Note：All the information in this draft can be edited by authors. And the entry will be online only after authors edit and submit it.

1. Introduction

Extracellular vesicles (EVs) have emerged as a new class of nanocarriers, triggering significant interest and enthusiasm. Extraordinary efforts have been made to develop new techniques that would make it possible to manufacture EV-based drug formulations for the treatment of various diseases, including cardiovascular diseases ^{[1][2][3][4][5][6]}[1–6], regenerative disorders ^[7][8][7,8], infectious diseases [9], cancer ^{[10][11][12][13][14][15][16][17][18][19]}[10–19], as well as autoimmune [20] and neurological disorders ^[17][21][17,21]. EVs are short- and long-distance mediators of intercellular communication that offer distinct advantages, uniquely positioning them as highly effective drug nanocarriers. They comprise various types of nanovesicles, including exosomes (30–120 nm), microvesicles (MVs) (50 nm–1μm), and apoptotic bodies (500–1000 nm) ^[22][23][24][22–24]. Notably, EVs consist of cellular membranes with multiple adhesive proteins on their surface ^[25][26][25,26] that enable efficient cell entry and delivery of therapeutic cargo.

The unique properties of EVs can be attributed to their biogenesis. Exosomes are initially produced by invagination of the endosomal membrane to create multivesicular bodies (MVB) [27]. In contrast, exosomes’ close relatives, MVs, are greater in size and bud directly from the plasma membrane. Therefore, exosomes and MVs originate from endosomal and plasma membranes, respectively. Apoptotic bodies form during the apoptotic process, when the cellular cytoskeleton breaks up, causing the membrane to bulge outward [28]. Different techniques have been developed for the characterization of EVs. Among them are nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS), that provide information about: count (NTA) and size distribution (NTA and DLS); flow cytometry, western blotting, and mass spectrometry (MS) that can be used to characterize biochemical content of EVs; and several microscopy techniques Atomic Force Microscopy (AFM) and Cryogenic Transmission Electron Microscopy (CryTEM) that make it possible to assess EV morphology [29]. The structure, biogenesis and composition of EVs have been extensively described in several excellent reviews ^{[7][18][21][30][31][32][33][34][35][36]}[7,18,21,30–36].

Similar to artificial nanocarriers, EVs can improve the fundamental characteristics of a free drug, such as its stability and solubility, and protect the drug against degradation in the bloodstream [31]. Relatively tight lipid bilayers in EV membranes can provide a sustained and prolonged release of the incorporated drug. Furthermore, contrary to most synthetic nanocarriers, EVs can cross biological barriers, including the blood brain barrier (BBB), making them especially valuable for the treatment of neurodegenerative disorders. It has been shown that EVs can cross the BBB from the brain to the bloodstream ^[37][38][37,38], as well as from blood to the CNS in vitro ^[39][40][39,40] and in vivo ^[41][42][43][41–43] under pathological conditions. However, whether EVs cross the BBB in the absence of pathology is still debated. Furthermore, these natural nanocarriers have low immunogenicity (especially, autologous EVs) and low cytotoxicity, which are usually substantial impediments for conventional synthetic nanoparticles. Finally, some types of EVs exert tissue tropism that makes it possible to target their formulations to specific cell types or migration towards inflamed tissues ^[44][45][44,45]. It is worth mentioning that bioinspired nanocarriers may have unique biological activity which is reflective of their origin, i.e., parent cells, that provides additional therapeutic efficacy to the incorporated drug [43]. These attractive features have contributed to the growing interest in EVs and inspired numerous studies aimed at their introduction to the field of drug delivery.

Despite these advantages, the clinical translation of EVs has been greatly slowed down due to a number of drawbacks, including upscaling processes of isolation and purification, as well as the lack of a means of efficiently loading these natural nanovesicles with therapeutics. Reliability, reproducibility, and donor-donor variations of EV formulations are still of significant concern. Furthermore, EV functional heterogenicity and limited yields represent serious obstacles for their future applications. Thus, depending on the mechanism of EV release, they may contain different proteins, active proteasomes, and even organelles (e.g., mitochondria) [46]. Inadequate targeting is another challenge for the clinical translation of different EV-based drug formulations. Herein, we will discuss how these hurdles can be overcome to introduce this unique biomimetic drug delivery system to the clinic.

2. Implications Related to Biological Activity Inherited from EVs Origin

The biological activity of EVs released by various types of cells is vast and promising. Their ability to impact cells depends largely upon their protein markers and their cargo, which mimic the properties of their origin. Isolated EVs taken directly from specific types of cells, such as fibroblasts, neuronal cells, macrophages, and even cancer cells have a wide array of both pathogenic and therapeutic activities, largely depending their host cells. Therefore, one should pay special attention to the source of EVs and possible unwanted biological activity inherited from their parent cells. For example, EVs derived from diseased cells may contribute to the ability of a pathogen to spread throughout the body and evade the immune system [47]. Tumor-derived EVs are well-documented to express specific immune system markers such as MHC Class I and II molecules, death receptor ligands (FasL) and many others. The expression of these markers enables EVs to interact directly with prominent immune system cells such as T cells, B cells, and NK cells to encourage oncogenic activity and inhibit the immune system processes. Melanoma-derived EVs express FasL, which activates the Fas/FasL pathway to induce lymphocyte apoptosis, allowing tumors to evade cell-mediated cell death [47]. Next, EVs may contain prominent mediators that encourage angiogenic activity, metastasis, and mRNA transfer, leading to growth within the tumor microenvironment. Thus, gliomas, i.e., human brain and spinal cord tumors, express an oncogenic form of the epidermal growth factor receptor, EGFRvIII [48]. In mice, EVs containing EGFRvIII were shown to be released into the blood and fuse with tumor cells lacking EGFRvIII, conferring oncogenic activity upon previously benign cells. Moreover, cancer cell-derived EVs were shown to transport oncoproteins, including antigen MelanA/Mart-1 (melanoma), carcinoembryogenic antigen (CEA) (colon carcinoma), and HER2 (breast cancer) [49]. Finally, EVs can carry cancer-related miRNAs. Specifically, large amounts of small RNAs such as let-7, miR-1, miR-15, miR-16 and miR-375, which play an important role in cancer, were found in EVs [50]. Furthermore, Li at al. [51] studied the mechanism underlying the association between EVs and hypoxia during cancer progression. It was suggested that cancer cell-derived EVs mediate miRNA transfer and promote prometastatic behavior. Thus, oral squamous cell carcinoma (OSCC) cells secreted miR-21-rich EVs that ultimately contributed to the migration and invasion of OSCC cells [52]. In addition, miR-29a-3p carried by EVs from OSCC cells promoted M2-type macrophages polarization, and such macrophages enhanced the proliferation and migration of OSCC cells [53]. Hence, in many cases, it is preferable to use “clean” EVs without interior content that would not induce unwanted effects in patients. One approach to achieve this is to develop methods for the removal of the cargo of naive EVs without significant changes of the structure and content of their membranes. For example, Jang et al. [54] suggested using exosome-mimetic nanovesicles produced by the breakdown of monocytes via a serial extrusion through filters. These cell-derived nanovesicles should be depleted of their internal content inherited from parent cells.

Interestingly, EVs released by mesenchymal stem cells (MSCs) may deliver a bioactive cargo that inhibits or promotes tumor growth ^[55][56][57][55–57]. Thus, some studies indicated that MSC-derived EVs can play several roles in tumorigenesis, angiogenesis, and metastasis ^[42][58][42,58], although other studies showed tumor-suppressing effects ^{[59][60][61][62]}[59–62]. These inconsistencies may be attributed to the source of parent MSCs, specifically, whether MSCs were obtained from cancer patients or healthy individuals [63]. Accordingly, nonmodified EVs may possess specific properties that would be beneficial to their therapeutic outcomes. For example, MSC-derived EVs have received much attention as potential therapeutic agents with regenerative properties ^{[64][65][66][67][68][69][70]}[64–70], including protective effects in models of myocardial ischemia/reperfusion injury ^[65][66][71][65,66,71], pulmonary vascular disease [72], chronic myocardial infarction [73], and stroke ^{[68][74][75][76]}[68,74–76]. Furthermore, EVs released by neural stem cells (NSCs) are known to promote neural tissue regeneration and functional recovery by releasing paracrine factors. In a recent report, Zhang et al. [77] demonstrated that the treatment of parent NSCs with interferon-gamma (IFN-γ) induced a generation of altered EVs that exerted improved therapeutic effects in an ischemic stroke rat model. Likewise, EVs derived from NSCs were shown to preserve and restore photoreceptors, decreasing apoptosis during retinal degeneration in rats [78]. Finally, EVs, particularly those produced by immune cells, are known to have immune-modulating, protective, and regenerative effects in conditions such as cardiovascular disease, atherosclerosis, and stroke [79]. Obviously, this additional biological activity may improve the therapeutic outcomes of drug-loaded formulations and should be considered when bio-inspired formulations are developed. For example, our earlier investigations demonstrated that naive EVs released by regenerative anti-inflammatory subtype of M2 macrophages produced synergistic neuroprotective effects in mouse models of Parkinson’s disease [43]. These effects were subtle but could be beneficial when added to the effects of incorporated therapeutics. Overall, these developments indicate that EVs can implement more than only inert carrier functions by being biological response modifiers. Further tailoring EVs may provide biologically active carriers that may be modified in accordance with the disease and produce, for example, the cytotoxic effects of EVs released by M1 macrophages for cancer treatment, or the neuroprotective effects of EVs released by M2 macrophages for the treatment of neurodegenerative disorders, and enhance the outcomes of their therapeutic cargo.

3. Improving Functional Heterogenicity and Yields of EVs Nanocarriers

EVs consist of various types of nanovesicles, namely exosomes, MVs, and apoptotic bodies ^[22][23][24][22–24]. It has shown to be difficult to separate EVs and MVs, mainly due to overlapping vesicle sizes and proteins expressed on their surface. Therefore, in most cases, a mixture of EVs and MVs is used to produce drug formulations [24]. It should also be noted that the absolute separation and definition of various EVs based on their size or biogenesis has yet to be established beyond doubt, and there is currently no consensus on markers that distinguish the origin of these vesicles once they have left the cell [80].

EVs can be isolated from conditioned cell culture media or bodily fluids by different methods, including differential centrifugation, filtration paired with centrifugation, concentration paired with ultracentrifugation, immunoaffinity chromatography, size exclusion chromatography, and polymer-based precipitation. Each isolation technique has advantages and disadvantages that should be considered in terms of being reproducible, specific, and feasible [81]. Differential ultracentrifugation combined with density gradient centrifugation are considered the “gold standard” for isolating EVs. This process involves applying a centrifugal force to a solution containing EVs, e.g., a conditioned cell culture media or biological fluids. It is worth noting that the type, quantity, and quality of EVs isolated by this method is sensitive to the g force, rotor type, angle of rotor sedimentation, radius of centrifugal force, pelleting efficiency, and solution viscosity. Gradient centrifugation requires extensive (62–90h) centrifugation time [82], but provides a more uncontaminated EV isolate than ultracentrifugation alone. Of note, ultracentrifugation is associated with morphological alterations and partial aggregation of vesicles. Immunoaffinity chromatography is a more efficient method for isolating EVs as compared to differential ultracentrifugation and density gradient ultracentrifugation [81]. It requires a single easy step without using harsh chemicals. However, this method provides a relatively low yield and can be used for small volumes only. In addition, because this method of EV isolation depends on antibody recognition of EV proteins, only a subset of all EVs (those expressing the antibody-recognized protein) can be captured. Size exclusion chromatography (SEC) preserves the integrity and biological activity of EVs using gravity flow when vesicle structure and integrity remain intact. This is a fast and easy procedure that requires a small sample volume. However, a low concentrated sample needs an additional process for enrichment. Finally, polymer precipitation is relatively easy to use and does not require specialized equipment or a lengthy run time. However, it has been shown that this method coprecipitates nonvesicular contaminants such as lipoproteins, as well as polymer material [83]. Thus, Patel et al. [84] compared four EV isolation techniques for yield and purity. The polymer-based precipitation method had the maximum yield, followed by size-exclusion chromatography and differential ultracentrifugation. The immunoaffinity-based isolation method yielded the fewest EVs. Importantly, a high yield of EVs was accompanied by contaminations with serum proteins and chemical impurities, including high salt concentration, Sodium Dodecyl Sulfate (SDS), or Polyethylene glycol (PEG) contaminations after polymer-based precipitation. These issues may be addressed by pre- and post- isolation steps. Pre-isolation involves the removal of subcellular particles such as lipoproteins. Post-isolation involves removal of the polymer, typically by using a Sephadex G-25 column [82]. Therefore, considering large-scale clinical manufacturing [85], a level of segregation EVs from copurifying components may influence the functionality and therapeutic activity of the final product.

The translation of EV-based formulations into clinical practice requires compliance with existing regulatory frameworks [86]. EVs are a fairly heterogeneous population in terms of their biochemical composition, size, and the source [87]. Thus, the standardization and effective purification of large amounts of these nanovesicles is a critical, but still considerable, challenge. Specifically, the manufacturing of homogeneous drug nanoformulations, and production and quality control, are crucial requirements. Moleirinho et al. [88] developed a purification method using semicontinuous multicolumn chromatography, a robust, scalable and efficient tool for EV purification. Besides the higher recoveries obtained with the continuous system when comparing with batch chromatography, the EV properties were maintained during the purification process regarding their size and morphology. A fast and reliable method of isolating serum EVs was reported by Navajas et al. [89]. Using size-exclusion chromatography with qEV columns (Izon, Christchurch, New Zealand), a homogeneous population of EVs in terms of size, morphology, and protein composition was obtained.

Another challenge that has critical implications for the use of EV-based formulations is whether the sufficient number of these carriers can be generated [90]. Indeed, the EV yield per cell will impact the final production cost, as well as having clinical applications. In this respect, the choice of parent cells is very important. For example, MSCs are known to produce large numbers of EVs, suggesting that these cells may be efficient for EV production in a clinically applicable scale ^[65][91][65,91]. Several reports have indicated that specific treatments of EV producing cells could considerably increase the yield of these natural nanocarriers. For example, culturing dendritic cells (DCs) for a prolonged time [92] or at low pH [93] increased EV production up to ten-fold. Furthermore, the addition of neutral and cationic-bare liposomes enhanced EV secretion in a dose-dependent manner [94]. However, the possible contamination of EVs with liposomes is a serious concern associated with this method. Gao et al. [95] reported high yield of EVs using nitrogen cavitation that instantly disrupted neutrophils to form nanosized membrane vesicles. The authors indicated that this approach made it possible to increase the manufacture of EVs by 16-fold. Another option is to break parent cells, for example, monocytes/macrophages with simultaneous loading with anticancer agents, followed by the isolation of EV-like nanoparticles [54]. In attempting to mimic the function of EVs with nanovesicles, Jang et al. [54] utilized human U937 monocytic cells to produce nanovesicles with the ability to carry large amounts of therapeutics. While maintaining the plasma membrane proteins of the targeted cells, the drug-loaded nanovesicles were able to efficiently induce tumor cell death and increase the production yield of chemotherapeutics in relation to naturally occurring EVs by 100-fold. Of note, one should consider that the alteration of cell culture conditions can certainly increase yield, but the impact on the biological effect of EVs has to be crucially assessed for biosafety reasons.

Next, the mass production of EVs by membrane fusion with lipid-based materials was suggested in several reports ^[96][97][98][96–98]. The manufacture of large quantities of drug nanocarriers was achieved via a membrane extrusion technique [96] that allowed up to a 43-fold increase in the numbers of vesicles postisolation. The production of hybrid EVs was also proposed by Rayamajhi et al. [97]. EVs from mouse macrophages were hybridized with synthetic liposomes that increased the yield and retention of the EV functional properties. The manufacture of hybrid EVs was also suggested by De La Peña et al. [98]. This group utilized coated liposomes as artificial EVs, and discovered that the obtained nanocarriers functioned as naturally occurring EVs and efficiently activated immune responses [98]. Chemically-induced membrane blebbing was suggested for the fast production of large numbers of EV-like vesicles [99]. Different chemical agents, for example, sulfhydryl, paraformaldehyde, or dithiothreitol, were shown to lock the cell in a fixed physiological state and promote the release of vesicles from a plasma membrane to the conditioned media.

A different approach for the upscaling production of EVs was reported in a study conducted by Li et al. [100]. Instead of manufacturing EVs from animal cells, the authors utilized biocompatible bovine milk EVs (mEVs) that can be obtained inexpensively in large quantities [100]. mEVs were loaded with doxorubicin (Dox) and decorated with hyaluronan (HA), in order to direct them to CD44-overexpressing tumor cells. HA is a CD44-specific ligand which ensures that the EVs are directed to the cell membrane of the specified tumor cells. mEVs were able to deliver chemotherapeutics to tumor-specific cells in vitro and trigger apoptosis [100]. Crashed grapes were also suggested as an abounded source for EV-like nanoparticles [101]. Thus, the oral administration of EV-like nanovesicles from grapes facilitated intestinal regeneration in a mouse model of colitis that was induced by exposing mice to dextran sodium sulfate in drinking water. The EV-like nanovesicles prevented the colitis-associated reduction of both intestinal length and villus height. As a result, mice treated with grape-derived EV-like nanoparticles lived twice as long as untreated mice.

References

1. Chong, S.Y.; Lee, C.K.; Huang, C.; Ou, Y.H.; Charles, C.J.; Richards, A.M.; Neupane, Y.R.; Pavon, M.V.; Zharkova, O.; Pastorin, G.; et al. Extracellular Vesicles in Cardiovascular Diseases: Alternative Biomarker Sources, Therapeutic Agents, and Drug Delivery Carriers. J. Mol. Sci. 2019, 20, 3272.
2. Boulanger, C.M.; Loyer, X.; Rautou, P.E.; Amabile, N. Extracellular vesicles in coronary artery disease. Rev. Cardiol. 2017, 14, 259–272.
3. Zamani, P.; Fereydouni, N.; Butler, A.E.; Navashenaq, J.G.; Sahebkar, A. The therapeutic and diagnostic role of exosomes in cardiovascular diseases. Trends Cardiovasc. Med. 2019, 29, 313–323.
4. Jia, G.; Sowers, J.R. Targeting endothelial exosomes for the prevention of cardiovascular disease. Biophys. Acta Mol. Basis Dis. 2020, 1866, 165833.
5. Tikhomirov, R.; Donnell, B.R.; Catapano, F.; Faggian, G.; Gorelik, J.; Martelli, F.; Emanueli, C. Exosomes: From Potential Culprits to New Therapeutic Promise in the Setting of Cardiac Fibrosis. Cells 2020, 9, 592.
6. Wei, J.; Hollabaugh, C.; Miller, J.; Geiger, P.C.; Flynn, B.C. Molecular Cardioprotection and the Role of Exosomes: The Future Is Not Far Away. Cardiothorac. Vasc. Anesth. 2020, doi:10.1053/j.jvca.2020.05.033.
7. Ramasubramanian, L.; Kumar, P.; Wang, A. Engineering Extracellular Vesicles as Nanotherapeutics for Regenerative Medicine. Biomolecules 2019, 10,
8. Akbari, A.; Jabbari, N.; Sharifi, R.; Ahmadi, M.; Vahhabi, A.; Seyedzadeh, S.J.; Nawaz, M.; Szafert, S.; Mahmoodi, M.; Jabbari, E.; et al. Free and hydrogel encapsulated exosome-based therapies in regenerative medicine. Life Sci. 2020, 249, 117447.
9. Kumar, S.; Zhi, K.; Mukherji, A.; Gerth, K. Repurposing Antiviral Protease Inhibitors Using Extracellular Vesicles for Potential Therapy of COVID-19. Viruses 2020, 12, 486.
10. Bell, B.M.; Kirk, I.D.; Hiltbrunner, S.; Gabrielsson, S.; Bultema, J.J. Designer exosomes as next-generation cancer immunotherapy. Nanomedicine 2016, 12, 163–169.
11. Moore, C.; Kosgodage, U.; Lange, S.; Inal, J.M. The emerging role of exosome and microvesicle- (EMV-) based cancer therapeutics and immunotherapy. J. Cancer 2017, 141, 428–436.
12. Masaoutis, C.; Mihailidou, C.; Tsourouflis, G.; Theocharis, S. Exosomes in lung cancer diagnosis and treatment. From the translating research into future clinical practice. Biochimie 2018, 151, 27–36.
13. Liu, C.; Su, C. Design strategies and application progress of therapeutic exosomes. Theranostics 2019, 9, 1015–1028.
14. Xie, X.; Wu, H.; Li, M.; Chen, X.; Xu, X.; Ni, W.; Lu, C.; Ni, R.; Bao, B.; Xiao, M. Progress in the application of exosomes as therapeutic vectors in tumor-targeted therapy. Cytotherapy 2019, 21, 509–524.
15. D’Agnano, I.; Berardi, A.C. Extracellular Vesicles, A Possible Theranostic Platform Strategy for Hepatocellular Carcinoma-An Overview. Cancers 2020, 12, 261.
16. Scavo, M.P.; Depalo, N.; Tutino, V.; de Nunzio, V.; Ingrosso, C.; Rizzi, F.; Notarnicola, M.; Curri, M.L.; Giannelli, G. Exosomes for Diagnosis and Therapy in Gastrointestinal Cancers. J. Mol. Sci. 2020, 21, 367.
17. Chung, I.M.; Rajakumar, G.; Venkidasamy, B.; Subramanian, U.; Thiruvengadam, M. Exosomes: Current use and future applications. Chim. Acta 2020, 500, 226–232.
18. Jurj, A.; Zanoaga, O.; Braicu, C.; Lazar, V.; Tomuleasa, C.; Irimie, A.; Berindan-Neagoe, I. A Comprehensive Picture of Extracellular Vesicles and Their Contents. Molecular Transfer to Cancer Cells. Cancers 2020, 12, 298.
19. Tran, P.H.; Xiang, D.; Nguyen, T.N.; Tran, T.T.; Chen, Q.; Yin, W.; Zhang, Y.; Kong, L.; Duan, A.; Chen, K.; et al. Aptamer-guided extracellular vesicle theranostics in oncology. Theranostics 2020, 10, 3849–3866.
20. Sousa, C.; Pereira, I.; Santos, A.C.; Carbone, C.; Kovacevic, A.B.; Silva, A.M.; Souto, E.B. Targeting dendritic cells for the treatment of autoimmune disorders. Colloids Surf. B Biointerfaces 2017, 158, 237–248.
21. Ha, D.; Yang, N.; Nadithe, V. Exosomes as therapeutic drug carriers and delivery vehicles across biological membranes: Current perspectives and future challenges. Acta Pharm. Sin. B 2016, 6, 287–296.
22. Vader, P.; Mol, E.A.; Pasterkamp, G.; Schiffelers, R.M. Extracellular vesicles for drug delivery. Drug Deliv. Rev. 2016, 106, 148–156.
23. Villa, F.; Quarto, R.; Tasso, R. Extracellular Vesicles as Natural, Safe and Efficient Drug Delivery Systems. Pharmaceutics 2019, 11, 557.
24. Batrakova, E.V.; Kim, M.S. Using exosomes, naturally-equipped nanocarriers, for drug delivery. Control. Release 2015, 219, 396–405.
25. Thery, C.; Amigorena, S.; Raposo, G.; Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Protoc. Cell Biol. 2006, 30, 3–22.
26. Thery, C.; Ostrowski, M.; Segura, E. Membrane vesicles as conveyors of immune responses. Rev. Immunol. 2009, 9, 581–593.
27. Witwer, K.W.; Thery, C. Extracellular vesicles or exosomes? On primacy, precision, and popularity influencing a choice of nomenclature. J. Extracell. Vesicles 2019, 8, 1648167.
28. Liu, D.; Kou, X.; Chen, C.; Liu, S.; Liu, Y.; Yu, W.; Yu, T.; Yang, R.; Wang, R.; Zhou, Y.; et al. Circulating apoptotic bodies maintain mesenchymal stem cell homeostasis and ameliorate osteopenia via transferring multiple cellular factors. Cell Res. 2018, 28, 918–933.
29. Van der Pol, E.; Coumans, F.A.; Grootemaat, A.E.; Gardiner, C.; Sargent, I.L.; Harrison, P.; Sturk, A.; van Leeuwen, T.G.; Nieuwland, R. Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J. Thromb. Haemost. 2014, 12, 1182–1192.
30. Vlassov, A.V.; Magdaleno, S.; Setterquist, R.; Conrad, R. Exosomes: Current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biophys. Acta 2012, 1820, 940–948.
31. Turturici, G.; Tinnirello, R.; Sconzo, G.; Geraci, F. Extracellular membrane vesicles as a mechanism of cell-to-cell communication: Advantages and disadvantages. J. Physiol. Cell Physiol. 2014, 306, C621–C633.
32. Aryani, A.; Denecke, B. Exosomes as a Nanodelivery System: A Key to the Future of Neuromedicine? Neurobiol. 2016, 53, 818–834.
33. Anand, S.; Samuel, M.; Kumar, S.; Mathivanan, S. Ticket to a bubble ride: Cargo sorting into exosomes and extracellular vesicles. Biophys. Acta Proteins Proteom. 2019, 1867, 140203.
34. Elsharkasy, O.M.; Nordin, J.Z.; Hagey, D.W.; de Jong, O.G.; Schiffelers, R.M.; Andaloussi, S.E.; Vader, P. Extracellular vesicles as drug delivery systems: Why and how? Drug Deliv. Rev. 2020, doi:10.1016/j.addr.2020.04.004.
35. Tschuschke, M.; Kocherova, I.; Bryja, A.; Mozdziak, P.; Volponi, A.A.; Janowicz, K.; Sibiak, R.; Piotrowska-Kempisty, H.; Izycki, D.; Bukowska, D.; et al. Inclusion Biogenesis, Methods of Isolation and Clinical Application of Human Cellular Exosomes. Clin. Med. 2020, 9, 436.
36. Dai, H.I.; Vugmeyster, Y.; Mangal, N. Characterizing Exposure-Response Relationship for Therapeutic Monoclonal Antibodies in Immuno-Oncology and Beyond: Challenges, Perspectives, and Prospects. Pharmacol. Ther. 2020, 108, 1156–1170.
37. Matsumoto, J.; Stewart, T.; Sheng, L.; Li, N.; Bullock, K.; Song, N.; Shi, M.; Banks, W.A.; Zhang, J. Transmission of alpha-synuclein-containing erythrocyte-derived extracellular vesicles across the blood-brain barrier via adsorptive mediated transcytosis: Another mechanism for initiation and progression of Parkinson’s disease? Acta Neuropathol. Commun. 2017, 5, 71.
38. Winston, C.N.; Goetzl, E.J.; Akers, J.C.; Carter, B.S.; Rockenstein, E.M.; Galasko, D.; Masliah, E.; Rissman, R.A. Prediction of conversion from mild cognitive impairment to dementia with neuronally derived blood exosome protein profile. Alzheimers Dement. Diagn. Assess. Dis. Monit. 2016, 3, 63–72.
39. Chen, C.C.; Liu, L.; Ma, F.; Wong, C.W.; Guo, X.E.; Chacko, J.V.; Farhoodi, H.P.; Zhang, S.X.; Zimak, J.; Segaliny, A.; et al. Elucidation of Exosome Migration across the Blood-Brain Barrier Model In Vitro. Mol. Bioeng. 2016, 9, 509–529.
40. Kuroda, H.; Tachikawa, M.; Yagi, Y.; Umetsu, M.; Nurdin, A.; Miyauchi, E.; Watanabe, M.; Uchida, Y.; Terasaki, T. Cluster of Differentiation 46 Is the Major Receptor in Human Blood-Brain Barrier Endothelial Cells for Uptake of Exosomes Derived from Brain-Metastatic Melanoma Cells (SK-Mel-28). Pharm. 2019, 16, 292–304.
41. Sun, D.; Zhuang, X.; Xiang, X.; Liu, Y.; Zhang, S.; Liu, C.; Barnes, S.; Grizzle, W.; Miller, D.; Zhang, H.G. A novel nanoparticle drug delivery system: The anti-inflammatory activity of curcumin is enhanced when encapsulated in exosomes. Ther. 2010, 18, 1606–1614.
42. Yang, T.; Martin, P.; Fogarty, B.; Brown, A.; Schurman, K.; Phipps, R.; Yin, V.P.; Lockman, P.; Bai, S. Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio. Res. 2015, 32, 2003–2014.
43. Haney, M.J.; Klyachko, N.L.; Zhao, Y.; Gupta, R.; Plotnikova, E.G.; He, Z.; Patel, T.; Piroyan, A.; Sokolsky, M.; Kabanov, A.V.; et al. Exosomes as drug delivery vehicles for Parkinson’s disease therapy. Control. Release 2015, 207, 18–30.
44. Haney, M.J.; Klyachko, N.L.; Harrison, E.B.; Zhao, Y.; Kabanov, A.V.; Batrakova, E.V. TPP1 Delivery to Lysosomes with Extracellular Vesicles and their Enhanced Brain Distribution in the Animal Model of Batten Disease. Adv. Healthc. Mater. 2019, 8, e1801271.
45. Yuan, D.; Zhao, Y.; Banks, W.A.; Bullock, K.M.; Haney, M.; Batrakova, E.; Kabanov, A.V. Macrophage exosomes as natural nanocarriers for protein delivery to inflamed brain. Biomaterials 2017, 142, 1–12.
46. Marcoux, G.; Duchez, A.C.; Cloutier, N.; Provost, P.; Nigrovic, P.A.; Boilard, E. Revealing the diversity of extracellular vesicles using high-dimensional flow cytometry analyses. Rep. 2016, 6, 35928.
47. Van der Pol, E.; Boing, A.N.; Harrison, P.; Sturk, A.; Nieuwland, R. Classification, functions, and clinical relevance of extracellular vesicles. Rev. 2012, 64, 676–705.
48. Al-Nedawi, K.; Meehan, B.; Micallef, J.; Lhotak, V.; May, L.; Guha, A.; Rak, J. Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Cell Biol. 2008, 10, 619–624.
49. Andre, F.; Schartz, N.E.; Movassagh, M.; Flament, C.; Pautier, P.; Morice, P.; Pomel, C.; Lhomme, C.; Escudier, B.; le Chevalier, T.; et al. Malignant effusions and immunogenic tumour-derived exosomes. Lancet 2002, 360, 295–305.
50. HValadi; Ekstrom, K.; Bossios, A.; Sjostrand, M.; Lee, J.J.; Lotvall, J.O. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Cell Biol. 2007, 9, 654–659.
51. Li, L.; Li, C.; Wang, S.; Wang, Z.; Jiang, J.; Wang, W.; Li, X.; Chen, J.; Liu, K.; Li, C.; et al. Exosomes Derived from Hypoxic Oral Squamous Cell Carcinoma Cells Deliver miR-21 to Normoxic Cells to Elicit a Prometastatic Phenotype. Cancer Res. 2016, 76, 1770–1780.
52. Kogure, T.; Lin, W.L.; Yan, I.K.; Braconi, C.; Patel, T. Intercellular nanovesicle-mediated microRNA transfer: A mechanism of environmental modulation of hepatocellular cancer cell growth. Hepatology 2011, 54, 1237–1248.
53. Cai, J.; Qiao, B.; Gao, N.; Lin, N.; He, W. Oral squamous cell carcinoma-derived exosomes promote M2 subtype macrophage polarization mediated by exosome-enclosed miR-29a-3p. J. Physiol. Cell Physiol. 2019, 316, C731–C740.
54. Jang, S.C.; Kim, O.Y.; Yoon, C.M.; Choi, D.S.; Roh, T.Y.; Park, J.; Nilsson, J.; Lotvall, J.; Kim, Y.K.; Gho, Y.S. Bioinspired exosome-mimetic nanovesicles for targeted delivery of chemotherapeutics to malignant tumors. ACS Nano 2013, 7, 7698–7710.
55. Coffman, L.G.; Choi, Y.J.; McLean, K.; Allen, B.L.; di Magliano, M.P.; Buckanovich, R.J. Human carcinoma-associated mesenchymal stem cells promote ovarian cancer chemotherapy resistance via a BMP4/HH signaling loop. Oncotarget 2016, 7, 6916–6932.
56. Melzer, C.; von der Ohe, J.; Hass, R. Concise Review: Crosstalk of Mesenchymal Stroma/Stem-Like Cells with Cancer Cells Provides Therapeutic Potential. Stem Cells 2018, 36, 951–968.
57. Vakhshiteh, F.; Atyabi, F.; Ostad, S.N. Mesenchymal stem cell exosomes: A two-edged sword in cancer therapy. J. Nanomed. 2019, 14, 2847–2859.
58. Qi, J.; Zhou, Y.; Jiao, Z.; Wang, X.; Zhao, Y.; Li, Y.; Chen, H.; Yang, L.; Zhu, H.; Li, Y. Exosomes Derived from Human Bone Marrow Mesenchymal Stem Cells Promote Tumor Growth Through Hedgehog Signaling Pathway. Physiol. Biochem. 2017, 42, 2242–2254.
59. Wu, S.; Ju, G.Q.; Du, T.; Zhu, Y.J.; Liu, G.H. Microvesicles derived from human umbilical cord Wharton’s jelly mesenchymal stem cells attenuate bladder tumor cell growth in vitro and in vivo. PLoS ONE 2013, 8, e61366.
60. Bruno, S.; Camussi, G. Role of mesenchymal stem cell-derived microvesicles in tissue repair. Nephrol. 2013, 28, 2249–2254.
61. Bruno, S.; Collino, F.; Deregibus, M.C.; Grange, C.; Tetta, C.; Camussi, G. Microvesicles derived from human bone marrow mesenchymal stem cells inhibit tumor growth. Stem Cells Dev. 2013, 22, 758–771.
62. Takahara, K.; Ii, M.; Inamoto, T.; Nakagawa, T.; Ibuki, N.; Yoshikawa, Y.; Tsujino, T.; Uchimoto, T.; Saito, K.; Takai, T.; et al. microRNA-145 Mediates the Inhibitory Effect of Adipose Tissue-Derived Stromal Cells on Prostate Cancer. Stem Cells Dev. 2016, 25, 1290–1298.
63. Roccaro, A.M.; Sacco, A.; Maiso, P.; Azab, A.K.; Tai, Y.T.; Reagan, M.; Azab, F.; Flores, L.M.; Campigotto, F.; Weller, E.; et al. BM mesenchymal stromal cell-derived exosomes facilitate multiple myeloma progression. J. Clin. Investig. 2013, 123, 1542–1555.
64. Lee, C.; Mitsialis, S.A.; Aslam, M.; Vitali, S.H.; Vergadi, E.; Konstantinou, G.; Sdrimas, K.; Fernandez-Gonzalez, A.; Kourembanas, S. Exosomes mediate the cytoprotective action of mesenchymal stromal cells on hypoxia-induced pulmonary hypertension. Circulation 2012, 126, 2601–2611.
65. Lai, R.C.; Yeo, R.W.; Tan, K.H.; Lim, S.K. Exosomes for drug delivery—A novel application for the mesenchymal stem cell. Adv. 2013, 31, 543–551.
66. FArslan; Lai, R.C.; Smeets, M.B.; Akeroyd, L.; Choo, A.; Aguor, E.N.; Timmers, L.; van Rijen, H.V.; Doevendans, P.A.; Pasterkamp, G.; Lim, S.K.; et al. Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury. Stem Cell Res. 2013, 10, 301–312.
67. Katsuda, T.; Tsuchiya, R.; Kosaka, N.; Yoshioka, Y.; Takagaki, K.; Oki, K.; Takeshita, F.; Sakai, Y.; Kuroda, M.; Ochiya, T. Human adipose tissue-derived mesenchymal stem cells secrete functional neprilysin-bound exosomes. Rep. 2013, 3, 1197.
68. Xin, H.; Li, Y.; Chopp, M. Exosomes/miRNAs as mediating cell-based therapy of stroke. Cell. Neurosci. 2014, 8, 377.
69. Ilmer, M.; Vykoukal, J.; Boiles, A.R.; Coleman, M.; Alt, E. Two sides of the same coin: Stem cells in cancer and regenerative medicine. FASEB J. 2014, 28, 2748–2761.
70. Emanueli, C.; Shearn, A.I.; Angelini, G.D.; Sahoo, S. Exosomes and exosomal miRNAs in cardiovascular protection and repair. Pharmacol. 2015, 71, 24–30.
71. Kang, K.; Ma, R.; Cai, W.; Huang, W.; Paul, C.; Liang, J.; Wang, Y.; Zhao, T.; Kim, H.W.; Xu, M.; et al. Exosomes Secreted from CXCR4 Overexpressing Mesenchymal Stem Cells Promote Cardioprotection via Akt Signaling Pathway following Myocardial Infarction. Stem Cells Int. 2015, 2015, 659890.
72. Lee, Y.; el Andaloussi, S.; Wood, M.J. Exosomes and microvesicles: Extracellular vesicles for genetic information transfer and gene therapy. Mol. Genet. 2012, 21, R125–R134.
73. Ibrahim, A.G.; Cheng, K.; Marban, E. Exosomes as critical agents of cardiac regeneration triggered by cell therapy. Stem Cell Rep. 2014, 2, 606–619.
74. Jeyaseelan, K.; Lim, K.Y.; Armugam, A. MicroRNA expression in the blood and brain of rats subjected to transient focal ischemia by middle cerebral artery occlusion. Stroke 2008, 39, 959–966.
75. Liu, F.J.; Lim, K.Y.; Kaur, P.; Sepramaniam, S.; Armugam, A.; Wong, P.T.; Jeyaseelan, K. microRNAs Involved in Regulating Spontaneous Recovery in Embolic Stroke Model. PLoS ONE 2013, 8, e66393.
76. Lusardi, T.A.; Murphy, S.J.; Phillips, J.I.; Chen, Y.; Davis, C.M.; Young, J.M.; Thompson, S.J.; Saugstad, J.A. MicroRNA responses to focal cerebral ischemia in male and female mouse brain. Mol. Neurosci. 2014, 7, 11.
77. Zhang, G.; Zhu, Z.; Wang, H.; Yu, Y.; Chen, W.; Waqas, A.; Wang, Y.; Chen, L. Exosomes derived from human neural stem cells stimulated by interferon gamma improve therapeutic ability in ischemic stroke model. Adv. Res. 2020, 24, 435–445.
78. Bian, B.; Zhao, C.; He, X.; Gong, Y.; Ren, C.; Ge, L.; Zeng, Y.; Li, Q.; Chen, M.; Weng, C.; et al. Exosomes derived from neural progenitor cells preserve photoreceptors during retinal degeneration by inactivating microglia. Extracell. Vesicles 2020, 9, 1748931.
79. Batrakova, E.V.; Kim, M.S. Development and regulation of exosome-based therapy products. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2016, 8, 744–757.
80. Witwer, K.W.; Buzás, E.I.; Bemis, L.T.; Bora, A.; Lässer, C.; Lötvall, J.; Nolte-‘t Hoen, E.N.; Piper, M.G.; Sivaraman, S.; Skog, J.; et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. Extracell. Vesicles 2013, 2, 20360.
81. Tauro, B.J.; Greening, D.W.; Mathias, R.A.; Ji, H.; Mathivanan, S.; Scott, A.M.; Simpson, R.J. Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods 2012, 56, 293–304.
82. Taylor, D.D.; Shah, S. Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes. Methods 2015, 87, 3–10.
83. Taylor, D.D.; Zacharias, W.; Gercel-Taylor, C. Exosome isolation for proteomic analyses and RNA profiling. Methods Mol. Biol. 2011, 728, 235–246.
84. Patel, G.K.; Khan, M.A.; Zubair, H.; Srivastava, S.K.; Khushman, M.; Singh, S.; Singh, A.P. Comparative analysis of exosome isolation methods using culture supernatant for optimum yield, purity and downstream applications. Rep. 2019, 9, 5335.
85. Xu, R.; Greening, D.W.; Zhu, H.J.; Takahashi, N.; Simpson, R.J. Extracellular vesicle isolation and characterization: Toward clinical application. J. Clin. Investig. 2016, 126, 1152–1162.
86. Lener, T.; Gimona, M.; Aigner, L.; Borger, V.; Buzas, E.; Camussi, G.; Chaput, N.; Chatterjee, D.; Court, F.A.; del Portillo, H.A.; et al. Applying extracellular vesicles based therapeutics in clinical trials—An ISEV position paper. Extracell. Vesicles 2015, 4, 30087.
87. Chevillet, J.R.; Kang, Q.; Ruf, I.K.; Briggs, H.A.; Vojtech, L.N.; Hughes, S.M.; Cheng, H.H.; Arroyo, J.D.; Meredith, E.K.; Gallichotte, E.N.; et al. Quantitative and stoichiometric analysis of the microRNA content of exosomes. Natl. Acad. Sci. USA 2014, 111, 14888–14893.
88. Moleirinho, M.G.; Silva, R.J.S.; Carrondo, M.J.T.; Alves, P.M.; Peixoto, C. Exosome-based therapeutics: Purification using semi-continuous multicolumn chromatography. Purif. Technol. 2019, 224, 515–523.
89. Navajas, R.; Corrales, F.J.; Paradela, A. Serum Exosome Isolation by Size-Exclusion Chromatography for the Discovery and Validation of Preeclampsia-Associated Biomarkers. Methods Mol. Biol. 2019, 1959, 39–50.
90. Nordin, J.Z.; Lee, Y.; Vader, P.; Mager, I.; Johansson, H.J.; Heusermann, W.; Wiklander, O.P.; Hallbrink, M.; Seow, Y.; Bultema, J.J.; et al. Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties. Nanomedicine 2015, 11, 879–883.
91. Witwer, K.W.; van Balkom, B.W.M.; Bruno, S.; Choo, A.; Dominici, M.; Gimona, M.; Hill, A.F.; de Kleijn, D.; Koh, M.; Lai, R.C.; et al. Defining mesenchymal stromal cell (MSC)-derived small extracellular vesicles for therapeutic applications. J. Extracell. Vesicles 2019, 8, 1609206.
92. Lamparski, H.G.; Metha-Damani, A.; Yao, J.Y.; Patel, S.; Hsu, D.H.; Ruegg, C.; Le Pecq, J.B. Production and characterization of clinical grade exosomes derived from dendritic cells. Immunol. Methods 2002, 270, 211–226.
93. Ban, J.J.; Lee, M.; Im, W.; Kim, M. Low pH increases the yield of exosome isolation. Biophys. Res. Commun. 2015, 461, 76–79.
94. Emam, S.E.; Ando, H.; Lila, A.S.A.; Shimizu, T.; Ukawa, M.; Okuhira, K.; Ishima, Y.; Mahdy, M.A.; Ghazy, F.S.; Ishida, T. A Novel Strategy to Increase the Yield of Exosomes (Extracellular Vesicles) for an Expansion of Basic Research. Pharm. Bull. 2018, 41, 733–742.
95. Gao, J.; Wang, S.; Wang, Z. High yield, scalable and remotely drug-loaded neutrophil-derived extracellular vesicles (EVs) for anti-inflammation therapy. Biomaterials 2017, 135, 62–73.
96. Jhan, Y.Y.; Prasca-Chamorro, D.; Zuniga, G.P.; Moore, D.M.; Kumar, S.A.; Gaharwar, A.K.; Bishop, C.J. Engineered extracellular vesicles with synthetic lipids via membrane fusion to establish efficient gene delivery. J. Pharm. 2020, 573, 118802.
97. Rayamajhi, S.; Nguyen, T.D.T.; Marasini, R.; Aryal, S. Macrophage-derived exosome-mimetic hybrid vesicles for tumor targeted drug delivery. Acta Biomater. 2019, 94, 482–494.
98. De la Pena, H.; Madrigal, J.A.; Rusakiewicz, S.; Bencsik, M.; Cave, G.W.; Selman, A.; Rees, R.C.; Travers, P.J.; Dodi, I.A. Artificial exosomes as tools for basic and clinical immunology. Immunol. Methods 2009, 344, 121–132.
99. Thone, M.N.; Kwon, Y.J. Extracellular blebs: Artificially-induced extracellular vesicles for facile production and clinical translation. Methods 2020, 177, 135–145.
100. Li, D.; Yao, S.; Zhou, Z.; Shi, J.; Huang, Z.; Wu, Z. Hyaluronan decoration of milk exosomes directs tumor-specific delivery of doxorubicin. Res. 2020, 493, 108032.
101. Ju, S.; Mu, J.; Dokland, T.; Zhuang, X.; Wang, Q.; Jiang, H.; Xiang, X.; Deng, Z.B.; Wang, B.; Zhang, L.; et al.; Welti, R.; Mobley, J.; Jun, Y.; Miller, D.; Zhang, H.G. Grape exosome-like nanoparticles induce intestinal stem cells and protect mice from DSS-induced colitis. Ther. 2013, 21, 1345–1357.