Extracellular vesicles encompass a large heterogeneous family of membrane-bound structures, such as microvesicles, exosomes, and apoptotic bodies. The Heine group initially named all vesicles derived from plasma membrane exosomes (1). Earlier studies by the Turbide group reported that reticulocyte culture releases exosomal vesicles that can be harvested by ultracentrifugation. Further analysis of these vesicles revealed the presence of transferrin receptors, nucleoside transporters, glucose transporters, acetylcholinesterase, and Na⁺-independent amino acid transporter (2). Exosomes are capable of transferring miRNAs and mRNAs between cells (3). Almost all cell types release exosomes with varying molecular compositions and biogenesis pathways. Several bodily fluids, such as saliva, urine, breast milk, amniotic fluid, synovial fluid, serum, and ascites fluid, contain exosomes under physiological and pathological conditions. Exosomes were previously considered cellular waste generated due to cell damage and homeostasis. However, they have been extensively investigated after revealing the antitumor potential of exosomes carrying MHC-I and MHC-II (4).

Exosomes range from 30–150 nm in size (5, 6). Later research ascertained their genesis to be from the inward budding of endosomes, producing multivesicular bodies with intraluminal vesicles. Subsequently, proteins are incorporated into the invaginating membrane, and cytosolic components are engulfed and enclosed within intraluminal vesicles. The contents of exosomes can differ depending on the source and physiological conditions of the cells that release them (7). The fusion of multivesicular bodies with the plasma membrane results in the release of intraluminal vesicles into the extracellular space, giving rise to “exosomes” (2, 8, 9). This is primarily an endosomal sorting complex required for transport (ESCRT)-dependent processes. However, it reportedly functions independently of ESCRT complexes (10, 11). Alternatively, they may also be trafficked to the lysosomes for degradation. These exosomes are mini versions of parent cells and have emerged as critical mediators in inter-cell communication, functioning as vehicles delivering a complex cargo of proteins, lipids, and nucleic acids from the parent cells to other distant cells. Upon reaching recipient cells, they interact either via surface ligands or through the delivery of activated receptors or epigenetic modulation of the recipient cells via the cargo of bioactive cells, consequently modulating the physiology of recipient cells. Thus, exosomes play distinct roles in a multitude of physiological processes, such as immune response, cell differentiation, signal transduction, and antigen presentation. Various cells secrete exosomes; the cargo is unique to its cellular origin and thus can be used as a biomarker. Their ability to package biomolecules within them has facilitated their use as drug delivery systems (12–15).

This review focuses on one of the major physiological roles of exosomes: innate immune regulation. To maintain body function and homeostasis, protection against foreign agents is essential. This function is effectively performed by the immune system, a complex network of cells and organs that protects the organism against infectious agents, such as bacteria, viruses, and other pathogens. This is achieved through a combination of innate and adaptive immunity. Innate immunity is an antigen-non-specific, evolutionarily conserved system in most multicellular organisms, whereas lymphocytes mediate adaptive immunity through antigen specificity and memory. The innate immune system forms the first line of defense and comprises a network of cells, including monocytes/macrophages, dendritic cells, neutrophils, and natural killer cells, facilitating the earliest interactions between the host and pathogens. Upon entry of a foreign object, pattern-recognition receptors on immune cells, such as Toll-like receptors (TLRs), RIG-I-like receptors, and certain DNA sensors, such as cGAS, recognize various molecular signatures of invaders called pathogen-associated molecular patterns (PAMPs), which include numerous molecules, such as lipopolysaccharide (LPS) from gram-negative bacteria, peptidoglycans from gram-positive bacteria, and unmethylated CpG DNA from bacteria and viruses. Upon recognition of the entry of an invader, cell-cell communication is critical for swiftly spreading the message of infection and enabling the innate immune system to mount a broad response against the pathogen. Until recently, cytokines and chemokines have been extensively studied for their role as messengers in innate immunity. However, recent research has revealed that exosomes are also vital in this communication (16). This review mainly focuses on the mechanisms by which exosomes mediate the innate immune response of the host to pathogens (viruses, bacteria, and parasites) as well as act against their cells via autoimmunity. We also focus on using exosomes as diagnostic biomarkers and therapeutic agents. We also focus on using exosomes as diagnostic biomarkers and therapeutic agents ( Figure 1 ).

Exosomes are released from pathogens and host cells. They modulate stimulatory or suppressive effects on the innate immune system through exosome-mediated intercellular communications. They are crucial in immune regulation, including antigen presentation, immune activation, immune suppression, and immune tolerance. Their immune activator or suppressor role depends mainly on the source of the exosomes and their biomolecular content. Exosomes derived from healthy human plasma samples contain various RNA species, such as mRNAs and noncoding regulatory RNAs, within these circulating vesicles (17–19). Pathways related to NF-κB activation and TLR cascades differ between exosomal mRNAs from naïve cells compared with those from LPS-stimulated cells, indicating significant changes in adaptive and innate immune processes. Exosomes are also carriers of critical soluble mediators such as cytokines. After LPS stimulation, RAW 264.7 mouse macrophages exhibited increased levels of cytokines, predominantly chemokines. Ten of the 16 cytokines secreted by LPS-stimulated RAW 264.7 cells were from cell-derived exosomes (20).

The immune system is vital for the protection of the body and the maintenance of homeostasis. However, this system must be tightly regulated for the normal physiological functioning of the body to avoid response to non-threatening and beneficial antigens (for instance, microbiota), to distinguish self from non-self, and from over-reactivity to antigens (allergy or hyper-immunity). Despite stringent control, the immune system can sometimes react to its cells, tissues, and other components, contributing to autoimmunity. Exosomes also engage in these processes, which are detailed in the subsequent sections. Common auto immune thyroid diseases (AITDs) like Hashimoto’s thyroiditis and Graves’ diseases are class of immune disorders caused by the irregular infiltrations of lymphocytes and over productions of autoantibodies to cause the hypothyroidism or hyperthyroidism (52) and genetic along with environmental factors were acknowledged for contributing to development of AITDs (53, 54). Irregular activation/production of autoantibodies like thyroid peroxidase antibodies (TPOAb), thyroglobulin antibodies (TGAb) and thyrotropin receptor antibodies (TRAb) cause thyroid dysfunction, which greatly affect the quality of life (54–56). So, it is clear that over production of autoantibodies and its governance by regulatory T-cell (Tregs) and T-helper-17 cells (Th17) is prime in controlling immune-induced thyroid pathogenesis. The micro communications between T-cells and AITDs are mainly controlled by plasma microvesicles (57). Intercellular communication mediated by exosomes is helpful in regulating innate and adaptive immunity by delivering the target antigens, inflammatory factors presentation and killer proteins. Recent studies have reported that the crucial role of apoptosis in the maintenance of tissue homeostasis and the pathogenesis of immune diseases and AITDs were known for dysregulation of apoptotic signaling pathways (58). A study reported that excess iodide could induced apoptosis through TNF-related apoptosis-inducing ligand and death receptor expression (59), Iodinization increased TPOAb and TGAb, which lead to increased apoptosis via the Fas-mediated pathway (60). B cell-human lymphoblastoid cell lines derived exosomes enriched with Fas Lignad (FasL), which lead to activation of apoptosis in CD4+ T cells by Fas/FasL interaction, so exosomes can be a could be associated with cell apoptosis in Hashimoto’s thyroiditis (61). Immuno-modulatory actions and immune responses of cells are fine-tuned by exosomal surface protein markers like HSP60 and HSP70, which binds to various receptors like TLR, CD40, and Cd9 of the immune cells (62). However, specific mechanism of trans-communications and molecular switch at receptor level yet to be unraveled.

Graves’ disease is the most common cause of hyperthyroidism with manifestation of thyrotoxicosis and Graves’ ophthalmopathy and highest incidence in 30-50 aged women (63). An interesting studies by Rossi and others found that Dichlorodiphenlytrichloroetahns (DDT) stimulated thyroid follicular cells have active role in exosome mediated transferring the thyrotropin releasing hormones (TSHR) which involved in production of autoantibodies to the TSHR leading to Graves’ diseases (64). As mentioned before epigenetic modification (DNA methylation, histone modifications and noncoding RNAs(ncRNAs) interference) also plays a main role in pathogenesis of Graves’ diseases (65, 66), recent studies revealed that nucleic acids present in exosomes retain their pathological function in a recipient cell after direct transfer exosomal cargoes (67) and different stages of Graves’ diseases patients showed a different noncoding RNAs in exosome compared to healthy (68). Till now, exosomes related AITDs are still in sporadic stage. Till now, understanding of exosomes related AITDs are still in its infancy. Consequently, further research is required to elucidate the snapshot mechanism of exosomes in AITDS thereby delivers the deep understanding of AITDs, diagnosis and prognosis.

Drug induced liver injury (DILI) is common and almost all types of medicines. Recovery from DILI may require discontinuation medicines, hospitalization, or even liver transplantation (69, 70). Exosome are an attractive therapeutics and drug delivery vehicle, recently the hepatocyte and MSCs derived exosomes/EVs have revealed that they are effective in hepatic regeneration in liver injury models (71–73). Hepatocyte exosomes shown to have ceramidase and sphingosine kinase 2 (SK2) in their compartment, increased sphingosine-1-phosphate (S1P) in hepatocyte, which promotes proliferation hepatocytes (71). The MSC-EVs enriched in glutathione peroxidase 1 (GPX1) and knockdown of GPX1 reduced the protective effects of MSC-EVs in DILI model (74, 75). However, more preclinical experiments are still needed to understand roles of exosomes in DILI which can accelerate successful clinical translation of exosome therapy in DILI.

For end-stage organ diseases, transplantation becomes the only therapeutic option left with patients (143). Regardless of advancement in the field of medicine and surgery, substantial portion of patients continue to suffer various post-transplant complications and rejection of transplants. In recent years, EVs (including exosomes) have fascinated boundless attention of the scientist and surgeons as exosomes becoming a potential tool for understanding the transplanted organ physiology and diseases. Numerous studies have shown that exosomes released by various antigen presenting cells, which involves in humoral and cellular immune system (144).

The transplanted organ releases exosomes (allograft-exo) with donor major histocompatibility complex (MHC) molecules and they go out from the graft to the recipient’s lymphoid system (145, 146). These allograft-exo are internalized by recipient antigen-presenting cells (APCs), which leads to presence of donor MHC molecules on recipient APCs’ surface. This is called as semi-direct pathways in T cell-mediated rejection without direct contact between donor cells with recipient APCs (146–148). In a skin graft mice model, the donor MHC molecules are transported by EVs to the host’s lymphoid organs, then dendritic cells/B cells take up this donor MHC molecules (MHC-I and MHC-II) and presented to T cells, which leads to a direct inflammatory alloresponse in mice (146, 149). In case of solid organ transplantation, for example the heart transplant, recipient’s blood vessels are connected to the transplant during surgery. This leading to donor passenger leukocytes from graft may move to recipient, reported that donor dendritic cells was present with in the spleen of allogeneic heart transplanted mice (150), this was further reported in other studies that allo-MHC cross-dressed cells in lymphoid organs in vivo after cardiac transplantation (151, 152), allograft-exo may have been involved as well. Some studies show that allogeneic exosomes under certain circumstances involved in tolerance rather than rejection of allografts (153, 154). However, exosome involvement in organ transplantation still has not been elucidated and extensively studies are needed.

Inborn or innate immunity is inherited immunity consists of cellular and hormonal arms. The intercellular communication during the human genesis is first drafted by cell “language” communication system which is mediated by exosomes to maintain the rigidity of the cell. The effective immune response to foreign materials, like pathogen associated molecular patterns (PAMPS), danger associate molecular pattern (DAMPS), or life style associated molecular pattern (LAMPS), is controlled by exosome communication language. PAMPS and DAMPS are structurally diverse external molecules from pathogenic bacteria which includes, lipids, proteins, carbohydrates and material by products. Mega receptors in defense cell like leukocytes, first will have defense mechanism during the bacteria, virus, parasites and fungi invasion. Cellular recognition patterns such as Toll like receptors (TLRs) are expressed in various immune cells like leukocytes, macrophages, dendritic cells, B cells, T cells, and even non-immune cells like fibroblasts and epithelial cells (155). TLR first binds to PAMPS, and DAMPS thereby ignites the cell signaling cascades as combat mechanism. Under sterile inflammation condition, parenchymal and stromal cells communicate via extracellular vesicles through innate system. For example, during noxious gas inhalation, in lung, epithelial cell produces more exosomes with excess loading of microRNA17/221 (miRNA17/221) thereby promoting the local inflammation through the recruitment of M0 macrophage (156). Type 1 transmembrane protein, TLR has two domains like leucine-rich repeats (LRR) motif and Toll/LI-1R (TRR) as cytoplasmic domain in all immune cells and having different sub types like TLR-1, 2, 4, 5, 6 as cell surface members and TLR-3, 7, 8 and 9 as intracellular components (157). Among all these TLR-2 recognizes pathogens metabolites like lipoproteins/lipopeptides, peptidoglycan, glycosyphosphatidylinositol, phenol soluble modulin, zymosan and glycolipids (158). Double stranded RNA (dsRNA) of viruses will be recognized by TLR-3, while TLR-7 perceive GU rich single stranded RNA (ssRNA) but TLR-5 spots the bacterial flagellin (159–161). In tumor development and subsequent metastatic microenvironment, series of coordinated events are taking place between exosomes and TLR, thereby it plays an important role in the cancer development process. In cancer microenvironment scenario, TLR-8 plays important role by transmitting ECmiRNAs like miRNA 21 and miRNA 29a because of highest binding capacity with TLR-8 receptors thereby activates the receptors in immune cells and subsequently leads to the premetastatic inflammatory cytokines (162).

Despite the evidence that exosomes do play important role in the intercellular communications, the production and transport rely on the donor cells physiological conditions and receptor activations of receiver cells. TLR is known to be an importance factor in acute kidney diseases like urinary tract infections, ischemia-reperfusion (IR) injury, lupus nephritis and diabetic nephropathy (163). Micromanagement of exosomes in renal physiology have been studied both in vitro and in vivo condition. Miyazawa et al. first reported the role of exosome mediated aquaporin 1 and 2 (AQP-1, AQP-2) transport in collecting duct cells (164). Lipopolysaccharide (LPA) induced renal tubular epithelial cells showed the elevated level of exosome miRNA 19b-3p thereby induce the macrophage dependent tubulointerstitial inflammation (165). Extensive investigation on exosome mediated urolithiasis (kidney stone disease) remains under investigated, indeed, decreased the level of miRNA-21and Let-7 was noticed in lupus nephritis patients (166). However, genetic manipulations of specific transcriptomes aided with exosome engineering would shed better understanding of kidney stone disease (167).

Potential role of exosomes in immunomodulation disorder in blood cancer or the induction of hematological diseases in polycythemia vera (PV), essential thrombocythemia (ET) myelofibrosis (MF) (168). Chronic Myeloid leukemia (CML) cells (K562) derived exosomes promoted the angiogenesis, which was inhibited by dasatinib through inhibition of exosome release from K562 CML cells and their microenvironments (169). This in vitro study strongly supports hypothesis that microenvironment driven by K562 CML cells derived exosomes governs the clinical manifestation.

These exosomes also contains angiogeneic miRNAs such as mir-92 which helps in tumor cell migration during hypoxic condition (170). Disturbed HIF-1 pathways with upstream and downstream associated gene regulations by miR-1555, mir210, and mir135b also plays important role in migration of the CML cells by suppress or activation of factor inhibiting hypoxia inducible factor 1 (FIH-1). Taken altogether, cumulative factors such as EVs, growth factors cytokines and miRNAs play a fascinating role in the leukemogenesis, and cross-talks between various cell populations via each of the indicated components promote the formation of different types of hematological malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), lymphoma, and multiple myeloma (MM) (171).

The body uses autophagy to eliminate unhealthy cells so that it can replace them with new, healthier ones. In normal conditions, long-lived proteins and old organelles were degraded by autophagy for restoration of cellular contents. But under stressful conditions, for example hypoxic (lower oxygen) conditions, autophagy is activated to recycle molecules for providing energy and nutrients (172–174). Studies on exosomes have enlightened our understanding of their biological functions and recently several studies have shown that cancer exosomes play a great role in tumor progression and metastasis (175, 176). The studies have reported that knockdown of both autophagy related 16 Like 1 and autophagy related 5 in breast cancer cells shown to produce and release lesser exosome, this leads to decrease tumor metastasis (177, 178). Studies on G alpha interacting protein (GAIP) and GAIP interacting protein C-terminus have been shown involves in stimulation of biogenesis of exosome and autophagy flux in pancreatic tumor cells (179, 180). Inhibition of autophagy related 12–autophagy related 3, a complex plays important role in late step of autophagosome formation, reduce the exosome biogenesis by disrupting late endosome trafficking in multivesicular body (MVB) through interacting with Alix (172, 181). Several studies have well documented that autophagy, exosome plays important role in tumor progression (172, 182, 183). Autophagy and exosome release are robustly activated in tumors, which strongly showing that both these pathways are play a interplay in between and they are as part of hallmark of cancer cells.

Recently the scientists are focusing on inhibition of tumors progression and metastasis by targeting the autophagy and exosome biogenesis. A study reported that sulfisoxazole targeting the endothelin receptor A, which lead to inhibition of exosome biogenesis through increased degradation of MVB via the autophagy–lysosome pathway (184). A Study suggested that an inhibitor of ULKs (a serine/threonine protein kinase) leads to accumulation of immature early autophagosomal structures (185); ULK1 are involved in the trafficking of autophagy related 9, which is main player in intraluminal vesicle formation within amphisomes and autolysosomes (186, 187). Likewise other several inhibitors have been studied to inhibit biogenesis of exosomes (185, 188). Still several preclinical and clinical studies are required to understand the role of cancer exosome and autophagy and their targeting in cancer treatments.

Exosomes are released by cells, and their components provide a brief story about the microenvironment in and around the cell. These could be signals, exosomes presenting antigens, or even cancer-promoting factors. This property has previously been exploited for diagnostic purposes. The increased and highly stable exosome expression in patients with cancer renders it an up-and-coming player for cancer diagnosis. Various groups have used exosomes present in the plasma for diagnostic purposes. Different immunological markers of exosomes have been characterized in patients with chronic lung allograft dysfunction. Lung transplant recipients who displayed both the phenotypes of obstructive bronchiolitis obliterans syndrome and restrictive allograft syndrome had exosomes with distinct molecular and immunological profiles. Upon further testing, restrictive allograft syndrome samples were observed to have a higher concentration of pro-inflammatory factors, indicating severe allograft injury (189). Exosomes have emerged as prominent biomarkers for cancer diagnoses. Another group investigated the prominence of plasma-derived exosomal miR-19b in the diagnosis of pancreatic cancer. The results suggested that the levels of Exo-miR-19b normalized using miR-1228 were comparatively lower in patients with pancreatic cancer than in healthy individuals. This indicates that exosomes are promising candidates as biomarkers (190). This property of exosomes is utilized to diagnose various types of cancers, such as non-small cell lung cancer (NSCLC), prostate cancer, osteosarcoma, and cervical cancer. Lower expression of circulating miR-651 was observed in patients with cancer than in healthy individuals. Moreover, exosomes collected from HeLa cells were rich in CD63, CD9, and CD81 proteins, which are cancer cell hallmarks (191).

Long non-coding RNA (lncRNA) expression in urinary exosomes of NSCLC patients and healthy individuals as a potential lung cancer diagnosis has been explored. The results indicated that differential lncRNAs in urinary exosomes are NSCLC biomarkers because lncRNAs are enriched in specific pathways that might be involved in tumor cell proliferation and other processes associated with NSCLC pathogenesis (192). Liquid biopsy of exosomes isolated from patients with prostate cancer revealed that exosomes are enriched for genes that are hallmarks of prostate cancer, such as androgen receptor, kallikreins (KLK2), cyclin-dependent kinase inhibitor 1A (CDKN1A), KLK10, JUN, and B2M (β₂ microglobulin). These observations indicate that exosomes transport critical disease RNA transcripts and can be used as non-invasive diagnostic biomarkers (193). Exosomes of patients with prostate cancer had higher amounts of survivin, an apoptosis inhibitor, which is another biomarker for early detection of prostate cancer (194). These examples suggest that this is an untapped niche for early cancer diagnosis and has potential.

One of the first examples of exosome biodistribution in the body by in vivo positron emission tomography (PET) for non-invasive monitoring of copper-64 (64Cu)-radiolabeled polyethylene glycol (PEG)-modified exosomes was demonstrated with high imaging quality and quantitative measurements of blood residence and tumor retention. A fluorescent dye, amine-reactive Alexa Fluor 488 (NHS-Alexa Fluor 488), was conjugated to exosomes to form Alexa Fluor 488−exosome and Alexa Fluor 488−exosome−PEG. PEGylation confers an enhanced pharmacokinetic profile and higher tumor accumulation in exosomes compared with that of native exosomes. However, it also reduces premature hepatic sequestration and clearance of exosomes, thereby highlighting their improved therapeutic delivery efficacy and safety (195). Notably, this study provides crucial guidelines for obtaining precise and quantitative information on the biodistribution of exosomes via surface engineering, radiochemistry, and molecular imaging, which may aid future exosome research.

A technique was devised for in vivo neuroimaging and exosome tracking using gold nanoparticle labeling (196). Exosomes may be directly labeled with glucose-coated nanoparticles (GNP) without the need to label parent cells, and this labeling occurs through an active mechanism linked to the GLUT-1 (glucose transporter-1). Intranasal delivery is more effective than intravenous injection for brain accumulation. The noninvasive intranasal delivery route has multiple benefits and is a potential and effective treatment option for various CNS disorders. In contrast to the non-lesioned brain, the detection and accumulation of MSC-derived, GNP-labeled exosome labeling in the stroke region of the brain up to 24 h after administration was observed. Multifunctional exosomes for cancer theranostics were developed by electroporating urinary exosomes and ultra-small gold nanoparticles with Ce6 (chlorin e6) to obtain real-time fluorescence imaging and improve photodynamic therapy (197). The nanocomposites cloaked with exosomes exhibited enhanced long-term retention, biocompatibility, and penetrating behavior than that of free Ce6. A comprehensive study of the theranostic application of exosomes in cancer has been described by Ailuno et al. (198).

Circulating tumor exosomes with specific biomolecules have been recently used for detecting cancers and assessing therapy response and they are becoming more common as diagnostic targets (199–201). In recent times an ExoChip (with an anti-CD63 antibody), microfluidics device constructed to capture and collect specific exosomes, it showed that exosomes were significantly higher from individuals with cancerous diseases than healthy controls (202). Liquid biopsy with exosomal IGF-1R expression is used in individuals with lung cancers instead of invasive traditional tissue biopsies, which was achieved by microfluidics exosome analysis platform (203). A microfluidics chip is used for evaluation of circulating EpCAM-positive exosomes with plasma exosomes. EpCAM-positive exosomes was higher in individuals with breast cancer than healthy individuals (204). These studies and several other studies suggest that microfluidics-based exosome isolation and detection methods are more reliable (205) but still further discoveries are needed for its routine clinical translation.

The role of exosomes for therapeutic purposes requires further elucidation; nonetheless, their potential has been well-established. Exosomes did not exhibit toxicity upon injection, and nano-sized membrane-bound vesicles were well tolerated by the body. These properties make them excellent candidates for contact delivery because they do not undergo degradation. Both targeted and non-targeted exosome deliveries successfully alter protein expression in cancer cells (206).

There are several methods for isolating and purifying exosomes. Based on yield and purity, acoustic nanofilter (207), ExoSearch chip (208), immunoprecipitation (209), density gradient (210), precipitation kits, and ultracentrifugation methods are in use. A recent study demonstrated a separation method based on acoustofluidics by integrating acoustics and microfluidics approaches to isolate exosomes from blood directly [140]. This approach combines cell removal with exosome isolation. By integrating these modules into a single chip, exosomes were isolated with a blood cell removal rate of over 99.999% (211). Another study described a novel approach using microfluidic devices to isolate exosomes from whole blood, directly facilitating translation to the clinic (212). The choice of the device has a significant impact on the yield and purity of exosomes in the case of cell lines and complex biological fluids. The isolation of exosomes can be validated using nanosight tracking analysis, transmission electron microscopy, or western blotting. The revolutionized development of different approaches for exosome isolation and purification has made procedures more feasible for early diagnosis and therapeutics.

The off-target binding of drugs leads to several adverse effects (213). Several research groups are investigating targeted drug delivery to obtain high efficacy and low toxicity. Exosomes play a significant role in the targeted delivery of drugs and drug-like molecules. Similarly, paclitaxel (PTX)-loaded AS1411-chol exosomes (AS1411: a nucleolin-targeting aptamer) were delivered to target cancer cells with high efficiency in a recent study (214). Surface engineering, genetic engineering, chemical modification, and membrane fusion are some approaches adopted for the targeted delivery of exosomes (12). By using targeted surface engineering delivery and increasing exosome concentration at disease sites, this approach can reduce toxicity and increase therapeutic efficacy (215). In genetic engineering, ligands or homing peptides can be fused with TMP (5,10,15,20-tetrakis (1-methyl-4-pyridinio)), which is expressed on the surface of exosomes (216). In chemical modification, any functional group can be modified by another; for example, alkynes can be modified by an amine group. The exosomal lipid bilayer membrane can spontaneously fuse with other membranes using a membrane fusion approach.

Targeting exosomes to deliver chemotherapeutics, such as doxorubicin, to breast cancer tumor tissues in mice has been evaluated. The results were substantial as the treatment caused enhanced and rapid tumor regression without toxicity than that with standard therapy (217). Another study successfully used exosomes as a delivery vector to transport PLK-1 siRNA to bladder cancer cells in vitro, resulting in selective gene silencing of PLK-1 (218). ACTN4 is highly expressed in exosomes from patients with castration-resistant prostate cancer (CRPC). RNA interference-mediated ACTN4 downregulation significantly attenuates cell proliferation and tumor invasion, thereby confirming its role in prostate cancer development (219). Exosomes produced by adipose-derived stromal cells (ASCs) can also be used in prostate cancer therapy. ASC-derived exosomal miR-145 could reduce Bcl-xL activity and promote prostate cancer cell apoptosis via the caspase-3/7 pathway (220).

Phosphatidylserine (PS) expressed on the surface of exosomes is linked to T-cell immune suppression. Recently, a novel PS-binding molecule was developed that successfully blocked the immunosuppressive activity of human ovarian tumors and melanoma-associated exosomes. Upon treatment with ExoBlock, T-cell-mediated tumor suppression was significantly enhanced along with an increase in the number and function of CD4 and CD8 T-cells, which are involved in reducing tumor metastasis, thus providing promising antitumor therapy (221). Exosomes isolated from human umbilical cord MSCs were transfected with miR-3182. Exosomal miR-3182 significantly reduced cell migration and proliferation in triple-negative breast cancer (TNBC) cells. We also observed that miR-3182 loaded exosomes induced apoptosis in TNBC cells by downregulating mTOR and S6KB1 expression. Thus, this treatment can reduce the invasiveness of TNBC cells (222). Exosome therapy has also been used to treat other diseases.

In psoriasis, phospholipases on exosomes are associated with disease progression. Inhibition of phospholipases suppresses psoriasis progression (223). Inflammatory cytokines in the IL-23/Th17 axis and TNF-α signaling can be targeted. However, these can cause toxic side effects over an extended period (224). Alternatively, exosomes derived from MSCs have been used as potential treatments. This was tested in a mouse skin psoriasis model. The levels of pathology-associated IL-17, IL-23, terminal complement complex, and C5b-9 complex were reduced in the skin of mice treated with exosomes relative to that in the control (129).

Exosomes derived from adipose-derived MSCs (ADSCs) have therapeutic applications in hepatic I/R injury. ADSC-derived exosomes improved liver function by maintaining mitochondrial homeostasis through mitochondrial fission inhibition and promoting mitochondrial fusion and biogenesis. This may be attributed to the exosome-induced increase in the expression of mitochondrial fusion proteins such as OPA-1, MFN-1, and MFN-2. In contrast, they decreased DRP-1 and Fis-1 mRNA and protein expression related to mitochondrial fission. Additionally, exosomes significantly increase the expression of PGC-1α, NRF-1, and TFAM genes and proteins related to mitochondrial biogenesis (225). Another study in hindlimb ischemic animal models reported that MSC-derived extracellular vesicles activated VEGF receptors in endothelial cells, increased neovascularization at the site of ischemia, and accelerated recovery (226). In an associated study, extracellular vesicles derived from RAW 264.7, macrophages could also induce angiogenesis in vitro and in vivo (227). Exosomes derived from MSCs can target endogenous MSCs to enhance osteogenic differentiation and reduce adipogenic differentiation, which is a promising therapy for osteoporosis (228). Other possible treatment methods could be effective in initiating bone repair, regulating the immune response, and preventing bone resorption. Similarly, MSC-derived exosomes have also been used to treat inflammatory disorders, such as neurological disorders. This is accomplished using mRNAs, miRNAs, and immunosuppressive factors sourced from MSCs (229). Exosomes produced by MSCs stimulated by IFNγ reduced demyelination, decreased neuroinflammation, and upregulated the number of CD4, CD25, FOXP3, and regulatory T cells within the spinal cord. Moreover, exosomes reduce the levels of pro-inflammatory Th1 and Th17 cytokines; hence, exosomes can also serve as therapeutic targets for neurodegenerative disorders (230). Another promising study in this direction is from the Shetty group, which demonstrated that extracellular vesicles isolated from induced pluripotent stem cells effectively induce neurogenesis and treat neurodegenerative disorders in animal models (231). The hallmark of patients with pulmonary embolism is that pulmonary epithelial cells (PECs) are resistant to apoptosis. Mao et al. used exosomal miR-28-3p, which upregulated miR-28-3p expression and increased apoptosis by targeting apoptosis inhibitor 5 (API5) in PECs (229).

Milk-derived exosomes (mExosomes) are one of the most economical and promising drug delivery systems. Milk, an easily accessible raw material, can be prepared in substantially large amounts. Uniform particle size of ~100 nm, unique phospholipid layer (232), low immunogenicity, and inflammatory response (233, 234) make mExosomes an attractive research topic and an ideal vehicle for drug delivery in the future. The anticancer properties of drug-encapsulated mExosomes demonstrated in vitro growth inhibitory action in breast (T47D and MDA-MB-231) and human lung (A549 and H 1299) cancer cell lines (235). Animal studies on anti-tumor activity in mice bearing xenografts of A549 lung tumors exhibited low cytotoxicity and high bioavailability of mExosomes in the organs of the experimental model. A similar study reported that curcumin-loaded mExosomes exhibit enhanced anti-inflammatory and antitumor activities. This study also revealed that curcumin-loaded mExosomes have higher bioavailability than free curcumin (236).

RNA-based therapy has recently gained attention, but the challenge involves the delivery of highly unstable RNA molecules to biological systems. Gene expression studies have demonstrated the compatibility of mExosomes in delivering miR-148a-3p to hepatic (HepG2) and intestinal (Caco-2) cell lines (236), suggesting that mExosomes can be utilized as carriers of functional microRNAs. In a recent study (237), the mucus penetrability of siRNA-loaded exosomes was enhanced by adding a hydrophilic surface coating of PEG. PEGylated mExosomes exhibit improved penetrability and stability in an acidic gut environment. Bovine mExosomes are reportedly considered therapeutic agents against arthritis. Studies have demonstrated that spontaneous polyarthritis in IL-1Ra-deficient mice and collagen-induced arthritis is diminished by oral administration of mExosomes (238). However, the mechanism underlying the delayed disease onset remains unknown. Bovine mExosomes containing immunoregulatory miRNAs and anti-inflammatory proteins might have targeted inflammatory pathways. These are just a few examples of using exosomes for therapeutic purposes; further discoveries and breakthroughs have yet to be made.

Research on exosomes suggest that they are representative of the cellular condition and play a crucial role in upregulating, stabilizing, or down-regulating that condition. Exosomes secreted from a diseased cell would support disease progression, such as promoting angiogenesis for cancer or enhancing receptor expression to support viral infection or enhance inflammation. Exosomes secreted from stem cells reflect their purpose of supporting regeneration, healing, and growth. This information indicates that exosomes could be the next-generation technology that could receive large-scale public acceptance owing to their success in non-invasive diagnostics and non-toxic, target-specific drug delivery techniques used in clinical trials.

PG, HM and B-CA contributed to the conception, writing, and discussion of this manuscript. All authors wrote the initial draft of the manuscript. All authors Contributed to the critical conclusion of the manuscript. All authors contributed to the article and approved the submitted version.