**2. Extracellular vesicles: definition and main characteristics**

Over time, the definition and role of EVs have been strongly questioned. Unanimously, considered as ranging from 20 to 200 nm to 10 μm in diameter, EVs can be differentiated into three major classes: exosomes, microvesicles (MVs), or ectosomes and apoptotic bodies. However, there are a limited number of studies on apoptotic bodies, so frequently the term EVs refers to exosomes and microvesicles [17]. Moreover, recent research underlined the possibility of subdividing EVs, for example, mitochondrial protein-enriched EVs or other categories of exosomes, based on their proteins and RNA profile (such as large or small exosome vesicles) [20–22].

EVs are regularly classified based on biogenesis, release pathway, size, content, and function [1, 6, 23]:

1.*Exosomes* are produced and secreted by all cell types and have a characteristic diameter between 30 and 150 nm [24]. The biogenesis and release pathway begin with early endosomes, deriving from inwardly budding of the plasma membrane of the cell. The same process will be applied then to the limiting membrane of the early endosomes, representing the second phase. The maturation of the early endosomes will lead to multivesicular bodies (MVBs) formation [23]. Both early endosomes and MVBs are participating in performing certain functions related to cellular material (especially proteins), like endocytic and trafficking functions [25]. Finally, MVBs present two possible routes of evolution: one refers to degradation of MVBs by lysosomes, including its components, and the second one to attaching MVBs to the plasma membrane of the cells and releasing its constituents, exosomes, in the extracellular space [6, 26, 27]. Even though the specific

factors that regulate these mechanisms are not well known, the most underlined pathway researched by studies is the endosomal sorting complexes required for transport (ESCRT) [28]. Based on the primary mechanism involved in the biogenesis of exosomes, ESCRT proteins, it is obvious that exosomes contain these proteins [29]. However, another mechanism involved independently is based on the sphingomyelinase enzyme, which was studied by researchers because cells without ESCRT mechanism can still produce CD63 positive exosomes, a protein from the tetraspanin family [30]. Exosomes also contain glycoproteins, low levels of proteins associated with endoplasmic reticulum and Golgi apparatus, cholesterol, ceramide, noncoding RNA, mRNA, miRNA, and cytosol [6, 24, 31].

Some well-known functions of exosomes are the facilitation of communication between cells, cell preservation, association with cancer evolution, stimulation of immune response, involvement in the functions of the nervous system (myelination, growth, and survival of the nerve cells, but also the progression of neurological diseases by containing pathogenic proteins, as a beta-amyloid peptide, superoxide dismutase and α-synuclein [24, 32–35]. Because of their constituents, exosomes are becoming more and more attractive for researchers to discover new implications in diseases and potentially new therapeutic methods [24]. For example, as already mentioned, exosomes contain α-synuclein, which is involved in Parkinson's disease [36]. New studies are concentrating on the association with glioblastoma, acute kidney disease, pancreatic or lung cancer, vaccines or other immunological uses, and diminishing tissue injury [37–41].

2.*Microvesicles* are a type of EVs measuring between 100 nm and 1 μm [1]. Their biogenesis and release pathway are still not well known. However, MVs are produced by outward budding of the plasma membrane of the cells, involving cytoskeleton elements (actin and microtubules and other cytoskeletal proteins like ARF6 and RhoA), molecular motors (kinesins and myosins) and fusions machinery (ESCRT, SNAREs, and tethering factors) [1, 42, 43]. The content of the MVs, largely determined by their biogenesis, is represented by proteins associated with cytosol and plasma membrane (especially tetraspanins), cytoskeletal proteins, integrins, glycosylated and phosphorylated proteins, and heat shock proteins [24, 44, 45]. In addition, MVs contain cholesterol, mRNA, miRNA, and cytosol [6]. Other specific markers helping in differentiation between MVs and exosomes need to be further studied [24]. Like exosomes, MVs participate in communication between cells, a particular characteristic being their ability to deliver proteins, lipids, or nucleic acids to another cell [1, 23]. Primarily, this function facilitates communication between healthy cells, but on the other hand, it can be a way to spread cancerous cells in the body, leading to metastasis [46]. That's why future studies must focus on this individuality of MVs, to develop potentially new therapeutic methods in cancer. Other possible purposes of MVs use in the future are, as already noted, the same as with exosomes [24].

A particular type of MVs is represented by *oncosomes*, which are secreted by the shedding of plasma membrane blebs of cancer cells [2, 47]. Even if their main characteristics are still not well known, some experimental studies on glioblastoma and prostate cancer have shown that their biogenesis is linked to serine/threonine kinase 1 (AKT1) and epidermal growth factor receptor (EGFR) pathways [48, 49]. Their size depends on the stage of cancer, reaching up to 1000 nm in the final stages, thus being

