**Table 2.** *Cont.*

Messenger RNAs (mRNAs). Increased levels of epidermal growth factor receptor variant type III (EGFRvIII) mRNA have been detected in the serum exosomes of glioblastoma patients, suggesting its use as a new glioblastoma diagnosis method instead of surgery [270]. Exosome Diagnostics, Inc. (Waltham, MA, USA) have developed methods for detecting one or more biomarkers in urine microvesicles in order to aid the diagnosis, monitoring and treatment of diseases such as cancer, especially prostate gland-related pathologies. Biomarkers, which are mRNAs of one or more isoforms of a large group of genes, facilitate the detection of prostate cancer by determining the fusion between SLC45A3 and BRAF genes in urinary microvesicles [311]. Recently, Dong and coworkers found that exosomal serum membrane type 1-matrix metalloproteinase (MT1-MMP) mRNA increases significantly in gastric cancer (GC) patients, which correlates with the tumor, lymph node and metastasis (TNM) stage and lymphatic metastasis. These findings indicate that exosomal MT1-MMP mRNA can be utilized as a biomarker for GC diagnosis and early treatment [312]. Similarly, exosomal heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) mRNA levels, which are remarkably higher in hepatocellular carcinoma (HCC) patients than in other groups, are associated with the Child-Pugh and TNM stage classification, portal vein tumor emboli and lymph node metastasis. This confirms that exosomal serum hnRNPH1 mRNA could be an effective marker of HCC [313]. Esophageal cancer-related gene-4 (Ecrg4) has been shown to be a tumor suppressor in several studies. Mao and colleagues have reported that serum exosomes contain higher levels of ECRG4 mRNA in healthy individuals than in their cancer counterparts, thus showing that exosomal ECRG4 mRNA can be used for cancer detection [314].

MicroRNAs (miRNAs) are small noncoding, double-stranded RNA molecules that degrade complementary mRNA sequences in target cells in order to inhibit protein translation. These molecules are reported to be abnormally expressed in several types of cancer, suggesting their role in the pathogenesis of human cancer [315]. Eight miRNAs, previously shown to be diagnostic markers of ovarian cancer, have been reported to be present at similar levels in biopsy specimens of ovarian cancer and circulating exosomes isolated from the same ovarian cancer patients [316]. With respect to lung tumors, Rabinowits and coworkers found similar miRNA patterns in plasma exosomes and tumor biopsies from lung adenocarcinoma patients. However, miRNA levels in lung cancer patients and control subjects differed significantly, indicating that circulating exosomal miRNA could be useful for lung adenocarcinoma screening tests [269]. Hepatocellular carcinoma (HCC) is a primary liver malignancy and a leading cause of cancer-related mortality worldwide. Exosomal miRNA-210 secreted by hepatocellular carcinoma cells is reported to promote angiogenesis by targeting SMAD4 and STAT6 in endothelial cells. Therefore, exosomal miRNA-210 could be used as a therapeutic target in anti-HCC therapy [109]. In this regard, circulating miRNAs in serum exosomes have potential as novel biomarkers for predicting hepatocellular carcinoma recurrence following liver transplantation [317]. In addition, Takeshita and colleagues reported that the sensitivity and specificity of serum miRNA-1246 in an esophageal squamous cell cancer (ESCC) diagnosis are 71.3% and 73.9%, respectively. Serum miRNA-1246, which closely correlates with the tumor, lymph node and metastasis (TNM) stage, has been shown to be a strong independent risk indicator of poor survival rates. Intriguingly, miRNA-1246 levels were found to be elevated in serum exosomes from ESCC patients but not in ESCC tissue samples, suggesting that exosomal serum miRNA-1246 could be a valuable diagnostic and prognostic biomarker of ESCC [318]. Circulating exosomal miRNA-17-5p and miRNA-92a-3p were found to be upregulated in colorectal cancer (CRC) patients. Their expression levels correlated closely with metastasis and chemotherapy resistance [319]. Moreover, exosomal miRNA-320d has been identified as a promising blood-based biomarker for distinguishing metastatic from nonmetastatic diseases in the serum of CRC patients. Therefore, these noninvasive biomarkers may have great potential to predict the clinical behavior of CRC and to monitor tumor metastasis [320,321]. Mitchell et al. reported that circulating miRNA-141 levels are strong diagnostic markers of prostate cancer [322]. Furthermore, exosomal serum miRNA-141 and

miRNA-375 have been found to correlate with tumor progression in prostate cancer [323]. The enrichment of the let-7 miRNA family in exosomes from AZ-P7a cells may reflect their oncogenic characteristics, including tumorigenesis and metastasis, suggesting that AZ-P7a cells release let-7 miRNAs via exosomes into the extracellular environment to maintain their oncogenesis [182].

Long noncoding RNAs (lncRNAs). Exosomes also contain lncRNAs, now characterized as potential diagnostic and prognostic biomarkers for a wide range of pathologies. These functional RNAs, which are longer than 200 nucleotides, do not code for proteins but, rather, bind to a variety of nucleic acids and proteins as a means to regulate gene expression at the transcriptional and/or translational level. Colon cancer-associated transcript 2 (CCAT2), a novel lncRNA transcript encompassing the rs6983267 SNP, is significantly upregulated in CRC tissues as compared to adjacent noncancerous tissues. The higher expression levels of CCAT2 are associated with a greater depth of local invasion, positive lymph node metastasis and advanced TNM stage [324]. Moreover, exosomal lncRNA and miRNA-217 are differentially expressed in the serum of colorectal carcinoma patients and correlate with tumor classifications (T3/T4), advanced clinical stages (III/IV) and lymph node or distant metastasis [325]. LncRNA 91H is known to play a prominent role in tumor development by enhancing tumor cell migration and invasion through the modification of heterogeneous nuclear ribonucleoprotein K (HNRNPK) protein expression. In addition, CRC patients with high lncRNA 91H expression demonstrate a higher risk of tumor recurrence and metastasis [326]. Interestingly, exosomes from healthy donors carry a significant amount of HOTTIP (HOXA distal transcript antisense RNA) transcripts in comparison to CRC patients, with a significant statistical correlation between low exosomal HOTTIP levels and poor overall survival rates. Therefore, lncRNA *HOTTIP* could be a viable biomarker for CRC patients to predict the postsurgical survival time [327]. Exosomal serum lncRNA HOTAIR (Hox transcript antisense intergenic RNA) and miRNA-21 expression levels were higher in patients with lymph node metastasis than those without. In addition, exosomal HOTAIR and miRNA-21 achieved a sensitivity and specificity of 94.2% and 73.5%, respectively, in differentiating the malignant from benign laryngeal disease, suggesting that the combined evaluation of their serum expression levels may be a valuable biomarker of laryngeal squamous cell carcinoma [328].

Proteins. Exosomal protein signatures have also been used as potent alternative diagnostic markers of cancer. The epidermal growth factor receptor (EGFR) localized to exosome membranes has been found to be a possible marker for lung cancer diagnosis [329]. In this regard, Jakobsen and coworkers reported that the EGFR is highly expressed on the exosomal surface by analyzing the extracellular vesicles secreted by lung cancer cells [330], indicating that the EGFR is a promising biomarker for diagnosing non-small cell lung cancer (NSCLC). The epidermal growth factor receptor variant type III (EGFRvIII) transcript was detected in serum exosomes from 25 spongioblastoma patients but was not found in serum exosomes from 30 normal control individuals. Therefore, exosomal EGFRvIII may provide diagnostic information for glioblastoma patients [270]. Similarly, Graner et al. reported that brain tumor exosomes can escape from the blood–brain barrier, with potential systemic and distal signaling and immune consequences, and that serum exosomes from brain tumor patients possess EGFR, EGFRvIII and TGF-beta [331]. A microfluidic chip was used to analyze exosomal protein types in the blood circulation of spongioblastoma patients. In this regard, Shao and colleagues found that circulating exosomes contain EGFR-VII, EGFR, PDPN and IDH1, which can be used to analyze primary tumor mutations and to indicate drug efficacy [332]. Urinary exosomal proteins have also been investigated as potential biomarkers for prostate and bladder cancers. Nilsson et al. showed that urinary exosomes in prostate cancer patients express prostate-specific antigen (PSA), prostate cancer gene-3 (PCA-3), transmembrane serine protease 2-erythroblast transformation-specific (ETS) transcription factor family member-related gene fusion (TMPRSS2-ERG) and other prostate cancer-related markers, indicating the potential for the diagnosis and monitoring of cancer patients [333]. In this respect, Chen and colleagues found that 24 urinary exosomal proteins

presented at significantly different levels in hernia (control) and bladder cancer patients. In particular, they revealed the strong association of TACSTD2 with bladder cancer and the potential of human urinary exosomes in noninvasive cancer diagnosis [334]. CD24, found in the MVB cytoplasm, is released into the extracellular environment via exosomes and is associated with the poor prognosis of ovarian carcinomas [335]. Logozzi and colleagues found that plasma CD63+ exosome levels are significantly higher in melanoma patients as compared to healthy control individuals [336]. This team recently showed that plasmatic exosomes from prostate cancer patients overexpress carbonic anhydrase IX (CA IX), as well as CA IX-related activity. In addition, CA IX expression correlated with intraluminal acidity in the plasmatic exosomes of these cancer patients [337]. The acidic microenvironment was reported to induce an upregulation of both the expression and activity of CA IX in cancer-derived exosomes, along with an increase in their production levels [338]. Finally, leucine-rich alpha-2-glycoprotein 1 (LRG1) expression levels were found to be higher in the urinary exosomes and lung tissue of NSCLC patients as compared to healthy individuals, indicating that LRG1 may be a candidate biomarker for noninvasive NSCLC diagnosis [309].

Lipids. Exosome lipidomics show great potential for the identification of suitable markers for cancer diagnosis. Recently, using an untargeted high-resolution mass spectrometry approach, our research group identified similarities between structural lipids differentially expressed in cancer stem cell (CSC)-derived exosomes and those derived from patients with malignant melanoma (MM) [339]. Our results showed significant metabolomic differences between exosomes derived from MM CSCs and those from differentiated tumor cells and, also, between serum-derived exosomes from patients with MM (MMPs) and those from healthy controls (HCs). We detected metabolites from different lipid classes, such as glycerophosphoglycerols, glycerophosphoserines, triacylglycerols and glycerophosphocholines. Interestingly, we found that PC 16:0/0:0 glycerophosphocholine expression was lower in both CSCs and MMPs in comparison with differentiated tumor cells and HCs, respectively, while lysophospholipid sphingosine 1-phosphate (S1P) levels were found to be lower in serum-derived exosomes from MMP patients than from HCs. These results indicate the importance of structural lipids detected in exosomes as biomarkers in the early detection of cancer and their potential in the determination of aggressiveness and therapeutic monitoring [339].

#### 4.1.2. Use of Exosomes for Molecular Diagnostics of Neurodegenerative Diseases

Recent evidence indicates the potential involvement of exosomes in the nervous system and highlights their role in transcription regulation, neurogenesis and plasticity [340]. Several central nervous system (CNS) cell types, such as neurons and glial cells, are known to communicate intercellularly by releasing EVs. However, these vesicles could also play a role in the development of neurodegenerative diseases. Parkinson's disease (PD) is a progressive neurodegenerative disorder that mostly affects the motor system. Proteomic profiling was used to differentially identify proteins expressed in serum exosomes from PD patients and healthy individuals [341]. In addition, Fraser and colleagues identified leucine-rich repeat kinase 2 (LRRK2) as a biomarker in urinary exosomes from PD patients that predicts the risk of the development of this disease among LRRK2 mutation carriers [342]. The aggregation of α-synuclein may play an important role in PD pathology. Exosomes have been shown to be able to transfer the α-synuclein protein to neighboring normal cells, thus possibly exacerbating PD pathogenesis [197].

Alzheimer's disease (AD), another neurodegenerative disorder, is now regarded as the most common casue of dementia. The early detection of exosome-associated tau, which is present in human cerebrospinal fluid (CSF) samples and is phosphorylated at Thr-181 (AT270), would be helpful for AD diagnosis [194]. In this regard, the T-tau, Ptau and neurofilament light (NFL) biomarkers could be used to differentiate effectively between AD patients and healthy subjects [343]. Exosomal lipids could also be used as promising biomarkers for AD diagnosis. In this respect, 10 lipids from plasma were able

to predict phenoconversion to AD within a two-to-three-year timeframe with over 90% accuracy [344]. predict phenoconversion to AD within a two-to-three-year timeframe with over 90% accuracy [344].

**Figure 3.** Exosome-associated molecules used for diagnosis and therapy. For instance, epidermal growth factor receptor variant type III (EGFRvIII) is associated with the classical glioblastoma (GBM) subtype [345]. MicroRNA (miRNA)-124 has been reported to enhance the chemosensitivity of GBM cells to temozolomide and to decrease GBM cell migration [346]. In addition, the delivery of miRNA-34a results in the inhibition of GBM cell proliferation, invasion, migration and tumurogenesis both in vivo and in vitro [347]. Lung cancer was also detected using exosomal biomarkers. In this context, Liu et al. found that miRNA-23b/10b-5p/21-5p were good candidates for its diagnosis [348], while Dejima and coworkers considered miRNA-21/4257/451a reliable biomarkers [349,350]. Other miRNAs such as homo sapiens (hsa)-miRNA-320d/320c/320b were suggested as potential biomarkers [351]. On the other hand, exosome miRNA-101/373 serum levels were found to be linked to aggressive breast carcinomas [352]. Other authors recommend miRNA-1246/21/223-3p as potential indicators of breast cancer [353,354]. Therapeutic quantities of doxorubicin (Dox) and cholesterol-modified miRNA 159 (Cho-miRNA-159) were delivered to triple-negative breast cancer (TNBC) cells and exhibited improved anticancer effects [355]. In addition, miRNA-204-5p and miRNA-21 efficiently inhibited cancer cell proliferation and increased chemosensitivity by specifically suppressing their target genes in human colorectal cancer cells [356,357]. Adipose-derived stromal cells (ASCs) were shown to be able to promote prostate cancer cell apoptosis via exosomal miRNA-145 through the caspase-3/7 pathway [358,359]. **Figure 3.** Exosome-associated molecules used for diagnosis and therapy. For instance, epidermal growth factor receptor variant type III (EGFRvIII) is associated with the classical glioblastoma (GBM) subtype [345]. MicroRNA (miRNA)-124 has been reported to enhance the chemosensitivity of GBM cells to temozolomide and to decrease GBM cell migration [346]. In addition, the delivery of miRNA-34a results in the inhibition of GBM cell proliferation, invasion, migration and tumurogenesis both in vivo and in vitro [347]. Lung cancer was also detected using exosomal biomarkers. In this context, Liu et al. found that miRNA-23b/10b-5p/21-5p were good candidates for its diagnosis [348], while Dejima and coworkers considered miRNA-21/4257/451a reliable biomarkers [349,350]. Other miRNAs such as homo sapiens (hsa)-miRNA-320d/320c/320b were suggested as potential biomarkers [351]. On the other hand, exosome miRNA-101/373 serum levels were found to be linked to aggressive breast carcinomas [352]. Other authors recommend miRNA-1246/21/223-3p as potential indicators of breast cancer [353,354]. Therapeutic quantities of doxorubicin (Dox) and cholesterol-modified miRNA 159 (Cho-miRNA-159) were delivered to triple-negative breast cancer (TNBC) cells and exhibited improved anticancer effects [355]. In addition, miRNA-204-5p and miRNA-21 efficiently inhibited cancer cell proliferation and increased chemosensitivity by specifically suppressing their target genes in human colorectal cancer cells [356,357]. Adipose-derived stromal cells (ASCs) were shown to be able to promote prostate cancer cell apoptosis via exosomal miRNA-145 through the caspase-3/7 pathway [358,359].

#### In many studies, exosomes have been used as delivery vectors for small-molecule *4.2. Use of Exosomes as Therapeutic Agents*

*4.2. Use of Exosomes as Therapeutic Agents*

therapeutic agents, as they are capable of traveling from one cell to another and of conveying their cargo in a biologically active form, thus acting as attractive gene and drug delivery vehicles [360]. Cancer cells internalize a significantly larger percentage of exosomes as compared to normal cells. HEK293 and MSC exosomes were therefore effectively used as delivery vectors to transport PLK-1 small interfering RNA (siRNA) to bladder cancer cells in vitro, resulting in the selective gene silencing of PLK1 [361]. In addition, the internalization of exosomes in tumor cells is ten times greater than that of liposomes of comparable size due to their lipid composition and surface proteins, indicating the superior specificity of exosomes for cancer targeting [362]. Furthermore, exosomes offer several advantages over standard delivery vehicles. For example, exosomes are able to cross biological barriers, such as the blood–brain barrier (BBB), have poor immunogenicity and can be cell-specific [363]. Therefore, exosomes could be next-generation nontoxic delivery In many studies, exosomes have been used as delivery vectors for small-molecule therapeutic agents, as they are capable of traveling from one cell to another and of conveying their cargo in a biologically active form, thus acting as attractive gene and drug delivery vehicles [360]. Cancer cells internalize a significantly larger percentage of exosomes as compared to normal cells. HEK293 and MSC exosomes were therefore effectively used as delivery vectors to transport PLK-1 small interfering RNA (siRNA) to bladder cancer cells in vitro, resulting in the selective gene silencing of PLK1 [361]. In addition, the internalization of exosomes in tumor cells is ten times greater than that of liposomes of comparable size due to their lipid composition and surface proteins, indicating the superior specificity of exosomes for cancer targeting [362]. Furthermore, exosomes offer several advantages over standard delivery vehicles. For example, exosomes are able to cross biological barriers, such as the blood–brain barrier (BBB), have poor immunogenicity and

can be cell-specific [363]. Therefore, exosomes could be next-generation nontoxic delivery tools that combine nanoparticle sizes with high capacity levels, making them powerful vectors for the treatment of a variety of pathologies [364].

Doxorubicin-loaded exosomes are transported to tumor tissues and reduce tumor growth in mice without any adverse effects observed from this equipotent free drug [365]. Tian and coworkers used mouse immature dendritic cells (imDCs) for exosome production due to their lack of immunostimulatory markers. Purified imDC-derived exosomes were gently mixed with doxorubicin (DOX) in an electroporation buffer and then examined under a transmission electron microscope to verify the recovery of their plasma membrane. After loading the therapeutic cargo, these vesicles successfully delivered DOX to the targeted cell nucleus, leading to the inhibition of tumor growth without overt toxicity [366]. In another study, exosomes derived from a brain endothelial cell line, bEND.3, were loaded with DOX and used to deliver the anticancer drug across the blood–brain barrier (BBB) for the treatment of brain cancer in a zebrafish model [367]. The membrane vesicles mediated the autonomous intercellular migration of anticancer agents through multiple cancer cell layers and enabled hydrophobic and hydrophilic compounds to significantly penetrate both spheroids and in vivo tumors, thereby enhancing their therapeutic efficacy [368]. Interestingly, chemotherapeutic agents epirubicin and paclitaxel increased miR-503 levels in exosomes released from human umbilical vein endothelial cells (HUVECs) as compared to control conditions and were demonstrated to induce antitumor responses during breast cancer chemotherapy [369].

Exosomes also have the potential to deliver oligonucleotides, such as mRNA, miRNA and various noncoding RNAs, as well as mitochondrial and genomic DNA, to other cells, thus offering considerable advantages as ideal delivery systems for gene therapy [370]. As with the incorporation of genetic material into living cells, Alvarez-Erviti and colleagues used electroporation to deliver short interfering siRNA analogs to the brain in mice via exosomes [363]. In addition, Wahlgren and coworkers used plasma exosomes as gene delivery vectors to transport exogenous siRNA to human blood cells. The vesicles successfully delivered the administered siRNA to monocytes and lymphocytes, leading to robust gene silencing of mitogen-activated protein kinase 1, thus suggesting exosomes as a new generation of drug carriers that enable the development of safe and effective gene therapies [371]. Similarly, Kamerkar et al. demonstrated a technique for the direct and specific targeting of oncogenic KRAS in tumors using electroporated MSC-derived exosomes with siRNA. This treatment suppressed cancer in multiple mouse models of pancreatic cancer and significantly increased overall survival rates [372]. The same method was used to load exosomes with miRNA to the epidermal growth factor receptor (EGFR) expressed in breast cancer cells, indicating that exosomes can be used therapeutically to target EGFR-expressing cancerous tissues with nucleic acid drugs [373]. Finally, endothelial cells treated with chemotherapeutic agents are reported to release more exosomes that contain miRNA-503. Given that miRNA-503 is downregulated in exosomes released from endothelial cells cultured under tumoral conditions, the introduction of miRNA-503 into breast cancer cells altered their proliferative and metastatic capacities by inhibiting both CCND2 and CCND3 [369].

Lee and colleagues demonstrated that exosomes derived from MSCs deliver specific miRNA mimics (miRNA-124 and miRNA-145) and decrease glioma cell migration and the stem cell properties of cancer cells, providing an efficient route of therapeutic miRNA delivery in vivo [374]. In addition, the intratumoral injection of exosomes derived from miRNA-146-expressing MSCs results in a considerable reduction in glioma xenograft development in a rat brain tumor model and decreases cell growth and invasion, suggesting that the export of specific therapeutic miRNA into MSC exosomes represents an effective treatment strategy for malignant glioma [375]. O'Brien and coworkers engineered EVs loaded with miRNA-134, which is substantially downregulated in breast cancer tissue as compared to healthy tissue. It has been demonstrated that miRNA-134-enriched EVs reduce STAT5B and Hsp90 levels in target breast cancer cells, as well as cellular migration

and invasion, and enhance the sensitivity of these cancer cells to anti-Hsp90 drugs [376]. Similarly, MSC-derived exosomes encapsulated with miRNA-379 were administered in breast cancer therapy in vivo. The results of this study show that miRNA-379-enriched EVs are potent tumor suppressors with an exciting potential as an innovative therapy for metastatic breast cancer [377]. Bovy et al. identified miRNA-503, whose expression levels are downregulated in exosomes released from endothelial cells cultured under tumoral conditions. Endothelial cells are able to transfer miRNA-503 via exosomes to breast cancer cells, thus impairing their growth and altering their proliferative capacity [369]. Breast cancer cells prime MSCs to secrete exosomes containing distinct miRNA contents, which promotes quiescence in a subset of cancer cells and confers drug resistance. According to this study, a novel therapeutic approach to target dormant breast cancer cells based on the systemic administration of MSCs loaded with antagomiRNA-222/223 resulted in the chemosensitization of cancer cells and increased survival rates [148].

Shtam et al. introduced two different anti-RAD51 and -RAD52 siRNAs into Henrietta Lacks (HeLa) cell-derived exosomes. These exosomes effectively delivered siRNA into the recipient cancer cells and caused strong RAD51 knockdown, providing additional evidence of the ability to use human exosomes as vectors in cancer therapy [378]. In a similar study, Shimbo and coworkers found that the transfer of miRNA-143 by means of MSC-derived exosomes decreases in the in vitro migration of osteosarcoma cells [379]. In addition, miRNA-122-transfected adipose tissue-derived MSCs (AMSCs) can effectively generate miRNA-122-encapsulated exosomes, which can mediate miRNA-122 communication between AMSCs and hepatocellular carcinoma (HCC) cells, thereby elevating tumor cell sensitivity to chemotherapeutic agents through the alteration of miRNA-122 target gene expression in HCC cells [380]. Usman and colleagues have described a strategy for generating large-scale amounts of exosomes for the delivery of RNA drugs, including antisense oligonucleotides (ASOs). They chose human red blood cells (RBCs), which are devoid of DNA, for EV production. RBC EVs were demonstrated to deliver therapeutic ASOs in order to effectively antagonize oncogenic micro-RNAs (oncomiRNAs) and to suppress tumorigenesis [381]. Exosomes could potentially deliver therapeutic proteins to recipient cells, with a recent study reporting the feasibility of using exosomes as biocompatible vectors that could improve the targeting and delivery of therapeutic proteins to specific cells in diseased tissues [382]. In addition, Haney et al. used a new method to treat Parkinson's disease (PD). In fact, catalase-loaded exosomes produce a potent neuroprotective effect on both in vitro and in mouse brains following intranasal administration. This result demonstrates the capacity of exosomes to load fully functional proteins and to treat specific disorders [383]. Several approaches have envisaged the utilization of specific conserved domains in order to enhance the loading of proteins. For instance, Sterzenbach and colleagues exploited late-domain (L-Domain) proteins and ESCRT machinery pathways to load Cre recombinase into exosomes. This protein was successfully delivered to neurons through a nasal route, a well-characterized noninvasive method to deliver exogenous proteins to the brain via exosomes [384]. Human ubiquitin was also used as a sorting sequence to deliver diverse proteins into exosomes such as EGFP and nHER2. Interestingly, C-terminal–ubiquitin fusion may act as an efficient signal sequence of antigenic proteins into exosomes, which could support the use of exosomes as vaccines [385].

#### **5. Conclusions**

A considerable number of physiological and pathological processes are undoubtedly governed or, at least, modulated by the intervention of exosomes. This places exosomes in a privileged position and optimizes their use as a potential tool in clinical applications for both diagnosis and therapy. Despite groundbreaking improvements, a number of limitations and challenges remain with regards to transforming exosome applications into clinical therapies. Further exploration of the molecular composition and function of exosomes, along with an appropriate cell source for exosome production according to the intended therapeutic use, will undoubtedly enhance the final outcome of any clinical applications

using these membrane vesicles. Taking into account the low biofluid volumes available for diagnosis application, standard and highly effective exosome isolation, purification, characterization and manipulation methods need to be developed to make these vesicles a clinical reality. Furthermore, the loading of exosomes without altering their functional efficacy and the natural characteristics of the donor cell are crucial for oncological research and their development. Finally, with research in exosome biology in its infancy, further studies to evaluate the possible impacts of exosomes in major preclinical models are required to assess the safety/toxicology issues and to ensure their safe and effective use in therapeutic settings.

**Author Contributions:** H.A. and L.M.: manuscript writing, H.A. and K.B.: figure artwork, F.M. and B.B.: manuscript review and J.A.M. and K.B.: manuscript writing and final approval of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was funded by the Spanish ISCIII Health Research Fund and the European Regional Development Fund (FEDER) through research grants PI12/01097, PI15/02015, PI18/00337 (F.M.), PIE16-00045 (J.A.M.), DTS19/00145 (J.A.M.) and PI18/00330 (K.B.). The study was also supported by the Ministry of Science, Innovation and Universities (MICIU, RTI2018-101309-B-C22, FEDER funds), by the Consejería de Economía, Conocimiento, Empresas y Universidad de la Junta de Andalucía (SOMM17/6109/UGR, FEDER Funds), the Chair of "Doctors Galera-Requena in cancer stem cell research" (CMC-CTS963) and the Junta de Andalucia Health and Families Department (CARTPI-0001-201). The CECEyU and CSyF of the Junta de Andalucía FEDER/European Cohesion Fund (FSE) provided the following research grants: 2016000073391-TRA, 2016000073332-TRA, PI-57069 and PAIDI-Bio326 (F.M.) and PI-0014-2016 (K.B.). K.B. was also on a Nicolas Monardes Regional Ministry of Health contract (0006/2018). H.A. held a Research Excellence PhD Fellowship (2UAE2020) from the National Center for Scientific and Technical Research (CNRST). L.M. was supported by the Mohammed VI Polytechnic University of Morocco.

**Institutional Review Board Statement:** Not applicable as this is a review article of the availableliterature and not a research study involving human participants.

**Informed Consent Statement:** Not applicable as this is a review article of the availablelitera-ture and not a research study involving human participants.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding authors.

**Acknowledgments:** Figures were created using Biorender.com. We also wish to thank Michael O'Shea for proofreading the review and María Tristán-Manzano for her help in generating the figures.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results.

#### **References**

