Extracellular vesicles as standard-of-care therapy: will fast-tracking the regulatory processes help achieve the goal?

ABSTRACT

Extracellular Vesicles (EVs) became a focus of clinical research when experimental and pre-clinical studies showed that they mimic their parent cells’ regenerative and therapeutic effects and their cargo carries disease-specific diagnostic and prognostic biomarkers. Since the publication of data forms an endpoint of the study, this review specifically focused on the published clinical trials done with EVs. For brevity, this review was restricted to the last 10 years. Unexpectedly, the literature search showed that very few clinical trials assessing the therapeutic applications of EVs were published in this period indicating that they have not reached their desired endpoint. Conversely, most studies showed the potential of EVs present in various biofluids as a promising source of diagnostic and prognostic biomarkers for various diseases, and predictive markers to assess the effectiveness of therapy. This stark difference in the numbers could perhaps be due to the time-consuming regulatory processes involved in the clinical-grade preparation and characterization of EVs, and the determination of their safety and effective dose regimens. One wonders whether fast-tracking regulatory affairs could help accelerate the therapeutic use of EVs. This aspect needs urgent attention.

1. Introduction

Any novel concept or finding having potential therapeutic value takes a long time to reach its actual clinical applications – Extracellular Vesicles (EVs) are no exception to this [1]. EVs, once considered cellular waste, gained prominent status as future off-the-shelf, cell-free biologics when experimental and pre-clinical studies revealed that they mimic the regenerative and therapeutic properties of the parent cells [1,2]. All types of cells, including prokaryotic ones, secrete EVs, and their participation in various physiological and pathological conditions is evident [3]. EVs mediate intercellular communication through the delivery of bioactive cargo to the recipient cells and organs and elicit diverse biological responses. Deng et al. (2024) have reviewed the impact of EVs in bone-organ cross-talk. Their compilation of evidence shows how EVs secreted by bone cells regulate the metabolic activities of distant organs and vice versa. Similar studies need to be done with EVs from other organs and tissues [4].

The EVs secreted by cells like mesenchymal stem/stromal cells (MSCs), induced pluripotent cells (iPSCs), embryonic stem cells (ESCs), platelets (PLTs), etc., are at the forefront of clinical studies for assessment of their therapeutic applications as well as for their use as biomarkers [5–8]. Recently, Liu and Su (2023) have proposed an interesting proposal of using EVs secreted by organoids (OEVs) as therapeutics. Since organoids tend to recapitulate some of the features of the native tissue architecture and physiological functions, these EVs could have better therapeutic potential [9].

Several experimental and preclinical studies have underscored the importance of EVs as biomarkers [6,10]. Body fluids like blood, saliva, urine, spinal fluid, etc. contain EVs [11]. These body fluids, referred to as “liquid biopsies,” can be collected non-invasively, and hence, form an important source of diagnostic samples. EVs carry several macromolecules such as various types of RNA species, DNA, proteins, lipids, cytokines, growth factors, organelles like mitochondria, etc [12], and this molecular profile often reflects the tissue or cells producing them. Developmental or physiological processes affect the signaling pathways prevailing in the parent tissues or cells and consequently could induce dynamic changes in the molecular composition of the EVs [13]. However, the disease-specific changes in the EV composition could be unique and could serve as diagnostic and prognostic markers of the particular disease/pathological condition and its progression [14]. Importantly, specific molecular changes in the EVs could also reflect the patient’s response to the therapy, and hence, this area of clinical research is advancing rapidly [15].

Several clinical trials using EVs are in progress (https://clinicaltrials.gov/), however, these studies are at various stages of progress, and hence, the analyzed data obtained in these studies are yet to appear in the public domain indicating that they have to reach the endpoint. To gain an insight into the current state of progress in clinical studies using EVs, this review specifically focussed on “published clinical studies using EVs”. The advantages of reviewing published clinical studies are two-fold: one, the trials have reached their endpoint, expected or otherwise, and hence, the data are presented in a final analyzed form, and two, the publications have passed the peer review, providing a reliable source of information. The review reveals that though the regenerative properties of EVs have been established in several high-powered experimental and pre-clinical studies, the clinical studies assessing their therapeutic potential are taking a long time to reach the endpoint, compared to those evaluating their utility as biomarkers. This slow pace could perhaps be related to the stringent time-consuming regulatory processes involved in their clinical-grade isolation, characterization, determination of their safety and effective dose regimens, and the route of administration. One wonders whether fast-tracking regulatory affairs could help accelerate the therapeutic use of EVs. Since the pace at which regulatory bodies work could be country-specific, there may not be a single solution that can be applied universally. However, clinicians and researchers involved in the field can form a country-specific consortium and closely interface with the regulators to work out the fast-tracking approval process. Nonetheless, this aspect needs urgent attention.

1.1. Extracellular vesicles (EVs)

Classically, EVs have been classified into 3 main categories: Apoptotic bodies (ABs or APO-EVs; 1–5 µM), microvesicles (MVs; aka ectosomes; 200–1000 nm), and exosomes (Exo; 30–150 nm). Apoptotic bodies, as the name suggests, are secreted by cells undergoing apoptosis and usually, this fraction is discarded and not considered useful for regenerative applications. However, several studies have shown that the infused MSCs undergo apoptosis in vivo and the ABs secreted by these apoptotic bodies (APO-EVs/apoVs) can exert reparative effects, suggesting that the ABs need to be revisited in the context of regenerative medicine [16]. MVs are generated by the outward budding and subsequent pinching of the plasma membrane, whereas Exosomes are produced as intraluminal vesicles in the lumen of multivesicular endosomes, which are secreted after they fuse with the cell surface [17,18]. The biogenesis of EVs has been depicted schematically in Figure 1.

Figure 1.

Biogenesis of extracellular vesicles (EVs).

The EVs are categorized into 3 main categories: Apoptotic bodies (1–5 µM), microvesicles (200–1000 nm), and exosomes (30–150 nm). 1] Apoptotic bodies are secreted by cells undergoing apoptosis 2] Exosomes are produced in the lumen of multivesicular bodies, which fuses with the cell surface for secretion, and 3] MVs are generated by the outward budding and subsequent pinching of the plasma membrane.

As per the nomenclature proposed by the International Society of Extracellular Vesicles [ISEV; 19], EVs are classified based on vesicle size: multivesicular-body derived EVs (classically called exosomes, EXOs) are small EVs (sEVs; 30–100 nm in diameter), whereas plasma membrane-shed microvesicles (MVs) are known as larger EVs (lEVS; 100–500 nm in diameter). However, most publications still use the classical terminology of MVs and Exo to describe them. Some publications, especially in the field of cardiac diseases/disorders, use the term circulating microparticles (cMPs) to describe EVs in general without making any distinction between large and small particles. EVs can be additionally described based on their biochemical composition, expression of specific markers, or cells of origin.

The International Society for Cell and Gene Therapy (ISCT) has published the criteria for “manufacturing and characterizing extracellular vesicles from umbilical cord-derived mesenchymal stromal cells for clinical testing” [20]. Although these guidelines are specifically aimed at umbilical cord-derived EVs, in principle, they apply to EVs from any other sources, as the EVs fall into the category of “investigational new product.” ISCT has also published a report on “Therapeutic advances with native and engineered human extracellular vesicles,” which gives an overview of the current state of the art in developing and advancing EV therapeutics [21]. This report shows that in addition to the native EVs, engineered EVs – whether modified to alter their composition after secretion or where the parent cells are engineered to modify the EV composition – are also forming a rapidly growing area of therapeutics [22,23].

2. Clinical studies using EVs

Clinical use of EVs has several facets. EVs can be used as therapeutics and biomarkers of disease/pathological conditions or as biomarkers for assessment of the effectiveness of therapy (Figure 2(a, b)). Although the therapeutic potential of EVs has been underscored in several experimental and pre-clinical studies, very few clinical studies on the therapeutic effects of EVs were published in this review period (n = 4, Table 3). On the other hand, the majority of publications reported the data obtained in the clinical studies using EVs as biomarkers for disease/pathological conditions or effectiveness of therapy (n = 42; Tables 1 and 2). This stark difference in the number of publications on therapeutic aspects vis-à-vis biomarker studies indicates that the application of EVs as the “standard-of-care” therapeutic could take much longer time than anticipated or hoped for.

Figure 2.

(a,b) The overall landscape of various types of clinical studies done with EVs is illustrated.

Clinical studies involving EVs fall into four main categories such as using EVs as biomarkers of disease status or progression, using EVs as biomarkers for assessing the effectiveness of therapies used to treat various types of diseases/conditions, use of EVs as therapeutics, safety, and dose-determination studies of EVs, and use of human EVs in preclinical models. This stratification shows that the majority of the published studies dealt with EVs as biomarkers of disease or pathological conditions and the effectiveness of the therapeutic intervention, indicating that the biomarker field is moving faster than the others.

Table 3.

A summary of the data obtained in the studies evaluating the therapeutic potential of EVs has been tabulated.

Sr no	Category of Disease/tissue	Disease/condition	Therapy	EV source	Conclusion	Reference number
1	COVID-19	Acute respiratory distress syndrome (ARDS) and multi-organ failure	Perinatal allogenic MSCs (100 × 106cells; n = 11) or one dose of MSCs (100 × 106 cells) followed by one dose of MSC-derived EVs (n = 8).	Perinatal allogenic MSCs	MSCs and their EVs are safe and can significantly reduce the serum levels of inflammatory markers in COVID-19 patients	[78]
2	COVID-19	ARDS in severe COVID-19	BM-MSC-derived EVs (named ExoFlo)	BM MSCs	No adverse events were seen. 60-day mortality was decreased, and ventilation-free days improved in patients treated with ExoFlo-15 compared with a placebo	[79]
3	Wound healing	Atrophic acne scars	human adipose tissue stem cell (Ad-SC)-derived exosomes and fractional CO2 laser (3 consecutive treatments)	Ad-MSCs	the exosomes-treated sides achieved a significantly more significant improvement than the control sides	[80]
4	Wound healing	Chronic venous ulcer (CVU)	autologous serum-derived EVs applied at wound edges three times a week for 2 weeks	autologous serum	the s-EVs-treated lesions displayed a higher percentage of granulation tissue and higher sloughy tissue reduction compared to the control group	[81]

Table 1.

The data obtained in the studies involving EVs as biomarkers of disease or pathological conditions has been tabulated.

Sr no	Category of Disease/tissue	Disease/condition	EV source	Conclusion	Reference number
1	Cancer	Multiple Myeloma (MM)	Serum and BM	CD44-harboring EVs in serum could be a predictive biomarker of the overall survival of MM patients. A level of CD44 higher than 280 ng/ml indicates a significantly increased risk of death.	[23]
2	Cancer	GVHD	Serum	CD3+CD8+ and CD3+ HLA-DR+EVs are helpful for monitoring and evaluation of acute GVHD	[24,25]
3	Cancer	Acute myeloid leukemia (AML)	circulating EVs	Blast-specific EVs might serve as biomarkers of minimal residual disease. Increased levels of endothelial-specific EVs having procoagulant activity indicate vascular injury and thrombogenicity in AML patients.	[26]
4	Cancer	Ischemic stroke in cancer patients	Circulating cancer-cell-derived EVs	Cancer cell-derived EVs-mediated coagulopathy results in ischemic stroke via TF-independent mechanisms	[27]
5	Cancer	Prostate cancer	Prostate cancer (PCa) cells	The number of EVs in PCa cells correlates with the disease stage.	[28]
6	Cardiac	stable coronary artery disease (CAD)	Plasma MVs	Increased levels of miR‐126 and miR-199a in circulating MVs predict a reduced risk of major adverse CV in patients with stable CAD	[30]
7	Cardiac	Acute coronary syndrome (ACS) experiencing sudden cardiac death (SCD).	Plasma EVs	miR-208b-3p and miR-143-3p present in plasma EVs may serve as promising biomarkers in predicting SCD in patients with ACS	[31]
8	Cardiac	Coronary artery disease (CAD)	Plasma EVs	lncRNA AC100865.1 (CoroMarker) present in the EVs is a specific biomarker for CAD	[33]
8	Cardiac	Familial hypercholesterolemia (FH)	Circulating microparticles (cMPs)	FH patients have higher levels of CD45+/CD3+ cMPs than no-FH patients.	[34]
9	Cardiac	FH patients having high cardiovascular risk (HCVR)	Circulating MPs	HCVR patients have higher numbers of platelet-derived CD142+/TSP1+ MPs as well as of tissue factor-rich monocyte-derived CD142+/CD14+ MPs than controls	[35]
10	Cardiac	older subjects with moderate-to-high cardiovascular disease (CVD) risk	Circulating MPs	MP shedding relates to CVD progression	[36]
11	Cardiac	Patients on Mediterranean Diet + nuts who had suffered cardiovascular events (CVE) or had not (no-CVE)	Circulating MPs	Reduced cMPs derived from activated platelets, leukocytes, and endothelial cells predict protection against CVE in patients at high CV risk profile treated with MedDiet supplemented with nuts and receiving up-to-date CV drug treatment.	[37]
12	Cardiac	Patients with acute phase of ST-elevation myocardial infarction (STEMI)	MPs from blood collected from obstructed arteries and Circulating MPs	The presence of CD66b+/CD62E+/CD142+ cMP in peripheral blood may be a sensitive marker of the thrombo-occlusive vascular process in the coronary arteries of STEMI patients	[38]
13	Cardiac	Adverse cardiovascular events (MACE) in STEMI patients	Circulating MPs	Monocyte-derived cMPs in the acute phase could predict CV death	[39]
8	Blood disorders	Pulmonary arterial hypertension (PAH) in β-thalassemia patients	RBC- and platelet-derived EVs from blood samples	Platelet-derived EVs could be considered a marker of chronic platelet activation in transfusion-dependent thalassemia/HbE patients who have undergone antiplatelet therapy. PS-bearing large RBC-EVs could be used as biomarkers to determine the pathology of RBCs in these patients.	[40]
9	Neural	Parkinson’s Disease (PD)	Plasma	tau and Aβ1–42 in plasma EVs are significant prognostic markers of cognitive function in PD patients.	[42]
10	Neural	Parkinson’s Disease (PD)	Plasma	miR‑34a‑5p is significantly up‑regulated in small EVs isolated from PD patients. miR‑34a‑5p levels are also associated with disease duration.	[41]
11	Neural	Patients suffering from various neurological disorders	Saliva, serum, plasma, CSF and urine	Heme oxygenase-1 (HO-1) present in the circulating EVs may contribute to the systemic manifestations of various neurological conditions	[43]
12	Neural	Multiple sclerosis (MS)	Serum	Several dysregulated EV miRNAs were identified relative to healthy controls in the serum of MS patients at various stages of the disease, but the study does not show the presence of any specific miRNA in the EVs	[44]
13	Kidney	Patients undergoing renal transplant	Urine	CD133+ EVs increased modestly after renal transplant, and these EVs express glomerular and proximal tubular markers reflecting the activity of CD133+ progenitor cells	[45]
14	Lung	Healthy individuals smoking E-cigarettes (30 puffs)	Blood	Smoking nicotine-containing e-cigarette vapor causes a significant increase in the levels of endothelial- and platelet-specific EVs, signifying vascular changes.	[46]
15	Liver	Non-alcoholic fatty liver disease (NAFLD)	Plasma	Hepatocyte-derived EVs present in the plasma could identify NAFLD patients at an early stage	[47]
16	Obesity	metabolic dysfunctions in obesity	Plasma	Increased levels of circulating MVs and EXOs correlate with BMI. MVs contain Macrophage Migration Inhibitory Factor (MIF), which triggers rapid ERK1/2 activation in macrophages	[48]

Table 2.

The data obtained in the studies using EVs as biomarkers for assessment of the effectiveness of therapy has been depicted in a tabulated form.

Sr no	Category of Disease/tissue	Disease/condition	Therapy	EV source	Conclusion	Reference Number
1	Cancer	Pediatric Acute Myeloid Leukemia (AML)	Chemotherapy	Plasma	Leukemia-derived EV-dsDNA could potentially be used as a biomarker to measure treatment response in pediatric AML	[51]
2	Cancer	Head and neck squamous cell carcinoma (HNSCC)	chemo-radiation therapy	Plasma	EV-associated TGFβ3 can be used as a predictor for response to chemoradiation therapy in HNSCC patients	[52]
3	Cancer	advanced hepatocellular carcinoma (HCC)	sorafenib + selective internal radiation therapy (SIRT) or sorafenib alone.	Plasma	EV-ACTR3 could differentiate between responders and non-responders receiving SIRT+ sorafenib therapy but not in patients receiving sorafenib alone.	[53]
4	Cancer	Melanoma	BRAF inhibitor monotherapy (e.g., dabrafenib) and/or combined therapy with BRAF and MAPK/ERK kinase (MEK) inhibitors (e.g., dabrafenib and trametinib).	Plasma	EV phenotyping could be used to monitor treatment responses	[54]
5	Cancer	Gastroesophageal junction adenocarcinoma	Neoadjuvant pembrolizumab-containing chemoradiation followed by surgical resection and adjuvant pembrolizumab	Plasma	PD-L1 expressing EVs may identify treatment responders	[55]
6	Cancer	Prostate cancer	dietary protein restriction (PR)	Plasma	EVs containing leptin receptor and increased ratio of pan-tyrosine to serine 312 phosphorylated forms of IRS1 in L1CAM+ EVs can be used to follow physiologic responses to dietary interventions	[56]
7	Cardiac	Coronary artery disease (CAD)	dietary nitrate supplementation combined with or without clopidogrel therapy	Circulating platelet-derived EVs	Reduced levels of platelet-derived CD41+ EVs indicate a response to clopidogrel	[57]
8	Cardiac	Subclinical atherosclerosis	rosuvastatin	LDL-EVs in serum	An increase of plasminogen and VWF levels in LDL-EVs correlated with LDL reduction	[58]
9	Cardiac	Ischemic heart disease	Intramyocardial injection of allogeneic adipose-derived stem cells (ADSC)	Plasma	Reduced levels of miR-126 in small EVs can potentially be used as a biomarker for improved cardiac function post-ADSC treatment.	[59]
10	Cardiac	Coronary artery bypass (CABG) surgery	Remote ischemic preconditioning (RIPC)	Serum	RIPC exerts its cardioprotective effect via the early release of EVs harboring cardioprotective miRNAs, including miR-21.	[60]
11	Cardiac	Coronary artery disease (CAD)	RIPC and antiplatelet therapy	EVs derived from platelets, leukocytes, endothelial cells, and erythrocytes	Decreased levels of circulating platelet-derived EVs indicate the effectiveness of RIPC in CAD patients taking conventional antiplatelet therapy.	[61]
12	Cardiac	Acute myocardial infarction (AMI),	Ticagrelor or clopidogrel	Plasma	Ticagrelor reduces post-AMI mortality by preventing the increase in platelet EVs, fibrinogen+, PS+, and leukocyte EVs in plasma	[62]
13	Neural	Alzheimer’s Disease (AD)	Exenatide	cerebrospinal fluid (CSF)	Exenatide produced a significant reduction of Aβ42 in the EVs of neuronal origin	[63]
14	Neural	Alzheimer’s Disease (AD)	Intranasal insulin	Plasma	Levels of pS312-IRS-1 and pY-IRS-1 in the circulating neuronal EVs correlate with cognitive changes in response to low-dose intranasal insulin in AD patients	[64]
15	Neural	Chronic major stroke.	Intravenous injections of autologous MSCs	Plasma	An increase in circulating EVs harboring miRNA-18a-5p correlates significantly with improvement in motor function	[65]
16	Blood-related	Blood transfusion in critically ill patients	Healthy volunteers injected with lipopolysaccharide and transfused with stored autologous RBC units collected 35 days earlier.	RBC units and blood of the participants	RBC-EVs produced during storage do not express RBC membrane markers that are associated with clearance.	[66]
17	Blood-related	Blood transfusion in critically ill patients	Transfusion with RBCs stored for longer periods	Plasma	Transfusion of RBC units stored for up to 35 days does not result in substantial changes in coagulation or immune parameters	[67]
18	Blood-related	Effect of storage on platelet functions	Storage	Plateletpheresis concentrates (PCs)	The number of CD62P+ PLTs inversely correlated with the increased PL-EV levels, indicating loss of platelet functions during storage.	[68]
19	Blood-related	History of venous thromboembolism (VTE)	Rosuvastatin	Plasma and platelets	Rosuvastatin-mediated decrease in plasma PPL activity does not correlate with the levels of total or platelet-derived EVs	[69]
20	Exercise	Healthy humans	Exercise-induced secretion of proteins contained in EVs	Plasma	EVs produced during exercise mediate tissue crosstalk and exert systemic biological effects.	[70]
21	Exercise	Obesity in sedentary youth	Acute aerobic exercise consisting of 6 weeks of resistance training (RT).	Circulating EVs in blood	Production of larger TSG101+ and CD63+ EVs with increased protein indicates a response to RT.	[71]
22	Exercise	Healthy young adults	Traditional combined exercise (TRAD) and high-intensity tactical training (HITT)	Skeletal muscle- and serum-derived extracellular vesicles	TRAD elicited protein-coding gene response, whereas HITT elicited differential expression of microRNA enriched in brain regions in the EVs	[72]
23	Diet	Young adults	Symbiotic beverage enriched with the kimchi-derived bacterium Leuconostoc holzapfelii (L. holzapfelii)	Stool and urine samples	microbiota-derived EVs can be used as surrogate markers in predictive diagnosis studies	[73]
24	Diet	Moderate risk of CVDs	Fish oil (1.9 g/d n-3 PUFAs) or control oil (high-oleic safflower oil) for 12	Plasma and platelets	n-3 PUFAs significantly decreased the numbers of circulating EVs, doubled their n-3 PUFA content, and reduced their thrombin generation ability. These data could have implications for the prevention and treatment of thrombosis	[74]
25	Diabetes	Type-2 diabetes	Pioglitazone	Plasma and adipose tissue	The beneficial effects of pioglitazone are mediated by adipose-specific miRNA regulation	[75]
26	Pregnancy	Recurrent pregnancy loss (RPL).	intravenous immunoglobulin (IVIG)	Plasma	Statistically significant increase in the circulating levels of EVs in patients receiving IVIG. The biological role of this increase is not clear.	[76]

Pre-clinical studies on “human EVs” in animal or cell culture models and safety studies form an important area of clinical research as the data obtained in these studies lay the foundation for further phase II/III clinical trials.

All these published clinical studies are described below.

2.1. Use of EVs as biomarkers

The high number of publications dealing with the use of EVS as biomarkers indicates that this aspect of clinical research on EVs is advancing at a rapid pace perhaps due to relatively lesser regulatory hurdles and also due to much lower financial burden compared to therapeutic use.

An ideal biomarker for any disease or pathological condition is expected to have high levels of accuracy, sensitivity, and specificity. In addition to these attributes, it also needs to be present in the clinical sample that can be obtained using minimally invasive procedures. Most of these expectations are fulfilled by blood or plasma/serum samples, and hence, the majority of the clinical studies on using EVs as a source of biomarkers focus on blood/plasma samples. However, several studies also use other body fluids or cancer tissues for the isolation or detection of EVs. The blood or body fluids used to detect biomarkers are called liquid biopsies. Isolation of EVs from the body fluids offers a distinct advantage – compared to the biofluids, the biomarkers present in the isolated EVs get enriched severalfold and thus facilitate their detection with enhanced sensitivity and accuracy.

The importance of EVs in cellular functions in disease onset and progression makes them an important source for biomarker discovery and diagnosis/prognosis. However, the low yield of EVs isolated from biofluids poses a challenge in terms of high throughput and sensitivity. Recent studies have used high-throughput proteomic and glycoproteomic analyses for performing EV profiling with high sensitivity [24,25]. Such analyses form a very powerful tool in biomarker discovery for early-stage disease detection.

EVs expressing specific surface markers or molecular profiles of their cargo can be used as biomarkers for two distinct purposes: one, as markers of disease status or progression, and two, as markers to determine the efficacy of any therapy or intervention. They can also be used to monitor relapse or drug resistance. These clinical studies clearly show that EVs, especially circulating or plasma-derived EVs, are an important source of diagnostic and prognostic biomarkers and need to be studied in further detail to make them globally applicable.

2.1.1. Biomarkers of disease/pathological conditions (Table 1)

EVs acquire specific macromolecular profiles as a result of biochemical changes precipitated by the disease or pathological conditions. Several studies have endeavored to identify these specific changes in the EVs isolated from patients’ samples, compared to those from matched control subjects. These studies need to be done on larger cohorts, preferably in multi-centric trials, to make the identified molecule(s) a universally accepted biomarker of that particular disease. Several studies to detect biomarkers in EVs have been done, amongst which, cancer- and cardiac disease-related studies prevail.

2.1.1.1. Cancer

Early detection, patients’ response to the treatment, prognosis of disease-free survival, staging, detection of relapse, etc. form crucial aspects of cancer management. Identification of specific biomarkers predicting these aspects would substantially contribute to the medical management of cancer patients. The most ideal biomarker of cancer would be the one that can detect cancer at a very early stage even before the patient becomes symptomatic. The term “cancer” covers a broad range of neoplastic diseases, and hence, the identification of a very specific biomarker for a particular type of cancer is a tough job. But efforts made in this direction would eventually help in achieving this goal.

Multiple myeloma (MM) is a hematological malignancy of plasma cells in the bone marrow. MM cells are dependent on the cytokines secreted by the bone marrow stromal cells (BMSCs). CD44 is an adhesion molecule required by the MM cells to adhere to the BMSCs and induce IL-6 from them. CD44 is also known to cause drug resistance in MM cells [26]. Harshman et al. [27] characterized the proteome of EVs isolated from serum samples collected from newly diagnosed Multiple Myeloma (MM) patients (n = 233) and healthy donors. They found that the CD44 primarily localizes in the EVs present in the MM patients’ sera and increased levels of CD44 in the serum-EVs, especially higher then 280 ng/ml, indicate the significantly increased risk of death. The data suggest that the levels of CD44-harbring EVs could form a predictive biomarker of the overall survival of MM patients.

Graft vs Host disease (GVHD) is one of the major complications in allogeneic hematopoietic stem cell transplantations (HSCT). Using a serum-EV assay previously developed in their laboratory, Oba et al. [28,29] measured T-cell-specific EVs in 20 patients (n = 13 children, n = 7 adults) undergoing HSCT, and correlated their number with GVHD. They found that CD3⁺CD8⁺ EVs and CD3⁺ HLA-DR⁺EVs increased in GVHD, and this increase was associated with persistent GVHD. They conclude that these EVs could help in monitoring and evaluating acute GVHD. Although the work is commendable, the utility or the advantage of the complicated assay used in this study over the more standardized and universally accepted standard flow cytometry is unclear. Also, this study needs to be done on larger cohorts.

Acute myeloid leukemia (AML) is characterized by excessive proliferation of nonfunctional blast cells. Minimal residual disease (MRD) is a primary cause of relapse in these patients, and therefore, there is a need to develop sensitive diagnostic tools to detect MRD at an early stage. Also, many AML patients develop venous thromboembolism (VTE). EVs are known to promote cancer progression and EVs bearing tissue factor (TF) cause prothrombotic state in cancer patients. Tzoran et al. [30] examined whether circulating EVs can be used as biomarkers of disease progression and procoagulant activity in AML. EV concentration, cell origin, and expression of coagulation proteins were determined in the blood samples of newly diagnosed AML patients (n = 42) and healthy controls (n = 24) using a flow cytometer. The blood samples were collected at diagnosis, nadir, and remission. They found that the reduction in the number of blast-specific EVs correlated with post-remission survival. They suggest that blast-specific EVs might serve as biomarkers of MRD and an increase in endothelial-specific EVs having procoagulant activity could indicate vascular injury and thrombogenicity in AML patients.

EVs play an important role in coagulopathy in cancer patients resulting in ischemic stroke. Bang et al. [31] determined the levels of circulating cancer cell marker+ and TF+ EVs in TF-bearing EVs. Levels of EVs expressing various cell-specific markers were determined in the sera of patients with ischemic stroke with or without active cancer (n = 155 and 25, respectively), cancer patients without stroke (n = 32), and healthy controls. They found that the levels of cancer cell-derived EVs (CD326⁺ and CD326⁺CD142⁺) were significantly higher in the sera of patients having cancer-related stroke and correlated with levels of D-dimer and with TF⁺ EVs and platelet-derived EVs. These levels decreased upon treatment with anticoagulation therapy. However, the authors mention that the patients need to be followed for more extended periods to establish the use of cancer-cell-derived EVs as predictive biomarkers of the onset of stroke in cancer patients.

Unlike the previously described studies done with liquid biopsies, Agarwal et al. [32] enumerated the number of EVs in prostate cancer (PCa, n = 10) and benign prostate hyperplasia (BPH, n = 6) tissue biopsies using a transmission electron microscope. They found that the number and density of EVs were significantly higher in the PCa cells than in the BPH cells. Since the number of EVs in the PCa cells positively correlated with the Gleason score, the authors concluded that the EV number could potentially become a biomarker in the staging of PCa. This study needs to be done on a much larger cohort to get statistically significant data among the various grades of PCa. Also, whether these EVs are specific to prostate cancer needs to be determined by using specific markers. The technique used in this study is very specialized, so its routine clinical use may pose technical difficulties. Although electron microscopy offers the advantage of magnification and higher resolution, the ability to discriminate between artifacts arising during sample preparation and actual sub-cellular structures needs specialized training. Also, the exorbitant cost involved in setting up this facility could be a major deterrent in its routine use in clinical diagnostics.

2.1.1.2. Cardiac

Cardiovascular disease (CVD) is one of the leading causes of death worldwide. Early detection and diagnosis of CVD could help in reducing the mortality and morbidity associated with it. Several clinical trials aim to identify a biomarker in EVs – referred to as microparticles (MPs) in most of these studies – for early detection of CVD.

miRNAs – a class of noncoding RNAs – regulate gene expression post-transcriptionally by binding to specific mRNAs and inducing their degradation or translational repression. miRNAs present in the EVs are promising disease-specific biomarkers. Several studies have investigated the presence of cardiac disease-specific miRNA in circulating EVs and shown that these miRNAs indicate cardiac injury [33]. Increased levels of vascular and endothelial-specific miRNAs in EVs, but not in plasma, were found to be associated with cardiovascular events in patients with stable coronary artery disease (CAD) [34]. They also found that miR-126 was present in CD31⁺/CD42b⁻ endothelial cell-derived MVs, whereas miR-199a was detectable in CD31⁺/CD42b⁺ platelet-derived MVs.The authors reported that the increased levels of miR‐126 and miR-199a in circulating MVs predict a reduced risk of major adverse CV in patients with stable CAD. Likewise, Huang et al. [35] analyzed the miRNAs present in the plasma EVs as biomarkers for early diagnosis of patients suffering from Acute Coronary Syndrome (ACS) experiencing sudden cardiac death (SCD). They found a positive correlation of miR-208b-3p and miR-143-3p levels with myoglobin, which is an early indicator of acute myocardial infarction. The authors conclude that miR-208b-3p and miR-143-3p present in plasma EVs may serve as promising biomarkers in predicting SCD in patients with ACS. They also point out that these miRNAs can be used in postmortem forensic investigations to determine the cause of death due to ACS.

In addition to miRNAs, non-coding RNAs (ncRNA) such as small ncRNAs, circular RNAs, and long ncRNAs are also known to get selectively sorted into EVs and modulate various biological processes in recipient cells. These ncRNAs also serve as diagnostic and prognostic biomarkers of diseases [36]. Yang et al. [37] identified lncRNA AC100865.1 (referred to as CoroMarker) as a differentially expressed marker from the microarray analysis of plasma samples collected from male patients with or without coronary artery disease (CAD; n = 15 in each; 5 samples from each set were pooled to get 3 CAD and 3 Non-CAD samples). The predictive value of CoroMarker was further confirmed using samples collected from 221 CAD patients and 187 control individuals. The authors conclude that the CoroMarker present in the EVs is a specific biomarker for CAD. This study underscores the importance of studying RNA category biomarkers in the EVs, as these are likely to be more stable.

EVs secreted from various tissues or cells carry specific surface markers that are associated with the activated state of cells or tissue injury. These changes can be quantified by using flow cytometry and the change in their altered levels can be used as biomarkers of the disease. Suades et al. [38] characterized the circulating microparticles (cMPs) in patients with stable statin-treated heterozygous familial hypercholesterolemia (FH) and examined whether the phenotype correlates with atherosclerotic plaque burden. Age/sex-matched non-FH patients were used as comparators. They found that FH patients with subclinical lipid-rich atherosclerotic plaques had significantly higher levels of CD45⁺/CD3⁺ cMPs, indicative of higher endothelial activation and higher pro-inflammatory profile, than in non-FH patients. Also, the levels of these cMPs could differentiate between FH patients with lipidic or non-lipidic plaques. FH Patients are at high cardiovascular risk (HCVR) and develop premature coronary artery disease. Suades et al. [39] investigated whether HCVR patients having lipid-rich atherosclerotic lesions have circulating prothrombotic MPs. They found that HCVR patients have higher numbers of platelet-derived CD142⁺/TSP1⁺ MPs as well as of tissue factor-rich monocyte-derived CD142⁺/CD14⁺ MPs than controls. Since the number of circulating tissue factor-rich MPs correlated with subclinical atherosclerotic plaque burden detected by MRI, the authors propose that these MPs could be used as biomarkers of silent atherothrombotic disease and serve as prognostic markers of cardiovascular risk. Chiva-Blanch et al. [40] assessed whether MPs of vascular origin would predict future cardiovascular events (CVE) in older subjects with moderate-to-high cardiovascular disease (CVD) risk under standard treatment. They found that the individuals who suffered a CVE showed increased MP shedding from lymphocytes and smooth muscle cells (SMC) after one year of intervention. The authors concluded that MP shedding relates to CVD progression. In a parallel study [41], they characterized the circulating MPs in patients who had suffered (CVE) or had not (no-CVE), a CVE during follow-up. These patients were selected from an ongoing Mediterranean Diet + nuts (MedDiet-nuts) arm of the PREDIMED clinical trial (ISRCTN35739639). They found that no-CVE patients showed decreased levels of MPs secreted by activated platelets, leukocytes, and endothelial cells. They conclude that in patients having high CV risk treated with MedDiet-nuts, reduced levels of these MPs are predictive of protection against CVE.

Increased levels of circulating MPs in the acute phase of ST-elevation myocardial infarction (STEMI) indicate microvascular obstruction. Saudes et al. [42] examined the phenotype of cMPs in the blood of STEMI patients. Samples were collected at percutaneous coronary intervention (PCI) from the obstructed coronary and peripheral circulation in STEMI patients (N = 40). Peripheral blood of age-matched patients recovering from STEMI [after 72 h] and control individuals (N = 20/group) served as controls. They found that in STEMI patients, especially those treated at short ischemic time, the number of annexin V-positive cMPs expressing CD66, CD62E, and CD142 originating from monocytes, endothelium, and activated vascular cells was higher in the affected coronary artery than in the peripheral blood. The authors concluded that the presence of these cMP in peripheral blood could form a sensitive biomarker indicative of the thrombo-occlusive vascular process in STEMI patients. In a parallel study, Chiva-Blanch et al. [41] determined whether cMPs could form a prognosis biomarker for adverse cardiovascular events (MACE) in STEMI patients. However, they did not find any differences in MP levels between patients with or without a MACE. However, the analysis showed that Annexin V-AV± cMPs carrying tissue factor were higher in patients who died because of CV, compared to patients who survived. The authors suggest that assessing the monocyte-derived cMPs in the acute phase could predict CV death.

2.1.1.3. Blood disorders

Pulmonary arterial hypertension (PAH) is commonly found in β-thalassemia patients. EVs shed by activated platelets and plasma membranes of abnormal RBCs are associated with thrombotic risk. Manakeng et al. [43] determined the numbers of phosphatidylserine (PS)-bearing RBCs and EVs in the blood samples of splenectomized TDT-β-thalassemia/HbE patients (n = 11 with PAH and n = 14 without PAH; n = 15 normal subjects) by flow cytometry. They found that the number of large PS⁺ RBC-EVs and platelets and their EVs (pEVs) were significantly increased in β-thalassemia patients with PAH, but not in patients without PAH, compared to normal subjects. They suggest that p-EVs could be considered a marker of chronic platelet activation in transfusion-dependent thalassemia/HbE patients who have undergone antiplatelet therapy. Likewise, they propose that PS-bearing large RBC-EVs could be used as biomarkers to determine the pathology of RBCs in these patients.

2.1.1.4. Neurological disorders (NDs)

Neurological disorders (NDs) comprise heterogeneous diseases that affect the body’s autonomic, peripheral, as well as central nervous system. They include migraine, epilepsy, multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, etc. Mortality and disability associated with NDs have become a global public health challenge as a result of an increase in life expectancy and population aging. Most NDs lack a cure, and hence, early diagnosis would play an important role in slowing down the ND progression [44].

Cognitive decline is a significant problem in Parkinson’s Disease (PD) patients. Identifying biomarkers indicating the onset of cognitive decline could help better manage these patients. Chung et al. [45] investigated the role of EVs bearing α-synuclein, tau, and β-amyloid 1–42 (Aβ1–42) as biomarkers for cognitive dysfunction in the plasma collected from Parkinson’s Disease patients (PD; n = 116) and control subjects (n = 46). Although there was no significant difference in the number of tau and Aβ1–42 containing EVs between PD patients and controls, the Tau levels in EV showed a significant association with cognitive function. Also, EVs bearing tau and Aβ1–42 were significantly elevated in PD patients having cognitive impairment when compared to those with optimal cognition. They conclude that tau and Aβ1–42 bearing plasma EVs can be used as prognostic markers of cognitive function in PD patients. Grossi et al. [46] explored the possibility of using plasma EV-associated MicroRNA‑34a‑5p as a biomarker for PD. Using a serial ultra-centrifugation technique, they separated large, medium, and small EVs from the plasma of PD patients (n = 15) and age-matched control (n = 4). The analysis showed that the expression of miR‑34a‑5p was significantly up‑regulated in small EVs isolated from PD patients, and the levels were associated with disease duration. These results underscore the importance of examining the miRNA content of each EV subpopulation to identify any biomarker having potential diagnostic value. However, this study needs to be validated in larger cohorts.

Oxidative stress is a causative agent of several neurodegenerative diseases. Heme oxygenase-1 (HO-1) induces high levels of oxidative stress and, hence, plays a significant role in neurological diseases. High levels of HO-1 are found in the blood of subjects suffering from neurological disorders. Cressatti et al. [47] investigated the HO-1-containing EVs in various biofluids collected from human subjects (n = 85). They isolated astrocyte-specific [glutamate aspartate transporter 1 (GLAST)-enriched] and CNS neuron-specific EVs [L1 cell adhesion molecule protein (L1CAM)-enriched] to examine whether the EVs harboring HO-1 protein are released by the central nervous system (CNS) neurons into the periphery. Saliva, serum, plasma, CSF, and urine samples were collected, and HO-1 was assayed in the EVs isolated from these biofluids using ELISA and Western blot. They found that EVs from all the biofluids examined contained full-length HO-1 protein, and most EV-associated HO-1 was derived from CNS. Based on these data, the authors propose that HO-1 present in the circulating EVs could potentially be used as a biomarker of neurological conditions. This possibility needs to be validated using samples from patients suffering from specific neurological conditions.

Disease-modifying treatments such as antipsychotic medication and psychological interventions given at the early stage of multiple sclerosis (MS) can slow disease progression and avert long-term disability. Cuomo-Haymour et al. [48] studied the miRNAs in the serum EVs of multiple sclerosis (MS) patients and matched controls to identify stage-specific differentially expressed miRNAs and investigated whether these miRNAs could serve as biomarkers of MS. They identified several dysregulated EV miRNAs in MS patients relative to healthy controls. However, the study fails to provide any specific miRNA signature in the EVs that can be used as a definitive biomarker. Hence, further research is needed to identify disease- and stage-specific biomarkers for MS.

2.1.1.5. Kidney

Necrosis and apoptosis of renal tubular epithelial cells due to ischemia-reperfusion injury are responsible for delayed graft function leading to morbidity and a decrease in graft survival. The renal function post-transplant is monitored by a percutaneous allograft biopsy, which is an invasive procedure. The development of noninvasive methods for the evaluation of renal function would improve the management of renal transplant patients. Urinary EVs derived from the cells of the nephron indicate kidney function. Dimuccio et al. [49] isolated CD133⁺ EVs from the urine of patients undergoing renal transplants (n = 25) and age-matched normal subjects (n = 20). They found that the urine of normal subjects, but not of patients suffering from kidney failure, contained CD133⁺ EVs. The levels increased modestly after the renal transplant. They also found that CD133⁺ EVs expressed glomerular and proximal tubular markers, suggesting that perhaps these EVs reflect the activity of CD133⁺ progenitor cells in renal homeostasis. They suggest that the CD133⁺ EVs could indicate the regenerative potential of progenitor cells after acute kidney injury.

2.1.1.6. Lung

Smoking nicotine-containing E-cigarettes, though considered effective for smoking cessation, can lead to pathological changes in the lungs such as increased airway inflammation, increased airway obstruction, vascular changes, and arterial stiffness. In a randomized, double-blind, crossover study, Mobarrez et al. [50] investigated the effect of smoking e-cigarettes, with and without nicotine, on vascular responses. They measured endothelial- (CD62⁺) and platelet-specific (CD41⁺CD62⁺CD154⁺) EVs in the blood samples collected from healthy young volunteers (n = 17) who were asked to take 30 puffs of E-cigarettes, with or without nicotine, in 30 minutes. They found that smoking nicotine-containing e-cigarette vapor caused a significant increase in the levels of endothelial- and platelet-specific EVs, signifying vascular changes. Based on their data, the authors suggest that prescribing the use of E-cigarettes to overcome smoking addiction should be done cautiously.

2.1.1.7. Liver

Non-alcoholic fatty liver disease (NAFLD) remains asymptomatic in the early stages, and hence, a blood-based biomarker could help identify these patients. Nakao et al. [51] examined whether the number of circulating hepatocyte-specific EVs (containing CYP2E1 and ASGR) could be used as a biomarker for NAFLD and whether these EVs decrease after weight loss surgery. They examined EVs in paired plasma samples collected before and after weight loss surgery (n = 56). They found that the plasma levels of hepatocyte-derived EVs were significantly elevated in NAFLD patients and decreased after weight loss surgery. The number of these EVs also correlated with the severity of the disease. The study indicates that hepatocyte-derived EVs present in the plasma could identify NAFLD patients at an early stage.

2.1.1.8. Obesity

Dysregulated production of adipokines is associated with obesity. Amosse et al. [52] examined the role of fat-derived EVs in metabolic dysfunction in obesity. They isolated microvesicles (MVs) and exosomes (EXOs) from the plasma samples collected from metabolic syndrome patients and quantified the soluble factors present in them. They found that an increase in the levels of circulating fat-derived MVs and EXOs correlated with BMI. They further showed that these MVs contain Macrophage Migration Inhibitory Factor (MIF), which triggered ERK1/2 activation in the macrophages. The data suggest the role of EVs in the development of obesity-associated metabolic complications.

Overall, these published studies show that EVs form a very powerful tool as biomarkers of various diseases and pathological conditions. Importantly, they also serve as an excellent tool to assess the effectiveness of the therapy. The field appears to be moving at a rapid pace. However, more intensive and focused efforts need to be put into it using larger, homogenous patient cohorts, and global collaborations to undertake multi-centric multi-national trials for the identification of very specific biomarker(s) for a specific disease condition and stage. Such endeavor would also help in the determination of therapeutic efficacy leading to more effective medical care. Since the EV membranes protect the macromolecules present within them from the enzymes present in the plasma and stabilize, the EVs seem to form a very promising tool in biomarker discovery. Additionally, plasma is a minimally invasive source compared to solid biopsies, making it an ideal source for the isolation of EVs.

2.1.2. EVs as biomarkers for assessment of effectiveness of therapy (Table 2)

Precision medicine and personalized medicine are some of the recently evolved concepts. These concepts are based on the fact that most disease conditions, especially cancer, show a spectrum, and each patient’s condition may require a specific therapeutic approach. To ensure the effectiveness of the therapy being administered it is necessary to judge whether it is showing the expected results. Such indication of success or failure would help in making a timely decision to continue or change the regimen.

2.1.2.1. Cancer

The term cancer covers a wide range of diseases that not only show tissue-specific variation but also cells-specific and patient-specific characteristics. The advent of molecular biology has revealed cancer heterogeneity at sub-cellular levels. This complex situation mandates that specific biomarkers need to be identified to judge the effectiveness of the therapy [53]. Also, specific biomarkers indicating the emergence of drug resistance during the treatment need to be identified so that a timely change in the therapeutic regime can be made.

Acute Myeloid Leukemia (AML) is a heterogenous hematological malignancy that can become life-threatening if left untreated. The heterogeneity arises due to various mutations in functional categories such as cell signaling, proliferation, epigenetic regulation, etc [54]. These mutations could be used to develop targeted therapies and also to assess the treatment efficacy. Kontopoulou et al. [55] isolated double-stranded DNA (dsDNA) from the plasma-derived EVs of pediatric AML patients (n = 29; at initial diagnosis or during treatment) and healthy donors (n = 5). They found that the dsDNA samples isolated from EVs showed leukemia-specific mutations in the primary leukemia cells. After treatment, the number of EVs decreased, and the leukemia-specific mutations were not detectable in the dsDNA isolated from post-treatment EVs. The authors suggest that leukemia-derived EV-dsDNA could potentially be used as a biomarker to measure treatment response in pediatric AML. However, these data need to be validated using a large patient cohort.

Transforming growth factor-beta (TGFβ) signaling plays a role in cancer progression and radio- and chemo-resistance by inducing epithelial-mesenchymal transition (EMT) and maintaining the cancer stem cells. Rodrigues-Junior et al. [56] quantified TGFβ3 protein in the EVs in plasma collected from patients suffering from head and neck squamous cell carcinoma (HNSCC, n = 38) treated with cytotoxic chemo-radiation therapy. They found that the TGFβ3 levels were significantly higher in the EVs collected from the plasma of non-responders than those isolated from the responders, and high levels of TGFβ3 in Annexin V-EVs correlated with the worst progression-free survival. They suggest that EV-associated TGFβ3 can be used as a predictor for response to chemoradiation therapy in these patients.

Shuen et al. [57] performed proteomics on the EVs collected from the plasma of advanced hepatocellular carcinoma (HCC) patients undergoing sorafenib + selective internal radiation therapy (SIRT; n = 25) or sorafenib alone (n = 20). Patients were classified as responders or non-responders based on the changes in alfa-fetoprotein (AFP) and imaging or overall survival. Proteomic analysis of EVs showed that ACTR3 (Actin-related protein 3) – a protein known to promote tumor development – was significantly higher in the EVs isolated from responders compared with non-responders, and their level significantly correlated with response, suggesting that plasma EV-ACTR3 could differentiate between responders and non-responders receiving SIRT+ sorafenib therapy, but not in patients receiving sorafenib alone.

EVs are heterogeneous in nature and exhibit multiple phenotypes, which reflect their biological functions. Wang et al. [58] studied the phenotypic changes in the plasma EVs of melanoma patients to assess the feasibility of using the plasma EV phenotypic evolution to determine their treatment responses. Using a multiplex EV phenotype analyzer chip (EPAC), they could detect cancer-specific EV phenotypes in the plasma of melanoma patients. Long-term follow-up of patients receiving targeted therapy (n = 8) showed that the EV profiles indicate the development of drug resistance. Although their data indicate the possible use of EV phenotyping for monitoring treatment responses, the authors suggest that further studies are needed to get a conclusive correlation between EV phenotype and clinical data.

Anti-programmed death (PD)-1 targeting immune checkpoint inhibitors in combination with chemotherapy have formed first-line therapy in the treatment of several cancers. Zhu et al. [59] explored whether plasma EVs could identify responders to treatment in patients with gastroesophageal junction adenocarcinoma treated with pembrolizumab (anti-PD-1 antibody)- and chemoradiation followed by surgical resection and pembrolizumab (n = 28). They found that an elevated plasma level of PD-L1⁺EVs was significantly associated with higher pathologic response. They suggest that levels of PD-L1-EVs may help in identifying the treatment responders.

Since dietary protein restriction (PR) is known to increase insulin sensitivity and suppress the growth of prostate cancer in animal models, Eitan et al. [60] examined the effect of PR on the biomarkers present in the plasma EVs of men with prostate cancer. They found that the levels of leptin receptors increased in the L1CAM⁺ plasma EVs from patients in the PR group. They further found that PR increased the ratio of pan-tyrosine (pY) to serine 312 phosphorylated (pSer312) forms of the insulin receptor signal transducer protein, IRS1 (Y/S IRS1 ratio) in L1CAM⁺ EVs, indicating improved insulin sensitivity. They suggest that these EV-associated biomarkers can be used to follow physiologic responses to dietary interventions in humans. The effect of PR on tumor growth was not examined in this study.

Collectively, these studies underscore the potential of using EVs as biomarkers to assess the response to therapy in cancer patients. This aspect is very important to determine the effective medical management of these vulnerable patients and decide whether to continue the treatment regimen or change it.

2.1.2.2. Cardiac conditions

Cardiovascular disease (CVD) is one of the leading causes of death worldwide. Early diagnosis plays an important role in reducing the mortality rate. Identification of CVD-specific biomarkers would help in identifying high-risk people and help in early diagnosis. Platelet-derived EVs express phosphatidylserine that stimulates thrombin generation and stabilizes fibrin clot formation leading to CAD, therefore, these EVs could be used as biomarkers of CAD.

Nitric Oxide plays an essential role in maintaining platelet homeostasis. Dietary nitrates elevate circulating levels of nitrite. Burnley-Hall et al. [61] performed a randomized, double-blind study to assess the effect of dietary nitrate supplementation (SiS nitrate gel vs. placebo) combined with (n = 10) or without (n = 10) clopidogrel therapy on circulating EVs in coronary artery disease (CAD) patients. They found that dietary nitrate reduced the number of platelet-derived CD41⁺ EVs in the plasma of CAD patients on clopidogrel therapy and increased their response to clopidogrel. They suggest that dietary nitrate supplementation could be helpful to reduce the risk of clot formation in CAD patients.

Verbree-Willemsen et al. [62] examined the changes in LDL-EVs in serum collected from patients with subclinical atherosclerosis (n = 666) and treated with rosuvastatin versus placebo. They found that two years of rosuvastatin treatment was strongly associated with increased levels of plasminogen and VWF levels in LDL-EVs. The change of LDL-EV-plasminogen level correlated with LDL reduction. Since the rosuvastatin-mediated increase in LDL-EV coagulation proteins differs from the serum LDL levels, the authors conclude that LDL-EVs can be used as an intermediate biomarker for statin therapy and coagulation.

Traxler et al. [63] investigated the effect of intramyocardial injection of allogeneic adipose-derived stem cells (ADSCs) on cardiac function in patients with ischemic heart disease. They studied miRNAs in the small EVs after ADSC (n = 10) or placebo treatment (n = 5). They found that after 12 months of treatment, the expression of miR-126 decreased significantly in patients receiving ADSC, but not in those receiving a placebo, suggesting that the reduction of miR-126 in small EVs can potentially be used as a biomarker for improved cardiac function post-ADSC treatment.

Remote ischemic preconditioning (RIPC) protects the heart against ischemia/reperfusion injury. Two clinical studies have examined the involvement of EVs in the protective effects of RIPC. Frey et al. [64] examined whether RIPC in anesthetized patients undergoing coronary artery bypass (CABG) surgery (n = 58, randomized into RIPC or sham) results in the release of EVs harboring cardioprotective miRNAs in the bloodstream. They found that five minutes after RIPC, EV concentration increased in the RIPC group, and the expression of 26 miRNAs, including the cardioprotective miR‐21, was found to increase in them. The authors conclude that RIPC exerts its cardioprotective effect via the early release of EVs harboring cardioprotective miRNAs. Reddel et al. [65] investigated the changes in EVs derived from platelets, leukocytes, endothelial cells, and erythrocytes and compared the data with global coagulation parameters and fibrinolytic factors in patients suspected of having CAD receiving RIPC and antiplatelet therapy (n = 60; RIPC = 31, sham = 29). They found that RIPC, but not the sham treatment, significantly decreased the circulating platelet-derived CD41+ CD6+ EVs. The number of PS+ CD62P+CD45+ CD11b+ CD144+ CD31+/CD41– or CD235a+ EVs was not affected. The data suggest that RIPC reduces the number of platelet-derived EVs in the circulation of CAD patients undergoing antiplatelet therapy. They also found that clinical variables such as the use of statin, diabetes, and hypertension alter the effectiveness of RIPC.

In acute myocardial infarction (AMI), various cells, such as activated platelets, leukocytes, and endothelial cells, release PS-exposing EVs having procoagulant properties. Ticagrelor is known to reduce mortality after AMI compared to clopidogrel, but the mechanism involved in this difference was not known. Gasecka et al. [66] compared the number of EVs and their procoagulant activity in the plasma of AMI patients (n = 60) treated with ticagrelor or clopidogrel. The numbers of EVs from activated platelets (CD61⁺, CD62p⁺), fibrinogen⁺, phosphatidylserine (PS⁺), leukocytes (CD45⁺), endothelial cells (CD31⁺, 146⁺), and erythrocytes (CD235a⁺) were determined by flow cytometry. EV procoagulant activity was measured by a fibrin generation test. They found that the numbers of platelet EVs, fibrinogen⁺, PS⁺, and leukocyte EVs were lower on ticagrelor than on clopidogrel after 6 months. The number of endothelial EVs and EV procoagulant activity did not differ between patient groups. The authors conclude that ticagrelor reduces post-AMI mortality by preventing the increase in the number of EVs in plasma.

Overall these studies indicate that the number, phenotype and molecular composition of serum EVs can be utilized as biomarkers for various aspects of cardiac diseases.

2.1.2.3. Neural

Patients suffering from neurological diseases show cognitive impairment and loss of motor functions. Although these diseases are difficult to cure, improvement in these functions would improve the quality of life of these patients. Several drugs are being tried to improve the cognitive ability of AD patients. Preclinical studies have suggested that exenatide, a glucagon-like peptide-1 (GLP-1) agonist used in the management of type 2 diabetes mellitus, has neuroprotective and disease-modifying effects in AD. Mullins et al. [67] conducted a double-blind, randomized, placebo-controlled Phase II clinical trial to assess the safety and tolerability of exenatide in participants (n = 18) likely to develop AD based on cerebrospinal fluid (CSF) biomarkers. They found exenatide to be a safe and well-tolerated drug, but it didn’t produce any difference in clinical and cognitive measures except for a significant reduction of Aβ42 in the EVs of neuronal origin. However, the study was terminated early, so firm conclusions could not be drawn. Insulin resistance is implicated in cognitive impairment in AD patients. Hence, Mustapic et al. [68] examined the potential of neuronal-enriched EV as biomarkers to assess cognitive changes in AD patients in response to intranasal insulin. They isolated neuronal-enriched EVs (CD171⁺) from plasma samples of subjects (n = 56) with probable AD in a placebo-controlled trial of intranasal insulin (20 or 40 IU). They found that in participants treated with 20 IU insulin, EV biomarkers of insulin resistance (pS312-IRS-1, pY-IRS-1) showed strong positive correlations with cognitive changes, especially in ApoE ε4 non-carriers. They concluded that levels of pS312-IRS-1 and pY-IRS-1 in the circulating neuronal EVs correlate with cognitive changes in response to low-dose intranasal insulin in AD patients.

Stem cell therapy is considered a potential regenerative strategy for patients with neurological diseases. Bang et al. [69] investigated the levels of circulating EVs and trophic factors after intravenous injections of autologous MSCs in patients with chronic major stroke treated with autologous MSC (n = 39) or standard treatment (n = 15). Serial samples were collected before and 3 months post-therapy, and microRNAs and trophic factors in the EVs were quantified. They found a significant increase in the number of plasma-EVs within 24 hours after the intravenous injection of MSCs (1 × 10⁶ cells/kg). The number of circulating EVs correlated with improved motor function after adjustment of various clinical parameters. MicroRNAs related to neurogenesis, but not the tropic factors, significantly increased EVs in the plasma of patients receiving MSC therapy. Specifically, the levels of miRNA-18a-5p, which is linked to the nerve growth factor receptor signaling pathway and axonal guidance, increased in the EVs from the MSC group. The authors concluded that the increase in the circulating EVs correlated significantly with improved motor function. However, the reason behind the unresponsiveness of some of the treated patients was not apparent.

2.1.2.4. Blood-related

The EVs derived from platelets and RBCs are important factors to be considered in transfusion medicine, as they could function as determinants of the quality of the stored blood and platelets. Stored RBC units contain high concentrations of RBC-EVs, and hence, after transfusion, the number of EVs in circulation increases. These RBC-EVs cause adverse effects in critically ill patients. Therefore, van Manen et al. [70] investigated whether these RBC-EVs exhibit membrane markers that are associated with the clearance of RBCs. Six volunteers were injected with Escherichia coli lipopolysaccharide and transfused with 35-day stored autologous RBC units. EV analysis was done on the RBC units and the volunteers’ blood before and after transfusion. They found that only PS⁺RBC-EVs were present in the RBC units. Before transfusion, the volunteers’ samples contained PS⁺ and CD59⁺ RBC-EVs, but the expression of the other RBC membrane markers was much lower. Two hours post-transfusion, the concentration of RBC-EVs increased 2.4-fold but decreased toward baseline levels. Therefore, the authors concluded that RBC-EVs present in the stored units do not express clearance-associated RBC membrane markers.

Several studies have shown that transfusion with RBCs stored for more extended periods is associated with increased mortality in critically ill patients. Norris et al. [71] measured various coagulation, immune parameters, and EV markers in critically ill patients (n = 100) receiving RBC transfusion. They found that levels of protein C, factor V, and PS+ CTLA-4+ (CD152) EVs in the plasma of patients receiving fresh RBC units (stored less than 8 days) and those receiving standard storage age RBC units were significantly different. The coagulation and EV markers and all cytokines tested showed no difference between study arms. However, after transfusion, 6 coagulation parameters, 15 cytokines, and 7 EV parameters changed significantly. The authors concluded that transfusion of stored RBC units (up to 35 days) does not change coagulation or immune parameters.

Platelet storage often leads to a loss of functionality. Platelet-derived extracellular vesicles (PL-EVs) are present in plateletpheresis concentrates (PCs). Black et al. [72] analyzed PL-EVs in 42 PC samples to examine whether these EVs influence the quality of PCs. In addition to the number of EVs present in the PCs, they measured the functionality of PLTs in response to thrombin by flow cytometry. They found that the externalization of CD62P, indicating platelet functionality, was significantly decreased during storage, and also, the number of CD62P⁺ PLTs inversely correlated with the increased PL-EV levels. The authors suggest that the analysis of PL-EVs could be used as a quality control parameter for stored PCs.

Ramberg et al. [73] investigated the effect of rosuvastatin treatment on plasma procoagulant phospholipids (PPL) activity and levels of EVs in patients with a history of venous thromboembolism (VTE). Patients with a history of VTE (n = 245) were asked to stop anticoagulant treatment and randomized to either 20 mg/day of rosuvastatin treatment or no treatment for 28 days. Plasma samples were collected before and after the treatment, and PPL activity and EV levels were determined. They found that rosuvastatin-mediated decrease in plasma PPL activity did not correlate with the levels of platelet-derived EVs. Although this is a negative finding, clinicians and researchers working in the field need to know about it so that unnecessary duplication of research work can be avoided.

2.1.2.5. Exercise

Exercise plays a vital role in regulating metabolism. Physical inactivity leads to decreased insulin sensitivity, reduced lipid clearance, loss of muscle mass, and accumulation of visceral adiposity. Three clinical studies examining the potential of EVs as biomarkers to assess the effect of exercise on human health were found.

Whitham et al. [74] characterized the exercise-induced secretion of proteins contained in EVs in healthy humans. They found that following a 1-hr cycling exercise, there was an increase in the circulating EVs containing more than 300 proteins without having a signal peptide sequence. Using a mouse model, they showed that the exercise-induced EVs tend to localize in the liver and can transfer their protein cargo. The data indicate that EVs produced during exercise mediate tissue crosstalk and exert systemic biological effects.

The importance of exercise in preventing and managing obesity is well known. Pierdoná et al. [75] evaluated whether changes in EVs after acute aerobic exercise could identify the responders following resistance training (RT). They studied the circulating EVs in the blood samples collected from sedentary, obese young subjects (n = 11) at pre- and post-acute exercise. After 6 weeks of RT, the subjects were stratified into responders or non-responders based on changes in insulin sensitivity. The authors found that there was a significant increase in EV production in both groups. However, EV size distribution analysis showed that the blood samples from responders predominantly contained larger EVs (sized 150–300 nm) having higher protein yield, whereas those from non-responders contained smaller EVs (sized 50–150 nm) with lower protein yield. Their data suggest that the production of large TSG101⁺ and CD63⁺ EVs indicates the response of obese young subjects to acute exercise.

Lavin et al. [76] performed transcriptomics in the skeletal muscle- and serum-derived EVs before and after traditional combined exercise (TRAD) and high-intensity tactical training (HITT) in healthy young adults (n = 40). EV analysis showed that TRAD evoked a consistent protein-coding gene response, whereas HITT led to an enrichment of brain-related microRNA. Their data indicate that such analyses could be potentially used to understand mechanisms of exercise response and adaptation.

2.1.2.6. Diet

A combination of probiotics and prebiotics confers health benefits by modulating the intestinal microbiome. The EVs secreted by these intestinal microbes can help in determining the effect of diet on the gut microbiome. Yang et al. [77] assessed the effect of a symbiotic beverage enriched with the kimchi-derived bacterium L. holzapfelii on the intestinal microbiota and composition of EVs isolated from stool and urine samples in healthy Korean adults (n = 20; 9 males and 12 females). They found that stool- and urine-derived EVs showed significant alterations in four and eight microbial genera, respectively, while only minor changes were observed in the gut microbiome. These findings suggest that microbiota-derived EVs can be potentially used as surrogate markers in diagnostic studies.

In a randomized, double-blind, placebo-controlled study, Bozbas et al. [78] investigated the effects of fish oil on the circulating EVs in subjects with moderate risk of CVDs (n = 40). The subjects were given either fish oil- (1.9 g/d n-3 PUFAs) or control oil-containing (high-oleic safflower oil) capsules for 12 weeks, and the number and fatty acid composition of circulating and platelet-derived EVs (PDEVs) were determined. They found that administration of n-3 PUFA-containing capsules significantly decreased the numbers of circulating EVs having twice the amounts of n-3 PUFA content and reduced their ability to generate thrombin. The authors concluded that these data could have implications for the prevention and treatment of thrombosis.

2.1.2.7. Diabetes

Pioglitazone, a widely used drug for the treatment of type 2 diabetes (T2D), acts via peroxisome proliferator-activated receptor-g (PPARg) and increases insulin sensitivity leading to a durable reduction in HbA1c. Nunez Lopez et al. [79] examined whether pioglitazone treatment alters the molecular cargo of circulating adipocyte-derived EVs in type 2 diabetes (T2D) patients. They conducted a 3-month trial in T2D patients (n = 24). The patients were treated with pioglitazone (45 mg/day) or placebo, and the levels of miRNAs were measured in plasma-derived and adipose tissue-derived EVs. They found that the levels of 5 miRNAs (i.e., miR-7-5p, miR-20a-5p, miR-92a-3p, miR-195-5p, and miR-374b-5p) were significantly downregulated in EVs in response to pioglitazone treatment, whereas an opposite effect was seen for miR-195-5p in subcutaneous adipose tissue. Changes in miRNA expression correlated with improved insulin sensitivity. Their data suggest that the beneficial effects of pioglitazone are perhaps mediated by the regulation of adipose-specific miRNA.

2.1.2.8. Pregnancy

Recurrent pregnancy loss (RPL) is the most common pregnancy complication. Immunological disturbances appear to be a risk factor in RPL, and hence, various immune-therapeutic have been tried in this context. Several studies have suggested that intravenous immunoglobulin (IVIG) might improve pregnancy outcomes in women with RPL. Since in pregnancy disorders, changes in the number and composition of placental and non-placental EVs have been reported, Jørgensen et al. [80] studied the effects of IVIG or placebo (albumin) on plasma EVs in patients suffering from RPL (n = 39). They found a statistically significant increase in the levels of circulatory sEVs in patients receiving IVIG; however, the biological role of this increase has not been clarified in the study.

2.2. Therapeutic applications of EVs: (Table 3)

Recently, Duong et al. [81] have reviewed ongoing clinical trials investigating the therapeutic application of EVs. They found 73 active trials and noted that a majority of them are using MSC-derived EVs. Also, among the various disease conditions being treated with EVs, the trials related to COVID-19 and acute respiratory distress syndrome (ARDS) were the most common ones. My search of published clinical trials using EVs as therapeutic revealed an unexpected finding – compared to the use of EVs as biomarkers, only 4 clinical studies were published on therapeutic applications of EVs out of which 2 were on COVID and 2 were on wound healing. This low number of publications indicates that perhaps most of the ongoing trials on therapeutic aspects of EVs have not reached their endpoint. It is also possible that the cost involved in the clinical trials is prohibitively high, and hence, not many groups take up the challenge. In addition to the cost involved in setting up GMP-grade setup, the expertise needed, the time frame required for final analysis, etc., stringent parameters led down for biomanufacturing of EVs, the time-consuming regulatory requirements for manufacturing clinical-grade EVs could be one of the critical contributing factors to this low publication rate [20].

2.2.1. COVID

The COVID pandemic has created interest in the clinical applications of MSC-derived EVs. Two publications have already appeared in the literature. However, whether these fast-paced publications happened due to as the accelerated regulatory approvals, considering the gravity and urgency of the situation needs to be determined. If found to be so, this model can be implemented for application of EVs in therapeutics.

Acute respiratory distress syndrome (ARDS) and multiorgan failure caused by severe inflammation are the leading causes of mortality in COVID-19 patients. MSCs and their EVs are known to have anti-inflammatory properties. In a randomized control trial, Zarrabi et al. [82] evaluated the safety and therapeutic efficacy of perinatal allogenic MSCs and their EVs in COVID-19 patients with ARDS. The patients either received two consecutive injections of MSCs (100 × 10⁶cells; n = 11) or a combination of MSCs (100 × 10⁶ cells) and MSC-derived EVs (n = 8). Control (n = 24) groups received only the standard treatment. They found that mortality was reduced in MSC(n = 3) and MSC+EV (n = 0) groups as compared to the control (n = 8). MSC + EV infusion significantly decreased inflammatory cytokines such as IL-6, TNF-α, IFN-γ, and CRP. Based on these data, the authors concluded that MSCs and their EVs are safe and can significantly reduce the serum levels of inflammatory markers in COVID-19 patients. In a prospective phase 2 randomized placebo-controlled trial, Lightner et al. [83] determined the safety and efficacy of bone marrow mesenchymal stem cell (BM-MSC)-derived EVs (named ExoFlo) in COVID-19 patients having ARDS. The patients were randomized into three groups and infused with a placebo, ExoFlo (10 ml), or ExoFlo (15 ml) on days 1 and 4. They did not find any treatment-related adverse events in the patients. 60-day mortality was decreased, and ventilation-free days improved in patients treated with ExoFlo-15 compared with placebo.

2.2.2. Wound healing

Even though in healthy individuals the wounds usually heal by natural processes, several persons develop chronic non-healing wounds or abnormal scarring. In recent years, the topical application of EVs has been tried for wound-healing purposes.

Atrophic acne scars often show an incomplete recovery due to inflammation-induced damage to the dermal matrix. These scars are also psychologically damaging and can cause social disability. In a prospective, double-blind, randomized study, Kwon et al. [84] assessed the safety and efficacy of 3 consecutive treatments with human adipose tissue stem cell (Ad-SC)-derived exosomes combined with fractional CO₂ laser for acne scars in 25 patients. Post-laser treatment, Ad-SC-derived exosomes were applied on one side of the face, and a control gel was used on the other side. They found that the exosomes-treated sides achieved a significantly more significant improvement than the control sides as determined by a substantial reduction in ECCA (échelle d’évaluation clinique des cicatrices d’acné) score. The authors concluded that a combined treatment comprising exosomes and fractional CO₂ laser would provide synergistic effects on the atrophic acne scars.

In most patients suffering from chronic venous ulcer (CVU), the ulcer remains unhealed and forms a chronic condition. In a pilot study, Gibello et al. [85] investigated whether treatment with autologous serum-derived EVs (s-EVs) would improve the healing process. The patients were treated with s-EVs three times a week for 2 weeks. They found that the s-EVs-treated lesions displayed a higher percentage of granulation tissue and higher sloughy tissue reduction than the control group. They attribute this therapeutic effect of sEVs to the increase in microvascular proliferation evoked by transforming growth factor-β1 present in the sEVs.

2.3. Safety studies: (Table 4)

Table 4.

Summarizes the data obtained in the safety studies done with EVs.

Sr no	Category of Disease/tissue	Study subjects	Therapy	EV source	Conclusion	Reference number
1	Delayed wound healing	Healthy individuals	Paired punch biopsies created in the skin were treated with allogenic platelet-derived EVs or placebo	Allogenic platelets	The application of allogenic pEVs was safe as the wounds treated with pEVs and placebo healed normally.	[84]
2	Lung diseases	Healthy individuals	Nebulization with various doses of EVs	Adipose tissue-derived MSC (haMSCs)-EVs	Nebulization of EVs was safe as no serious adverse events were observed	[85]

Intricate mechanisms involved in the intercellular cross-talk between EVs and the recipient tissues or cells pose a challenge in the development of EV-based therapeutics. The payload carried by the EVs varies according to cell type, culture conditions, and the signaling pathways prevailing in the parent cells [86,87]. Coupled with these factors, variable protocols followed for the isolation of EVs also complicate the matter, making standardization of EV preparations a difficult task. These aspects warrant stringent safety studies to be done with even EVs isolated from natural sources before the clinical trials are initiated.

EVs of activated platelets are known to release various growth factors, cytokines, and extracellular matrix modifiers. Johnson et al. [88] assessed the safety of allogeneic, platelet-derived EVs (pEVs) as a potential therapeutic for delayed wound healing in healthy persons (n = 11). A punch biopsy wound was created in the skin of each inner arm and treated with pEVs or a placebo. They found that the application of allogenic pEVs was safe, and the time required for wound closure in the pEV-treated and control wounds was comparable in both groups. To prove the therapeutic efficacy of the pEVs, this study needs to be done in patients with delayed wound healing.

Administration of drugs via nebulization increases the bioavailability of the drug in the lungs. In a phase I single-arm study, Shi et al. [89] explored the safety of nebulized human adipose tissue-derived MSC (haMSCs)-EVs in healthy volunteers. The study included five cohorts of healthy volunteers (3–6 subjects/cohort, total n = 24) who received a single nebulization of haMSC-EVs (range − 2.0 × 10⁸ to 16.0 × 10⁸ EVs). The volunteers tolerated the treatment well, and no serious adverse events were observed. These findings suggest that nebulization of haMSC-EVs could be a promising therapeutic strategy in pulmonary diseases.

2.4. Preclinical studies of “human EVs” in animal or cell culture models: (Table 5)

Table 5.

Summarizes the data obtained in the studies using “human EVs” in animal or cell culture models.

Sr no	Source of EVs	Objective	Model system used	Experiment	Conclusion	Reference Number
1	EVs collected from patients suffering from acute coronary syndrome (ACS; n = 30) after percutaneous coronary intervention (PCI).	To test the cardio-protective properties of EVs	In vitro model – Human microvascular endothelial cells (HMEC-1) + cardiomyocyte H9c2 cells co-culture. Ex vivo model – isolated rat hearts	Cardioprotective effects of EVs on hypoxia/reoxygenation damage and ischemia/reperfusion injury (IRI) were investigated.	PCI leads to the loss of cardioprotective effects of EV from ACS patients by reducing Dusp6 mRNA and upregulation of stress- and cell-cycle-related genes in them	[86]
2	EVs isolated from patients undergoing RIPC (n = 10) exposed to two different anesthetics,	Effect of two different anesthetics, namely, isoflurane or propofol against hypoxia-induced apoptosis	H9c2 cells	After 6 hours of EV treatment, the H9c2 cells were cultured for 18 hours in normoxic or hypoxic atmospheres.	Propofol adversely affects the cardioprotective effects of EVs isolated post-RIPC.	[87]
3	EVs isolated from human atrial explant-derived cells or vehicles.	To examine whether EVs isolated from human heart explant-derived cells can prevent postoperative Atrial fibrillation (AF) after open chest surgery	Rats	Rats were treated with a trans-epicardial injection of EVs isolated from human atrial explant-derived cells or vehicles	EV treatment abrogated the pericarditis-associated AF by reducing the infiltration of inflammatory cells and the production of pro-inflammatory cytokines.	[88]
4	Exosomes isolated from the plasma of untreated Acute Myeloid Leukemia (AML) patients and healthy subjects.	To examine whether exosomes present in the plasma of AML patients affect antitumor activities of immune cells.	NK 92 cell line	NK92 cells were treated with exosomes isolated from AML patients.	Exosomes isolated from the plasma of untreated AML patients block the antileukemic action of NK cells by delivering inhibitory ligands to the cognate receptors, thereby reducing their therapeutic potential.	[89]

Although the studies included in this part use EVs isolated from human samples, their effect has been assessed in preclinical in vitro or in vivo models. The outcome of these studies is expected to lead to further clinical trials and, hence, form an essential part of clinical trials. Surprisingly, similar to the publications on therapeutic applications of EVs, the number of publications on pre-clinical trials on human EVs was also less (n = 4). Efforts to initiate and complete well-designed pre-clinical trials need to be strengthened so that they could pave a way for the therapeutic applications of EVs. Femminò et al. [90] studied the cardio-protective properties of EVs collected from patients suffering from acute coronary syndrome (ACS; n = 30) after percutaneous coronary intervention (PCI). The cardioprotective effects of EVs on hypoxia/reoxygenation damage and ischemia/reperfusion injury (IRI) were investigated in vitro [Human microvascular endothelial cells (HMEC-1) + cardiomyocyte H9c2 cells co-culture] and ex vivo (isolated rat hearts). They found that both EV-naïve (PCI + sham) and EV-RIPC [collected from patients with PCI+ remote ischemic pre-conditioning (RIPC)] failed to drive cardio-protection both in vitro and ex vivo due to the loss of Dusp6 mRNA and upregulation of stress- and cell-cycle-related genes in them. The authors conclude that PCI leads to the loss of cardioprotective effects of EV from ACS patients.

Remote ischemic preconditioning (RIPC) protects the heart following ischemia/reperfusion, depending on the anesthetic used. Abel et al. [91] examined whether EVs isolated from patients undergoing RIPC (n = 10) exposed to two different anesthetics, namely, isoflurane or propofol, protect H9c2 cardiomyoblasts against hypoxia-induced apoptosis. After 6 hours of EV treatment, the H9c2 cells were cultured for 18 hours in normoxic or hypoxic atmospheres. They found that RIPC-EVs, but not the control ones, decreased the apoptosis of H9c2 cells. A prior isoflurane exposure of H9c2 cells in vitro increased the protection by RIPC- and control-EVs, while propofol abolished the protective effect. Based on these data, they conclude that propofol adversely affects the cardioprotective effects of EVs isolated post-RIPC.

Atrial fibrillation (AF) is a common complication of open chest surgery. AF happens due to the inflammation in the pericardial space. Using a rat model, Parent et al. [92] tested whether EVs isolated from human heart explant-derived cells can prevent postoperative AF. Rats were randomized into the sham operation group or sterile pericarditis induction group and treated with a trans-epicardial injection of EVs isolated from human atrial explant-derived cells or vehicles. They found that EV treatment abrogated the pericarditis-associated AF by reducing the infiltration of inflammatory cells and the production of pro-inflammatory cytokines. EV treatment also attenuated atrial fibrosis and hypertrophy post-pericarditis by suppressing fibroblast proliferation. The authors suggest that injection of EVs during open-chest surgery could have anti-inflammatory effects and prevent post-operative AF.

Adoptive cell therapy is being actively investigated in patients with refractory/relapsed acute myeloid leukemia (AML). Exosomes present in the plasma of patients with AML can inhibit antitumor activities of adoptively transferred immune cells by transferring suppressive molecules to them. Hong et al. [93] show that exosomes isolated from the plasma of untreated AML patients affect the antileukemic action of NK cells by delivering inhibitory ligands to the cognate receptors, thereby reducing their therapeutic potential.

3. Future perspective

EVs mediate intercellular communication by delivering bioactive molecules and evoking diverse biological responses in the recipient cells or tissues via cellular uptake, interaction with cellular receptors, or modulation of the microenvironment. The highly promising data obtained in experimental and pre-clinical studies raised a strong possibility that soon the EVs would become cell-free, off-the-shelf therapy for several disease conditions [7].

However, contrary to this expectation, a review of recently published clinical studies showed that very few studies were related to the therapeutic aspects of EVs, while most of them dealt with the use of EVs as biomarkers of disease or pathological conditions and the effectiveness of therapeutic intervention. The lower number of therapeutic trials of EVs could be due to various reasons, including the rigorous regulatory issues involved in the therapeutic applications, stringent parameters required for manufacturing and characterization of EVs [20], cost involved in setting up GMP facilities, long time-frames required for final analysis, etc [8,94]. Undoubtedly, if EVs have to become the future cell-free therapy, aka nanomedicine, there is an urgent need to do double-blind placebo-controlled clinical trials of GMP-grade EVs having proven safety profiles [95]. Perhaps, one of the ways by which these studies could be brought to their desired endpoint, is to accelerate the regulatory processes so that the clinical trials can be done at a faster pace and the field can move forward. This aspect needs urgent attention and if found to be the major deterrent to performing clinical trials, appropriate steps may be taken by the stakeholders by having a close interface with the regulators.

In the area of biomarker research also, there is a need to have very sensitive and reproducible methods for macromolecular profiling of the EVs. Having large publicly available datasets of EV specimens collected from healthy individuals would also help in identifying the disease- and/or stage-specific changes in the composition of EVs.

4. Conclusion

Despite a plethora of literature underscoring the therapeutic potential of EVs in experimental and preclinical studies, the dream of using EVs as “standard-of-care” therapy could take a long time to realize. On the other hand, clinical studies using EVs as biomarkers of disease conditions or the effectiveness of therapy have progressed well and yielded promising results.

Funding Statement

Financial support was provided by Symbiosis Centre for Research and Innovations, Symbiosis International (Deemed University).

Article highlights

• Published clinical trials on EVs: This review specifically focuses on the recently published clinical trials done with EVs.

• EVs as therapeutics: The highly promising data obtained in experimental studies raised a strong possibility that EVs would soon become cell-free, off-the-shelf therapy for several disease conditions.

• Unrealized dream: Despite a plethora of literature underscoring the therapeutic potential of EVs in experimental and preclinical studies, the dream of using EVs as “standard-of-care” therapy could take a long time to realize.

• Slow pace of therapeutic use of EVs: Only four clinical trials assessing the therapeutic applications of EVs were published in this period indicating that they have not reached their desired endpoint.

• EVs as disease biomarkers: On the other hand, clinical studies using EVs as biomarkers of disease conditions or the effectiveness of therapy have progressed well and yielded promising results. The publications related to using EVs as biomarkers show that the EVs present in various biofluids are a very promising source of diagnostic and prognostic biomarkers for different diseases, and importantly, predictive markers to assess the effectiveness of therapy.

• Fast-tracking of regulatory affairs: The slow pace of publications involving EVs as therapeutics makes one wonder whether fast-tracking regulatory affairs could help accelerate the therapeutic use of EVs. This aspect needs urgent attention.

Author contributions

Vaijayanti Kale: Conceptualization, Literature Search, Writing & Editing.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.