key: cord-0978919-ru8xqqj4
authors: Kang, Ju-Seop
title: Chapter 20 The potential of exosomes as theragnostics in various clinical situations
date: 2020-12-31
journal: Exosomes
DOI: 10.1016/b978-0-12-816053-4.00020-1
sha: c3389d43f03cee6e8d20a4e5da3d1754cd003a6c
doc_id: 978919
cord_uid: ru8xqqj4

Abstract Exosomes are membrane-bound cargo measuring 30–140nm comprised of a lipid bilayer containing various proteins, RNAs, DNAs, and bioactive lipids that can be transferred between cells. They have been shown to be produced and released by many different types of healthy and diseased cells. Exosomes are secreted by all types of cells in culture, and are also found in various body fluids including blood, saliva, urine, and breast milk. Exosomes are essential for healthy physiological as well as pathological processes. In addition to their normal function, exosomes are involved in the development and progression of various diseases, potentiating cellular stress and damage. Pathogens take advantage of exosome release from infected host cells by manipulating host-derived exosomes to evade the immune system responses. Exosomes are involved in other pathological conditions such as neurodegenerative diseases, liver diseases, heart failure, cancer, diabetes, kidney diseases, osteoporosis and atherosclerotic cardiovascular disease. Hence, we can exploit exosomes as biomarkers and vaccines and modify them rationally for therapeutic interventions including tissue engineering. Further studies on exosomes will explore their potential and provide new methodology for effective clinical diagnostics and therapeutic strategies: such uses can be called exosome theragnostics. This chapter reviews the potential theragnostic (diagnostic and therapeutic) application of exosomes in major organ systems in clinical fields.

magnetic resonance imaging findings. Plasma exosomes derived from prostate acinar cells were investigated as new biomarkers for diagnosis of prostate cancer (see [12] ). Proteomics profiling of exosomes showed potential biomarkers of the disease (see [13] ). Patients who experienced recurrence of hepatocellular carcinoma after liver transplantation showed increased levels of a miRNA biomarker in serum exosomes (see [14] ).

Exosomes have also been evaluated as biomarkers for various non-cancer diseases of multiple organs including the central nervous system (CNS) (see [15] ), liver (see [16] ), kidney (see [17] ), lung (see [18] ), and arteries (see [19] ). In the CNS, tau-mediated neuropathology is the result of extracellular accumulation of abnormally processed tau protein (see [20] ). In the M1C cell neuroblastoma tauopathy model, tau protein appeared to be spread via exosomes, and AT270 phosphorylated-tau was increased in the cerebrospinal fluid (CSF), a biomarker for early stages of Alzheimer's disease (AD). The elevated levels of AT270-phosphorylated tau in the CSF seen in mild to moderate cases of sporadic AD resulted from selective abundance of phosphorylated tau in the exosome portion of the CSF relative to total CSF tau. The increase in CSF phosphorylated tau level with attack of AD was associated with an increase in the exosome-associated portion in the CSF. In another study, levels of autolysosomal proteins (cathepsin D, lysosomal-associated membrane protein 1, and ubiquitinylated proteins) in neuron-derived serum exosomes distinguished patients with pre-clinical AD from matched controls and patients with frontotemporal dementia (see [21] ). Altered microRNA profiles in CSF/blood exosomes associated with neurodegenerative disorders are possible new biomarkers in the early diagnosis of AD and PD (see [22] ), and the fact that exosomes can deliver siRNA offers a therapeutic potential in AD (see [23, 24] ). Proteomics profiling of serum exosomes identified proteins that were abundant in patients with PD compared with healthy subjects (see [25] ).

With regard to lung disease, exosomes isolated from bronchoalveolar lavage fluids of patients with asthma compared with healthy subjects showed different miRNA profiles (see [18] ). Exosomes are released from the key cells implicated in asthma such as mast cells, eosinophils, dendritic cells (DCs), T-cells, and bronchial epithelial cells. These in turn can provoke the activation or repression of other asthma-related cells and stimulate allergic responses (see [26, 27] ). The DC-derived exosomes (Dex, dexosome) have costimulatory molecules on their surfaces that can stimulate allergen-specific Th2 cells (see [28, 29] ). The eosinophil-derived exosomes also have important roles in the modulation of asthma, and their numbers are increased in asthmatic patients (see [30, 31] ). The exosomal miRNA content in patients with severe asthma was significantly different compared with healthy subjects (see [32] ). The dysregulated miRNAs were associated with pathways related to airway integrity as well as being correlated with certain clinical features such as eosinophil count or FEV1 (see [33] ). In a different study, the exosomal miRNA profile in patients with severe asthma was related to the TGF-ß and ErbB signaling pathway and focal adhesion (see [34] ).

Exosomes may also serve as vaccines for allergic diseases (see [35] ). Exosomes isolated from the bronchoalveolar lavage fluid of mice after respiratory exposure to the olive pollen allergen induced tolerance and protection against allergic sensitization in mice (see [31] ).

Increased miR-192 levels in serum exosomes predicted the development of heart failure after acute myocardial infarction (see [36] ). Finally, urinary exosomes have been used as starting material for diagnostic biomarkers for renal, urogenital, and systemic diseases (see [17, 37] ).

Despite the number of studies showing the relevance of EV biomarkers with diverse diseases, the results of individual studies have shown inconsistent trends. Methodological differences in EV purification may explain this contradiction (see [38] ). For a given application, it is mandatory to inspect the method in terms of its sensitivity and specificity including quality control measures under well-defined settings.

Exosomes are preferential candidates for use in vaccines for infectious diseases such as toxoplasmosis, diphtheria, tuberculosis, and atypical severe acute respiratory syndrome (SARS). It has been reported that immunization using DCs with Toxoplasma gondii antigens (T-Ag) in healthy mice induced protection against a virulent strain of T. gondii after oral application, but it was difficult to obtain a sufficient amount of DCs pertinent to vaccination (see [39] [40] [41] ). Murine bone marrow-derived DCs pulsed in vitro with intact diphtheria toxin (DT)-released exosomes after injection into mice showed induction of IgG2b and IgG2a responses specific for DT (see [42] ). Infection with Mycobacterium tuberculosis excites macrophages to stimulate the release of exosomes, and it should be noted that exosomes containing M. tuberculosis peptide-MHC-II complexes can induce antimicrobial T-cell responses (see [43, 44] ). Exosomes as vaccination materials have also been studied in SARS-related coronavirus (CoV), an infection that causes a fatal atypical pulmonary disease. Kuate et al. [35] found that exosomes with the SARS-CoV spike S protein produced neutralizing antibody titers, which was further reinforced by priming with the SARS-S exosome vaccine and then boosting with the presently applied adenoviral vector vaccine (see [35] ).

Stimulating a potent and general cytotoxic T lymphocyte (CTL) immune reaction has therapeutic potential for various diseases, including viral infections. For example, inducing anti-Ebola virus (EboV)-specific CTL immunity could have benefits in both therapeutic and preventive settings (see [45] ). In fact, stimulation of virus-specific CTLs has been recognized in survivors of acute EboV infections (see [46] ), and virus-specific CTL immunity plays a crucial role in protection in several nonhuman primates, including macaques (see [47] ). Furthermore, transfusion of CD8 + T lymphocytes from mice infected with mouse-adapted EboV to naïve recipient mice defended them against EboV infection (see [48] ). Consistently, a powerful CTL-related immunity response could also have pertinent therapeutic effects with influenza viruses A (Flu) (see [49] ) and hepatitis C (HCV) virus infections (see [50] ). Anticoli et al. [45] suggested an exosome-based vaccine platform to design exosomes in vivo with the E7 protein of human papilloma virus (HPV). This method involves intramuscular injection of a DNA vector encoding HPV-E7 fused at the C-terminus of an exosome-anchoring Nef mutant protein (Nef mut ). Human immunodeficiency virus type-1 (HIV-1) Nef mut is a 27-kDa protein (see [51] ) connecting with raft microdomains at cellular membranes (see [52] ). Nef mut lacks several anti-cellular effects generally caused by wild-type Nef, including CD4 downregulation, increase of HIV-1 infectivity, PAK-2 stimulation, and MHC Class I down-regulation, and is found in exosomes at very high levels (see [53, 54] ). In this alignment, the ≈11-kDa E7 protein produced both potent and effective antigen-specific CTL immunity. To establish the general application of this technology, immunogenicity studies were performed with an array of viral products of various origins and sizes including EboV, West Nile Virus NS3 and HCV NS3. All antigens were stable upon fusion with Nef mut , and were transferred into exosomes at levels compared to Nef mut . When injected into mice, DNA vectors expressing the various fusion products produced a clearly detectable antigen-specific CD8+ T cell response with sufficient cytotoxicity to kill peptide-loaded and/or antigen-expressing syngeneic cells (see [45] ).

DCs are the most competent cells at presenting antigens, and are the only antigen-presenting cell able to stimulate naïve T cells, creating the adaptive immune reaction (see [55] ). Indeed, we can define cancer immunosurveillance as a stage of stepwise results leading to the effective killing of cancer cells by T cells: specifically, DC capturing and processing of tumor neoantigens is the first phase, a process that depends on molecular signals such as pro-inflammatory cytokines, co-stimulatory ligands, dying tumor cells-derived molecules, and gut microbiome products (see [56] ). Accordingly, potent DC-based cancer vaccinations have been researched for some time; some positive results using these technologies have emerged, such as Sipuleucel-T immunotherapy for castration-resistant prostate cancer (see [57] ). However, the diverse application of DC-based cancer vaccines shows some main limitations (see [58, 59] ). Fig. 1 describes DC-based immunotherapeutic strategies. Dexosome (Dex)-based cancer vaccines have recently emerged as an alternative that may overcome some of these obstacles. First, the Dex molecular component is simple to analyze, thus enabling the rigid definition of validation parameters (see [60] ). Second, Dex components are more plentiful in peptide-MHC class II complexes, allowing for higher yields (see [58, 60] ). Third, Dex compared with DC can tolerate longerterm frozen storage, for up to 6 months (see [58] ). In addition to these merits, the immunosuppressive tumor microenvironment often inhibits antigen presentation and T cell stimulation by DCs, but this should not affect Dex (see [61, 62] ). Finally, Dex are not associated with most of the risks related to the administration of viable cells, such as generation of immune dysfunction or microvascular occlusions (see [63] ). Tumor peptide-pulsed Dex produced in vivo CTL priming, tumor growth repression, and tumor remission. Indeed, single intradermal injections stimulated significant tumor growth repression after a week, and 40-60% of the animals were tumor-free after 60 days (see [62] ). Furthermore,

DCs-based immunotherapeutic strategies: (1) to harvest peripheral blood mononuclear cells, (2) to generate immature DCs with cytokine stimulation, (3) to mature DCs by sensing the presence of a potential pathogen ("stress signal") via detection of PAMPs (exogenous signal) or infection-induced alteration in self-markers (endogenous signal), (4) cancer cells brought into contact with dendritic cells are consumed, thereby imprinting the dendritic cells with the cancer marker (5) to transfer activated antigen-presenting DCs back to the patients, (6) to stimulate robust anti-tumor immune effector cells such as T cells and NK cells. these cell-free immunotherapeutic vaccines were more potent than directly administered viable DC vaccines, with which only 20% of the mice were tumorfree after 60 days. These differences may illustrate the exosomes' resistance to the immunomodulatory effects of the tumor microenvironment, which can block the ability of DCs to present antigens (see [62] ). In the past decade, several successful clinical trials were performed assessing the feasibility, safety and efficacy of Dex-based cancer vaccines in patients with non-small cell lung cancer (NSCLC) (see [64] ) and metastatic melanoma (see [65] ), and in general the results were promising. In both trials, the patients received four doses of vaccine that consisted of autologous Dex loaded with several different MHC class II peptides. Vaccine production was shown to be practical, and the therapy was well tolerated with only minor grade 1-2 adverse events (see [64, 65] ). A more recent phase II clinical trial evaluated the use of IFN-γ-Dex, Dex derived from IFN-γ-stimulated mature DC, as maintenance immunotherapy after the use of first-line chemotherapy in patients with advanced NSCLC (see [66] ). This study showed the feasibility of production and safety of IFN-γ-Dex, with only one of 26 patients developing a grade 3 hepatotoxicity. This trial did not show any objective tumor response among clinical outcomes, according to the Response Evaluation Criteria in Solid Tumors. However, it did show that the patients with the longest progression-free survival had a notable improvement in NK cell function after Dex treatment, showing that Dex can stimulate the NK cell arm of antitumor immunity in patients with advanced NSCLC (see [66] ).

Tumor-derived exosomes also function as an antigen delivery system, capable of blocking tumor development in a CD4+ and CD8+ T cell-dependent pattern (see [67] ). Because of this, cell-free vaccines based on the use of tumorderived exosomes are another possibility for clinical application. However, the isolation of tumor-derived exosomes is inconvenient and has a low preparation efficiency, with a low yield from in vitro culture of the patients' tumor cells (see [68] ). However, malignant effusions from patients with melanoma are rich in exosomes, which can transmit tumor antigens to DCs, which in turn stimulate tumor-specific CTL capable of an effective in vitro antitumor response (see [69] ). A phase I clinical trial examined the effects of exosomes harvested from the ascites of patients with advanced CRC as immunotherapy, and showed that a combination of tumor exosomes with GM-CSF permitted a more effective induction of systemic anti-tumor immunity and CTL responses compared with tumor exosomes alone. The patients treated with only tumor exosomes showed no therapeutic response, while one patient with stable disease and one patient with a minor clinical response were observed in the group treated with ascites-derived exosomes combined with GM-CSF (see [68] ). Despite the attempts engaged thus far, Dex-based immunotherapy as a novel cancer control remains a highly encouraging possibility. Dex are proficient mediators of immune responses and the technical simplicity of managing their immunostimulatory characteristics (via the donor DC) along with their advantages over whole cell-based applications, confirms their therapeutic promise (see [60] ).

Of particular interest to this section, exosomes can serve as both promoters of tumor growth and invasion by establishment of an immunosuppressive microenvironment and as agents for cancer immunosurveillance by inducing antigen presentation and stimulating destruction of tumor cells by CD4+ (see [70] ) and CD8+ (see [69] ) T cells and by components of the innate immune system, such as NK cells (see [71] ) (Fig. 1 ).

The most important characteristic for a successful nanocarrier is satisfactory in vivo behavior. The development of EVs as delivery systems requires comprehension of their in vivo kinetics after administration. However, understanding of extracellular behavior, pathways of cell uptake, and subcellular paths of EVs remains obscure. EVs released by various types of cells can be found and are relatively stable in the blood circulation and biological fluids. This suggests that EVs are more slowly cleared and remain longer than synthetic nanocarriers in biological systems. However, different in vivo pharmacokinetic studies have shown that when EVs are injected into the circulation, they are rapidly cleared. EVs derived from B16 melanoma cells and splenocytes underwent rapid clearance and showed a very short half-life, approximately 2 min, after intravenous administration in mice (see [72, 73] ). Increased levels of fetuin-A in urinary exosomes correlated with acute urinary injury (see [74] ).

Exosomes and microvesicles participate in a large variety of body processes. They are carriers of concentrated genetic and proteomic information, and thus are believed to play important roles in cell-to-cell communication. Secreted vesicles can carry their messages in different ways. Firstly, they may stimulate recipient target cells via ligands expressed on their surface. For example, it has been verified that antigen-presenting exosomes derived from DC induce the T cell-mediated immune response in vivo (see [75] ). In addition, ligand-receptor signaling via exosomes can also play a role in other regulatory processes, such as angiogenesis (see [76] ), hemostasis (see [77] ), cancer progression (see [78] ), and metastasis (see [79] ). Secondly, secreted EVs may transfer surface receptors from one cell to another by fusion with the plasma membrane of target cells (see [80, 81] ). With this mechanism, HIV increase susceptibility to infection by transferring CD4 receptors from infected cells to intact cells (see [82] ). EVs appear to have multiple obvious advantages, such as high delivery capacity, innate targeting properties, and low immunogenic potentiality, which position them as efficient biological delivery systems for therapeutics ranging from small molecules to macromolecular nucleic acids and proteins (see [83] ) ( Table 1) .

Despite the development of various methodologies for EV-based delivery, a major hurdle is the lack of standardized, efficient, and reasonable approaches for isolation of EVs. Isolation methods need to be validated when considering the reproducibility, yield, purity, and functional properties of EVs for its general application. Also, there is no validated standard procedure for storage time, which needs to be carefully evaluated for EV-based products (see [88] ). Fig. 2 shows the overall scheme of different modalities for using exosome-based formulations (see [89] ).

Also, the loading efficiency of therapeutics into EVs remains inadequate. The relatively tight and ordered lipid bilayer impedes efficient loading of drugs into EVs without reducing membrane integrity. Such damage might alter the immune-oriented characteristics of EVs and make them visible to the mononuclear phagocyte system (see [89] ). Therefore, ideal loading methods should not only possess high loading efficiency but also preserve the structural integrity of EVs and the functional integrity of therapeutics (see [83] ).

A growing area of interest is the application of diverse nanotechnologybased DDS such as liposomes, polymeric nanoparticles, dendrimers, and magnetic nanoparticles (see [90] ). These delivery systems are being used to deliver various types of cargo including chemotherapeutics, anti-inflammatory drugs and miRNAs (see [91, 92] ). The nano-sized diameters of these DDS facilitate delivery through the blood and lymphatic systems with effective drug-loading capacity (see [93] ). Moreover, our understanding has progressed regarding the 

-Nanoscale vesicles for biocompatibility and stability in body fluids.

-Naturally derived low immunogenicity.

-Stealth capacity against immune system.

-Inherent target properties with reduced off-target effects.

-Ability of guiding therapeutic cargo across biological barriers, especially BBB.

-Capacity to be loaded with specific small molecules such as miRNA and drugs.

-Unique composition allows direct membrane fusion with target cell, for efficient cell uptake.

-Safe in clinical trials.

-Low inherent toxicity.

-Secretion and uptake mechanism, composition, and biological functions are not yet understood.

-Impact on the target cell is unknown.

-Isolation techniques with high efficiency and robust yield are lacking.

-Scalable production difficult-there are no optimal purification methods: largescale production is expensive and challenging.

-Efficient loading methods without damaging EV integrity are lacking.

-In vivo data have been less studied, and in vivo tracking requires further studies.

-Clinical studies on therapeutic delivery are lacking.

-Vesicles with heterogeneous constituents can be immunogenic. in vivo pharmacokinetic behaviors of EVs such as circulatory half-life, tissue distribution in vivo, cellular uptake, and intracellular fates. This understanding is crucial to the clarification of the biological functions of exosomes and practical application of exosome-based therapeutics. To define the pharmacokinetics of exosomes clearly, the first step includes the evaluation of tissue distribution in vivo, in other words the so-called biodistribution of exosomes. Several labeling methods with small lipophilic fluorescence dyes have been introduced and used for that purpose for in vivo tracking. Although the reliability of in vivo analysis would be decreased by the release of free dye from exosomes, this strategy is a useful approach to assess the localization of exosomes to tissues (see [94, 95] ). PKH67, a lipophilic fluorescent dye, was used to label highly metastatic B16F10 murine melanoma cell-derived exosomes that accumulated in the lung, bone marrow, spleen, and liver, enhanced endothelial permeability in the lung, and facilitated tumor metastasis to the lung (see [96] ). In addition to fluorescent dyes, lipophilic near-infrared dyes such as 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiD) and 1,1′-dioctadecyltetramethylindotricarbocyanine iodide (DiR) have been widely used for the imaging of exogenously administered exosomes. DiD-labeled MSC exosomes were well distributed to the spleen and liver after intravenous administration in normal mice (see [94] ). On the other hand, in a mouse model of acute kidney injury, these exosomes accumulated in the kidney, as well as the spleen and liver, after intravenous injection. This finding may explain how the intravenous administration of MSC-derived exosomes stimulates the recovery from cisplatin-induced acute kidney injury in SCID mice (see [97] ). In the study of in vivo behavior of DiR-labeled exosomes of various cell types such as B16F10 murine melanoma cells, C2C12 murine myoblast cells, bone marrow-derived DCs, and HEK293T human embryonic kidney cells, exosomes were mainly distributed in the liver, spleen, lung, and gastrointestinal tract after intravenous administration. Among these exosomes, B16F10 murine melanoma cell-derived exosomes mainly accumulated in the lung compared with the exosomes derived from the other two types of murine cells. The highest accumulation in the spleen and liver was observed with DC exosomes and C2C12 exosomes, respectively. HEK293T exosomes mainly accumulated in the liver after intravenous injection and the liver, pancreas, or gastrointestinal tract after intraperitoneal or subcutaneous injection, respectively (see [98] ). Because of its higher sensitivity and stability, the labeling of exosomes with radiotracer is a more appropriate method for quantitative assessment of the pharmacokinetics and tissue distribution of exosomes compared with labeling with fluorescence dyes or chemiluminescent proteins. For example, 111 In-labeled PC3 exosomes rapidly disappeared from blood circulation and were primarily distributed in the liver (12% injection dose [ID]/g at 24 h) after intravenous injection (see [99] ). Various types of cells recognize and take up exosomes. Therefore, identifying these cells is important for further exploration of the biology of exosomes and for the development of exosome-based therapeutics. The mouse DC-derived exosomes were picked up by macrophages in the spleen and liver (see [90] ), and exosomes derived from MDA-MB-231 breast cancer cells were taken up by macrophages in the lung and brain after intravenous administration, respectively. Exosomes derived from C2C12 cells, NIH3T3 cells, MAEC cells, and RAW264.7 cells were mainly picked up by macrophages in the liver after intravenous administration (see [100] ). These results suggest that macrophages are the main cells that actively take up exogenous exosomes (see [101] ). It has been predicted that exosomes are taken up by cells through the recognition of surface molecules on the membranes of the exosomes. Several studies investigated the molecules that may contribute to the in vivo pharmacokinetics of exosomes. An in vitro study proved that carbohydrate moieties on the membranes of exosomes contributed to the cellular uptake of exosomes (see [102] ). Exosomes derived from tumor cells that metastasized to the lung (MDA-MB-231 and 4175) or to the liver (BxPC-3 and HPAF-II) mainly gathered in the lung and liver, respectively. A proteomic analysis of exosomes showed high expression of integrins α6β4 and αVβ5, respectively. Exosomes collected from integrin β4-knocked down 4175 cells showed reduced accumulation in the lung. These results demonstrate that integrins play a key role in the pharmacokinetics and tissue distribution of exosomes in vivo (see [101] ). Exosomes derived from genetically modified immature DCs expressing the iRGD peptide showed a selective distribution to αv integrin-positive tumor tissues (see [103] ). In addition to delivery of small RNA molecules, which depend on intracellular delivery to perform intrinsic functions, EVs have also been applied to deliver chemotherapeutic agents with the aim to increase their efficacy and reduce adverse effects. One study involved the use of exosomes to deliver curcumin to treat an inflammatory disease (see [104] ). Exosomes are applied to form a complex with curcumin to enhance curcumin anticancer activity (see [105] ). The intravenous injection of integrin-targeted, dendritic cell-derived EVs with the chemotherapeutic doxorubicin led to significant repression of tumor growth compared to free doxorubicin in a mouse model of breast tumor. Moreover, doxorubicin when loaded into EVs was shown to cause less cardiac damage, which is otherwise its most important dose-limiting adverse effect (see [103] ). The advantage of exosomal doxorubicin versus liposomal doxorubicin involves the natural orientation of exosomal membrane proteins and their ability to interact with the receptors in the target cell plasma membrane (see [106, 107] ). Furthermore, exosome-encapsulated paclitaxel compared with free taxol was shown to be more effective in controlling the growth of Lewis lung carcinoma metastases, and it holds significant potential for the delivery of various chemotherapeutics to treat drug-resistant cancers (see [108] ). Repeated intraperitoneal injections of cisplatin-loaded EVs improved long-term survival of ovarian cancer-bearing mice as compared to free cisplatin, and intravenous injection of doxorubicin-loaded EVs delayed growth of established subcutaneous hepatic cancer (see [109] ). Importantly, these exosome-encapsulated drugs did not adversely affect liver or kidney function, which is frequently observed after administration of free drugs.

Regrettably, the movement of large proteins through the blood-brain barrier (BBB) is severely limited. In fact, 98% of all potent drugs that may be better options for various CNS diseases are not applied in the clinic because they cannot cross the BBB (see [110] ). Various nano-sized drug formulations have been developed to overcome this hurdle (see [111, 112] ). Parkinson's disease (PD) is known to be associated with brain inflammation, microglia activation and secretory neurotoxic activities, including reactive oxygen species (ROS) (see [113, 114] ). Samples of brain tissue from patients with PD have shown reduced levels of redox enzymes, catalase, and superoxide dismutase, and other antioxidants (see [115] [116] [117] ), which indicates an impaired defense against oxidative stress and neurodegeneration in these patients. Among these molecules, catalase is one of the most effective natural antioxidants: it scavenges one million free radicals through a catalytic reaction. Therefore, successful delivery of catalase into the brain may be an important and possible approach to PD therapy (see [84] ). EVs have been proposed as therapeutic delivery vehicles for the treatment of PD. Exosomes are readily taken up by neuronal cells in vitro, and a considerable number of exosomes was detected in PD mouse brain after intranasal administration. Catalase-loaded exosomes (ExoCAT) were shown to suppress microglial activation and protect neurons against ROS more efficiently compared to free catalase in in vitro and in vivo models of PD (see [84] ). Therefore, ExoCAT is a more adaptable strategy for treating inflammatory and degenerative disorders such as PD (see [84, 118] ). Although these reports are preliminary, the results have shown that exosomes are promising candidate drug delivery systems for the treatment of a variety of diseases.

Exosomes derived from human adipose tissue-derived mesenchymal stem cells (MSCs) are considered to have therapeutic value for treating AD (see [119] ). Exosomes can be used in vivo as a vehicle to carry active neprilysin (NEP), the most important enzyme for β-amyloid (Aβ) peptide degradation in the brain. MSC-derived exosomes also decrease intracellular and extracellular Aβ levels in the neuroblastoma cell line N2A in vitro. Therefore, human adipose tissue-derived MSC-originated exosomes are proposed as a potential therapy against AD from the point of view of their Aβ-degrading capacity. Recent studies have demonstrated that multipotent mesenchymal stromal cells (MSCs) hold great promise for neurovascular remodeling and neurological function recovery following a stroke. It has been demonstrated that MSC-derived exosomes have a neuroprotective effect against stroke due in part to changing the miRNA profile of exosomes during and after stroke (see [120] ).

When we consider the problems associated with many current nanoparticulate delivery systems, exosomes as a mimic of "natural delivery systems" are a potential alternative for delivery of the biological molecules described above.

Because of their small size and host-derived cellular product, these vesicles can avoid phagocytosis or degradation by macrophages and also circulate for long periods of time in the body. One of the interesting advantages of these delivery vehicles includes their ability to cross the BBB and arrive in the CNS (see [121] ). Insufficient knowledge of the nature of exosomes and their role in the pathophysiology of overall health and disease makes it complicated and difficult to predict long-term safety and therapeutic effects. In vivo trafficking of exosomes, their biological fate and their impact on targeted organs need to be understood with regard to therapeutic cargo loading and assembly for drug delivery (see [122] ). Currently, there is no distinct optimal purification technique for isolation of exosomes with high purity (see [123] ). The current isolation methods yield low quantities of exosomes, and their large-scale production for clinical studies and post drug approval is expensive and complicated (see [38] ). It is highly likely that future clinical use will demand hybrid designs of exosomes (see [124] ), and when combined with therapeutic cargo they may show undesirable effects. Even though extensive exosome biology is already known, exosomes comprise heterogeneous constituents and may show immunogenicity (stimulatory or suppressive) effects based on the nature of the donor cells. Exosomes provide immense promise and are a new therapeutic area for delivery of various synthetic and biological molecules in cellular therapy. Exosomes as drug delivery systems provide a major advantage as there is no undesired aggregation or homing of exosomes in the liver and/or first-pass effect before arriving in target sites. Well-characterized exosomes with long-term safety that deliver nucleic acids and therapeutic molecules between cells and through difficult-to-cross membranes such as BBB would have major practical significance. However, before these drug delivery systems become a therapeutic reality, components and processes including immune reactions need to be clarified (see [122] ). The emerging evidence that tumor cell-derived exosomes have unique properties may be used to develop an exosome-based drug delivery system that is better than synthetic drug carriers. However, some limitations and hurdles must be overcome before exosome-based drug delivery systems can be used in the clinic. Important issues still need to be autologous or can nonimmunogenic exosome factories. Therefore, a strategy needs to be developed for manufacturing vesicles for therapeutic application, with establishment of the producer cell type, physical methods to produce vesicular nanoparticles, enhancement of EV yield and scale-up, measurement of potency of EV-based products, and EV-inspired bioengineered artificial vesicles. Recently, a group of researchers developed a method to produce exosome-mimetic vesicles, which can overcome natural exosome limitations as like low drug-loading efficiency and low exosome production yields (see [125] ). These chemotherapeutic-loaded nanovesicles, which are 100-200 nm in diameter, were generated by breaking down cells by serial extrusion through filters with diminishing pore size (see [126] ). It was further suggested that these nano-vesicles with exosome-mimetic properties can be used as a platform for RNAi transfer to the cell cytoplasm (see [127] ). However, the high level of cholesterol, ganglioside, and sphingomyelin in exosomal membranes leads to a more rigid bilayer structure than that of their parent cells (see [128] ), which suggests that their fusion with lipid-based particles requires rough conditions (see [129] ), such as aggressive freeze-thaw processes (see [130] ). To avoid the need for such conditions, Yang et al. [131] designed a virus-mimetic fusogenic exosome platform to deliver membrane proteins to target cell membranes, involving integrated vascular stomatitis virus G protein, a viral fusogen (see [131] ). Interestingly, these methods allow easy exosome modification by fusing exosomes derived from modified cells with liposomes inserted with peptides, antibodies or polyethylene glycol.

Although there are currently many challenges in the treatment of cancer and other refractory diseases, exosomes, including exosome-mimetic nanovesicles, are considered valid diagnostic biomarkers and potential therapeutic tools. Moreover, along with chemical, physical, cellular and genetic engineering techniques, many existing exosome modification strategies are promising for application in various clinical situations (Fig. 2) .

Antigen-presenting cell exosomes are protected from complement-mediated lysis by expression of CD55 and CD59

The multifaceted exosome: Biogenesis, role in normal and aberrant cellular function, and frontiers for pharmacological and biomarker opportunities

Diagnostic and prognostic potential of extracellular vesicles in peripheral blood

Biomolecular characterization of exosomes released from cancer stem cells: possible implications for biomarker and treatment of cancer

Non-coding RNAs as clinical biomarkers for cancer diagnosis and prognosis

The clinical relevance of circulating, exosomal miRNAs as biomarkers for cancer

RNA expression patterns in serum microvesicles from patients with glioblastoma multiforme and controls

MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer

Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers

Epidermal growth factor receptor localized to exosome membranes as a possible biomarker for lung cancer diagnosis

Glypican-1 identifies cancer exosomes and detects early pancreatic cancer

Multiple recognition assay reveals prostasomes as promising plasma biomarkers for prostate cancer

Proteomic profiling of exosomes leads to the identification of novel biomarkers for prostate cancer

Identification of a bona fide microRNA biomarker in serum exosomes that predicts hepatocellular carcinoma recurrence after liver transplantation

Diagnostic and therapeutic potentials of exosomes in CNS diseases

Exosomes in the pathogenesis, diagnostics and therapeutics of liver diseases

Urinary exosomes: a novel means to non-invasively assess changes in renal gene and protein expression

Altered microRNA profiles in bronchoalveolar lavage fluid exosomes in asthmatic patients

Novel methodologies for biomarker discovery in atherosclerosis

Exosome-associated tau is secreted in tauopathy models and is selectively phosphorylated in cerebrospinal fluid in early Alzheimer disease

Altered lysosomal proteins in neural-derived plasma exosomes in preclinical Alzheimer disease

Altered microRNA profiles in cerebrospinal fluid exosome in Parkinson disease and Alzheimer disease

Prognostic serum miRNA biomarkers associated with Alzheimer's disease shows concordance with neuropsychological and neuroimaging assessment

SiRNA delivery with exosome nanoparticles

Identification of distinct circulating exosomes in Parkinson's disease

Intercellular communication by extracellular vesicles and their microRNAs in asthma

Role of microRNAs and exosomes in asthma

Exosomes with major histocompatibility complex class II and co-stimulatory molecules are present in human BAL fluid

Exosomes-nanovesicles with possible roles in allergic inflammation

Exosome secretion by eosinophils: a possible role in asthma pathogenesis

Exosomes from bronchoalveolar fluid of tolerized mice prevent allergic reaction

Exosomes in severe asthma: update in their roles and potential in therapy

LSC abstract-altered small RNA cargo in severe asthma exosomes

Altered circulating exosomal RNA profiles detected by next-generation sequencing in patients with severe asthma

Exosomal vaccines containing the S protein of the SARS coronavirus induce high levels of neutralizing antibodies

Circulating p53-responsive microRNAs are predictive indicators of heart failure after acute myocardial infarction

Exosomes in urine biomarker discovery urine proteomics in kidney disease biomarker discovery

Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes

Toxoplasma gondii antigen-pulsed-dendritic cellderived exosomes induce a protective immune response against T. gondii infection

Exosomes are an effective vaccine against congenital toxoplasmosis in mice

A vaccine based on exosomes secreted by a dendritic cell line confers protection against T. gondii infection in syngeneic and allogeneic mice

Exosomes from bone marrow dendritic cells pulsed with diphtheria toxoid preferentially induce type 1 antigen-specific IgG responses in naive recipients in the absence of free antigen

Mycobacterium tuberculosis synergizes with ATP to induce release of microvesicles and exosomes containing major histocompatibility complex class II molecules capable of antigen presentation

Exosomes released from M. tuberculosis infected cells can suppress IFN-γ mediated activation of naïve macrophages

An exosome-based vaccine platform imparts cytotoxic T lymphocyte immunity against viral antigens

Human Ebola virus infection results in substantial immune activation

CD8+ cellular immunity mediates rAd5 vaccine protection against Ebola virus infection of nonhuman primates

Functional CD8+ T cell responses in lethal Ebola virus infection

Mucosal immunization with integrasedefective lentiviral vectors protects against influenza virus challenge in mice

Current progress in development of hepatitis C virus vaccines

HIV-1 Nef: taking control of protein trafficking

HIV Nef is secreted in exosomes and triggers apoptosis in bystander CD4+ T cells

Genetic and functional analysis of the human immunodeficiency virus (HIV) type 1-inhibiting F12-HIVnef allele

A strategy of antigen incorporation into exosomes: comparing crosspresentation levels of antigens delivered by engineered exosomes and by lentiviral virus-like particles

The cell biology of antigen presentation in dendritic cells

Oncology meets immunology: the cancer-immunity cycle

Sipuleucel-T immunotherapy for castrationresistant prostate cancer

Exosomes for cancer immunotherapy

Dendritic cell-derived exosomes as immunotherapies in the fight against cancer

Dendritic cell-derived exosomes for cancer therapy

Dendritic cells in antitumor immune responses: I. Defective antigen presentation in tumor-bearing hosts

Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell derived exosomes

Immunotherapeutic potential of extracellular vesicles

A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer

Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial

Dendritic cell-derived exosomes as maintenance immunotherapy after first line chemotherapy in NSCLC

Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming

Phase I clinical trial of autologous ascites-derived exosomes combined with GM-CSF for colorectal cancer

Malignant effusions and immunogenic tumourderived exosomes

B lymphocytes secrete antigen-presenting vesicles

Dendritic cell-derived exosomes promote natural killer cell activation and proliferation: a role for NKG2D ligands and IL-15Rα

CD169 mediates the capture of exosomes in spleen and lymph node

Visualization and in vivo tracking of the exosomes of murine melanoma B16-BL6 cells in mice after intravenous injection

Exosomal Fetuin-A identified by proteomics: a novel urinary biomarker for detecting acute kidney injury

Indirect activation of naïve CD4+ T cells by dendritic cell-derived exosomes

Microparticles in angiogenesis: therapeutic potential

Microparticles, thrombosis and cancer

Exosomes from ovarian cancer cells induce adipose tissuederived mesenchymal stem cells to acquire the physical and functional characteristics of tumor-supporting myofibroblasts

TGF-β-induced epithelial to mesenchymal transition

Endothelial expression of autocrine VEGF upon the uptake of tumor-derived microvesicles containing oncogenic EGFR

Platelet-derived microparticles stimulate proliferation, survival, adhesion, and chemotaxis of hematopoietic cells

Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication

Recent advances on extracellular vesicles in therapeutic delivery: challenges, solutions, and opportunities

Exosomes as drug delivery vehicles for Parkinson's disease therapy

The complex ultrastructure of the endolysosomal system

Extracellular vesicles: novel promising delivery systems for therapy of brain diseases

Updated technology to produce highly immunogenic dendritic cell-derived exosomes of clinical grade: a critical role of interferon-γ

Applying extracellular vesicles based therapeutics in clinical trials-an ISEV position paper

Development and regulation of exosome-based therapy products

Extracellular vesicle mimetics: novel alternatives to extracellular vesicle-based theranostics, drug delivery, and vaccines

Drug delivery and nanoparticles: applications and hazards

Nanoparticles as drug delivery systems

Advances and challenges of liposome assisted drug delivery

Biodistribution of mesenchymal stem cell-derived extracellular vesicles in a model of acute kidney injury monitored by optical imaging

Extracellular vesicles as natural nanosized delivery systems for small-molecule drugs and genetic material: steps towards the future nanomedicines

Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET

Microvesicles derived from mesenchymal stem cells enhance survival in a lethal model of acute kidney injury

Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting

Exploration of the drug delivery potential of tumor-derived exosomes. (Doctor of Philosophy)

Cell type-specific and common characteristics of exosomes derived from mouse cell lines: yield, physicochemical properties, and pharmacokinetics

Pharmacokinetics of exosomes-an important factor for elucidating the biological roles of exosomes and for the development of exosome-based therapeutics

Mature dendritic cells pulsed with exosomes stimulate efficient cytotoxic T-lymphocyte responses and antitumour immunity

A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy

A novel nanoparticle drug delivery system: the antiinflammatory activity of curcumin is enhanced when encapsulated in exosomes

Phase II trial of curcumin in patients with advanced pancreatic cancer

Brain microRNAs and insights into biological functions and therapeutic potential of brain enriched miRNA-128

Targeted therapeutic delivery using engineered exosomes and its applications in cardiovascular diseases

Development of exosome-encapsulated paclitaxel to overcome MDR in cancer cells

Delivery of chemotherapeutic drugs in tumour cell-derived microparticles

Drug transport across the blood-brain barrier

Polymer nanomaterials

Nanotechnology applications and approaches for neuroregeneration and drug delivery to the central nervous system

Oxidative stress and antioxidant therapy in Parkinson's disease

Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains

Erythrocyte antioxidant enzymes in Parkinson's disease

Brain peroxidase and catalase in Parkinson disease

Transition metals, ferritin, glutathione, and ascorbic acid in parkinsonian brains

Exosomes derived from genetically modified DC expressing FasL are anti-inflammatory and immunosuppressive

Potential application of extracellular vesicles of human adipose tissue-derived mesenchymal stem cells in Alzheimer's disease therapeutics. In: Stem cell renewal and cell-cell communication

Extracellular vesicles in mesenchymal stromal cells: a novel therapeutic strategy for stroke

Exosomes as nanocarriers for immunotherapy of cancer and inflammatory diseases

Exosomes as therapeutic drug carriers and delivery vehicles across biological membranes: current perspectives and future challenges

A review of exosome separation techniques and characterization of B16-F10 mouse melanoma exosomes with AF4-UV-MALS-DLS-TEM

Monolayer coated gold nanoparticles for delivery applications

Exosome theranostics: biology and translational medicine

Bioinspired exosome-mimetic nanovesicles for targeted delivery of chemotherapeutics to malignant tumors

RNAi delivery by exosome-mimetic nanovesiclesimplications for targeting c-Myc in cancer

Microenvironmental pH is a key factor for exosome traffic in tumor cells

Re-engineering extracellular vesicles as smart nanoscale therapeutics

Engineering hybrid exosomes by membrane fusion with liposomes

Virus-mimetic fusogenic exosomes for direct delivery of integral membrane proteins to target cell membranes