membrane. LE membranes invaginate to form intraluminal vesicles (ILVs) within MVBs. During this process, some proteins are engulfed and packaged within the ILVs. ILVs then fuse with the cell's plasma membrane and release the vesicles into the extracellular space.

As reported previously, the formation of ILVs can occur either dependent or independent of the ESCRT complex. The ESCRT complex is a set of proteins that function together to facilitate the formation of MVBs, vesicle release, and protein cargo sorting [13–15]. ESCRT 0 has two subunits, HRS and STAM ½, which bind together and recognize specific ubiquitinated proteins in early endosomes. This leads to the recruitment of ESCRT 1 containing Tsg 101, Vps28, Vps37, and Mvb 12, which further recruits ESCRT II. ESCRT II is composed of four subunits, Vps22-EAP30, Vps36—EAP45 and Vps25—EAP20 which starts the invagination of endosomal membranes encapsulating different molecules/cargo such as proteins and nucleic acids.

### *Engineering of Extracellular Vesicles as Nano Therapy for Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.101149*

The ESCRT II subunit Vsp25 then binds with Vsp20 to activate and recruit ESCRT III. It deubiquitinates proteins and allows complete membrane invagination, generating ILVs. Other adaptor proteins such as Vps4 interact with ESCRT III to finally start budding of the membrane, ECSRT subunit removal, and cargo delivery. Hence, the ESCRT complex regulates the whole process of vesicle budding and cargo sorting into exosomes [16, 17]. In cancer, an increased amount of exosomes is often observed in the bodily fluids of cancer patients as a result of deregulation of exosomal formation and secretion [18]. Specifically, in breast cancer, the amount of exosomes released by the human tumor cell line B42 clone 16 was much larger than that released by the parental normal mammary epithelial cells (HMEC B42), as shown by Azmi et al. [19].

Exosomes are composed of a heterogeneous set of cytosolic, nuclear, mitochondrial, ribosomal, and membrane-bound proteins derived from donor cells [20]. Some of these proteins are conserved irrespective of their origin; therefore, they are considered exosomal markers such as tetraspanins, ESCRT proteins, and major histocompatibility complex (MHC) molecules [21]. In addition, some proteins are related to the phenotype of producing cells, such as cancer-derived exosomes, which in turn determines their biological mechanisms. The lipid bilayer membrane of exosomes contains transmembrane proteins, transporter proteins, adhesion molecules, and lipid raft-associated proteins. Exosomes contain nucleic acids such as DNA (ssDNA, mtDNA, dsDNA, and RNA (mRNA, miRNA, and lncRNA) [22]. Exosomal miRNAs and mRNAs are transferred from donor cells to recipient cells, thus modulating the latter's phenotype. Although there are numerous reports indicating the presence of DNA within exosomes, the mechanisms leading to this phenomenon remain unclear. Exosomes also exhibit an exclusive set of lipids distributed in their bilayers, such as sphingolipids, arachidonic acid, cholesterol, phosphatidylserine, and

#### **Figure 1.**

*Schematic representation of exosome biogenesis and composition. Exosomes originate from multivesicular bodies and shed into extracellular space packaging motley of proteins such as ESCRT associated protein, chaperones, along with ssDNA, RNA and dsDNA, miRNA and lncRNA.*

ganglioside [23, 24]. Lipids such as lysobisphosphatidic acid are abundant in the inner membranes of multivesicular bodies and play a crucial role in exosome formation [25, 26]. ExoCarta is a database containing all the data on exosomal content, with over 47,000 protein, mRNA, and lipid entries. Furthermore, ExoCarta is an excellent source of information for exosome characterization (**Figure 1**) [27].
