Applications of Exosomes

### **Chapter 6**

## Perspective Chapter: Tissue Specificity of Exosomes and Their Prospects as a Drug Delivery System

*Alisa Petkevich, Aleksandr Abramov and Vadim Pospelov*

### **Abstract**

This chapter reviewed the various sources of exosomes and their characteristics. Exosomes, which in the context of the proposed chapter are the synonym for extracellular vesicles up to 200 nm, play a pivotal role in cell to cell communication thus leading to the involvement of exosomes in inflammation and cancer development. This brings exosomes to the forefront of promising markers of a sub-clinical stage of the disease, which makes identifying exosome's source and destination one of the main goals in exosome research. However, due to some biogenesis features and technological difficulties, which are discussed further, identification of a certain exosome's address, or its specificity for a certain tissue or cell type, becomes a non-trivial task. The chapter covers the following questions: some of the main barriers on the way of testing tissue specificity hypothesis of exosomes, exosomes from synovial fluid and CSF and their features, exosomes from mesenchymal stem cells (MSCs) of different origins, and some membrane and cargo exosomal markers for tissue specificity and the prospect of exosomes as a drug delivery approach.

**Keywords:** exosomes, tissue specificity, synovial fluid, cerebrospinal fluid, mesenchymal stem cells of different origins, drug delivery

### **1. Introduction**

The term "exosome" has been known since the beginning of 1970s, when it was first used in papers published in "The Proceedings of the National Academy of Sciences (PNAS)" and dedicated to the transfer of DNA fragments between Drosophila or Neurospora cells. The term in that context had little in common with lipid bilayer vesicles, which may contain a wide variety of nucleic acids and proteins and was mainly related to DNA fragments that are not integrated into the exome and eliminated during meiosis [1]. The first use of the term "exosomes" in the extracellular vesicles context happened in 1980s, when Trams et al. in 1981 described vesicles that are produced directly by outward budding at the plasma membrane [2]. Nowadays, the ISEV (International Society for Extracellular Vesicles) consensus recommendation on nomenclature is to use "extracellular vesicle" as the "generic term for particles naturally released from the cell that is delimited by a lipid bilayer and cannot replicate" and to modify "EV" based on clear, measurable characteristics such as the cell of origin, molecular markers, size, density, function, etc [1]. In this paper we assume exosomes and extracellular vesicles (EV) as synonym terms. They are assumed to be a tool for intracellular communication and are promising biomarkers in different pathologies, including tumor growth. Another perspective trend for exosome' application is the use of the latter as a delivery system for various therapeutic targets. These microvesicles have some of the features of an ideal drug delivery system such as high biocompatibility, minimal toxicity, and tissue specificity. The latter feature may seem difficult to be objectively determined as far as the experimental design for confirmation or refutation of exosomes` tissue specificity is troublesome. This chapter proposes to discuss various aspects of the tissue specificity of exosomes, including some of the main barriers on the way of testing the tissue specificity hypothesis of exosomes, exosomes from synovial fluid and CSF and their features, exosomes from mesenchymal stem cells (MSCs) of different origin and some of membrane and cargo exosomal markers for tissue specificity the prospect of exosomes as a drug delivery approach.

### **2. Some of the main barriers on the way of testing the tissue specificity hypothesis of exosomes**

First of all, culturing difficulties should be admitted. The difference between cell lines and even the corresponding primary cell cultures is obvious and does not allow to extrapolation of data obtained on cell lines to corresponding cells of an organism without limitations. Barriers in experiments with exosomes on the primary cell cultures lie on the surface: during obtaining the ex vivo culture of human cells successive steps should be performed, including dissection and/or disaggregation of the tissue, which may be accompanied by the formation of vesicles as the result of mechanical destruction of cells, especially in working with dense tissues, requiring preliminary mechanical grinding [3]. Another barrier, and perhaps more serious, is time limits. It is well known, that the longer primary cell cultures exist the less they reflect the ex vivo state of the corresponding tissue. Anyway, the latter reason along with others such as changes in morphology and signaling under the influence of antibiotics, culture flask, absence of tissue architectonics, etc., is not unique to exosome experiments.

Secondly, in terms of biogenesis exosomes are secreted intraluminal vesicles (ILVs), which sequester specific proteins, lipids, and cytosolic components, whilst multivesicular bodies (MVBs), and late endosomes are a subset of specialized endosomal compartments rich in intraluminal vesicles. ILVs are generated by the inward budding of endosomal membranes and MVBs get transported to plasma membrane *via* cytoskeletal and microtubule network, after that they undergo exocytosis postfusion with the cell surface whereby the ILVs get secreted as exosomes [4]. The biology of this process does not allow us to make a firm assumption, that exosomes, even secreted by different cell types, have distinct superficial/membrane markers, identifying their specificity. Nevertheless, there is a probability of determining exosome's cell or tissue origin or tissue-specific engagement in disease by the presence of specific combinations of surface proteins and their abundance [5]. Anyway, there is always a possibility to suppose the origin of exosomes by their cargo, for example, by

### *Perspective Chapter: Tissue Specificity of Exosomes and Their Prospects as a Drug Delivery System DOI: http://dx.doi.org/10.5772/intechopen.111566*

miRNA profile, which is discussed later, but considering all of the above identification of exosome fraction of certain miRNA profiles is a non-trivial task. Moreover, the effects of exosomes from different sources might overlap with each other and, for example, might act complementary in eliciting inflammatory reactions, e.g. as has been observed for microvesicles from atherosclerotic plaques [6, 7].

Thirdly, partly due to exosomes' size, there are technical difficulties in identifying exosomal membrane markers especially when there is a complex of these markers. Currently, opportunities for the detection of exosomes are improving due to the new emerged technologies. Imaging flow cytometry is on the edge of these technologies, remaining one of the basic instruments for phenotype description. Imaging flow cytometry overcomes obstacles in traditional flow cytometry by including a CCD camera with a 60× objective, allowing detection of vesicles with sizes below 500nm through enhanced fluorescence with only a small number of fluorophore-labeled antibodies like two protein targets per exosome [5]. However, this area is growing rapidly, and flow cytometry kits based on beads carrying up to 37 exosomal protein markers on their surface are nowadays available. There are some alternative options for multiplex surface markers recognition on exosomes like proximity-dependent barcoding assay, converting the protein composition to DNA sequence information via bound antibody-DNA conjugates with the following decoding by NGS [5].

And, fourthly, the biological materials which are widely available for sampling are blood, urine, and saliva. Apparently, every of this biomaterial is a stock of everything from everywhere and it is extremely difficult to determine the origin of exosomes, isolated from these biomaterials. A major source of microvesicles in plasma is represented by platelets, other sources of exosomes in blood plasma are endothelial cells, smooth muscle cells, monocytes, lymphocytes, and erythrocytes [6, 8]. Berckmans René et al. in 2001 discovered, that among total exosomes, isolated from human plasma, 82% were platelet exosomes, 15% from erythrocytes, and 3% from leukocytes; in 2019 they discovered, that among total isolated plasma, 52% are platelet EVs, 29% erythrocyte Evs, 20% leucocyte Evs and a low concentration of EVs from (activated) endothelial cells (E-selectin, CD62E) can be detected [9]. The biofluids which allow assuming the specificity of the isolated exosomes are synovial fluid and cerebrospinal fluid (CSF), exosomes from these biomaterials are discussed below.

### **3. Exosomes from synovial fluid and CFS**

The biofluid, which is available for research in a limited number of cases, is synovial fluid. With a large synovial microvesicular pore radius reaching 40 nm, it can be assumed, that the exosomes, isolated from the synovial fluid, are mostly exosomes, produced by fibroblast-like synoviocytes and by chondrocytes, which constitute the synovial membrane and joint cartilage respectively [10].

Huang et al. investigated exosomes from synovial fluids of patients with different joint diseases: gout, rheumatoid arthritis (RA), axial spondyloarthritis (axSpA), and osteoarthritis (OA). The main goal of the experiment was not to identify tissue or cell-specific markers in exosomes but to determine markers, which would primarily allow differentiating these disease states in patients. However, this is valuable research in characterizing tissue specificity of exosomes, which include samples of synovial fluid of total of 100 patients. Twenty-five proteins were found highly expressed in gout uniquely, lysozyme C and protein S100-A9 included, whose bioinformatic analysis was significantly involved in "neutrophil degranulation" and "prion diseases". Along with differentially expressed proteins, there were thirtynine proteins highly expressed in axSpA uniquely and twenty-eight proteins in RA. In axSpA among others there were RNA-binding protein 8A and protein transport protein Sec24C included, whose bioinformatic analysis was significantly involved in "acute-phase response" and "citrate cycle". In RA, these uniquely expressed proteins included pregnancy zone protein (PZP) and stromelysin-1, whose bioinformatic analysis was significantly involved in "serine-type endopeptidase inhibitor activity" and "complement and coagulation cascade" [11]. Apparently, these molecular events may have distinct functional consequences: exosomes isolated from synovial fibroblasts, which were cultured in conditions mimicking OA, were able to induce MMP-13 and aggrecan expression in articular chondrocytes isolated from healthy synovial joints, suggesting in vitro this would lead to tissue degeneration [12]. Moreover, Esa et al. admit in their review that exosomes produced by both synovial fibroblasts and chondrocytes under OA-like conditions upregulate the release of pro-inflammatory cytokine cascades, including MMP-13, creating a "positive-feedback loop" that drives inflammation within the joint and ultimately leads to the damage of articular cartilage and a loss of structural integrity [13]. By the way, it is interesting to note, that, unlike exosomes from MSCs of different origins, exosomes from healthy individuals or individuals with OA do not differ either in the concentration (OA: 1.18 × 1010 particles/ ml, n = 6; non-OA: 1.59 × 1010 particles/ml, n = 6) or in the size (OA: 0.128 μm, n = 6; non-OA: 0.127 μm, n = 6) [14].

Another biofluid that is also available in a limited number of cases is cerebrospinal fluid. Taking into account that CSF is produced by ependymal cells and permeability of blood-brain barrier to hydrophobic molecules and small non-polar molecules, it is possible to assume exosomes, isolated from CSF, are specific for the cells, making up and supporting functioning the central nerve system, such as neurons, astrocytes, microglia, and oligodendrocytes [15]. On the other hand, it should be admitted exosomes can cross BBB in blood-brain direction, which makes them a promising approach for the target delivery of therapeutic agents [15, 16]. The ability of exosomes to cross the blood-brain barrier (BBB) in the opposite direction makes them a highly attractive source of biomarkers originating in the CNS that could be isolated from the blood [17].

Otake et al. detected 14,807 genes in CSF exosomes, of which 4580 genes were commonly detected among four individuals, including neuron-enriched genes such as TUBB3 and CAMK2A. Gene Ontology analysis and pathway analysis with these genes revealed functional enrichment of ubiquitin-proteasome pathway, oxidative stress response, and unfolded protein response in CSF from patients with amyotrophic lateral sclerosis. These pathways are related to pathomechanisms of amyotrophic lateral sclerosis [18]. Along with common exosomal protein markers, expressed on the surface of CSF exosomes and in mRNA exosomal content, there are such proteins as NCAM, L1CAM, SOD1, α-synuclein Aβ42, total tau, TDP-43, and pT181-tau [16, 18]. The presence of SOD1, which is one of the most studied causes of amyotrophic lateral sclerosis, in exosomes secreted from motor-neuron-like NSC-34 cells overexpressing human wild-type or mutant SOD1 provided the first evidence for the secretion and cell-to-cell transmission of SOD1 in the context of ALS [16]. TDP-43 is assumed to facilitate a prion-like spread of its misfolded species [19]. NCAM is a neuronal cell adhesion protein, which is involved in cell-cell and cellmatrix interactions, and L1CAM is an axonal glycoprotein that plays an important role in nervous-system development and its mutations cause neurological syndromes known as CRASH [19, 20].

*Perspective Chapter: Tissue Specificity of Exosomes and Their Prospects as a Drug Delivery System DOI: http://dx.doi.org/10.5772/intechopen.111566*

### **4. Exosomes from mesenchymal stem cells (MSCs) of different origin**

Mesenchymal stem cells (MSCs) are the subject of intense research as they are a potential therapeutic tool for several clinical applications and among others one of the most available options to study stem cells, what is one of the main reasons why exosomes from these cells seem to be mostly described and studied. Thus, exosomes from different variants of mesenchymal stem cells seem to be well described and characterized.

In 2021 González-Cubero et al. described the phenotype of adipose tissue-derived mesenchymal stem cells (ASCs-derived) exosomes: from 37 exosomal surface epitomes 31 were detected and 6 were undetected. Among the detected ones were CD3, CD4, CD19, CD8, HLA-DRDPDQ, CD56, CD105, CD2, CD1c, CD25, CD49e, ROR1, CD209, CD9, SSEA-4, HLA-ABC, CD63, CD40, CD11c, CD81, CD41b, CD86, CD326, CD133/1, CD29, CD69, CD45, CD31, CD20, CD14, while CD3+, CD45+, CD56+, HLA-ABC, and HLA-DRDPDQ were particularly strongly enhanced in samples with ASCsderived exosomes (99.99% ±0.06%, 55.45% ±6.36%, 88.68% ±4.29%, 84.66% ±5.99%, 59.98% ±7.45%, respectively). However, CD42a, CD44, CD62P, CD142, CD146, and MCSP were undetectable [20]. Like ASCs-derived exosomes, it was shown, that exosomes from BMSC do not express CD146 and CD42a. However, CD1c, CD2, CD3, CD4, CD14, CD20, CD25, CD31, CD40, CD45, CD49e, CD56, CD69, CD133/1, and CD326 also were undetectable in exosomes from BMSC [21].

Moreover, 1 year in 2020 Wang et al. compared the exosomes, isolated from bone marrow-derived MSC (BM-MSC), umbilical cord-derived MSC (UC-MSC), and adipose tissue-derived MSCs (AT-MSC). They found that AT-MSCs produced exosomes more intensively, as far as the concentration of exosomes in the supernatant, collected for the same time period, was higher than that of BM-MSC or UC-MSC exosomes [22]. However, simultaneously in 2020 Xu et al. showed this is not a strict regularity: during the experiment, they got supernatant with the density 2.38 × 1011/mL in exosomes from BMSCs; 1.08 × 1011/mL in exosomes from ADMSCs and 1.75 × 1011/ mL in exosomes from UCMSCs [23]. In the research of Wang et al., exosomes from all three different tissue sources were studied with TEM, typical cup-shaped vesicles were observed and no differences in shape among the exosomes were noted [22]. Xu et al. showed there is sometimes possibly a slight difference in the size distribution of exosomes from BMMSCs, ADMSCs, and UCMSCs: in the case of BMMSCs, exosomes were round or dish-shaped with a diameter of 40–100 nm, the average particle diameter of exosomes was 70.3 nm. Exosomes from ADMSCs were uniform in size with a diameter of 30–100 nm with the average particle diameter within 95 nm and the majority of exosomes were 72.8 nm, while the UCMSCs exosomes were round in shape with a diameter of 10–90 nm and most of the particles had diameters of about 80.6 nm [23].

A detailed proteomic analysis revealed 771, 457, and 431 proteins in exosomes from BM-MSC, AT-MSC, and UC-MSC, respectively; comparison of the three types of exosomes revealed 355 common proteins, and 341, 23, and 37 proteins unique to the exosomes from BM-MSC, AT-MSC, and UC-MSC, respectively. In terms of biological process, proteins from BM-MSC exosomes were mainly involved in granulocyte activation and regulation of cell migration, whereas proteins from AT-MSC exosomes were enriched in leukocyte activation involved in immune response and UC-MSC exosomes along with leukocyte activation proteins involved in immune response were enriched in proteins of collagen metabolic process. As for molecular function, AT-MSC exo and UC-MSC exo proteins were both significantly enriched in cell adhesion molecule binding, whereas BM-MSC exo proteins were mostly involved in protein complex binding and integrin binding. Along with protein cargo, Wang et al. examined membrane markers of isolated exosomes and identified some membrane proteins, that are differentially expressed: ATP2B1 and ATP1A1 showed high expression in AT-MSC exosomes, whereas ITGA2 and LRP1 showed low expression. LTGB3 and SLC44A1 showed low expression in UC-MSC exosomes. In contrast, ADAM9, ADAM10, CD81, CACNA2D1, NOTCH2, and HLA-A showed high expression in BM-MSC exosomes [23]. There is a strong data, exosomes of all three sources— BMMSCs, ADMSCs, and UCMSCs—show highly expressed exosomes specific markers CD63, HSP70, CD81, and CD9 [21, 23].

Exosomes from MSCs are known to express another protein, a milk fat globule- epidermal growth factor-factor VIII (MFGE8), a glycoprotein that bridges externalized phosphatidylserine (PS) on the apoptotic cell surface to alphaVbeta3 or alphaVbeta5 integrins on the phagocyte. The expression of this protein has certain functional consequences in exosomes: their administration increases macrophage uptake of apoptotic bodies in the border zone of infarction and is associated with reduced proinflammatory response, increase in neovascularization, lower infarct size, and an improvement in cardiac function [24].

Three years before that in 2017 Mead B. et al. discovered the difference in membrane proteins expression between exosomes from BMSC and fibroblasts: more CD11c+ and CD63+ exosomes were detected on the BMSC exosomes (20.3% ± 8.3%, 81.7% ± 12.3%, respectively) compared to fibroblast exosomes (7.7 ± 0.7, 49.6 ± 2.4, respectively), whereas more CD29+ and CD81+ exosomes were detected on fibroblast exosomes (32.4% ± 0.75%, 39% ± 3.3%, respectively) compared to BMSC exosomes (20.5% ± 1.9%, 15.3% ± 10.6%, respectively) [24]. On the surface of AT-MSCs exosomes along with an abundance of well-known exosomal markers CD63, CD9, and CD81, there was revealed the expression of CD105, an MSC marker, as well as CD44, CD29, CD49e, and melanoma-associated chondroitin sulfate proteoglycan (MCSP). In addition, MSCexosomes were found to be preferentially distributed in the damaged kidneys of mice with glycerol-induced AKI compared to in the healthy kidneys of control mice [25].

Exosomes from BM-MSCs and ADSCs show similar profiles, which are positive for CD105, CD73, CD90, and CD44; negative for CD45, CD31, and CD34 [26]. Nevertheless, there is a difference in functional capabilities of exosomes derived from BM-MSCs and ADSCs, the latter has a more significant neprilysin (NEP) activity: NEP-specific enzyme activity accounted for 38.3 ± 4.5% of total enzyme activity of ADSCs exosomes while BM-MSCs showed weak or undetectable NEP enzyme activity. Katsuda et al. calculated NEP-specific activity after the subtraction of fluorescence in the presence of thiorphan and they demonstrated that all ADSCs exhibited NEP-specific enzyme activity [27]. This makes ADSCs exosomes a promising approach for Alzheimer's disease treatment. This difference between exosomes from ADSCs and BM-MSCs is also determined in protein expression: immunoblot analysis revealed that the NEP protein expression level in ADSCs was ~4-fold higher than that of BM-MSCs [26].

### **5. Some membrane and cargo exosomal markers for tissue specificity**

As it was already mentioned above, identifying the source of a certain exosome with its membrane markers is a non-trivial task, requiring consideration of the combination of different proteins and their abundance.

Skogberg et al. in their study of exosomes from human thymic epithelial cells (TEC) revealed the typical mTEC-associated cytokeratins K5, K14, and in both cells *Perspective Chapter: Tissue Specificity of Exosomes and Their Prospects as a Drug Delivery System DOI: http://dx.doi.org/10.5772/intechopen.111566*

and exosomes, while the typical cortical thymic epithelial cell (cTEC) associated cytokeratin K8 and, for example, involucrin, a marker for late-stage mTEC differentiation, and CLAUDIN-1 were only identified in cells. Amidst the markers, which were found only in exosomes and not in cells, there were classical exosomal markers such as TSG101, CD82, CD63, MFG-E8, FLOTILIN-1 and immunoproteasome subunits PSMB9 and PSMB10, while PSMB8 was found in both, cells and exosomes. Other proteins, which were identified in cells and in exosomes from these cells, were CP, ALDOC, COL6A1, LAMA2, SRI, HSPG2, TSN, AOC3, SLC34A2, and F13A1 [28].

Mathivanan et al. also revealed some markers, which are specific for colorectal cancer cells (LIM1215) and can be identified both in cells and in exosomes. Amidst others, these are A33 antigen, cadherin-17, carcinoembryonic antigen, and ephrin-B1 and -B2, cell type-specific proteins associated with the gastrointestinal tract. Comparing proteomes of LIM125-derived exosome with previously published proteomes of human urinary exosomes and mast cell-derived exosomes, they found 31 proteins to be common between all of three exosomal proteomes, whereas 79 and 96 proteins were in common between LIM1215-mast cell and LIM1215-urine data sets. The 31 common proteins include Alix, transferrin, actins (α, β, and γ), RAB5B, RAB5C, EH-domain containing 4, heat shock proteins, annexins A6 and A11, and ADP-ribosylation factor 1 among others. Moreover, they found, that LIM125-derived pure exosome proteins are enriched with tetraspanin-containing proteins (p-value 0.0001) when compared with the entire human proteome and were the first to report the presence of phospholipid scramblase 3 in exosomes [29].

Saheera et al. in their recent review admit, exosomes from cardiomyocytes are enriched with proteins, which play critical roles in cardiomyocyte growth and survival like heat shock proteins (Hsp) like Hsp20, Hsp60, and Hsp70. Furthermore, these exosomes are known to be loaded with such inflammatory factors responsible for cardiac remodeling as IL-6 and TNF-α. Among others, these exosomes include GLUT4, GLUT1, and lactate dehydrogenase, different miRNAs, namely miR-320 and miR-126 [29]. The specificity of exosomes is possible to be used in target delivery purposes: the delivery of exosomes, expressing cardiac-targeting peptide on their membrane, in H9C2 cells was 16% greater than that of exosomes, which did not express this peptide, whereas the delivery of the exosomes of these two types was not different in HEK 293 cells exosomes expressing cardiac-targeting peptide (CTP)- Lamp2b on their membrane (CTP-Exo) was generated by introducing vectors encoding CTP-Lamp2b into HEK 293 cells. Moreover, compared with exosomes without cardiac-targeting peptide on its membrane, the in vivo delivery of exosomes to the hearts of mice was increased by 15% with CTP-Exo (P = 0.035) [30].

In exosomes from ECC1 cells, which are the most accurate resemblence of the endometrial luminal epithelium, Greening et al. found 14 proteins, which are essential for embryo implantation. As exosomal protein cargo, there were complement decayaccelerating factor (CD55, Rsc 7.1), perlecan (HSPG2, Rsc 5.9), and EGFR (Rsc 5.1), which are highly regulated at the time of blastocyst apposition and attachment [31, 32]. In general, it should be emphasized exosomes participate not only in tissue-specific processes like blastocyst apposition and attachment but in common processes like inflammation, cancer development, and cell senescence. Saheera et al. in their review, dedicated to exosomes' role in cell aging, admit senescence-associated exosomes could transfer many molecules and could accelerate the aging process or associated pathologies in an autocrine, paracrine, and endocrine fashion. Moreover, senescence-associated exosomes can intensify the aging process by cargos transfer between cells that may be recruited to increase the exosome release observed during cellular senescence.

Exosomes from older individuals were shown to have MHC-II expression on monocytes and they are taken up faster by B cells in older individuals when compared to young, and as a result, the levels of circulating exosomes could be reduced [32].

It is worth it to note in some cases exosomes may be one of the features causing graft rejection. Sharma et al. in 2018 revealed a higher expression of some proteins in exosomes isolated from patients with complications compared with patients without complications. They collected serum samples from patients who had undergone lung (n = 30), heart (n = 8), or kidney (n = 15) transplantations. Using western blot along with CD9 identified tissue-associated lung SAgs, collagen V (Col-V) and K-alpha 1 tubulin (Kα1T), heart SAgs, myosin and vimentin, and kidney SAgs, fibronectin and collagen IV (Col-IV). Lung transplant recipients diagnosed with bronchiolitis obliterans syndrome had exosomes with higher expression of Col-V (4.2 fold) and Kα1T (37.1fold) than stable. Heart recipients with coronary artery vasculopathy had a 3.9-fold increase in myosin and a 4.7-fold increase in vimentin compared with stable. Exosomes in kidney transplant recipients diagnosed with transplant glomerulopathy 2-fold increased expression of fibronectin and 2.5-fold increased in Col-IV compared with exosomes from stable patients [33]. This is not a unique case of exosomes involvement in heart pathology processes: exosomes from macrophages exposed to diabetic milieu (high glucose or db/db mice) significantly increase inflammatory and profibrogenic responses in fibroblast (in vitro) and cardiac fibrosis in mice [34].

Some of the features which are specific for exosomes of certain origins are listed in the **Table 1**.

### **6. Exosomes as a promising approach for drug delivery**

Exosome delivery is a novel nanoscale delivery system with many advantages, such as biocompatibility, biodegradability, less toxicity, specificity to the target cells, small size, promotes plasma membrane fusion, among different cells, longer half-life, low-uptake machinery, capability to pass contents from one cell to another cell, low immunogenicity and the unique feature that they have more tendency to accumulate


*Perspective Chapter: Tissue Specificity of Exosomes and Their Prospects as a Drug Delivery System DOI: http://dx.doi.org/10.5772/intechopen.111566*



### **Table 1.**

*Some of the surface markers and proteome particularities of the exosomes of a certain origin.*

in the cancerous cell than normal cells [35]. Other features making exosome a promising delivery system are innate stability, the ability to cross biological barriers, and enhanced loading capability of biological molecules [36]. It should be noted, due to the preferential homing of exosomes for their source cells, cancer exosomes should not or should be used as drug carriers with particular attention because they may promote tumor invasion or epithelial-mesenchymal transition, or they may transfer tumor resistance genes to tumor cells [37].

Their superior tissue-homing capabilities have been identified such as unidirectional synaptic transfer of microRNA from T cells to antigen-presenting cells. Moreover, depending on the integrin expression pattern of the parent cells, different mammalian tumor exosomes were shown to preferentially target healthy cells in the

*Perspective Chapter: Tissue Specificity of Exosomes and Their Prospects as a Drug Delivery System DOI: http://dx.doi.org/10.5772/intechopen.111566*

predicted tissue. As it was mentioned above, cancer exosomes, like exosomes from sarcoma cells, show preferential tumor homing. As for the biodistribution, exosomes accumulate primarily in the liver, lung, spleen, and gastrointestinal tract and this distribution in some cases, like with systemic exosome administration, is not specific [38]. Nevertheless, depending on the origin of exosomes, the biodistribution may be changed: dendritic-cell-derived exosomes are preferentially taken up by the spleen, melanoma-cell-derived exosomes accumulate more prominently in the liver [38]. Despite a shorter half-life compared with liposomes, reaching a maximum of 60 minutes, exosomes were superior in escaping stress-relaxing environments and had a comparatively longer circulation half-life [38, 39].

Among others, there are three relatively simple and effective options for loading cargo into exosomes: electroporation, passive transport of the target during incubation, and sonication. Electroporation is a well-known method for different genetic structures loading into cells has the same principle in exosome loading: pores are created in the exosomal membrane by applying an electric field to a suspension of exosomes facilitating the movement of cargo into the lumen of the exosomes. A very simple and nevertheless effective way of cargo loading is a simple incubation of exosomes with the cargo: curcumin was efficiently loaded into exosomes after only 5 min of incubation at 22°C [40]. Another method to load cargo into exosomes is sonication with the same basic idea as electroporation, which is making pores in the bilipid exosomal membrane and cargo loading into exosomes via these pores. An accurately chosen regimen of sonication does not cause significant changes in the structure and content of exosomal membranes [40]. Thus, the appropriate method for cargo loading should be chosen based on the exosomes concentration, preliminary procedures like method of isolation and exosome storage condition and the loading target. Other methods of loading cargo into exosomes are transfection, freeze-thaw cycles, extrusion, surfactant treatment, and hypotonic dialysis.

Exosomes, being biodegradable nanoparticles, have successfully encapsulated bioactive molecules such as curcumin, paclitaxel, neurotoxin-I, and dexamethasone. Additionally, exosomes are utilized as drug delivery vesicles for multiple disease models of cancers, diabetes, and brain diseases such as Alzheimer's, prions, and Parkinson's [41].

Due to the biocompatibility of exosomes, various routes of administration are possible such as intravenous, intraperitoneal, oral, intranasal, and intratumoral. The exosomes have been considered a transporter of biomolecules, including lipids, proteins, enzymes, transmembrane proteins, cytoskeletal proteins, and genetic material. Exosomes were shown to effectively deliver an antibody-drug conjugate (trastuzumab-emtansine) to cancer cells in HER2-positive cancer [42]. Barok et al. showed that antibody-drug-conjugated exosomes bound to HER2+ cancer cells with growth inhibition and activation of caspases-3 [42]. Another example of successful use of cancer-related exosomes in cancer treatment is loading of modification of inhibitor of apoptosis protein survivin-survivin-T34A, which is a dominantnegative mutant of survivin—into exosomes isolated from melanoma cell lines. These exosomes were shown to effectively induce apoptosis in cancer cells [43, 44]. Kooijmans et al. anchored anti-epidermal growth factor receptor nanobodies to the surfaces of exosome vesicles via glycosylphosphatidylinositol to improve the interactions between exosomes and epidermal growth factor receptor-expressing tumor cells [45].

Elucidation of the mechanisms underlying protein and RNA sorting in exosomes may have great potential for developing various therapeutic applications. Although

in clinical trials exosomes are commonly used as diagnostic and/or prognostic and/ or predictive markers they are more than viable candidates for targeted drug delivery innovation due to the various benefits mentioned above [41].

### **7. Conclusion**

Working with exosomes isolated from biological fluids we do not havestrong arguments allowing us to firmly assume the tissue origin of the isolated exosomes. This is due to the biogensis features of exosomes and methodological difficulties in performing experiments to identify the tissue specificity of exosomes. All this explains a few data, allowing us to compare membrane proteins and protein and nucleic acids cargo of exosomes of a certain origin except the research on cell lines, which have a wide range of limitations in the extrapolation of obtained data to the corresponding tissue. However, exosomes remain a promising diagnostic approach for different pathologies and gain more interest as therapeutic agents delivery systems because of their biocompatibility, safety, and tissue-homing capabilities. Further research in exosomes biology would provide a big future for the application of these biomolecules for different aspects of clinical medicine.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Alisa Petkevich1 \*, Aleksandr Abramov2 and Vadim Pospelov2

1 N.N. Blokhin Russian Cancer Research Center, Moscow, Russia

2 Scientific and Practical Center of Specialized Health Care for Children Named After V.F. Voino-Yasenetskiy, Moscow, Russia

\*Address all correspondence to: pa.alisa26@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Perspective Chapter: Tissue Specificity of Exosomes and Their Prospects as a Drug Delivery System DOI: http://dx.doi.org/10.5772/intechopen.111566*

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[21] González-Cubero E, González-Fernández ML, Gutiérrez-Velasco L, Navarro-Ramírez E, Villar-Suárez V. Isolation and characterization of exosomes from adipose tissue-derived mesenchymal stem cells. Journal of Anatomy. 2021;**238**(5):1203-1217. DOI: 10.1111/ joa.13365

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[23] Xu H, Wang Z, Liu L, Zhang B, Li B. Exosomes derived from adipose tissue, bone marrow, and umbilical cord blood for cardioprotection after myocardial infarction. Journal of Cellular Biochemistry. 2020;**121**(3):2089-2102. DOI: 10.1002/jcb.27399

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[39] Herrmann IK, Wood MJA, Fuhrmann G. Extracellular vesicles as a next-generation drug delivery platform. Nature Nanotechnology. 2021;**16**:748-759. DOI: 10.1038/s41565-021-00931-2

[40] Conlan RS, Pisano S, Oliveira MI, Ferrari M, Mendes PI. Exosomes as reconfigurable therapeutic systems. Trends in Molecular Medicine. 2017;**23**(7):636-650. DOI: 10.1016/j. molmed.2017.05.003

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[42] Barok M, Puhka M, Vereb G, Szollosi J, Isola J, Joensuu H. Cancerderived exosomes from HER2-positive Cancer cells carry Trastuzumab-Emtansine into Cancer cells leading to growth inhibition and caspase activation. BMC Cancer. 2018;**18**:504. DOI: 10.1186/ s12885-018-4418-2

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[44] Huda MN, Nafiujjaman M, Deaguero IG, et al. Potential use of exosomes as diagnostic biomarkers and in targeted drug delivery: Progress in clinical and preclinical applications. ACS Biomaterials Science & Engineering. 2021;**7**(6):2106-2149. DOI: 10.1021/ acsbiomaterials.1c00217

[45] Butreddy A, Kommineni N, Dudhipala N. Exosomes as naturally occurring vehicles for delivery of biopharmaceuticals: Insights from drug delivery to clinical perspectives. Nanomaterials (Basel). 2021;**11**(6):1481. DOI: 10.3390/nano11061481

### **Chapter 7**

## Perspective Chapter: Exosomes – The Surreptitious Intercellular Messengers in the Body

*Naveen Soni, Jitender Jangra, Megha Chaudhary, Gargi Nandi and Bhawana Bissa*

### **Abstract**

Exosomes are secret intercellular messengers in the body, carrying crucial information from different organs. Different cargos can be packaged in exosomes including DNA, RNA, and proteins. The type of exosomal cargo can vary according to the tissue type, its pathophysiological state, and circadian rhythm. Therefore, exosomes have an immense potential to be utilized for diagnostic purposes if the conundrum of their cargo can be understood. Recent advances in exosome isolation and characterization have made it possible to define disease-specific cargo carried by these tiny messengers. We attempt to highlight disease-relevant exosomal cargos for diagnostic purposes.

**Keywords:** exosomes, cancer, neurodegeneration, cardiovascular, miRNA

### **1. Introduction**

Cells are the most unique and well-established micro-machines that assemble and make efficient metabolic molecules and pathways to maintain homeostasis. Exosomes are one of those cellular secretions that carry hidden information about proteins, RNAs, DNAs, and metabolites of secretory cells. Certain amounts of these molecules are packaged in the exosomes during their synthesis and secreted from the cell in a normal process. These exosomes are released in the extracellular environment and get captured by the nearby cells. Exosome content carrying information of parent cells makes the physiological changes in the recipient cell, making them an intercellular messenger of genetic and metabolic information.

Extracellular vesicles are the membrane-enclosed form of cell cargo with dynamic size, variety, and diversity. These can be distributed in three types based on the diameter of the vesicle; microvesicles (100-1000 nm), exosomes (30-150 nm), and apoptotic bodies (50-5000 nm) [1]. Exosomes were first recognized in rat ovum and algae in the 1950s [2, 3]. Soon after this, the detection of EVs was also done in plants and fungi [4, 5]. At that time EVs were not recognized well, instead, they were thought to assist in removing garbage from the cell. Later in 1996, Raposo declared that antigenspecific MHC-2 containing vesicles from B-lymphocytes induces an antigen-specific response in T-cells, clarifying that EVs are not garbage anymore [6]. EVs were also

discovered in bat thyroid follicular cells by Nunez and Gershon [7]. This was the first chapter to explain the proximity of multivesicular bodies near the limiting membrane, and their fusion with the membrane to release them into luminal space. EV secretion is the ancient feature, followed by archaea, prokaryotes, and eukaryotes that play a significant role in cell-cell communication [8, 9].

### **2. Exosome biogenesis**

Exosomes like vesicles (ELV) or exosomes are synthesized in the endosomal compartments in a well-regulated way, and stress, mutation, and alteration in the microbiological environment may change the generation and secretion of exosomes. This regulation is maintained by the multiple proteins such as RAB, SNAREs, and cytoskeletal proteins [10]. Endosomes are synthesized by the invagination of the cell membranes. A naive endosome is non-judgmental with no decided fate and is called an early endosome. It either can fuse with the available endocytic vesicles containing cargo for export/degradation/recycling or can mature into late endosomes [11, 12]. Late endosomes are slightly more acidic than early endosomes, which might affect exosome production. A study by Logozzi has revealed that when cells are grown in acidic pH, the amount of exosome synthesis also increases as compared to the buffered medium [13]. Within the late endosome, the inward budding of the membrane synthesizes intraluminal vesicles that accumulate cytosolic content such as nucleic acid, proteins, metabolites, ions, and lipids. At this stage, the whole organelle containing ILV is considered as MVBs (Multi Vesicular Bodies). The pre-decided fate of MVB is not clear whether it will fuse with lysosome or autophagosome or exocytose to deliver in luminal space.

Exosomes are synthesized in the MVBs in the form of ILVs. Biogenesis of exosomes necessitates enrichment of CD9 and CD63 tetraspanin molecules and assembly of ESCRT (Endosomal Sorting Complex Required for Transport) complex at the site [14–16]. ESCRT is the preferred route of ILV formation but if ESCRT is necessary for cargo selection and exosome secretion is still controversial. ESCRT consists of four ESCRT protein complexes: ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III. Another AAA ATPase Vps4 complex works together with the ESCRT-III to deform and amputee endosomal membrane [17]. These protein complexes show a high degree of cooperativity while sorting cargoes, vesicle budding, and MVB biogenesis [18]. The whole process of cargo selection, vesicle formation, and cargo incorporation occurs simultaneously. ESCRT-0 complex first initiates the recognition of microdomains on the endosomal membrane where ubiquitinated proteins are sequestrated. This occurs through the recognition by the HRS protein of ESCRT-0 to TSG101 of ESCRT-I. This initiates the invagination of the membrane, considering that all the proteins assigned for their secretion/degradation are clustered. ESCRT-0 complex interacts with the ESCRT-I and ESCRT-II and a wide-neck vesicle is formed inside the endosome [19]. Vesicle maturation marks the deubiquitination of clustered proteins. At this stage, ESCRT-0, ESCRT-I, and ESCRT-II units disassociate from the site and ESCRT-III assembles at the site. Snf7 protein, a unit of ESCRT-III, forms an oligomeric assembly and recruits ALIX (ALG-2 interacting protein X) at the site that stabilizes the ESCRT-III and promotes vesicle budding [20]. This complex narrows down the neck of the newly forming vesicle and then interacts with the AAA ATPase Vps4 complex, which is the key energy providing protein in releasing the vesicle from the endosomal membrane. Newly synthesized vesicle containing cargo now disassemble from the endosomal membrane and accumulate in the MVBs.

*Perspective Chapter: Exosomes – The Surreptitious Intercellular Messengers in the Body DOI: http://dx.doi.org/10.5772/intechopen.110779*

Another route for exosome production is the ceramide pathway, an ESCRT-Independent pathway. Microdomains present on the endosomal membrane are enriched with sphingomyelinases (SMases). These SMases cleave sphingomyelin lipid of the membrane, remove phosphocholine moiety and incorporate ceramide. These ceramides in the membrane induce lateral phase separation and amalgamate the microdomains of the membrane [21]. Consequently, a negative curvature is formed that promotes budding. Tetraspanins such as CD9, CD63, and CD81 are highly enriched in the exosome membrane and assist in protein sorting and exosome biogenesis. These tetraspanins containing microdomains are the specialized structure involved in an assortment of receptors and signaling molecules in the plasma membrane [22].

### **3. Exosome secretion**

Rab protein is the largest family of small GTPases that governs the switch of GTP hydrolysis. More than 60 members of the Rab family are present in the intracellular membrane, serving as the main regulator of vesicle secretion [23]. Rab GTPases cover a major portion of membrane trafficking by its interaction with SNAREs, motor proteins, and coat proteins. The activation of GTPase activity is regulated by the GEFs (Guanine nucleotide exchange factors). The study also demonstrates that the interaction of SNARE with Rab induces the release of exosomes [24]. Rab proteins are the key molecules that determine the size of exosomes and regulate MVB docking at the plasma membrane, such as Rab27a, and intracellular distribution of MVBs for exosomal traffickings, such as Rab27b [25, 26]. Rab27a and Rab27b interact with their respective effector proteins, Slp4 and Slac2b, respectively, to transfer MVBs from the perinuclear to the periphery area of the cell [25]. Abolishing these interactions leads to decreased exosome release and inhibited breast cancer cell invasion and migration [27]. Additionally, some factors such as HSP90 and lysosome-associated protein transmembrane-4B (LAPTM4B) also transfer MVBs toward the periphery to promote their secretion [28, 29]. KIBRA interacts with Rab27a and enhances its retention, while some other GEFs such as MADD and Fam45a control exocytosis [26, 30, 31]. Rab11 and Rab35 are majorly involved in the endosome recycling pathway, and also assist in exosome secretion and cargo selection. Loss of function in Rab11 and Rab35 results in exosome accumulation in the cells [32]. But a similar study declaring that Rab11a and Rab7 remain uninvolved during the exosome biogenesis process is still controversial. The same study also shows that Rab7 enhances the release of exosomes containing Alix and syntenin in breast cancer cells but its knockdown does not affect exosome release in HeLa cells [25, 33]. Some small GTPases such as Rab2a, Rab5a, and Rab9a also increase exosome secretion [25]. These diversified functions of Rabs modulate the exosome biogenesis machinery and its secretion out of the cells. HRS, STAM1, and TSG101 silencing decrease the exosome release in dendritic cells [34].

### **4. Exosome cargo**

The exosome content is solely dependent on the extracellular environment and intracellular metabolic activities that may vary at any stage of the cell. Exosomes are loaded with RNA, DNA, lipids, and proteins with different concentrations and types. This specificity can be changed from cell to cell even with the same environment. Many studies are ongoing and have been done to get exosome content. For now, few databases are accessible to collect information about the exosome cargo. These are exoRBase, Exocarta, EVpedia, Vesiclepedia, EV-TRACK, and ExoBCD [35–42]. For now, >9700 proteins, >3400 mRNA, >2800 miRNA, and > 1100 lipid data in EVs have been identified [39].


Different mechanisms have been proposed for the loading of RNAs in the EVs. For example, specific sequences within the 3′ UTR act as "zip-code" to export certain specific RNAs in the EVs. These "zip-codes" are about ~25 nt in length, such as the binding site for the miR-1289 carries by another mRNA containing the "CTGCC" sequence on its stem-loop structure [55]. It has also been seen that certain miRNAs carrying four nucleotide sequence motif "GGAG" interact with the hnRNPA2B1, which enhances their sorting in the EVs [56]. In addition, post-transcriptional modifications of miRNAs determine their fate of retention, such as uridylated 3′-end of miRNAs are sorted into the EVs, while adenylated 3′-end keeps them to stay within the cell [57]. Another mechanism is based on the nSmase2 activity, which if, overexpressed, releases more amount of miRNAs by enhancing exosome production [58]. Apart from this, the role of argonaute protein in the loading of RNAs in EVs is still a controversial statement. Some studies support that the knockdown of argonaute protein decreases certain specific miRNAs in the EVs. Whether argonaute is found in the EVs or the MVBs or in the endosomes, is still a complex scenario in the EV research [59, 60].

c.Proteins: The information about the protein cargo within the EVs is still unclear due to the differences in cell types, culture conditions, and isolation procedures. Only a fraction of common EV proteins can be identified that are generally found

### *Perspective Chapter: Exosomes – The Surreptitious Intercellular Messengers in the Body DOI: http://dx.doi.org/10.5772/intechopen.110779*

in the endosomal pathway, like Alix, tetraspanins, and some ESCRT proteins. In addition, proteins for exosome secretion such as Rab27a, Rab11b, and ARF6 are commonly found in the EVs. Interestingly, most of the EVs contain tetraspanins (CD9, CD63, CD81, CD82, CD86), antigen-presenting molecules (MHC-1 and MHC-II), transcription factors (Wnt, Notch, hedgehog), transport and fusion proteins (GTPase and flotillin), heat-shock proteins (Hsp20, Hsp27, Hsp60, Hsp70, Hsp90), cell-surface peptidases (CD13 and CD26), and signaling receptors like EGFR [36, 61–64]. Exosome composition is mostly decided by the cell type it is derived from. A drug-resistant cell secretes exosomes containing MDR-proteins such as ABCB1, ABCC2, ABCG2, and p-glycoproteins to enhance the tumor environment more resistant to drugs [65, 66].


### **5. Exosomes in different biological fluids**

Exosomes are very small molecules that formed within endosomes *via* different ESCRT-dependent processes [78]. Their sizes range from around 30 nm to 150 nm. These EVs are secreted into the various body fluids, such as blood, urine, saliva, breast milk, ascites effusions, nasal secretions, tears, amniotic, synovial, lymphatic, cerebrospinal, and seminal fluids by the various cell types found within the body, including red blood cells, B cells, T cells, mast cells, platelets, endothelial cells, fibroblasts, adipocytes, epithelial cells [79–85]. Exosomes present in them move through these fluids to other areas or interact with other cells to carry out a variety of biological functions, including the modulation of immune response [86, 87], antigen presentation [88, 89], and the transfer of RNA and proteins [90, 91], intercellular communication, non-classical protein secretion [92], and transmission of pathogenic cargo [93–95]. Exosomes are typically obtained from various body fluids using ultracentrifugation [96] based on the sedimentation principle, which yields a very pure exosomal fraction that is recognized as the gold standard. Size exclusion filtration [97] or chromatography [98] is a different procedure that involves filtering through a number of filters with pores smaller than 100 nm and then centrifuging (100,000 g) to concentrate the exosomes. The biological function and integrity of the exosome are maintained using this method. Using a solid support magnet or flow

cytometry, immune affinity capture [99] involves binding specific micro-beads to bio-fluids containing exosomes and separating the exosome-bound micro-beads from the bio-fluids. Exosome isolation is also done using kit-based techniques, such as the precipitation method ExoQuick [100] and the microfluidic technology (ExoChip) [101] based on the immunoaffinity methodology. The sample source and intended use of the exosomes may determine which of these various techniques and procedures to adopt, each of which has advantages and disadvantages of its own. Exosomes contain a variety of nucleic acids to perform various biological functions. The lipid bilayer's DNA, RNA, lipids, proteins, and metabolites keep them stable and allow for long-term storage. Even yet, the microenvironment and the type of cell to which an exosome is delivered determine what is contained within the cargo. As a result, the stability of different biomolecules within the exosome and their enrichment make them appropriate for a range of therapeutic and diagnostic uses. Exosome vesicles are primarily extracted from the serum, plasma, CSF, and urine and are the form that has been examined the most. As of now, exosome vesicles produced from particular fluids have more precise identification and validation than whole body fluid [78]. For instance, Kalra et al. [102] isolated EVs from plasma and demonstrated the depletion of highly abundant plasma proteins [103, 104]. As a result of their cargo and diverse features, exosomes are transformed by cells and may play a role in the progression of various diseases. As a diagnostic biomarker in the early detection and prognosis of diseases, these changed content (proteins or miRNAs) revealing distinct [78] profiles in exosomes are being different from the exosomes released by the normal/healthy cells. Another arm of exosomes is their therapeutic role for different purposes such as vaccination, biological targeting, and drug delivery tools, using a variety of therapeutic materials, including siRNA, antagomirs, g-RNA (siRNA), recombinant proteins, and anti-inflammatory drugs [105].

### **6. Exosomes in diseases**

Exosomes in disease pathophysiology have recently attracted a lot of attention from researchers. Literature studies have shown that due to the potential ability of cell-to-cell communication among homozygous and heterozygous cell types, exosomes acts as a mediator for maintaining healthy physiological conditions [106]. In addition to their regular role, these exosomes are manipulated by the pathogen to infect the host cell activity [107] and act to potentiate stress and damage [106]. Exosomes have been discovered to play a role in the onset and progression of a number of diseases, including cancer, autoimmune disorders, neurodegenerative diseases [108], cardiovascular diseases, liver diseases [109], and genetic diseases, among many others.

### **6.1 Exosomes in tumor microenvironment, metastasis, and angiogenesis**

Cancer is one of the oldest and deadliest diseases in the world. The ability of cancer cells to communicate with other cells is achieved primarily through exosome vesicles to maintain normal physiological conditions and trigger disease progression. These vesicles help cells to communicate between homotypic and heterotypic cells. In homotypic exosome transfer, exosome content and signaling capabilities allow cancer cells to progress and transmit cell growth, transformation, and survival signals to other cancer cells [110]. Various autocrine signaling pathways Akt/PI3K and MAP

### *Perspective Chapter: Exosomes – The Surreptitious Intercellular Messengers in the Body DOI: http://dx.doi.org/10.5772/intechopen.110779*

kinase are involved in its progression [111]. As heterotypic exosome transfer involves all stages of cancer development and progression, tumor spread is driven by its local tumor microenvironment (TME). TME is composed of different cell types, including endothelial cells, fibroblasts, and immune cells. This tumor microenvironment enables a variety of cancer cell-derived exosomes, such as CAF-derived exosomes (CDE) and fibroblast-derived exosomes, to sustain proliferation, evade growth suppression, evade immune recognition, and activate invasion and metastasis cascades. It helps in regulation, resisting cell death, initiation of angiogenesis, promotion of cell proliferation, and deregulation of cell energetics through juxtracrine and paracrine signaling interactions [112, 113].

In cancer metastasis, primary tumor cells migrate to another part of the body where they multiply and form new tumors. There are various stages in this process: vascular invasion, extravasation, tumor latency, and formation of macro- or micrometastases [114]. The process of metastasis is modulated by EMT, ECM remodeling, activity of the immune system, and alteration in tumor micro-environment [115, 116]. However, exosomes play a significant role during metastasis, as it influences tumor roles and primarily contributes to the formation of the pre-metastatic niche that determines specific organotrophic metastasis [114, 117]. During the invasion, the primary tumor releases various factors (microRNAs, EGFR signaling ligands, EMT inducers, etc.) that promote invasion [118–120]. For example, miR-10b is transported and released by exosomes and promotes the metastatic properties of breast cancer cells [121]. Another, miR-23a inhibits E-cadherin synthesis in lung cancer and melanoma cells, thereby inhibiting the release of TGF-1 supporting EMT-promoting effects [50, 51]. EGFR signaling factors include ligands such as amphiregulin, tissue-type plasminogen activator, and/or annexin II, and significantly increase cancer cell invasion [122]. Exosomes secrete EMT inducers such as vimentin, snail, and twist in urothelial cell lines while reducing E-cadherin and catenin expression through the TGF-1 pathway [123]. These exosomes have properties that drive exosome organotropism in cancer cells, and ITG**α**6β4 and -**α**6β1 are associated with lung metastasis, and ITG**α**vβ5 is associated with liver metastasis. Related, ITGβ3 is related to brain metastasis [124]. Exosomes also exhibit stromal cell proliferation, cancer cell migration and survival, and ECM remodeling that increases tumor cell resistance to apoptotic signals. This, along with the effect of chemokines and growth factors, leads to the formation of a new microenvironment for cancer cells, immune cells, and other stromal constituents that is referred to as the PMN [122, 125, 126]. For the initiation of the metastatic process, an adequate blood supply to the tumor facilitates the entry of tumor cells into the bloodstream [127]. Thus, angiogenesis provides an opportunity for tumor growth by supplying cancer with oxygen, nutrients, and metabolite replacement [127]. Exosomes can transport various biomolecules such as microRNAs, DNA fragments, proteins, lipids, and even pharmacological compounds from donor cells to recipient cells [128]. Therefore, noncoding RNAs, especially long noncoding RNAs (lncRNAs) and microRNAs, play important roles in regulating angiogenesis [129]. In addition, exosomes can interact with target cells such as endothelial cells (EC) as well as immune cells to initiate and promote angiogenesis. The uptake of tumor-derived exosomes by normal endothelial cells activates angiogenic signaling pathways and stimulates new blood vessel formation [130]. Exosomes migrate to the cell periphery and invade advanced pseudopods. After complete remodeling, neighboring ECs likely transport exosomes to other ECs and other cells within the TME (tumor microenvironment) *via* nanoparticle structures [131].

### **6.2 Exosomes in neurodegenerative diseases**

The majority of human neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, have aggregation of aberrant proteins as a common mechanism [132]. The existence of vesicles in the CSF has proven that these EVs are involved in the pathogenic spread of harmful proteins [132].

Alzheimer's disease is a neurological disorder caused by the disparate modification of Amyloid beta (Aβ) peptide and tau protein. Exosomes carry proteases, APP and its C-terminal fragments (CTFs-APP) that are caused by gamma and β secretase within the early endosomes, ultimately exports Aβ into the exosomes [133]. Exosomes provide a unique pathway for removing Aβ from cells. However, they make Aβ more prone to aggregation and, therefore, could endanger neighboring cells [134]. Another protein called TAU is crucial for accelerating tubulin assembly into microtubules and preserving their structural integrity. Tau's protein biological activity is compromised by hyperphosphorylation, which also results in defective microtubule stabilization and the formation of neurofibrillary tangles that impact neuronal connection and function [135, 136]. The mechanism of exosome-based release of Tau protein helps microglia to spread damaging tau protein [137, 138].

Parkinson's disease is mostly caused by an accumulation of clumped or misfolded alpha-syn nuclei, which affect the cells' ability to function as neurons [139]. Exosomes are thought to protect against neuronal cytotoxicity and prevent intracellular protein aggregation by excreting alpha syn nuclei outside of cells. This could result in an increase in the concentration of harmful alpha syn nuclei in extracellular space. The exosomes can take up both big and small alpha syn structures and cause various forms of downstream mediated toxicity from healthy neuronal cells [140]. These aggregates can kill the other target cells [141–145]. In addition to these, there are several exosomal miRNAs that contribute to the progression of PD pathogenesis. MiR-7 binding to the 3' UTR of SNCA mRNA suppresses transcription, which results in miR-7 loss. This loss of miR-7 is what causes greater -syn upregulation, aggregation, and dopaminergic neuron death in the brain of PD patients [146]. Another mi-RNA, miR-4639-5p has been upregulated, which negatively controlled the post-transcription of DJ-1 to cause significant oxidative stress and neuronal death in PD patients [147]. These all suggest a multi-functional role of exosomes in PD pathogenesis.

### **6.3 Exosomes in kidney diseases**

The role of exosomes in acute and chronic kidney disease is highly specialized. Studies have shown that cell-to-cell communication between different regions of the kidney and organs amplifies kidney damage [148]. This exosome vesicle release contains proteins from different regions of the nephron fragment, including the thick ascending limb of the Henle loop, the distal tubule, and the collecting duct [149]. Due to their different origins, they have different protein content than their origin and serve as biomarkers for certain diseases [150]. The extracellular vesicles release from podocyte mediate communication between glomeruli and renal tubules, whereby alterations in communication outside the vesicles can affect podocytes and cause tubular damage/injury [151]. Studies suggest that there is upregulation of CD2AP mRNA and downregulation of Wilms tumor protein 1 (WT1) in extracellular vesicles are potential biomarkers of podocyte injury. This mechanism contributes to damage amplification, development of tubule-interstitial fibrosis, and progression of CKD [152]. In acute chronic kidney disease, urinary exosomes miRNAs reflect the state of

### *Perspective Chapter: Exosomes – The Surreptitious Intercellular Messengers in the Body DOI: http://dx.doi.org/10.5772/intechopen.110779*

injury and fibrosis by the release of miR-9a, miR-16, miR-200a, and miR-141 [153]. A specific transcriptional repressor for activating transcription factor 3 (ATF3) was increased in sepsis-induced AKI [154]. In chronic kidney diseases such as diabetic nephropathy, there is a high glucose concentration in renal cells that cause changes in exosome composition and trafficking, further modifying and damaging intact cells [155]. Bioinformatics analysis revealed high levels of miR-133b and miR-342 in urinary exosomes of patients with diabetic nephropathy type 2 (T2DN) [156]. In addition, there are specific miRNAs such as miR-let-7i-3p, miR-24-3p, and miR-27b-3p, whose downregulation is involved in Wnt/β-catenin signaling, leading to T2DN pathogenesis [157]. Exosomes overexpress cellular repressors of multiple genes such as envoplakin, villin 1, prominin 1, and E1A-stimulated gene 1 (CREG1), causing autosomal dominant polycystic kidney disease (PKD) with abnormal morphological and proliferative changes [158, 159]. Circulating extracellular vesicles may lead to intra-organ crosstalk that shows an impact on autoimmune kidney diseases such as systemic lupus erythematosus, anti-phospholipid syndrome, and ANCA-associated vasculitis. For instance, circulating extracellular vesicles may encourage coagulation, thrombosis, and immune-mediated renal pathological conditions [160]. Placentaderived extracellular vesicles carrying anti-angiogenic factors that are released into the maternal circulation in pre-eclampsia may cause proteinuria and glomerular endothelial dysfunction [161].

### **7. Exosomes in cardiovascular diseases**

Cardiovascular diseases are major global diseases that affect the circulatory system [162]. Exosomes produced from the cardiac cells are one of the components in the body that keep cardiac under hypoxia and improve heart function [163]. These exosomes show changes in their states under various cardiovascular pathophysiology conditions and maintain homeostasis primarily during stress signals [164, 165]. Numerous diseases, such as cardiac fibrosis, ischemic heart diseases, heart failure, myocardial infraction, and cardiac hypertrophy, exhibit changes in cargo and protein content and serve as a biomarker for physiological changes [162]. It has been demonstrated that the pattern of fibroblast gene expression is regulated by cardiac cell-derived exosomes [166]. On external stimulation, cardiac fibrosis results in a sustainable remodeling of the extracellular matrix (ECM) through non-canonical Wnt and ERK1/2 pathways, as well as JNK pathways [167]. These pathways are promoted by the WNT-5a-enriched exosomes resulting in IL6 production and fibrosis [168]. Exosomes serve as intercellular communication (regulates intimal integrity) and myocardium remodeling in conditions such as ischemic heart disease and myocardial infarction respectively allowing injured cells to communicate with distant normal cells [162]. Exosomes derived from fibroblasts promote the RAS system and activate angiotensin II in cardiomyocytes that accelerate in cases of cardiac hypertrophy [169].

### **8. Diagnostic potential of exosomes**

Exosomes are small EVs (Extra vesicles) of size 30-150 nm in diameter secreted by both normal cells and diseased cells into the different body fluids such as plasma, saliva, bronchial lavages, urine, and many others [170]. These fluids having exosomes contain different biomolecules including RNA, DNA, proteins for their intercellular

communication, and transportation [171, 172]. There is differential expression of exosomal RNA and proteins derived from normal cells and diseased cells [173]. This exosomal protein and nucleic acid emerged as next-generation biomarkers for different pathology conditions such as neurodegenerative diseases, cardiovascular, kidney diseases, cancer, and others.

### **8.1 Proteins and cargo as diagnostic marker**

In cancer cells, the protein content of exosomes varies between healthy cells and diseases, and it resembles a variety of conditions associated with cancer, liver, kidney, and brain diseases [174]. Exosome-specific protein serves as a biomarker for disease pathology. For instance, distinct protein expression of different fluids acts as a biomarker. In breast cancer, serum-derived exosomes show enrichment of ADAM10, metalloprotease, CD9, Annexin-1, and HSP70 [175] proteins, and plasma-derived exosomes show diagnostic potential for fibronectin and developmental endothelial locus-1 (Del-1) [176]. In lung cancer, expression of CD151, CD171, and tetraspanin 8 is higher in serum exosome [177]. Glypican-1 (GPC1)-positive exosomes serve as potential biomarkers in early-stage pancreatic cancer [178] and CD26, CD81, S1C3A1, and CD10 could be used as a potential biomarkers for hepatic damage [179].

Apart from cancer, other diseases also have significant alteration in exosomes profile and lead to different expression of proteins act as a biomarker for diagnostic potential. In neurodegenerative diseases such as Parkinson diseases elevated expression of different proteins such as PrPc (glycoprotein) [180], DJ-1 [181] (plasma neural-derived), OxiDJ-1 [182] (urine-derived), and Tau Protein (neuron-derived) could be a marker for PD diagnosis. Other potential biomarkers such as a decreased expression of C1q derived from serum exosome and more of blood-derived Apolipoprotein A1 (Apo A1), clusterin, complement C1r subcomponent, and fibrinogen gamma chain exosomal expression levels in the plasma of PD subjects may serve as a biomarker for the diagnosis of PD [183, 184]. In Alzheimer's diseases, human serum-derived exosome shows an elevated expression of Cathepsin-D, LAMP-1, ubiquitinylated protein [185]. Downregulation of SNAP-25 [186] marks the synaptic loss during the progression of AD and HSP70 [185, 187] shows dysfunction and neurodegeneration.

The kidneys play a crucial role in the human body's homeostasis regulation and maintenance [188]. The release of exosomes from various parts of the kidney facilitates cell-to-cell communication that has an impact on the physiology of the kidney [189]. The proteins and nucleic acids contained by exosomes carry through the urine serve as a non-invasive diagnostic biomarker for renal diseases. For instance, the protein level of Fuetin-1 and AQP2 have been identified as potential biomarkers for acute kidney injury (AKI) [153, 190]. There is an increased amount of neutrophil gelatinase-associated lipocalin (NGAL) and activating transcription factor 3 used as a marker for early diagnosis in sepsis-induced AKI [154]. The non-invasive biomarker for PKD is demonstrated by the elevated expression of the urinary exosome proteins such as villin-1, periplakin, and envoplakin in ADPKD (autosomal dominant polycystic kidney diseases) [158]. However, increased expression of AQP-2 and AQP-5 in exosomes in chronic diseases like diabetic nephropathy can be used as a biomarker to diagnose T2DN [191].

### **8.2 Nucleic acid as diagnostic and prognostic biomarker**

Exosomes secreted from diseased cells contain different biomolecules than the healthy ones. Therefore, the basic nucleic acid content also varies with the diseases

### *Perspective Chapter: Exosomes – The Surreptitious Intercellular Messengers in the Body DOI: http://dx.doi.org/10.5772/intechopen.110779*

and mainly circulating microRNAs are being focused to carry out effective diagnosis and prognosis of numerous diseases [192]. For example, in breast cancer, the plasmaderived exosomes show the elevated expression of miR-1246 and miR-21 compared to healthy individuals [193, 194]. These serum-derived exosome shows miR-21 levels, which differentiate between metastatic and non-metastatic breast cancer [195]. Apart from these, upregulation of miRNAs, such as miR-223-3p, miR-16, miR-27a/b, miR-152, miR-199a-3p, miR-340, miR-376a, miR-410, and miR-598 [196–198], shows the presence of breast tumor. In non-small cell lung cancer (NSCLC), there is upregulation of different exosome miRNAs subset of 4 miRNAs (miR-378a, miR-379, miR-139-5p, and miR-200b-5p) and six miRNAs (miR-151a-5p, miR-30a-3p, miR-200b-5p, miR-629, miR-100, and miR-154-3p) respectively [199, 200]. Other than this, plasma-derived miRNA-9 and miRNA-15 can distinguish a metastatic and aggressive state of tumor, thus having high potential as a diagnostic marker for NSCLC [201]. However, in the diagnostic marker for hepatocellular carcinoma, there is upregulation of miRNA-21 in the plasma, which distinguishes patients from healthy individuals [202]. On the other hand, the cancer malignancy in hepatoblastoma is mediated by a panel of miRNAs involving miR-21, miR-34a, miR-34b, and miR-34c in plasma which are verified as diagnostic and prognostic tool [203]. High levels of miRNA-10b, miR-21, miR-30c, and miR-181a and decreased let-7a levels are seen in pancreatic ductal adenocarcinoma patients as compared to a healthy individual [204].

In neurodegenerative diseases such as Parkinson's and Alzheimer's disease, blood and peripheral fluids also contain exosomes synthesized from nerve cells that are passed through the BBB (blood-brain barrier). These fluids show different miRNAs expression in patients and healthy controls. miRNAs from serum are the non-invasive and feasible approach to determining biomarkers for neurodegenerative disease. Exosomal miRNAs derived from plasma, CSF, and serum are being either upregulated or downregulated in different pathophysiological conditions. Certain serum-derived miRNAs such as miR-15a-5p, miR-18b-5p, miR-30e-5p, miR-93-5p, miR-106a-5p, miR-143-3p, miR-335-5p, miR-361-5p, and miR-424-5p are upregulated in comparison with healthy individuals and some of the miRNAs such as miR-15b-3p, miR-342-3p, and miR-1306-5p are downregulated in AD patients [205, 206]. CSF-containing exosomes also show potential diagnostic miRNAs such as upregulation of miR-125b-5p and downregulation of miR-16-5p and miR-451a [207]. These differently regulated miR-NAs from different fluids improve the early onset and late-onset diagnosis and prognosis of AD. Similarly, in Parkinson's disease, several miRNAs derived from different fluids have differentially regulated miRNAs. Serum-derived exosomes have upregulated miR-24, miR-195, and miR-29a [208]. In plasma-derived exosomes, elevated level of miR-331-5p and let-7e-5p is observed. Some miRNAs like miR-10a-5p, miR-151a-3p, let-7f-5p, and many more are seen upregulated in CSF-derived exosomes [209]. Not only the upregulated miRNAs from different fluids show diagnostic potential but the downregulated miRNAs compared to healthy controls also act as diagnostic markers. For example, in PD patients, CSF-derived exosomal miRNAs show downregulated expression such as miR-27a-3p, miR-423-5p, miR-22-3p, miR-1, miR-22, miR-29, miR-374, miR-119a, miR-28 [210]. Some miRNAs such as miR-505 and miR-19b derived from plasma and serum respectively also show downregulation in PD patients [211].

Kidney diseases include AKI (acute kidney injury), chronic kidney diseases, diabetic nephropathy, polycystic kidney diseases, and various others. Exosomes isolated from urine contain differential biomarkers in form of microRNAs. In the AKI condition, urinary exosomes show various miRNAs for different conditions such as AKI progression including miR-16, miR-24, and miR-200c [212]. Also, miR-210 predicts AKI mortality

in ICU patients [213]. In sepsis-induced AKI, there is decreased expression of miR-376b, which acts as a potential biomarker for diagnosis [214]. Certain serum-derived exosomes reportedly decreased miR-24, miR-23a, and miR-145 expression in post-myocardial infarction AKI pathogenesis [215]. There are other serum-derived miRNAs that show a change in expression from healthy and AKI-diseased individuals including miR-101, miR-127, miR-210, miR-126, miR-26b, miR-29a, miR-146a, miR-27a, miR-93, and miR-10a [216]. Other kidney diseases, such as Diabetic Nephropathy, miR-192 is a master miRNA regulator of DN [212, 217]. Expression of miR-130 and miR-145 is upregulated, while miR-155 and miR-424 have reduced levels in diabetic patients with microalbuminuria, acting as a biomarker [218]. miR-415 derived from urinary exosome shows elevated expression in albuminuria and glomerulosclerosis and acts early diagnostic biomarker. miR-126 and the miR-770 family are derived from urine and blood as a promising biomarker for DN progression [212]. Although in diabetic patients, some Urine exosomal miRNAs including miR-192 and miR-21 show upregulated expression while reduced miR-30b levels which altered kidney function [219–222]. In type 2 diabetic nephropathy, the most upregulated miRNAs are MiR-34a and miR-320c which acts as a biomarker, and sediment miR-95 and miR-631 also reflect the severity and prognosis of type 2 DN [223–225]. Apart from these, certain other potential miRNAs biomarkers include miR-15b, miR-636, miR-34a, and miR-4534 in urine [226, 227] and miR-638 in serum. The ratio of albumin–creatinine shows an effect on miR-103a suggesting miR-103a as a dynamic biomarker reflecting pathological status and treatment response [228]. The role of EV miRNAs like miR-3907 upregulation in circulation predicts Autosomal Dominant Polycystic Kidney Disease progression [229]. Diagnosis shows other serum-derived miRNAs including miR-17 family members (miR-20a, miR-93, and miR-106a) show a significant decrease in expression after hemodialysis [230]. Apart from these, in some chronic kidney diseases such as hypertensive nephropathy, Lupus Nephritis, kidney immune diseases, and many others, the role of serum and urine-derived miRNAs show a prominent role in diagnosis, prognosis, and disease progression.

In cardiovascular diseases, circulating EVs miRNAs, miRNA-425, and miRNA-744 acts as a novel biomarkers for cardiac fibrosis [231, 232]. Also, miR-30d is associated with deleterious cardiac remodeling and the expression of fibrosis and

### **Figure 1.**

*(A) Multivesicular bodies can either merge with the lysosome or the autophagosome or be secreted out of the cell as a secretome. (B) Major pathways of exosome biogenesis (C) model structure of exosomes/EVs that carry certain proteins and receptors.*

*Perspective Chapter: Exosomes – The Surreptitious Intercellular Messengers in the Body DOI: http://dx.doi.org/10.5772/intechopen.110779*

### **Figure 2.**

*Schematic representation of exosome isolation and diagnostic importance in different types of disorders.*

inflammation-related genes [233]. In both coronary artery diseases and acute myocardial infraction, several exosomal miRNAs including miR-1, miR-133a, miR-208a, miR-423-5p, miR-499, miR-126, miR-21, and miR-29b show increased expression which potentially acts as a diagnostic biomarker as well as a prognostic marker for left ventricle remodeling [234–238]. However certain miRNAs including miR-423-5p [237], miR-499 [235], and miR-29b [239] essentially for AMI. miR-122 andmiR-199a [240] have elevated expression and miR-145 [241], miR-146a [242], miR-30c/d show downregulation that acts as diagnostic marker for CAD [243]. In contrast, miR-21, miR-199a miR-27a, and miR-30c/d show elevated levels, thus having a diagnostic potential of cardiac hypertrophy [244, 245]. However in heart failure diseases, the increased expression of miR-1254, miR-106a-5p, and decreased expression of miR-328 are other potential miRNA molecules apart from miRNA included in AMI, CAD, and cardiac hypertrophy, which act as diagnostic biomarker [246–248] (**Figures 1** and **2**).

### **Author details**

Naveen Soni, Jitender Jangra, Megha Chaudhary, Gargi Nandi and Bhawana Bissa\* Department of Biochemistry, Central University of Rajasthan, Ajmer, Rajasthan, India

\*Address all correspondence to: bhawana.bissa@curaj.ac.in

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*Perspective Chapter: Exosomes – The Surreptitious Intercellular Messengers in the Body DOI: http://dx.doi.org/10.5772/intechopen.110779*

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### **Chapter 8**

## Perspective Chapter: Clinical Application of Exosome Components

*Mengyuan Hou, Jingwu Li, Zhiwu Wang and Yankun Liu*

### **Abstract**

Exosomes belong to a subpopulation of EVs that carry different functional molecular cargoes, including proteins, nucleic acids, metabolites, and lipids. Notably, evidence has demonstrated that exosomes participate in bidirectional cell–cell communication and act as critical molecular vehicles in regulating numerous physiological and pathological processes. Since the specific contents within exosomes carry the information from their cells of origin, this property permits exosomes to act as valuable biomarkers. This chapter summarizes the potential use of exosome components in diagnosing, prognosis, or monitoring and treating multiple cancers and other non-neoplastic diseases. We also discuss the deficiency of basic applications, including the limitations of research methods and different research institutions and the differences generated by specimen sources. Thus, a better understanding of the problem of exosome detection may pave the way to promising exosome-based clinical applications.

**Keywords:** exosomes, cancer, dieases, diagnosis, prognosis, therapy

### **1. Introduction**

Exosomes are a subpopulation of EVs secreted by the endocytosis process, with a diameter ranging from 30 to 150 nm [1]. Exosomes are naturally secreted by all kinds of cells and carry diverse functional molecular cargoes, including proteins, lipids, nucleic acids, enzymes, and metabolites to promote intercellular communication. As one of the types of cargo of exosomes, nucleic acids include DNA, messenger, and noncoding RNA. Among all the molecular cargoes, proteins, and nucleic acids are the most abundant contents in exosomes [2]. They have biological functions and are selectively packaged into exosomes. As exosomes are naturally secreted by all kinds of cells and are commonly detected in bodily fluids, including blood, urine, saliva, and cerebrospinal fluid [3], the application potential of exosomes in clinical tumor diagnosis and therapy is promising. In this chapter, we will discuss the bioactive exosomal contents, focusing on proteins, noncoding RNAs, and DNA to better clarify their roles in disease development and the potential application of exosome cargoes (**Figure 1**).

**Figure 1.** *Application of exosome components in diseases.*

### **2. Exosome protein**

With the recent development of isolation methods of exosomes and the applications of protein spectrum, the roles of exosome proteins in the diagnosis, prognosis, and treatment of diseases have been demonstrated in many medical fields, especially for cancer [4]. Exosomal proteins include (1) Membrane transport and fusion-related proteins, such as annexin (Anx II), Rab-GTPase, and heat shock proteins (HSPs), including Hsp60, Hsp70, and Hsp90; (2) Four-transmembrane cross-linked proteins, namely CD9, CD63, CD81, CD82, CD106, Tspan8, intercellular adhesion molecule-1 (ICAM-1); (3) Multi-vesicular bodies (MVBs)-related proteins, for instance, ALIX and TSG101; (4) Other proteins, like integrins, actin, and myosin [5].

### **2.1 Application of exosomal proteins in tumors: Diagnosis, prognosis, and treatment**

### *2.1.1 Lung cancer*

Numerous studies have focused on the clinical application of protein components in circulating exosomes from lung cancer patients. Yet, blood derivatives are the biofluids of choice for metabolomic clinical studies due to their low invasiveness and wealth of biological information [6]. Of note, some exosomal surface proteins, like CD91, CD151, and CD171, have been investigated to be used as biomarkers for early diagnosis of lung cancer [7, 8]. Of these, exosomal CD91 showed a high sensitivity

### *Perspective Chapter: Clinical Application of Exosome Components DOI: http://dx.doi.org/10.5772/intechopen.110856*

for diagnosing stage I and II lung adenocarcinoma (LUAD) patients [7]. Notably, exosomal CD151 and CD171 have been demonstrated to be upregulated in LUAD and can distinguish the subgroups of lung cancer [8]. Besides that, as a diagnostic biomarker, exosomal epidermal growth factor receptor (EGFR) and programmed death-ligand 1 (PD-L1) are considered to be the compact surface plasmon resonance (SPR) biosensor in lung cancer diagnosis [9]. Moreover, plasma exosomal proteins have also been reported to be served as prognostic and therapeutic biomarkers. As such, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), placental alkaline phosphatase (PLAP), EGFR, ALIX (ALG-2-interacting protein X), and epithelial cell adhesion molecule (EpCAM) in circulating exosomes have been detected by extracellular vesicle array and correlated with overall survival (OS) of non-small cell lung cancer (NSCLC), which are recognized to be potential prognostic biomarkers for NSCLC [10]. Wang et al. also revealed that hypoxia-induced exosomes delivering pyruvate kinase M2 (PKM2) transmit cisplatin resistance to sensitive NSCLC cells. Additionally, Wang et al. found that exosomal PKM2 might be a potential biomarker and therapeutic target for cisplatin resistance in NSCLC [11]. Therefore, these exosomal proteins for lung cancer diagnosis, prognosis, and treatment must be further validated in larger cohorts.

### *2.1.2 Gastric cancer*

Gastric cancer (GC) is one of the most malignant cancers worldwide [12]. Importantly, exosomal proteins are specific diagnostic, prognostic, and therapeutic biomarkers for GC [13]. Various methods, such as liquid chromatography– tandem mass spectrometry (LC–MS/MS), have detected the proteomic profile of exosomes from the serum of GC patients. For example, a study reported that tripartite motif-containing protein 3 (TRIM3) protein in GC patients' serum exosomes was lower than in healthy donors using LC–MS/MS [14]. The higher exosomal transforming growth factor beta 1 (TGF-β1) expression of GC has been analyzed to be associated with tumor-node-metastasis (TNM) stage and lupus nephritis (LN) metastasis by enzyme-linked immunosorbent assay (ELISA) [15]. In addition, the high expression of exosomal TGF-β1 correlated with forkhead box protein 3<sup>+</sup> (FOXP3<sup>+</sup> ) Treg cells in draining LNs, and the high percentage of FOXP3<sup>+</sup> Treg cells correlated with tumor size, Bormann type, tumor depth, and lymph node metastasis [15]. Furthermore, exosomal angiotensinogen (AGT), serpin family H member 1 (SERPINH1), and matrix metallopeptidase 7 (MMP7) have been demonstrated to perform well in predicting OS and be non-invasive prognostic biomarkers of GC [16]. Gastrokine 1 (GKN1) has also been identified to be secreted from HFE-145 gastric epithelial cells and can reduce tumor growth and tumor volume, which could be served as a therapeutic target for GC [17]. Likewise, these effective therapeutic proteins can be encapsulated into the exosomes and might prevent GC progression [18]. Thus, these exosomal proteins contribute to the development of GC.

### *2.1.3 Breast cancer*

Exosomal proteins play an essential role in diagnosis evaluation and prognosis prediction of breast cancer (BC) [19]. Notably, it has been demonstrated that the exosomal human epidermal growth factor receptor 2 (HER2) was significantly increased in BC patients compared with healthy donors [20]. In addition, exosomal CD82 was significantly decreased in BC tissues compared with healthy donors and benign breast disease tissues [21]. Likewise, exosomes in preoperative plasma contained a higher level of developmental endothelial locus-1 (Del-1) than the postoperative plasma, and the high Del-1 level in postoperation was associated with early relapse [22]. Another report identified 1107 exosomal proteins between metastatic BC cell lines MDA-MB-231 and non-cancerous epithelial breast cell lines MCF-10A [23]. Moreso, 87 proteins were associated with BC, and 16 were correlated to BC metastasis [23]. Among them, exosomal glucose transporter 1 (GLUT-1), glypican 1 (GPC-1), and a disintegrin and metalloproteinase 10 (ADAM10) may be served as BC potential biomarkers [23]. Exosomes carrying proteins, including Anx II, Wnt7a, and ephrin type-A receptor 2 (EPHA2) could stimulate BC's invasive, angiogenesis, and metastasis abilities [24–26]. Exosomal Anx II has stimulated angiogenesis and BC metastasis [24]. Further, exosomal Wnt7a has been found to promote lung metastasis of BC [25]. Exosomal EPHA2 has also been found to activate the AMP-activated protein kinase (AMPK) signal pathway via the Ephrin A1-EPHA2 forward signal that promoted the angiogenesis and metastasis of BC [26]. Therefore, these exosome proteins may serve as potential BC therapeutic targets.

### *2.1.4 Colorectal cancer*

Due to carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), and CA24-2 having poor specificity for the diagnosis of colorectal cancer (CRC) [27], novel biomarkers need to be explored to diagnose CRC development. In recent years, exosomal proteins have been explored for their potential clinical values to serve as tissue and/or liquid biopsy biomarkers to diagnose early CRC. For example, Sun et al. extracted the exosomes in tissues and plasma. They validated that the fibrinogen beta chain (FGB) levels and beta-2-glycoprotein1 (β2-GP1) levels of exosomes in CRC tissue were significantly higher than those in paracancerous tissues [28]. Notably, the areas under the receiver operating characteristic (ROC) curve of plasma exosomal FGB and β2-GP1 as CRC biomarkers were 0.871 and 0.834, respectively, higher than those of CEA (0.723) and CA19-9 (0.614) [28]. Another oncogenic biomarker, exosomal GPC1 protein, was increased in tumor tissue and plasma of CRC patients [29]. However, GPC1 was also highly expressed in other tumor exosomes, which posed a challenge to the specificity of GPC1 in diagnosing CRC. In addition, some exosomal proteins were also proved to be carcinostatic biomarkers. For example, Jiang et al. verified that the level of exosomal angiopoietinlike protein 1 (ANGPTL1) was significantly decreased in CRC tissues compared with paired normal tissues and inhibited CRC metastasis to the liver [30]. Likewise, an exosome protein profile result demonstrated that the adherence-related proteins were enriched in the primary CRC cell (SW480) exosomes [31]. The metastatic factors (MET, S100 calcium-binding protein A8 (S100A8), S100A9, Tenascin-C(TNC)), signal transduction molecules (Ephrin B2 (EFNB2), Jagged1 (JAG1), SRC, Traf2 and Nck-interacting kinase (TNIK)), and lipid raft and lipid raft-associated components (Caveolin-1 (CAV1), Flotillin-1 (FLOT1), Flotillin-2 (FLOT2), Prominin 1(PROM1)) were enriched in the metastatic CRC cell (SW620) exosomes, which were associated with tumor progression and poor prognosis [31]. Interestingly, most exosomal proteins correlate with epithelial mesenchymal transition (EMT), migration, invasion, and angiogenesis. Therefore, exosomal proteins may be biomarkers for predicting CRC metastasis.

### *2.1.5 Pancreatic cancer*

Pancreatic cancer (PC) is one of the most lethal malignant neoplasms worldwide [32]. Existing tumor markers, such as CA19–9, cannot reasonably predict the occurrence and progression of PC, while exosomal proteins may play decisive roles in the occurrence and development of PC [33]. Excitingly, exosomal GPC1 has also been found in PC. Notably, the area under the ROC curve of GPC1 circulating exosomes (from the serum of pancreatic ductal adenocarcinoma (PDAC), benign pancreatic disease patients, and healthy donors) was 1.0, and the sensitivity and specificity of exosomal GPC1 were 100% [33]. Moreover, the level of circulating GPC1 exosomes was associated with tumor burden and the OS of PC patients [33]. Besides that, Xie et al. also observed that the high expression of exosomal CD44v6 (CD44 variant isoform 6) and C1QBP (complement C1q binding protein) was associated with a poor prognosis and a higher risk of postoperative liver metastasis of PDAC [34]. Costa-Silva et al. also found that exosomal migration inhibitory factor (MIF) was highly expressed in PDAC patients, which promoted liver pre-metastatic niche formation and metastasis [35]. The expression of exosomal MIF in PDAC liver metastasis was significantly higher than those without liver metastasis [35]. Furthermore, exosome survivin-T34A (T34A) enhances the sensitivity of gemcitabine to PC cells [36]. Importantly, these biomarkers illustrate their potential value in predicting the occurrence and development of PC.

### *2.1.6 Liver cancer*

Hepatocellular carcinoma (HCC) is one of the most common forms of cancer [37]. Although serum α-fetoprotein (AFP) has been widely applied as a biomarker for diagnosis and dynamic monitoring of HCC, it is not elevated in each HCC patient [38]. As a result, exosomal proteins have been searched for diagnosis, prognosis, and treatment of HCC. Fu et al. reported that high exosomal small mother against decapentaplegic family member 3 (SMAD3) protein level was positively related to tumor size and TNM stage and correlated negatively with disease-free survival (DFS) [39]. Sun et al. also found that HCC patients with high plasma exosomal S100A4 had a poor prognosis, which promoted HCC cell metastasis by activating the signal transducer and activator of transcription 3 (STAT3)/OPN signal pathway [40]. Moreso, downregulated C-Type Lectin Domain Family 3 Member B (CLEC3B) in HCC-derived exosomes promoted migration, invasion, and EMT of HCC cells via AMPK and vascular endothelial growth factor (VEGF) signals. Furthermore, the downregulation of CLEC3B in exosomes suppressed VEGF secretion in both HCC cells and endothelial cells (ECs), eventually inhibiting angiogenesis [41]. Therefore, these studies suggest that exosomal proteins play different roles in HCC development.

### *2.1.7 Prostate cancer*

Prostate cancer (PCa) is a life-threatening disease among men worldwide [42]. Notably, prostate-specific antigen (PSA) has often been used as a diagnostic biomarker for PCa [43]. However, due to PSA's lack of sensitivity and specificity, new biomarkers are urgently needed to assist in diagnosing, prognosis, and treating PCa [43]. A study reported that plasmatic exosomes expressing CD81 and PSA reached 100% specificity and sensitivity in distinguishing PCa patients from healthy donors [44]. Except for

the blood exosomes, urine exosomal prostate cancer antigen 3 (PCA3) and transmembrane protease serine 2:ERG (TMPRSS2:ERG) derived from PCa patients can also be used as non-invasive diagnostic biomarkers and monitor cancer patients' PCa status [45]. Another study reported that the urine exosomes integrin alpha-3 (ITGA3) and integrins beta-1 (ITGB1) in metastatic PCa patients were higher than PCa and benign prostatic hyperplasia (BPH) [46]. Plasma exosomal aldo-keto reductase family 1 member C3 (AKR1C3) have also been demonstrated to be associated with the OS of PCa patient, which is recognized to be a potential prognostic biomarker for PCa [47]. Furthermore, Krishn et al. demonstrated that αvβ3 integrin was transferred to β3-negative recipient cells by exosomes derived from PCa patient plasma and can be identified as a therapeutic target for PCa [48].

### **2.2 Application of exosomal proteins in other diseases: Diagnosis, prognosis, and treatment**

### *2.2.1 Cardiovascular disease*

In recent years, many studies have paid more attention to the roles of exosomal proteins in cardiovascular diseases (CVD). Notably, exosomal proteins from different cell origination play critical roles in cardiac cell development [49]. For instance, GLUT1, GLUT4, and lactate dehydrogenase (LDH) functioned in ECs for glucose transport and metabolism in cardiac cell-derived exosomes [50]. Proteins carried by exosomes using *in vitro* cultures of neonatal cardiac fibroblasts under normoxic conditions are known to be associated with the extracellular matrix, cytoskeleton, mitochondrial, and nucleotide-binding [51]. Exosomal milk fat globule epidermal growth factor VIII (MFGE8) could activate phagocytic signaling and efficiently clear dead cells, promoting cardiac recovery after injury [52]. Additionally, *ex vivo*, *in vivo*, and *in vitro* studies using settings of ischemia-reperfusion found that exosomal HSP70 transmitted cardioprotective signals to the heart by activating the toll-like receptor 4 (TLR4) downstream signal pathway [53]. Exosomal-associated human antigen R (HuR) has also been recently reported to increase inflammatory and profibrogenic responses *in vitro* and *in vivo* using diabetic heart models [54]. Thus, exosomal HuR might be a therapeutic target to alleviate cardiac inflammation and fibrosis in diabetes [54]. Hence, exosomal proteins can affect CVD by regulating metabolism, macrophage engulfment, and inflammatory and profibrogenic responses.

### *2.2.2 Respiratory system disease*

Exosomes released from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induced tissue factor (TF) expression that may drive thrombosis [55]. The EVs TF activity was related to disease severity and mortality and could be a prognostic biomarker and therapeutic for coronavirus disease 2019 (COVID-19) [55]. Monosialodihexosyl ganglioside (GM3)-enriched exosomes may contribute to the pathological processes related to COVID-19 and provide the most significant repository on the plasma lipidome and metabolome distinct to COVID-19 [56]. Moreso, *in vitro* studies have demonstrated that EV proteins, such as TNC, insulinlike-growth-factor-binding protein 7 (IGFBP7), fibrillin-1 (FBN1), alpha-2 collagen chain (I) (COL1A2), alpha-1 collagen chain (I) (COL1A1), and lysyl oxidase homolog 1 (LOXL1), secreted by fibroblasts might contribute to idiopathic pulmonary fibrosis (IPF) [57]. Furthermore, exosomes also transferred proteins to distant recipient

cells and were expected to be a new drug delivery system and a novel therapeutic target [58].

### *2.2.3 Nervous system diseases*

Parkinson's disease (PD) and Alzheimer's (AD) are classic chronic neurodegenerative diseases [59]. Previous studies have reported few specific blood biomarkers in diagnosing PD and AD [60]. This is, to some extent, likely attributed to the lack of biomarker specificity [60]. For example, exosomal DJ-1 and α-synuclein in plasma failed to distinguish between PD patients and healthy donors [60]. However, the two proteins in plasma neural-derived exosomes could distinguish PD patients and healthy donors [60]. Besides the circulating specimen, the urine exosomal Ser(P)- 1292 LRRK2 (leucine-rich repeat kinase 2) was considered a biomarker associated with PD progression [61]. In addition, the t-tau and p-tau levels derived from neuron exosomes of mild-AD groups were significantly higher than age-matched controls and mild cognitive impairment groups [62]. Likewise, exosomal growth differentiation factor-15 (GDF-15) derived from bone mesenchymal stem cells (MSCs) was confirmed to alleviate SH-SY5Y cell damage of AD by activating AKT/GSK-3β/β-catenin pathway [63]. Thus, exosomal proteins from different specimen sources may be used as potential biomarkers for diagnosis and prognosis of nervous system diseases.

### **3. Exosome RNA**

Exosomal RNAs include miRNAs, circRNAs, lncRNAs, and mRNAs [64]. Additionally, evidence has established that exosomes play a significant role in tumorigenesis and tumor progression by transferring miRNAs, circRNAs, and LncRNAs [1]. These exosomal RNAs are biomarkers and therapeutic targets for human diseases, particularly malignant tumors.

### **3.1 Application of exosomal RNAs in tumors: diagnosis, prognosis, and treatment**

### *3.1.1 Lung cancer*

It has been reported that free RNA molecules secreted by tumor cells will degenerate in the bloodstream [65]. The exceptions are cell-free microRNAs that can be detected in cancer patients' blood plasma or serum [65]. Relevant RNA molecular information, such as exosomes, may also be obtained in EVs [66]. The existing literature also shows that exosomal RNAs play critical roles in different stages of the development cascade of cancer. For instance, serum exosomal miR-96 and miR-23a were upregulated in lung cancer and could be used as a biomarker for diagnosing lung cancer [67, 68]. Besides circulating exosomal miRNAs, exosomes released from bronchoalveolar lavage fluid could also serve as biomarkers for early lung cancer diagnosis. For instance, exosomal miR-126 and Let-7a from bronchoalveolar lavage fluid were significantly higher in LUAD patients than in healthy donors [69]. Exosomal miRNAs could be used as a prognostic biomarker for NSCLC development, such as miR-23b-3p, miR-10b-5p, and miR-21-5p were found to be independently associated with poor OS of NSCLC [70]. Moreso, exosomal circ-002178 was enriched in plasma exosomes from LUAD patients and could be delivered into CD8+ T cells to induce PD1 expression [71]. Exosomes from NSCLC patient serum were enriched with circSATB2, which has high sensitivity and specificity for clinical detection and is related to lung cancer metastasis [72]. Furthermore, Lv et al. verified that exosomal long intergenic non-coding 00662 (LINC-00662) promoted proliferation, invasion, and migration of NSCLC by the miR-320d/E2F1 axis, indicating that LINC-00662 may be a potential therapeutic target for lung cancer [73].

### *3.1.2 Gastric cancer*

As discussed earlier, several circulating RNAs are excellent as potential diagnostic markers of GC. Circulating exosomal miR-1290 has been reported to be upregulated in various malignant cancers, including GC [74], LUAD [75], epithelial ovarian cancer [76], and PCa [77]. Kumata et al. observed that the miR-23b in exosomes from the plasma of GC patients was significantly lower than that of the healthy donors [78]. Many studies have also confirmed that exosomal circRNAs act as molecular sponges of miRNAs to regulate the proliferation, invasion, metastasis, and angiogenesis of GC cells [79–81]. Moreso, the circSHKBP1, elevated in GC tissues and serum, promoted GC progression by sponging miR-582-3p to increase HuR expression and suppress HSP90 degradation [79]. In contrast, exosomal circRELL1 has been reported to inhibit the progression of GC via the circRELL1/miR-637/EPHB3 axis [80]. Furthermore, exosomal lncRNA HOTTIP was also found to be a GC diagnostic biomarker associated with poor OS of GC patients [82]. In summary, these exosomal RNAs may serve as potential biomarkers for GC.

### *3.1.3 Liver cancer*

Much evidence has demonstrated that exosomal RNAs are involved in the growth, metastasis, and angiogenesis of HCC cells and could be used as diagnostic and prognostic biomarkers and therapeutic tools in HCC [83, 84]. Notably, exosomal miRNAs have been studied as the potential diagnostic biomarker for HCC. For example, serum exosomal miR-122, miR-148a, and miR-1246 were significantly higher in HCC patients than in liver cirrhosis and healthy donors [85]. Interestingly, exosomal miR-101, miR-106b, miR-122, and miR-195 were significantly lower in HCC serum than in chronic hepatitis B (CHB) [86]. A study also reported that high serum exosomal miR-638 was associated with HCC recurrence, suggesting the potential of exosomal miR-638 as a reliable biomarker for prognostic monitoring [87]. Another study reported that serum exosomal circPTGR1 was upregulated in HCC patients and associated with the TNM stage and OS. Moreover, exosomal circPTGR1 has promoted the proliferation, invasion, and migration of HCC via the miR-449a-MET axis [88]. Beyond miRNAs, some lncRNAs in exosomes may also be served as the promising diagnostic marker for HCC. Sun et al. indicated that the serum exosomal LINC-00161 was higher in HCC patients than in healthy donors, suggesting that LINC-00161 could be a potential diagnostic biomarker for HCC [89].

### *3.1.4 Breast cancer*

Most BC patients are hormone-dependent [90]. Increasing evidence demonstrated that exosomes play an essential role in breast tumorigenesis and progression by transferring miRNAs and LncRNAs [1]. Triple-negative breast cancer (TNBC) refers to the expression of estrogen receptor (ER), progesterone receptor (PR), and HER2

### *Perspective Chapter: Clinical Application of Exosome Components DOI: http://dx.doi.org/10.5772/intechopen.110856*

being negative in BC tissue [91]. Notably, miR-30b was associated with recurrence, and miR-93 was abundant in ductal carcinoma in situ (DCIS) [92]. Circulating exosomal miR-373 has been enriched in TNBC, and serum exosomal miR-373 was higher in ER-negative and PR-negative patients than in patients with hormonereceptor-positive patients [93]. In contrast, exosomal miRNAs, such as miR-16, were particularly enriched in estrogen-positive BC patients [92]. These studies remind us that the expression level of miRNAs in exosomes may contribute to the luminal classification of BC. Beyond miRNAs, circPSMA1 in exosomes acted as a "miRNAs sponge" to absorb miR-637 [94]. It could transmit migration and proliferation capacity to recipient cells to promote TNBC cell proliferation, migration, and metastasis via the miR-637/Akt1/β-catenin (cyclin D1) axis [94]. The lncRNA DARS-AS1 delivered by exosomes has been found to effectively inhibit TNBC cell growth and liver metastasis [95]. Thus, different RNA types can play different roles in TNBC development. To date, drug resistance is a significant obstacle to BC treatment [96]. Many pieces of evidence have demonstrated that exosomes regulate drug resistance for BC by delivering RNA. For instance, Han et al. demonstrated that miR-567 delivered by exosomes increased the sensitivity of BC cells to trastuzumab [97]. Similarly, lncRNA H19 could be transferred via exosomes to sensitive cells, leading to doxorubicin resistance in BC [98]. Thus, these studies strongly suggest that exosomal RNAs are known to act as biomarkers for BC development and drug resistance.

### *3.1.5 Colorectal cancer*

Exosomes carry and deliver specific molecules and have been found to mediate crosstalk between primary cancer sites and metastatic cancer loci [31, 84]. Exosomal miR-10a derived from SW480 cells inhibits human lung fibroblast migration and inflammatory factors releases, transferring the metastasis suppression signal to primary CRC [99]. Further studies have established a pair of human liver fibroblast cell lines to confirm the regulation function of miR-10a-5p between primary CRC and metastatic liver loci [100]. As mentioned above, abnormal expression of exosomal RNAs in peripheral blood can also be considered emerging diagnostic, prognostic, and therapeutic biomarkers of CRC. For instance, exosomal miR-1229 is significantly upregulated in the serum exosomes from CRC patients and was associated with tumor size, TNM stage, lymphatic metastasis, and poor OS [101]. Zeng et al. also found that miR-25-3p could form a pre-metastatic niche via stimulating angiogenesis and vascular permeability in CRC [102]. Circulating exosomal circRNAs could also serve as strong diagnostic biomarkers for CRC. Serum exosomal circ-0004771, circPNN, and circFMN2 levels were significantly upregulated in CRC patients [103–105]. Exosomal circ-133 was also significantly upregulated in the plasma of CRC patients and associated with hypoxia and cell metastasis by miR-133a/GEF-H1/RhoA axis [106]. Moreover, the mechanisms for targeting dysfunctional exosomal LncRNAs are being developed to treat CRC. For example, exosomal LINC-00659 has been found to promote CRC cells proliferation, invasion, migration, and EMT by miR-342-3p/ ANXA2 pathway, suggesting that LINC-00659 could work as a potential biomarker for selecting a suitable treatment strategy [107]. Essential to the development of improved therapeutic strategies is a mechanistic understanding of exosome-mediated cell communications. Furthermore, MSCs -derived exosomes have been used as carriers to deliver anticancer agents in CRC [108]. Notably, this therapeutic effect is based on direct cell–cell communications and indirect communications mediating by cell secretome [109].

### **3.2 Application of exosomal RNAs in other diseases: Diagnosis, prognosis, and treatment**

### *3.2.1 Cardiovascular disease*

As mentioned earlier, exosomal RNAs secreted from senescent cells are considered to be associated with CVD and vascular aging [110]. miR-155 contained in exosomes is transferred from smooth muscle cells to endothelial cells to induce endothelial injury and promote atherosclerosis [111]. In diabetic cardiomyopathy, miR-320 is enriched in diabetic patients and could inhibit Hsp20 in endothelial cells, exerting an anti-angiogenic function [112]. Additionally, exosomal LOC100129516 has been found to ultimately alleviate the progression of atherosclerosis and decrease the cholesterol level via the PPARγ/LXRα/ABCA1 pathway [113]. Therefore, these studies suggest that the pathologic role of exosomes involves RNA delivery and could contribute to developing diabetic cardiomyopathy via different cell signals.

### *3.2.2 Autoimmune disease*

Autoimmune diseases may be related to genetic, environmental, hormonal, and immunological factors [114]. Mounting evidence indicates that exosomes play an important role in autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and ankylosing spondylitis (AS). For example, it was demonstrated that urinary exosome miR-146a level was significantly increased in SLE patients compared with healthy donors. Its expression level was closely related to renal injury index, like proteinuria, histological features, and lupus activity [115]. It was also verified that exosomal circEDIL3 suppresses inflammation-induced angiogenesis and ameliorates RA via the miR-485-3p/PIAS3/STAT3 pathway [116]. In addition, the expression of serum exosomal nuclear paraspeckle assembly transcript 1 (NEAT1) in RA patients was higher than that of healthy donors. Moreover, NEAT1 has promoted the progression of RA by downregulating miR-144-3p and upregulating Rho-associated protein kinase 2 (ROCK2), suggesting that NEAT1 may be a potential biomarker and therapeutic target for RA [117]. The levels of exosomal circRNAs, like circ-0110797, circ-0097378, circ-0122309, circ-0058275, and circ-0008346 in AS, were significantly down-regulated compared with healthy donors, providing more optional biomarkers for the early diagnosis of AS [118].

### **4. Exosome DNA**

Circulating tumor DNA (ctDNA) fragments are released by tumor cells into the bloodstream. The information on genomic alterations identified in tumors, including point mutations, rearrangements, amplifications, and even gene copy variations, could be identified by analyzing ctDNA molecules [119]. Although cancer detection by monitoring ctDNA is an area of active investigation, identifying very low amounts of ctDNA in blood samples with variable amounts of free DNA (cfDNA) remains challenging. To date, the studies focused on exosome DNA (exoDNA) are fewer than those on exosome RNA and protein [120]. It is revealed that a variety of cancer-derived DNA markers in exosomes by high throughput genome and transcriptome comparative analyses, including copy number, point mutation, insertion, deletion, and gene fusion. ExoDNA has great potential for disease diagnosis, prognosis assessment, and treatment *Perspective Chapter: Clinical Application of Exosome Components DOI: http://dx.doi.org/10.5772/intechopen.110856*

monitoring. For instance, methylation tests of exoDNA and/or cfDNA derived from the gastric fluid can be used to diagnose GC. In fact, the difference between exoDNA and cfDNA is that the former derives from living cells, the latter from dead ones [121]. Urine from bladder cancer patients contained significant amounts of exoDNA compared with healthy donors [122]. Bernard et al. studied that the increased level of exoDNA was significantly associated with disease progression. Moreover, PC patients with metastases and detectable ctDNA at baseline status had poor progression-free survival (PFS) and OS compared with patients without detectable ctDNA [123]. Degli Esposti et al. also demonstrated that neuroblastoma patients with high tumor mutation load values in exoDNA had a worse outcome than those with lower values [124]. Importantly, the advantage of exoDNA is that they are stable enough to be analyzed retrospectively from frozen bio-banked samples [125]. Meanwhile, exoDNA widely exists in various body fluids and can be easily collected [126]. Therefore, exoDNA may be an ideal liquid biopsy method and a novel tumor marker. However, due to the uncertainty of technical methods and high cost, research on exoDNA is relatively limited.

### **5. Problems and prospects of exosomes**

Exosomes can be isolated from multiple sources, including cell culture medium, body fluids, and tumor tissues [28, 127, 128]. Thus, the components of exosomes have potential applications in diagnosis and prognosis for cancer and other diseases. Nevertheless, the basic application of exosomes is at an early stage and restricts their clinical application. Furthermore, considering that the tissue collection method is a non-invasive procedure, patient compliance may also limit the clinical application of exosomes.

### **5.1 Various isolation methods of exosomes**

Exosomes can be extracted by differential ultracentrifugation (UC), density gradient fractionation, polymeric precipitation, microfluidics techniques, and immunoaffinity isolation [129]. The major problem is the different methods will cause significant differences in the composition and content of exosomes. In addition, the low amounts of components in exosomes led to difficulties in quantification. To date, differential UC is the most commonly used isolation method to harvest highly purified exosomes from a cell culture medium [130]. Polymeric precipitation requires little hands-on time but produces the highest contamination [131]. Additionally, immunoaffinity isolation is based on the characteristic surface proteins on certain exosomes [132]. Antibodies conjugated with beads can select the desired exosomes (immuno-enrichment) or trap unwanted exosomes (immuno-depletion) [129]. This selection process makes it possible to clarify unique exosome populations while undoubtedly leading to lower yields [129]. Each method has advantages and limitations and varies in the quantification of exosome size [133]. As exosomes diagnostic and prognostic platforms become available, there will be requirements for clinical application and manufacturing standards development.

### **5.2 Differences in research institutions**

A biomarker that can be used in clinical applications should meet the following premise: There should be no statistically significant differences between different

detection institutions, detection methods, and researchers. However, it is not the case in the current situation of studying exosomes. For example, a report indicated that surface protein CD47 in circulating exosomes was higher in healthy than BC patients [134]. However, exosomal CD47 in BC patients was reported to be significantly higher than those in healthy controls [19]. Thus, the standards of specimen source, collection, and process in different institutions should first be well established.

### **5.3 The differences generated by sources of specimen**

Specimens for clinical application could be obtained from blood, tissues, and other biological fluids, like urine, hydrothorax, ascites, and cerebrospinal fluid [6, 28]. Among all these specimens, serum and plasma are the most suitable for reflecting healthy or diseased conditions, genetic variations, environmental factors, lifestyle, nutrition habits, and drugs [6]. They can also provide important information at a systemic level [6]. Nevertheless, the exosomal contents from plasma and serum simultaneously and place have been identified to differ in the stability and composition of metabolome and lipoproteome [6]. Thus, the criterion should be established for using different specimens, and collection tubes, even among the same blood matrix.

### **6. Conclusions**

In summary, technological improvements and our understanding of exosomal proteins, nucleic acids, and their exosomal content profiles may provide diagnostic, prognostic, and therapeutic clues for diseases in the future.

### **Acknowledgements**

This work was supported by the Natural Science Foundation of Hebei Province [no: H 2021105019] and the Key Laboratory of Precision Medical Testing in Tangshan [no: 2021TS009b]. The authors thank AiMi Academic Services (www.aimieditor. com) for English language editing and review services. The authors thank Professor Zhengang Wu reviewed and revised the manuscript before submission.

### **Conflict of interest**

The authors declare no conflict of interest.

*Perspective Chapter: Clinical Application of Exosome Components DOI: http://dx.doi.org/10.5772/intechopen.110856*

### **Author details**

Mengyuan Hou1 , Jingwu Li2 , Zhiwu Wang3 and Yankun Liu4 \*

1 North China University of Technology, Tangshan, China

2 Cancer Institute, Tangshan People's Hospital, Tangshan, China

3 Department of Chemoradiotherapy, Tangshan People's Hospital, Tangshan, China

4 Department of Medical Molecular Diagnosis, Tangshan People's Hospital, Tangshan, China

\*Address all correspondence to: rmyy\_lyk@163.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### **Chapter 9**

## Perspective Chapter: Development of Exosomes for Esthetic Use

*Byong Seung Cho and Diane Irvine Duncan*

### **Abstract**

While there are thousands of peer-reviewed papers on exosomes, most of the work has been done in the medical field. Studies and clinical trials on exosome-related products for the esthetic industry have just begun to be a regular occurrence. One of the reasons for this is a lack of regulatory approval for any exosome use. The FDA does not regulate topical cosmetic use, while only a few exosomes are registered on the International Cosmetic Ingredient Dictionary (ICID) of the Personal Care Product Council (PCPC), so most esthetic providers are utilizing exosomes in this manner. Clinical uses for exosomes in esthetic practice include the treatment of burns, active acne, atopic dermatitis, and chronic skin irritations. When used in combination with energy-based device treatment, exosomes reduce inflammation and redness, improve the rapidity of healing for laser and microneedling patients, and reduce the tendency for fibrosis and thick hypertrophic scar formation when used topically. Byong Cho is the CEO & CTO of ExoCoBio, one of the four largest exosome companies globally. He has developed a large research, development, and GMP manufacturing facility just south of Seoul, Korea. His topic, the development of exosomes for clinical esthetic use, will take us through the process of developing a safe and cost-effective biological regenerative product while staying in line with regulatory limitations.

**Keywords:** regenerative, esthetic, exosome, secretome, scar treatment, resurfacing, hair restoration, burn treatment

### **1. Introduction**

I first learned about exosomes in early 2016. I heard the word "exosome" for the first time from a close friend and professor of biology. After that, for several months, I did basic research and study on papers and patents related to exosomes [1]. Through this, I found that exosomes can develop a completely new biotech technology, and in particular, I became very interested in "stem cell-derived exosomes" for regeneration and dermatological applications. During 6 months in 2016, I was able to secure basic knowledge about it. In addition, it was identified that stem cell exosomes have great potential as a next-generation regeneration plus anti-inflammation technology. Further, I became very interested in Dr. Sai Kiang Lim, and her brilliant discovery, who discovered stem cell-derived exosomes for the first time in the world [2].

Additional factors that made me interested in exosomes in 2016 include my background. I had more than 15 years of experience in investing in biotechnology and commercialization in venture capital. After putting together 2 biotech startups in early 2002, I was the first venture capital investor in Asia who supported the commercialization of botulinum toxin technology. I have continued to have a deep interest and experience in the field of medical esthetics since then, and I thought that I found "something new" about the commercialization potential of stem cell-derived exosomes. I have been continuously trying to find and commercialize new technologies in the field of "regenerative medicine or aesthetics" for more than 10 years before knowing about exosomes.

My vision was to fill an unmet need for our global aging population. While there are botulinum toxins, dermal fillers, & energy-based devices, not all of them would be appropriate for very senior people. We would need something new—a regenerative esthetics or regenerative medicine treatment option that might be able to reverse certain aspects of aging. Also, stem cell-derived exosomes can be used to treat incurable or very difficult skin diseases like dermatitis, psoriasis, scleroderma, skin fibrosis, and so on, based on the dual and synergistic function of regeneration and anti-inflammation. This premise of exosome therapy could certainly expand the field of regenerative dermatology.

### **2. As a pioneer in the field, what were your first steps in developing exosomes?**

After learning about exosomes, the most important tasks for me were (1) establishing a business plan, (2) licensing and developing exosome technology, (3) financing to create a successful exosome startup, (4) registering adipose stem cell-derived exosomes as a cosmetic ingredient, and (5) finding a partner for marketing and sales in the US.

While exploring numerous scientific papers and patents on exosomes around the world [3, 4], I sought what kind of business to do with exosomes. In particular, the biggest challenge was to determine whether stem cell exosomes would become a major technology in the field of "regenerative medicine or regenerative aesthetics" in the future. And, since then I had no exosome technology on my own, and to license stem cell exosome technology, I read all the publications of Dr. SK Lim and directly contacted her to discuss technology licensing and to have her as a scientific advisor for 6 months in 2016. While studying other scientists' technologies and patents [5, 6], I was able to build a solid business plan at the time of ExoCoBio's establishment. Of course, since exosome technology is in its infancy, and I thought there would be many changes in the future, I planned to develop various types of exosome-based esthetic technologies and products in my 5-year business plan (**Figure 1**).

In the commercialization of new technology such as exosomes, the most important thing was a series of financing to support the business plan. With my early ideas, I made lots of calls to my VC friends to pre-market the business plan for 6 months. At that time, I had about 15 years of experience in the venture capital industry and successful commercialization and IPO (Initial Public Offering) of two biotech companies. With this background, I felt I could incorporate ExoCoBio in Jan 2017. Just after 3 months, I was able to raise about USD 12 million in March 2017 through Series A financing and angel financing from multiple venture capital firms and individuals. It was the biggest financing as Series A funding for a biotech startup in South Korea.

Our first office was small. After setting up my business plan and team building, ExoCoBio was incorporated in a tiny office of about 270 SqFt outside Seoul, *Perspective Chapter: Development of Exosomes for Esthetic Use DOI: http://dx.doi.org/10.5772/intechopen.111846*

### **Figure 1.**

*Clinical evaluation of the skin brightening effect of ASC-exosomes.*

South Korea, in Jan 2017. The company successfully raised about \$2 million from individual investors to get the first office and ExoCoBio got a small and humble laboratory in a university to start to develop our own manufacturing process of cell culture, TFF process, in vitro tests, and others.

One of the most significant jobs in 2017 was to register our own exosomes based on adipose stem cells (ASCE) to the ICID of the PCPC in the US. Since I knew that cosmetic registration was critical to commercialize this new exosome technology, and, I wanted to be the first in the world, the registration process was started immediately after the financing around April 2017. It took about 9 months which was longer than expected, because there was no predicate ever of cosmetic exosomes. With all the efforts, ExoCoBio became the first to have the International Nomenclature Cosmetic Ingredient (INCI) name and, still now, ExoCoBio has proudly the biggest number of exosome registration in the ICID.

The last job to develop a successful exosome business in 2017 was to find a partner who can do market this new technology and sell our products even before we have an actual product of ASCE 2 years ago. So, I was introduced to BENEV Inc. in California, US, which had about 20 years of experience in growth factor-based esthetic products worldwide. When I first met Mr. Ethan Min at his office in May 2017, in Mission Viejo, CA, we did talk a lot about our experiences and business plan including the product concept for several hours. Very fortunately, we had the same mind! He gave me important insight into the future of regenerative esthetics based on exosomes and then we initiated to collaborate to be the first and to create a new industry.

### **3. Were there any sources (say bone marrow) that you tried and discarded? Why choose ADSCs for cosmetic use?**

This is a very good question. It seems that many scientists and doctors have a misunderstanding about the source and quality of exosomes, especially about adipose stem cell-derived exosomes (ASCE).

So far, ExoCoBio has been focusing on adipose stem cell-derived exosome technology.

In 2019, ExoCoBio conducted an internal comparison of three types of stem cell exosomes. In other words, we cultured three types of adipose tissue-derived stem cells, umbilical cord-derived stem cells, and Wharton Jelly-derived stem cells and isolated exosomes from each. Then, when in vitro efficacy tests were done, there was no significant difference in efficacy. Through this series of internal studies, it was determined that adipose stem cell exosomes are the most commercially superior in terms of efficacy and cost.

Adipose stem cell exosomes produced by ExoCoBio have been proven with 8 scientific publications [7–12] and 48 patents including 8 US patents, that they will be the most effective in the esthetic field. For example, our clinical trial demonstrated that skin with acne scars was improved by the combined use of exosome derived from adipose tissuederived mesenchymal stem cells with fractional CO2 laser would provide synergistic effects on both the efficacy and safety of atrophic acne scar treatments [11].

As of March 11, 2023, there are 5609 "stem cell exosome" papers searched in Pubmed, including 625 "adipocyte stem cell exosome" papers, which is a significant proportion. I believe absolutely that ASCE has been scientifically validated. ExoCoBio is the only company that has performed a double-blinded, randomized, and split-face clinical study based on any kind of stem cell exosomes in the world. Also, according to a paper published by Xu, H. et al. in 2019 (41), it is known that adipose stem cell exosomes have the highest regenerative effect and contain the most growth factors for cardioprotection and anti-apoptotic effects than other exosome sources (**Figure 2**).

**Figure 2.** *Effects of ASC-exosomes on skin.*

### *Perspective Chapter: Development of Exosomes for Esthetic Use DOI: http://dx.doi.org/10.5772/intechopen.111846*

Some important discoveries made in the field of stem cells over the past 20 years, and as a result, several pros and cons were found. Exosomes derived from stem cell has been studied and the results of published articles demonstrated that exosome can overcome the cons of stem cell [13]. Stem cells and exosomes are different in many ways. In addition, adipose stem cell exosomes have the following advantages:


### **4. Were there any processes production wise that you tried and changed?**

In my view, there are a few important decisions to make or still to develop something to have better exosomes. ExoCoBio has tried to improve some parts in the last few years.

First, we need to think about the purity and impurities of exosomes. This is also related to regulatory affairs. During the last two decades or more, multiple clinical studies [17, 18] have shown that stem cells, including allogeneic stem cell treatments, are generally safe. So, it is very natural that stem cell-derived exosomes are believed to be generally safe, even though we need to do an extensive toxicological evaluation in the future. How many impurities can a product contain and retain safety? For example, any blood-derived exosomes may naturally contain lots of lipoproteins or proteins aggregate during the isolation process. Though not toxic, the presence of these may dilute the efficacy of the exosome product.

Second, the choice of a storage buffer is very important to maintain the potency of exosomes in the long term. For many companies and scientists, exosomes including stem cell exosomes are produced and stored in a phosphate-buffered saline (PBS) or similar simple buffer, which can easily degrade the quality of exosome in a short time, as published in the Journal of Extracellular Vesicles by Dr. Samir Andaloussi et al. in 2022. Dr. Andaloussi discovered that exosomes are drastically fractured at all temperatures of +4 ~ −80 degrees in Celsius tested. This may be a reason why many liquid exosome products currently available on the market have limited or less efficacy in the field. ExoCoBio has been focused on developing its proprietary formulation for long-term stability.

Third, lyophilization is the best process for the long-term storage of exosomes at present. Combined with a specific formulation of storage buffer, lyophilization can extend and keep the quality of exosomes shelf stable for 2 years. Two different groups of scientists have proven that they could produce exosomes in compliance with the GMP procedure a few years ago [19, 20]. Liquid or frozen exosomes are stable only for a limited time or only in a special formulation. A challenge in clinical practice is the storage of exosomes in a cryo tank or a special freezer that generates temperatures of −80 degrees Celsius. If the cryo tank runs out of nitrogen, or the power is off during the weekend, the costly exosomes are no longer usable. Stability on the shelf at room temperature or in a standard refrigerator can help a clinic maintain safety standards more easily than with demanding specialized laboratory equipment.

Fourth, pure exosomes are efficacious, but the effects could be further improved. From our studies, exosomes are found to be significant "seeds" or triggers for regenerative or anti-inflammatory effects, because exosomes contain a huge number of growth factors, cytokines, short-chain RNAs, peptides, and lipids. However, for example, when we added specific amino acids or other small peptides, efficacy was better for regeneration as well immune modulation in our experiments. Furthermore, due to the high cost of pure exosomes, market acceptance has been limited. ExoCoBio has decided to change its product strategy and become the first company to develop a combination of formulation processes in order to stimulate the potency of stem cell-derived exosomes. In this way, we could provide quality products based on ASCE. ExoCoBio is very proud to help people suffering from a variety of problematic skin conditions in addition to regular cosmetic uses around the world.

### **5. A similar question for extraction. Did you try ultracentrifugation and then discard that method? Why?**

Since the beginning of 2019, ExoCoBio has conducted various studies on the method of isolating exosomes. There are about 9 different methods to extract

exosomes from conditioned media of stem cell culture. The most frequent way is ultracentrifugation, well known by many publications at that time [21].

We saw the advantages of ultracentrifugation for exosome isolation as follows:


However, the long-term goal of ExoCoBio was to provide high quality exosomes at an affordable price. From that point of view, there were these disadvantages of ultracentrifugation for mass manufacturing of exosomes as follows:


Therefore, ExoCoBio decided to utilize another technology and process, namely tangential flow filtration (TFF). This is a membrane-based separation technique used to separate and concentrate biological molecules and particles from a liquid sample. It involves passing the sample across a semipermeable membrane under pressure, allowing smaller molecules or particles to pass through the membrane while retaining larger molecules or particles on the membrane surface.

TFF could be used for a variety of applications that can process the large volume of stem cell conditioned media, including:


• Harvesting and concentrating cells or cell debris from a culture or fermentation broth.

Actually, at that time, ExoCoBio found that TFF was very similar to the serial filtration method used in the first publication of Dr. SK Lim about 15 years ago [2]. To find exosomes, Dr. Lim performed a series of filtration with different pore sizes to track down the paracrine effect of stem cell-derived exosomes.

TFF is a versatile and scalable technique, which can be easily adapted to process large or small volumes of a sample. It can be combined with other separation techniques, such as chromatography, to achieve a higher degree of separation or purification. TFF is widely used in bioprocessing, biopharmaceutical production, and research applications.

### **6. Now that you have your new facility, tell us what your company is currently doing**

After 3 years and still ongoing about \$20 million investment, ExoCoBio built the world's largest GMP mfg. facility of ASCE production, named ExoGMP™ in Osong, South Korea. The purpose of ExoGMP is to produce intravenously injectable grade exosomes that are fully GMP-compliant, for regenerative medicine and regenerative esthetics as well. Though neither KFDA nor FDA approval for this use has been

**Figure 3.** *ExoGMP™ in Osong, South Korea.*

### *Perspective Chapter: Development of Exosomes for Esthetic Use DOI: http://dx.doi.org/10.5772/intechopen.111846*

achieved, the company plans to be ready with this type of product once regulatory approval has been achieved (**Figure 3**).

ExoCoBio has installed more than 200 instruments and equipment, trying to establish about 300 standard operating procedures (SOP). Our team is committed to the production of quality exosomes and finalizing all the qualifications to hopefully produce the first batch of clinical-grade exosomes in the second quarter of 2023. ExoCoBio plans to initiate a Phase 1 clinical study in 2025.

The future of exosomes in esthetic medicine looks strong. However, regulatory issues are still a hurdle. Currently, there are no approved uses for exosomes, either in the medical or esthetic field. While the FDA does not regulate topical cosmetic use, a provider cannot claim to be using the product "off label" if injecting into patients. To stay safe, all exosome use should remain as a topical cosmetic product until full regulatory approval has been obtained.

Many postulations regarding future uses for exosomes have been made. From curing cancer to the reversal of genetic mutations and epigenetic cellular change, the potential for exosome therapies appears to be broad and strong. A current challenge is reading the "message" or contents of each exosome. Targeted or programmed exosomes would be able to direct recipient cells to behave in a certain way. Because of the popularity of the term, many products claiming to have exosomes either have none or have a minimal amount. While exosomes are not living cells, proteins in the contents will degrade over time, so without proper storage formulation, long-term shelf stability is not possible. Exosomes are merely a vehicle for the message they contain. Once we can safely and cost-effectively tailor the directions for cellular change that these tiny particles carry, we can potentially direct recipient cells to repair, reverse such processes as methylation or senescence, and reacquire lost metabolic functions.

### **7. Plant-derived extracellular vesicles**

Plant exosomes, also known as extracellular vesicles, are small membrane-bound vesicles that are released by plant cells into the extracellular space. They are similar in structure and function to exosomes found in animals and other organisms. Plant exosomes contain various molecules such as proteins, lipids, and nucleic acids, which can be delivered to target cells and tissues to regulate various biological processes [22].

Research on plant exosomes is still relatively new. However, plant exosomes are involved in various physiological and developmental processes, such as cell-to-cell communication, stress response, and defense mechanisms. They have also been shown to play a role in inter-kingdom communication, where they can be taken up by other organisms such as fungi and bacteria [23]. In terms of anti-inflammation or immune modulation, edible *P. lobata*-derived exosomes promoted M2 macrophage polarization [24].

On Pubmed (pubmed.ncbi.nlm.nih.gov), we can find about 450 publications. One of the earliest publications is about multivesicular bodies (MVBs)-derived exosomes [23]. Further, one of the most recent publications is about the drug-delivery approach based on plant-derived exosomes for the treatment of inflammatory bowel disease and colitis-associated cancer [25]. In this publication, the isolation of plant-derived exosomes was done by ultracentrifugation mostly and it was found that the intake of plant miRNA may have a variety of effects on our bodies.

One of the R&D projects of ExoCoBio was to expand and apply ExoSCRT™ technology into plant- or microbial-derives extracellular vesicles or exosomes, to find

new material. In that way, I was very interested in rose stem cell (Callus) – derived EVs (RSCE), because (1) roses have been the most popular plant and a cosmetic ingredient for humans, (2) there has still no scientific discovery on the contents of the rose stem cell-derived exosomes. ExoCoBio has been researching to isolate and characterize RSCE for the last 3 years and found a few biological functions as follows (As of now, all the data on RSCE are unpublished, to be submitted for publication soon.):


**Figure 4.** *RSCE NTA analysis. Source: ExoCoBio Inc. (unpublished data).*

*Perspective Chapter: Development of Exosomes for Esthetic Use DOI: http://dx.doi.org/10.5772/intechopen.111846*

**Figure 5.** *RSCE TEM image. Source: ExoCoBio Inc. (unpublished data).*

### **Figure 6.**

*Collagen synthesis of RSCE in human dermal fibroblasts (HDF). \*note: Rose callus CM is the supernatant of the stem cell (callus) culture of rose. Source: ExoCoBio Inc. (unpublished data).*

One disadvantage of plant-derived exosomes is that there is no universal quality standard yet. Many of the previous studies were done before the announcement of the minimal requirements established by the International Society of Extracellular Vesicles (ISEV). So, we need to be cautious in evaluating the data and results and make sure how to isolate and characterize plant-derived EVs. Moreover, the biogenesis pathways of plant-derived exosomes are also not well defined yet. Classifying plantderived exosomes with the current terms used for animal EVs is still difficult due to a lack of current level of scientific discovery.

I believe that when we consider that microbes or bacteria are releasing extracellular vesicles and that kind of biology is universal across the kingdoms and species, it is very worth researching plant-derived EVs indeed for next-generation plant biology. ExoCoBio and other companies are trying to commercialize them as cosmetics or skincare products on the market.

### **8. Previous Writing**

### **8.1 What is skin?**

Our skin is a vital and complex organ that plays a crucial role in maintaining our overall health and well-being. Skin is the largest organ of the human body, and it is the outermost layer that covers and protects our entire body. It is a complex and multifunctional organ that serves many purposes. Some of the primary functions of the skin include:


### **8.2 The structure of skin**

The skin is the largest organ of the human body and has three main layers:


### **8.3 The skin aging**

Skin aging is a natural process that occurs as we age, and it is characterized by various changes in the skin's appearance, texture, and function. Some of the most common signs of skin aging include:


### **9. Current trend in skin rejuvenation and exosomes**

The trend in skin rejuvenation currently is towards non-invasive, minimally invasive, and more importantly, regenerative procedures that provide natural-looking results with little to no downtime. Patients are increasingly seeking out treatments that can address a variety of skin concerns, including wrinkles, fine lines, sun damage, and loss of volume, without the need for surgery or extensive recovery time. Some of the most popular non-invasive and minimally invasive treatments for skin rejuvenation include:


### **10. Adipose stem cells and aging**

Adipose stem cells, also known as adipose-derived stem cells (ASCs), are a type of stem cell found in adipose tissue (fat tissue). These cells have the ability to differentiate into various cell types, including adipocytes (fat cells), chondrocytes (cartilage cells), and osteocytes (bone cells). Adipose stem cells also have the strongest antiinflammatory and regenerative properties, which make them valuable in the field of regenerative medicine.

During aging, there is a gradual loss of adipose stem cells in the body including facial skin and scalp, which can contribute to various age-related health problems. This loss of stem cells is thought to be due to a combination of factors, including decreased stem cell proliferation and increased cell death.

As the number of adipose stem cells decreases with age, the body's ability to regenerate and repair damaged tissues also declines. This can lead to a range of skin and health problems, including slower wound healing, more inflammation in the skin and other organs, decreased muscle mass, and decreased bone density.

Scientists have been actively researching ways to preserve and replenish adipose stem cells in the body, in order to promote better health and slow down the aging

process. One promising approach involves the use of stem cell therapy, which involves the transplantation of stem cells into the body to replace damaged or depleted cells.

Recently, exosomes derived from adipose stem cells are being applied to treat a variety of diseases including dermatological and esthetic uses. Exosome esthetics refers to the use of exosomes in cosmetic procedures and treatments to improve the appearance of the skin, hair, and other parts of the body. In the field of esthetics, exosomes are used to stimulate the growth and regeneration of skin cells, reduce inflammation, and improve the overall appearance of the skin. Exosome-based treatments can be used to address a variety of cosmetic concerns, including fine lines and wrinkles, age spots, uneven skin tone, and acne scars. These treatments may involve the injection or topical application of exosomes directly to the skin or hair follicles. The exosomes can be derived from various sources, including mesenchymal stem cells, which are known to produce particularly potent exosomes with regenerative properties.

### **10.1 Exosomes for clinical applications**

Exosomes are nano-sized vesicles of 30–200 nanometers that are released by cells and contain a variety of biomolecules, including proteins, lipids, and nucleic acids such as cytokines, growth factors, & microRNAs. Exosomes play important roles in cell-to-cell communication and have been found to have a wide range of potential clinical applications.

Here are some examples of the clinical applications of exosomes:


small interfering RNAs (siRNAs), to cancer cells. Additionally, exosomes can be used to stimulate the immune system to attack cancer cells.

• Diagnosis and monitoring of diseases: Exosomes contain biomolecules that can serve as biomarkers for various diseases. This makes them a potential tool for the diagnosis and monitoring of diseases, such as cancer and neurodegenerative diseases.

### **10.2 The differences and combination between exosomes and current technologies**

Botulinum toxin is a neurotoxic protein produced by the bacterium *Clostridium botulinum*. This toxin is known to cause a severe form of food poisoning called botulism, which can be fatal in some cases. However, botulinum toxin has also been found to have therapeutic uses, particularly in the field of cosmetic and medical dermatology. In dermatology, botulinum toxin is used as a muscle relaxant to temporarily paralyze facial muscles and reduce the appearance of wrinkles and fine lines. The injection of botulinum toxin blocks the release of acetylcholine, a neurotransmitter that signals muscle contraction. This causes the targeted muscles to relax and reduces the appearance of wrinkles and fine lines.

Exosomes and botulinum toxins are two very different substances with distinct mechanisms of action and therapeutic applications. While both exosomes and botulinum toxins have potential applications in dermatology, they work through different mechanisms and have different therapeutic effects. Exosomes promote tissue repair and regeneration and have anti-inflammatory effects, while botulinum toxins are primarily used to reduce muscle activity and smooth wrinkles.

Dermal fillers are injectable substances used to restore volume and fullness to the face, reduce the appearance of wrinkles and fine lines, and enhance facial features. They are typically composed of a variety of materials, including hyaluronic acid, calcium hydroxylapatite, poly-L-lactic acid, and polymethylmethacrylate beads. Hyaluronic acid fillers are the most commonly used type of dermal filler. Hyaluronic acid is a naturally occurring substance found in the body that helps to hydrate and plump the skin. When injected into the skin, hyaluronic acid fillers can restore volume to the face, smooth out wrinkles and fine lines, and enhance facial features, such as the lips and cheeks. Mostly, dermal fillers are primarily used to restore volume to the face and reduce the appearance of wrinkles and fine lines. Only a few fillers have collagen-boosting effects, while exosomes can promote tissue repair and regeneration and have anti-inflammatory effects synergistically.

Energy-based devices for skin are non-invasive or minimally invasive devices that use various types of energy, such as light, radiofrequency, ultrasound, or laser, to improve the appearance of the skin. These devices can be used to address a range of skin concerns, including wrinkles, fine lines, sagging skin, hyperpigmentation, and acne scars. Some common types of energy-based devices for skin include:


Exosomes have been proven to work synergistically with these energy-based devices in dermatology. While exosomes give bio-stimulating signals to cells in the skin, energy-based devices provide manageable damage. Both mechanisms of action showed significant improvement to treat acne scars in combination with fractional CO2 laser and adipose stem cell exosomes recently.

### **11. Stromal vascular fraction (SVF), nanofat, and exosomes**

SVF, Nanofat, and exosomes have shown promise in skin rejuvenation and other regenerative medicine applications. Stromal vascular fraction (SVF) and exosomes are both derived from stem cells and have been investigated for their potential applications in regenerative medicine and skin rejuvenation. However, there are some key differences between the three:


One key difference between SVF/Nanofat and exosomes is that SVF/Nanofat contains a mixture of cell types, while exosomes contain only the molecules that are secreted by stem cells. This means that exosomes are a more focused approach to stem cell-based therapies, as they target the specific molecules that are responsible for promoting tissue repair and regeneration. Another difference between SVF/Nanofat and exosomes is that SVF/Nanofat is typically obtained autologously by processing

adipose tissue, while exosomes can be obtained from a variety of cell sources, including adipose-derived stem cells, bone marrow-derived stem cells, and mesenchymal stem cells. SVF/Nanofat is an autologous treatment while exosomes can be allogenic as "off-the-shelf products."

### **12. Key exosomes science and patents for dermatological use**

Exosome research is a rapidly evolving field with many scientists and researchers contributing to its development. There are two famous scientists in exosome research:


In last years, lots of scientists and companies have created key patents for esthetic and dermatological uses based on exosomes. They are stem cell, plant, & microbederived, as follows:


skin barrier, and/or the improvement of skin barrier function. The composition exhibits the effects of increasing the number of ceramides, dihydroceramides, and sphingoid bases, increasing the activities of enzymes that are involved in the synthesis thereof, and decreasing the activities of enzymes that are involved in the degradation thereof. In addition, the composition is able to restore skin barrier function by reducing TSLP, IL-4, and IL-13 which are closely associated with skin barrier damage, thus interrupting a vicious circle in which the lipids and proteins contributing to skin barrier decrease.


### **13. Exosomes manufacturing and quality standards**

Almost every cell is releasing exosomes so there are a huge number of types and sources of exosomes. So, it is very important to set up quality standards to produce exosomes for commercialization and clinical applications. There is currently no universally accepted quality standard for exosomes, as the field of exosome research is still evolving and the properties and characteristics of exosomes can vary depending on the source, isolation method, and intended use.

However, the ISEV has been trying to give minimal requirements on exosome standards from time to time. And there are generally accepted guidelines and best practices that have been proposed for the characterization and quality control of exosomes. These include:


In particular, to get the best quality of exosomes, the manufacturing process must be Good Manufacturing Practices (GMP)-compliant. The ISEV and the International Society of Cellular Therapy (ISCT) gave a guideline for GMP manufacturing, for example, which requires the creation of master cell banks (MCB) and working cell banks (WCB) to get consistent stem cell quality. Those MCB or WCB are required to initiate stem cell therapy worldwide, while blood-derived exosomes are limited in terms of consistent quality due to a lack of cell bank establishment.

While these guidelines can help ensure the quality and consistency of exosome preparations, it is important to note that the field of exosome research is still evolving and there is ongoing debate and discussion around the best practices for exosome characterization and quality control.

### **13.1 Exosome stability and long-term storage**

The stability of exosomes is an important factor that can affect their safety and efficacy. Exosomes are sensitive to environmental conditions such as temperature, pH, and oxidation, which can affect their structural integrity and functional activity. Therefore, proper storage and handling of exosomes are critical to maintaining their stability and potency.

Some factors that can affect exosome stability include:


Overall, proper storage and handling of exosomes are critical to maintaining their stability and potency.

### **13.2 Exosome-based aesthetic products**

A huge number of exosome-based cosmetic products are available on the market worldwide. The regulatory requirements for exosome-based cosmetic products vary depending on the country or region where the products are intended to be marketed. In general, exosome-based cosmetic products may need to comply with the following regulatory requirements:

• Ingredient safety: Exosome-based cosmetic products must use ingredients that are safe for use in cosmetics. The safety of the ingredients must be supported by data from safety assessments, including toxicology studies, clinical studies, and other relevant data.


In the United States, exosome-based cosmetic products are regulated by the Food and Drug Administration (FDA) or Personal Care Products Council (PCPC) as cosmetic products. The FDA does not require pre-market approval of cosmetic products.

To be a successful exosome product, it should be affordable with acceptable efficacy proved by non-clinical and clinical studies. The cost of exosome-based cosmetic products can vary depending on several factors, including the source of the exosomes, the manufacturing process, and the quality of the product. As exosome-based cosmetic products are still a relatively new technology, they may be more expensive than traditional cosmetic products. However, as the technology becomes more widely adopted, the cost of exosome-based cosmetic products may become more affordable.

It is important to note that affordability should not compromise safety and efficacy. Consumers should always choose exosome-based cosmetic products from reputable manufacturers, fully GMP-compliant and with lots of scientific publications, that have undergone rigorous safety and efficacy testing. The proven quality by a series of scientific publications and patents, rather than the simple number of exosomes, is the most important factor to choose products.

IN CONCLUSION, EXSOMES ARE NEW TECHNOLOGIES FOR ESTHETIC USE AND WE MUST CHOOSE SCIENTIFIC EVIDENCE-BASED EXSOME PRODUCTS.

*Perspective Chapter: Development of Exosomes for Esthetic Use DOI: http://dx.doi.org/10.5772/intechopen.111846*

### **Author details**

Byong Seung Cho1 \* and Diane Irvine Duncan<sup>2</sup>

1 ExoCoBio Exosome Institute, South Korea

2 Plastic Surgical Associates, Fort Collins, CO, United States

\*Address all correspondence to: ceo@exocobio.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by Sherin Saheera*

*Exosomes - Recent Advances from Bench to Bedsid*e is a comprehensive book exploring the field of exosomes, nanosized extracellular vesicles that regulate physiological and pathological processes. Organized into three sections, this book discusses various aspects of exosomes. The first section focuses on the use of exosomes derived from mesenchymal stem cells, including their applications in cardiovascular diseases and tissue engineering. The second section explores the role of exosomes in infectious diseases, encompassing immune reactions, pathogen transmission, and tuberculosis. The final section discusses the applications of exosomes such as drug delivery, signaling mechanisms, and even aesthetic purposes. This book provides valuable insights into the current understanding and potential applications of exosomes in diagnostics, therapeutics, and beyond.

### *Tomasz Brzozowski, Physiology Series Editor*

Published in London, UK © 2023 IntechOpen © 123dartist / iStock

Exosomes - Recent Advances From Bench to Bedside

IntechOpen Series

Physiology, Volume 20

Exosomes

Recent Advances From Bench to Bedside

*Edited by Sherin Saheera*