Unlocking the Potential of Extracellular Vesicles as the Next Generation Therapy: Challenges and Opportunities

Abstract

Background:

Mesenchymal stem cells (MSCs) have undergone extensive investigation for their potential therapeutic applications, primarily attributed to their paracrine activity. Recently, researchers have been exploring the therapeutic potential of extracellular vesicles (EVs) released by MSCs.

Methods:

MEDLINE/PubMed and Google scholar databases were used for the selection of literature. The keywords used were mesenchymal stem cells, extracellular vesicles, clinical application of EVs and challenges EVs production.

Results:

These EVs have demonstrated robust capabilities in transporting intracellular cargo, playing a critical role in facilitating cell-to-cell communication by carrying functional molecules, including proteins, RNA species, DNAs, and lipids. Utilizing EVs as an alternative to stem cells offers several benefits, such as improved safety, reduced immunogenicity, and the ability to traverse biological barriers. Consequently, EVs have emerged as an increasingly attractive option for clinical use.

Conclusion:

From this perspective, this review delves into the advantages and challenges associated with employing MSC–EVs in clinical settings, with a specific focus on their potential in treating conditions like lung diseases, cancer, and autoimmune disorders.

Introduction

In the adult body, many cells are capable of dividing and generating new copies of themselves, although they typically remain inactive. However, these cells can become active and divide when needed to replace lost cells due to normal turnover or tissue damage. Stem cells are a group of cells that possess the ability to self-renew, differentiate and carry out all of these functions therapeutically. Haematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) are the primary types of stem cells used for therapeutic purposes and have been used for over a decade, with bone marrow transplants being a common example. Stem cell populations are typically isolated from various tissues, including bone marrow, adipose tissue and umbilical cord, and have been utilised to treat patients with haematological cancers [1, 2].

It is worth mentioning that the second generation of stem cells, which are pluripotent stem cells (PSCs) such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), possess comparable abilities in extensive differentiation and can continue to expand in vitro without any limit [3]. Despite their potential in treating spinal cord injuries and creating cardiovascular progenitors for myocardial infarction, pluripotent stem cells (PSCs) are accompanied by safety concerns [4]. Reports suggest that PSC-derived products may give rise to tumours or ectopic tissue when residual PSCs or highly proliferative progenitors are present in the final product and are engrafted in a patient. Additionally, the use of human embryos has also sparked ethical debates due to the unregulated management of surplus embryos and associated genetic instability. Moreover, the reprogramming factors used for generating iPSCs have the potential to cause mutations or chromosomal abnormalities during prolonged in vitro culture or differentiation [5]. In recent years, engineering strategies have been employed to transform first and second-generation stem cells into next-generation therapies, with the aim of enhancing their specificity and efficacy as well as expanding their potential applications for new therapeutic areas. The next generation of stem cells can be categorised into two types based on their intended use: (i) those that act as delivery vehicles for therapeutic purposes, and (ii) those that serve as improved therapeutic agents. The next generation of stem cells has primarily focused on delivering medicinal drugs, and stem cell-derived extracellular vesicles are one of the next-generation therapies being utilised. Therefore, this review would focus on the challenges and application of extracellular vesicles (EVs) for the next generation of clinical therapy.

Extracellular vesicels is produced by stem cells act as communication tools within cells, regulating physiological conditions by transporting biomolecules locally or distantly. The scientific community has consistently emphasised the importance of EV RNA contents, which is evidenced by the detection of EV-related RNA in liquid biopsies, the alteration of EV-related RNA in disease states and the use of sensitive and specific liquid biopsies to measure EV-derived RNA contents in precision medicine.

EVs encompass three main subtypes based on their size and biogenesis. Exosomes, ranging from 40 to 150 nm in diameter, are formed through the inward budding of early endosomes, leading to multivesicular bodies (MVBs). These MVBs can subsequently fuse with lysosomes for degradation or with the plasma membrane, releasing exosomes to the extracellular environment. Microvesicles, larger in size with a diameter of 100–1000 nm, originate directly from the plasma membrane and are constantly released by almost all cell types, with additional triggering possible under abnormal conditions. On the other hand, apoptotic bodies, exceeding 1000 nm in diameter, are formed when apoptotic cells undergo fragmentation, facilitating the removal of cellular waste by macrophages. Initially believed to only store chemicals from dying cells, macrophages have been found to transfer these substances to healthy cells. Exosomes, in particular, have been extensively researched and were once considered to complement lysosomal and proteasomal degradation, playing a role in eliminating damaged membrane and cytosolic components [6–8].

EVs have various medical applications, including regenerative medicine and drug delivery, and both natural and manufactured EVs are under investigation [9–12]. EVs can evade the mononuclear phagocytic system and remain stable in circulation due to their negatively charged surfaces and the presence of the surface protein CD47 [13]. They can also cross biological barriers, suggesting that their lipid composition and protein content may provide intrinsic targeting capabilities. Despite the benefits of liposomal formulations for therapeutic delivery, there are several obstacles to deliver medication to target organs [10, 14, 15]. While there are advantages of using liposomal formulations for delivering therapeutics, achieving targeted medication delivery poses several challenges [9]. For example, liposomes have a brief half-life in the reticuloendothelial system (RES) and tend to accumulate in the spleen and liver, which restricts the amount of medication that reaches the intended site [10]. To overcome these limitations of liposomes, one of the various strategies is to switch to natural carrier systems for therapeutic distribution. EVs are being explored as a potential therapeutic delivery method that can overcome some of the limitations of synthetic drug delivery mechanisms like liposomes [16]. As potential carriers for drug delivery, EVs offer advantages such as increased biocompatibility, greater stability, and lower immunogenicity, which can be beneficial compared to traditional synthetic delivery vehicles such as liposomes and nanoparticles [17].

Several articles have highlighted that EVs can transport biological cargo to enhance angiogenesis, tissue healing and modulation of immune function, making them a promising acellular therapy for various diseases that can be further tailored for therapeutic delivery [9, 18]. By using EV producer cells to generate biological therapies and loading target ligands into or onto EVs, a drug delivery system can be developed that can potentially overcome the limitations of synthetic delivery systems. However, not all EVs produced by cells are suitable as drug delivery carriers as factors such as external proteins, size, productivity and intracavitary composition need to be carefully controlled for drug delivery-scale EVs. Over the past decade, dendritic cells derived from EV-based therapy have been employed in medication delivery and immunotherapy [17]. Alvarez-Erviti et al. demonstrated that dendritic cells, which are derived from EVs, can be used to deliver RNA interference (RNAi) to the brain following systemic injection by using RVG-targeted exosomes [14]. EVs, including dendritic cell EVs, have recently emerged as a novel shuttle for delivering therapies across biological barriers with high in vivo biocompatibility and minimal immunogenicity [19, 20]. Dendritic cell EVs have also demonstrated the ability to cross the blood–brain barrier (BBB), making them an attractive option for drug delivery in the future [21]. MSC–EVs are currently under intense investigation to replace MSCs for medication delivery. Bone marrow-derived MSCs (BM-MSCs) have been regarded as “manufacturers” of drug delivery EVs due to their ease of cultivation and extraction, low immunogenicity, and effectiveness in drug delivery [22–25].

Mesenchymal stem cells (MSCs) have been extensively studied due to their unique biological properties. These cells possess four key features that contribute to their therapeutic effects: (i) their ability to home to sites of inflammation following tissue injury when injected intravenously, (ii) their ability to differentiate into multiple cell types, (iii) their reduced immunogenicity and enhanced immunomodulatory functions, and (iv) their ability to secrete various bioactive molecules that can promote the recovery of damaged cells and inhibit inflammation [26]. EVs are a popular strategy for facilitating cell-to-cell communication through the transfer of biomolecules locally and systemically. Multiple sorting pathways are involved in cargo selection for EVs, with protein sorting being a key factor for determining the function and destination of EVs. Alterations in the cargo of EVs have been shown to have a significant impact on disease progression, highlighting the importance of understanding the cellular components and mechanisms that govern the loading process. Previous study identified the endosomal sorting complex required for transport (ESCRT) as a crucial player in sorting ubiquitin-labeled protein cargoes into vesicles [27]. The ESCRT machinery consists of four multi-protein complexes (ESCRT-0/-I/-II/-III), all of which play a role in sorting ubiquitinated cargo and are involved in terminating EV formation and budding [27]. During this process, protein 4, which is expressed by neural precursor cells, interacts with protein 1, which is an endosomal adaptor protein in EVs, to facilitate the dissemination of cargoes to the extracellular environment by fusing plasma membranes and releasing their exosomes [28]. The formation of intraluminal vesicles (ILVs) by budding is believed to be initiated by ESCRT I and II, with ESCRT III completing the process. A series of events lead to the sequestration of multivesicular body proteins into ILVs. Additionally, the formation of the multivesicular body is associated with detergent-resistant domains in exosome membranes, which are localised with a member of the tetraspanin protein family. The integration of Major Histocompatibility Complex Type II (MHC Type II) into tetraspanin CD9-containing lipid microdomains is required for sorting into exosomes. The fusion of the multivesicular body restricting membrane with the plasma membrane enables exosomes to be released from cells [29, 30].

There are various methods available for producing, isolating and purifying EVs. Standard T-flask and cell culture media can be used to secrete EVs from many cultured cells, but modifications to the culture environment can affect EV yield and characteristics. A previous study focused on the efficient production, isolation and purification of EVs, specifically for those derived from stem cells. It is important to note that there are multiple production platforms and modes available to produce different types of exosomes for various applications [31, 32]. Although there are limitations that need to be addressed such as optimising the characteristics of electric vehicles, large-scale production, and long-term storage, this review would focus on the development of next-generation stem cell-based therapies. Specifically, it would discuss the use of stem cell-derived EVs and the technologies involved in their production and storage as well as their advantages, disadvantages and associated challenges in various disease models for clinical applications.

Stem cell derived EVs in preclinical and clinical application

Preclinical studies of EVs application

Numerous studies have explored the potential of MSCs as a therapy for acute and chronic lung diseases as well as other respiratory conditions. While experimental studies have highlighted the benefits of MSC-based therapy for lung diseases, concerns have also been raised in terms of safety. These include the potential for transplanted MSCs to differentiate in an undesirable manner, leading to malignant transformation and vascular occlusion. Therefore, researchers shifted their focus to the secretomes or EVs produced by MSCs, which contain soluble factors that can mimic many desired therapeutic effects. The use of MSC-derived EVs as a treatment for lung diseases is a relatively new and constantly evolving field. Additionally, research has shown that MSC-derived EVs are effective in vitro by increasing macrophage phagocytosis, reducing inflammation and increasing ATP levels in human alveolar type 2 cells [33, 34].

Apart from that, cardiovascular diseases (CVD) are a global concern due to their high mortality rate. While conventional treatments have focused on using drugs such as ACE inhibitors to prevent progressive heart failure, current approaches have shifted towards using cellular and noncellular therapies. MSCs have been demonstrated to engraft following systemic or local treatment in animal models of cardiac injury, promoting repair of infarcted myocardium [35, 36].Transplantation of MSC has been reported to significantly increase capillary density and decrease collagen volume fraction in the myocardium in a rat model of dilated cardiomyopathy, leading to a decrease in left ventricular end-diastolic pressure and an increase in left ventricular maximum [37, 38]. Other studies have found that exosomes transported cardioprotective miRNAs such as miR93-5p and miR30a that prevent apoptosis in embryonic rat cardiomyocytes [39, 40].

Other than that, cirrhosis is a chronic clinical condition that can lead to liver failure. Liver transplantation is often considered the most viable option to reduce mortality, despite its highly invasive procedures. Cell-based therapy has been developed in both pre-clinical settings, making it an alternative option. Autologous bone marrow cell infusion therapy has been shown to ameliorate liver fibrosis and activate liver regeneration. Another study found that human mesenchymal stem cell-extracellular vesicle (huMSC-EV) decreased oxidative stress and apoptosis in liver injury via suppression of CCI₄ [41, 42]. Another important feature of MSCs is their low immunogenicity.

EVs are produced during cell activation, stress and apoptosis. They are involved in both healthy and pathological processes. A recent study suggested that the subpopulation of EVs provides numerous pieces of information on autoimmune-related diseases. Fernandez and colleagues found that EVs, which are derived from nervous and immune system cells, retain cellular memory such as inflammation, demyelination, axonal damage, astrocyte and microglia reaction, BBB permeability, leukocytes transendothelial migration, synaptic loss and neuronal death in multiple sclerosis (MS) [43]. Multiple sclerosis refers to a catastrophic central nervous system (CNS) illness that is characterised by autoimmune reactions to CNS antigens such as myelin basic protein, which results in neuronal destruction and the production of demyelinating plaques in the brain, spinal cord and optic nerves [44]. In animal models, experimental autoimmune encephalomyelitis (EAE) is considered the most common inflammatory demyelinating disease, which is mediated by T-helper (Th) cells, leads to T-cell and monocyte infiltration in the CNS associated with local inflammation. The capability of EV to cross BBB contributes to the spread of brain antigens to the periphery. Even worst, the injection of microglia-derived EVs into the CNS of EAE mice had increased inflammation and exaggerated the disease [45]. However, exosomes that are produced by IFNγ-stimulated MSCs (IFNγ-Exo) have shown to be an effective treatment for EAE. These findings support the use of MSC-derived exosomes as cell-free therapies for autoimmune and CNS diseases [46].

Besides that, in order to interact with the microenvironment and promote the growth and survival of tumours, malignant cells create more EVs than non-cancerous cells. The overexpression of Rho-ROCK signalling pathways in breast cancer, neuroblastoma, and gastric cancer, as well as the activation of carcinogenic pathways including EGFR and RAS, are just a few of the strategies used to do this [47–49]. These pathways boost EV uptake by cancer cells in addition to increasing EV synthesis. EV release may also be aided by lung cancer and hepatocellular carcinomas having higher levels of the YKT6 SNARE protein, which is controlled by miR-134 and miR-135b. EV release is also influenced by the hypoxic microenvironment that surrounds the tumour. Studies have revealed that miR-410 with EVs produced from human umbilical cord MSCs (hUC-MSC–EVs) can stimulate lung development. Further analysis revealed that miR-410 decreased the expression of the phosphatase and tensin homolog (PTEN) protein in lung adenocarcinoma cells via post-transcriptional modifications, leading to increased cancer cell proliferation. Additionally, overexpression of miR-1180 was found to increase chemoresistance towards cisplatin in ovarian cancer cells by inducing glycolysis through the activation of the Wnt signalling pathway [42]. Studies have also shown that MSC–EVs can regulate pathways involved in stem cell differentiation in cancer cell lines such as MG63 (osteosarcoma) and SGC7901 (gastric cancer) [50]. However, other studies demonstrated that hUC-MSC-EV could enhance the proliferation, migration and invasiveness of two breast cancer lines, MDA-MB-231 and MCF-7, while inducing drug resistance and increasing anti-apoptotic capability in gastric cancer cells [51, 52]. It would be beneficial to summarise the roles of stem cell-derived extracellular vesicles as protumrigenic and anti-tumorigenic factors in Tables 1 and 2.

Table 1.

Roles of Stem Cell-Derived Extracellular Vesicles as Protumorigenic Factors

Source of exosome	Exosome cargo	Target cancer	Target gene/pathway	Reference
Human umbilical cord-mesenchymal stem cell	miR-410	Lung Adenocarcinoma	↓PTEN protein expression	[53]
Bone marrow-mesenchymal stem cell	miR-1180	Ovarian Cancer Cell	Activation Wnt signalling pathway. Activation of aerobic glycolysis, pyruvate dehydrogenase kinase 1 (PDK1) gene	[54]
Human bone marrow-mesenchymal stem cell	Pathway	Osteosarcoma, Gastric Cancer	↑Hedgehog signalling pathway and its components namely Gli-1, Ptch-1, Shh, and Smo	[42]
Human umbilical cord-mesenchymal stem cell	Pathway	Breast Cancer	↑ERK pathway protein expression	[50]
Mesenchymal stem cell	Pathway	Gastric Cancer	Activation of calcium/calmodulin-dependant protein kinases and its downstream targets Raf/MEK/ERK kinase cascade. ↑multi-drug resistant proteins MDR, LRP and MRP	[51]

Table 2.

Roles of Stem Cell-Derived EVs as Antitumorigenic Factors

Source of exosome	Exosome cargo	Target cancer	Target gene/pathway	References
Bone marrow mesenchymal stem cell	miR-15a	Multiple Myeloma		[55]
Mesenchymal stem cell	miR-424	Ovarian Cancer	↓MYB transcriptional factor gene expression ↓ VEGF and VEGFR protein expression	[56]
Human liver stem cell	miR-15a, miR-181b, miR-320c and miR-874	Tumor Endothelial Cell	↓ITGB3, FGF1, EPHB4 and PLAU gene expression	[57]

Clinical studies of EVs application

Lung disease

With the recent outbreak of the COVID-19 pandemic, which can cause a wide range of clinical manifestations from asymptomatic infection to acute respiratory failure and death, numerous studies have been conducted on predictive biomarkers, protein carriers and antiviral drugs to treat the infection [58, 59]. Acute lung injury (ALI) is commonly associated with high rates of morbidity and mortality due to the rapid onset of lung failure and various direct or indirect damage to the lung parenchyma. However, studies have shown that MSC-derived EVs have a protective effect against ALI by promoting anti-inflammatory responses, which is evidenced by an increase in the level of phagocytic macrophages. This supports the role of EV-mediated mitochondrial transfer in this process [60, 61]. Studies have also demonstrated that the use of MSC-derived EVs can decrease inflammation in both the peribronchial and perivascular areas as well as reducing the number of goblet cells in chronic obstructive pulmonary diseases (COPD). These findings suggest that EVs may play a functional role in the pathogenesis of emphysema in COPD [62, 63]. Another study reported that miRNAs found in plasma-derived EVs are novel biomarkers for pulmonary disease due to significant differences that were observed in the size, concentration, distribution and phenotypic characteristics between EVs in the plasma of non-smokers, smokers and patients with COPD [64]. In conclusion, EVs are a promising biomarker for therapeutic applications in respiratory diseases due to their ability to transport bioactive cargo, which reflects the characteristics of their parental cells. Moreover, EVs are highly stable in bodily fluids, making them an attractive candidate for clinical use.

Cardiovascular disease

In a previous study, clinical trials using intracoronary injection of autologous BM-MSCs were effective in improving left ventricular function in patients who underwent primary percutaneous coronary intervention after the onset of acute myocardial infarction [65]. However, the study found that administration of EV reduced myocardial fibrosis only to a small extent through miR-133-a1, which is a gene that plays a role in proper skeletal and cardiac muscle function. The miR-133-al was found in extracellular vesicle-cardiovascular progenitor cell (EV-CPC), making it unlikely to increase its expression in EV-treated hearts. It was possible that EVs fostered an endogenous overexpression of miR-133-a1 in targeted cardiac resident cells [66]. Previous studies also reported that EVs could serve as a biomarker and treatment delivery tool for CVD due to increased platelet levels in secreted microvesicles of high-risk CVD patients [67, 68]. EVs present a promising therapeutic agent for CVD, given their low immunogenicity, low toxicity and ability to carry bioactive molecules to target cells.

Hepatocellular disease

Since EVs or exosomes are responsible for the remote therapeutic effects of MSCs, some studies have reported that EVs ameliorate liver fibrosis in cirrhotic mice, promote liver regeneration and hepatocytes proliferation, and also alleviate acute liver failure by diminishing macrophage activity [69, 70]. In another study, EVs also showed a crucial role in regulating the proliferation, immune escape and metastasis of hepatocellular carcinoma (HCC). They are also present in the circulation at the early stage of the disease, making EVs a biomarker for HCC detection [71]. EVs are crucial in mediating many signals among cells such as hepatocytes, stellate cells and other immune cells to perform important functions and maintain a homeostatic state in the normal healthy liver. By controlling the microenvironment and many signaling pathways in both the tumour and surrounding normal cells, EVs play a role in the development and progression of HCC [71].

Autoimmune diseases

Despite limited studies on the role of EVs in T-cell immune regulation, some reports have demonstrated their significance. EVs that are derived from regulatory T-cells (Treg) can stimulate other T-cells which are polarised to the Treg phenotype. Similarly, EVs which are derived from endothelial cells have the ability to modulate T-cell activation and alleviate chronic inflammation in tissues. In this case, MSCs release immunosuppressive exosomes in the form of anti-inflammatory miRNAs, which are used to treat autoimmune disease in clinical trials [72, 73]. Apart from MS, EVs are also effective in reducing the incidence of rheumatoid arthritis (RA) by increasing the level of circulating anti-inflammatory cytokine IL-10. Both exosomes and microparticles can exert an immunomodulatory response and alleviate symptoms by reducing T and B lymphocyte proliferation and TNFα levels [74]. EVs can reduce joint swelling via the release of paracrine signalling anti-inflammatory cytokines such as IL-1 receptor antagonist and inhibition of T-cell proliferation. In contrast, T-cell apoptosis and microRNAs cargo release from EV such as miR-192-5p, miR-548a-3p, miR-150-5p, miR-124a, miR-155, miR-17 and miR-34a are activated, thus alleviating RA symptoms [75–78]. Previous research has indicated that EVs can aid in various processes such as cellular proliferation and migration, maintenance of intestinal homeostasis, modulation of intestinal barrier function, and protection of lateral junction complexes in Crohn’s disease [79–81]. Crohn’s disease is an autoimmune disorder in which the immune system attacks healthy tissue in the gastrointestinal tract, leading to chronic inflammation. Despite the availability of a wide range of therapeutic drugs, many patients may not respond to treatment, with approximately 50% requiring surgery within a decade of diagnosis [82]. This highlights the need for new and non-invasive markers to aid in the diagnosis, prognosis and treatment of IBD. Studies have suggested that EVs that are derived from MSCs are important in treating IBD, with reports showing improved disease activity index scores and therapeutic effects in mitigating colitis [83–85]. Moreover, EV treatments have been linked to several pathological improvements of IBD, including immune modulation, oxidative stress reduction and apoptosis alleviation [74]. Collectively, EV-MSCs have been shown to reduce colonic inflammation in colitis, suggesting that the molecular cargo transported by exosomes plays a vital role in intercellular communication that governs colitis [86].

Cancer

The potential of EVs to stimulate the immune response as a cancer treatment has been extensively studied. In three different clinical trials, EVs have been used as vaccines to treat colorectal, melanoma, and lung cancer. These trials demonstrated that EVs were well-tolerated, safe, and not harmful, while also promoting anti-tumor responses such as the activation of Cytotoxic T lymphocytes and increased activation of T cells [87]. Following the failure of conventional treatments for GvHD, MSC–EVs have been used in a clinical experiment. Over the course of 2 weeks, patients received seven increasing doses of EVs; PBMCs were extracted, and cytokine response was tracked. Following the final MSC-EV treatment, the number of PBMCs that produce TNF, IL-1, and IFN was decreased by more than 50%. The pro-inflammatory cytokines IL-8, IL-6, and IL-17A decreased with treatment. The patients’ clinical symptoms, which included decreased vomiting and nausea and were stable 16 weeks after medication, also improved [88–90].

Moreover, extensive investigations have been carried out to explore the potential of EVs as a promising therapeutic approach for a diverse spectrum of diseases. Neurodegenerative disorders, encompassing Parkinson’s disease (PD), Alzheimer's disease (AD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS) [91, 92], and transmissible spongiform encephalopathies (TSE)—a fatal and pathogenic neurodegenerative disease [93], have emerged as key targets for EV-based therapies. Additionally, EVs hold therapeutic promise in addressing axial spondyloarthritis (AxSpA), a chronic condition impacting the spinal joints [94], as well as in treating neural ailments, such as spinal cord and nerve injuries [95]. Moreover, EVs have demonstrated significant wound-healing effects on corneal epithelial tissues [96], and their potential in promoting tendon-bone healing has important implications for soft tissue injuries, including anterior cruciate ligament injury and rotator cuff injury [97]. A comprehensive summary of the clinical applications of extracellular vesicles in various diseases is presented in Table 3.

Table 3.

Summary of application of EVs in clinical diseases

Diseases	Function of EV in disease	References
Lung diseases	Predictive biomarkers Reduction of lung injury Promoting anti-inflammatory activities	[60–64]
Cardiovascular disease	Serve as a biomarker Act as a treatment delivery tool Transportation of cardioprotective that prevents apoptosis	[65–68]
Hepatocellular disease	Decrease the inflammation Great therapeutic effect on target organs	[69–71]
Autoimmune disease	Promoting cell proliferation and migration Accessible for diagnostic, prognostic and therapeutic purpose Benefits on immune modulation, oxidative stress reduction and apoptosis alleviation Promote T-cells Ability to modulate T-cell activation and alleviate chronic inflammation Develop treatment strategies by increasing the level of circulating anti-inflammatory	[72–86]
Cancer	As protumorigenic factors Inhibit cellular proliferation, migration and invasion As antitumorigenic factors	[87–90]
Neurodegenerative disease	Induce an immunomodulatory effect and reduce neuroinflammation Biophysical activities for intercellular communications	[91–93]
Spines disease Axial spondyoarthritis (AxSpA)	Immunological regulation Tissue remodelling Cellular homeostasis	[94]
Neural diseases	Immune response regulation Inflammation reduction and cell-to-cell communication	[95]
Wound and tendon-bone healing	Facilitate tissue repair and regeneration Intercellular communication by transferring bioactive lipids, proteins, and nucleic acids Regulation of macrophage polarization, angiogenesis and bone metabolism	[96, 97]

Advantages of extracellular vesicles therapy

Cellular sorting and packaging

The potential benefit of utilising EVs for biotherapeutic delivery over synthetic carriers is that endogenous cellular machinery can be employed to generate and sort the necessary cargo inside EVs, which can be difficult to manufacture, store and load. Improved analysis of EV proteomic profiles and protein composition allows for engineering-based EV modifications for more efficient delivery [6]. RNA molecules are ideal for demonstrating the selective loading of cargo into EVs since their spectra often match those of the donor cells' RNA. A study showed that T-cells release extracellular vesicles that carry RNA cargo enriched in tRNA fragments, which activate the immune signal to enhance multivesicular bodies that contain specific tRNA fragments, while also inhibiting T-cell activation and cytokine production. Additionally, processes are required to deliver bioactive EV cargo into the cell. Some studies have suggested that different cell types use specific pathways to promote EV entry into the cells [92, 93].

Ability to cross biological barriers

The blood–brain barrier has been reported to impede the delivery of 98% of small molecules into the brain, which poses a challenge for CNS-based therapy. However, the use of EV has demonstrated great success in drug delivery due to their natural ability to penetrate anatomical barriers, retain stability, and sustain adequate binding effects [17, 93, 94]. EVs have been shown to successfully cross the BBB, which is evidenced by the presence of macrophage exosomes in mouse brain parenchyma. Interestingly, this can occur without binding with immune cells, leading to increased exosome levels that suggest an improved exosome–brain endothelium interaction [95]. Additionally, EV can minimally transport functional cargo from hematopoietic cells to the brain across the BBB during episodes of inflammation. The internalisation of EVs appears to be better than that of synthetic nanocarriers. Unlike artificial lipid nanoparticles that form islands at the cell surface with limited internalisation, EVs enter cells within minutes without first forming islands at the cell surface [91, 96, 97]. It should be noted that different recipient cell types can react differently with EVs from identical cell sources [98]. Since EVs are capable of transporting RNA cargo to recipient cells, they likely rely on endogenous mechanisms to transport cargo to the cytosol. EVs can accurately penetrate different levels of biological barriers, distribute cargo and activate a reaction in their target cells [9].

The potential of EVs to reduce the harmful effects of foreign chemicals when delivered to the body has made them an ideal source of therapeutic vehicles. EV have shown a mild immune response in blood and plasma transfusions, where large quantities of EVs were delivered to patients with no obvious side effects. EVs are considered reasonably safe when compared to virus-derived vehicles or cell treatments due to their non-replicative and non-mutagenic nature. This property has eliminated regulatory concerns regarding side effects or neoplasia development, and EVs do not appear to induce cellular toxicity or inflammation when they are exogenously engineered [6]. For example, the intravenous injection of C2C12 cell EVs with anchor peptides and splice switching oligos into mice with muscular dystrophy revealed the absence of cytotoxicity or inflammatory cells in the liver, kidneys or muscle [99]. To date, at least 11 clinical studies have been conducted to assess the safety and efficacy of various EV treatments before they can be used in clinics [6].

Milk exosomes have the potential to serve as an effective oral drug delivery system with numerous therapeutic applications because of their proven stability in an acidic environment, low cost, biocompatibility, stability, tumour targeting properties and non-toxicity [100–102]. The biochemical components of milk exosomes such as proteins, lipids and nucleic acids can significantly affect their ability to deliver therapeutic molecules. The proteins involved in milk exosome formation include testilin, which regulates membrane fusion, Rab GTPase that interacts with cytoskeleton proteins, and Alix and Tsg101 that play a role in endocytosis [103, 104].

EVs for targeted delivery

EVs possess natural targeting capabilities partly due to their lipid composition and protein content that result in their affinity for certain organs [13]. Modifying the endogenous cellular machinery to conjugate targeting molecules onto the surface of EVs can enhance their transport to a specific target location [6]. Researchers attempted to engineer EVs by modifying receptor-ligand binding, antibody-antigen binding and microenvironment-specific molecule binding [105]. For instance, exosomes that are engineered with hyaluronidase can destroy the extracellular matrix, leading to an increased T-cell population and enhanced permeability in the tumour environment [106].

Minimal immunogenicity and cytotoxicity

Naturally secreted EVs exhibit minimal immunogenicity and cytotoxicity compared to synthetically manufactured drug vehicles such as polymerised lipid nanoparticles that trigger harmful immunological response in vivo and mostly concentrate in the liver [10, 107–109]. In addition, EVs promote immune clearance due to their endogenous origin and excellent biocompatibility. However viral-based drug delivery systems such as adenoviruses, lentiviruses, retroviruses, lipid transfection reagents and nanoparticles are known to stimulate immune responses [110–112].

Comparison of EV between nanoparticle, stem cell and virus

Opsonisation is a crucial biological barrier that regulates the drug delivery system. In the biological system, synthetic nanoparticles can be quickly opsonised with proteins [113]. Early proteins that opsonise the pristine surface of synthetic nanoparticles can induce fast clearances by the mononuclear phagocyte system (MPS), leading to destruction via endosomal-lysosomal pathways within those cells [114]. When nanomedicines and protein conjugates are infused, they may elicit unexpected hypersensitivity responses, especially when they are given intravenously. The activation of the complement system appears to play a role in many of these negative responses [10]. Anaphylatoxins or complement peptides refer to fragments C3a, C4a and C5a that are produced as part of the activation of the complement system. Exposure of nanoparticles to blood via infusion can trigger complement-activation-related pseudoallergy (CARPA), which is an adverse immune overreaction (hypersensitivity) [115]. To avoid fast clearance, nanoparticle surface can be coated with a hydrophilic polymer layer such as poly(ethylene glycol) (PEG). However, the complex surfaces of EVs may require high specificity and selectivity for tissues and cells as well as intracellular routing for functional delivery. A mixture of natural proteins, carbohydrates and lipids is believed to potentially eliminate CARPA, despite the scarcity of experimental data [10]. The mechanism of absorption and intracellular routing of EVs can significantly influence drug carrier activities. Studies have shown that the uptake of synthetic systems can vary by cell type and can occur via both constitutive and inducible mechanisms [116]. However, it has also been reported that only 1–2% of the total amount of short interfering RNA (siRNAs) are able to escape endosomes and be absorbed [117], which suggests that synthetic systems may have compromised in vitro reliability [10].

Although MSC transplantation holds promise for future clinical applications, most current clinical trials remain in phase I or II. Clinical trials that involve autologous and allogeneic MSC products have raised concerns regarding tumorigenesis, cell death and inflammations [118]. In contrast, MSC EVs offer a non-invasive cell-free option that eliminates post-transplantation complications. Since MSC EVs are naturally stable, they have also been utilised to distribute bioactive substances [119]. However, for cell-based carriers, it is essential to maintain cargo loading with the presence of non-toxic chemicals and phenotypes. Besides, factors such as stem cell type, source, culture methodology as well as nanoparticle size, surface, electric charge and coatings can be determining factors for successful delivery [120].

Proteins such as transcription factors and cytokines in EVs can produce molecular signals, which will influence the activity of recipient cells. Both EVs and enveloped viruses are considered multicomponent transport units due to their ability to transfer RNAs that can activate pathogen identification receptors in target cells. Specifically, these receptors have been found to be triggered by viral genome fragments, virus-encoded short RNAs such as those produced by EBV, and specific host cell miRNAs [121]. EVs and viruses may share similar biogenesis pathways; however, only the latter can reproduce in cells. Crucially, EVs produced by infected cells are not natural since they can aid viral replication or boost antiviral defences. Understanding the morphology of EVs generated by infected cells as well as their cargo and pathomechanism is essential for fundamental virology research and translation into clinics [121]. Modifications of the viral vector that involve removing certain regions of their genomes have been linked to various adverse effects, including higher immunogenicity, inflammation, tissue degradation, toxin production, insertional mutagenesis and restricted transgenic capacity [122, 123]. In contrast to EVs, viruses require cell surface receptors to facilitate plasma membrane fusion and target certain cell types with specific receptors. The cellular tropism of viruses is defined by the selectivity of their receptors, which distinguishes them from EVs that may penetrate a broader range of cell types. Viruses rely on access to specific receptors and co-receptors to function that bind to envelope glycoproteins and cause significant molecular rearrangements, revealing the contents of the viral fusion complex. However, EVs are more versatile than viruses in terms of their entry mechanism, which include clathrin-dependent, caveolae-dependent and macropinocytosis [124, 125].

Challenges of extracellular vesicle in clinical platform

Heterogeneity of EVs

The heterogeneity of EVs is likely reflected in their size, content, functional impact on recipient cells, and cellular origin. The main idea of this content is that EVs, which are tiny vesicles with vary in size released by cells, possess a wide range of characteristics. These characteristics can be attributed to factors like their size, the substances they carry, the impact they have on interacting cells, and the specific type of cell they originate from. Essentially, EVs can differ in terms of their physical properties, molecular makeup, biological effects, and their cellular source.

Despite the remarkable benefits and potential of EVs, significant challenges need to be addressed before they can be used for clinical applications. Inconsistent isolation procedures, low drug loading efficiency and limited clinical-grade manufacturing make EV-based delivery difficult. These issues are due to inherited characteristics from parental origins that affect the extent of transport and targeting capabilities [105].

Scalability of EVs

Another important issue to address is the optimisation of therapeutic cargo for EVs [126]. Compared to other delivery vehicles such as liposomes, EVs have lower loading efficiency, which may be due to the varying content of parent cells during synthesis and a limited capacity for storing exogenous drugs [105, 127]. A previous study was conducted to optimise loading techniques and magnitudes [105]. The amount of saponin or hypotonic dialysis was found to increase by 11-fold compared to incubation, electroporation and extrusion methods [128]. This indicated that the loading efficiency depends on the loading capacity and hydrophobicity of the drugs, which is dictated by the lipid content of the EVs [105].

Most studies on the cellular absorption of EVs use fluorescent tags such as lipid dyes or fluorescently labelled proteins. While these techniques have yielded encouraging data on EV absorption, they do not accurately reflect the intracellular trafficking and processing of soluble cargo such as RNA molecules or cytosolic proteins [10]. For EVs to carry out their function, their cargo must reach the intracellular site of action. A previous study suggested that EVs follow a similar diffusion approach after uptake in adult dendritic cells, which may enable them to partially bypass degradative mechanisms. However, this observation may only be specific to dendritic cells, and it is uncertain whether EVs can escape degradative pathways in other cell types [129–131].

Stability of EVs

Despite these concerns, it is probable that a mechanism exists for EVs to evade degradative routes after uptake as functional effects have been observed through the transport of EV cargo. Nevertheless, the process by which functional content is released, which requires information to pass through the exosomal and endosomal membranes, remains unknown. Some studies have recommended the use of preservation techniques for stem cell-derived EVs such as cryopreservation, freeze drying or spray drying to enhance their absorption [129–132]. By preserving EVs for extended periods, cryopreservation enables a constant supply of EVs for clinical and commercial applications. To avoid cryoinjury to cells, it is essential to employ cryoprotectants. It has been observed that storing samples at − 80 °C can reduce EV concentration and purity, cause alterations in EV zeta potential, and shift EVs in size-charge plots, resulting in an increase in particle size and size variability. Additionally, exposing samples to freeze–thaw cycles can decrease EVs after the first cycle and increase particle size in a cycle-dependent manner [131].

Standardization of EV isolation and characterization methods

In addition, the lack of standardised protocols for EV isolation remains a major challenge [133–135]. Ideally, isolation techniques should be selective, convenient, cost-effective, reproducible, high-yield and time-saving. However, none of the reported protocols satisfy all of these model characteristics [105]. Therefore, large-scale production of EVs should focus on maximising cell cultures with a more efficient method.

The production of clinical-grade EVs is a complex process that requires strict adherence to quality control and standardisation. Careful selection of the starting material for EV production is essential to ensure the purity and quality of the EVs. While MSCs are commonly used as a source of EVs, other sources such as dendritic cells or platelets can also be used. In order to use EVs for clinical purposes, the isolation process used to extract them from the starting material must be standardised and improved. Ultracentrifugation, size-exclusion chromatography and ultrafiltration are common techniques for EV isolation [136].

It is crucial to validate the isolation method to ensure consistent size, shape and cargo of the EVs. This can be achieved through various techniques such as electron microscopy, nanoparticle tracking analysis and western blotting. Furthermore, the EVs should be characterised to determine their size distribution, surface markers and contents. Methods such as flow cytometry, mass spectrometry and RNA sequencing can be utilised for this purpose.

Regulatory issues and delivery

Translating these nanosystems into clinics is necessary to determine appropriate manufacturing strategies that ensure quality and yield [137, 138]. Unfortunately, there are no absolute state-of-the-art methods that meet the manufacturing requirements of Good Manufacturing Practice Grade (GMP) EVs. Issues on scalability, reproducibility, safety, potency, physical properties, surface charge and purification of the finished material should be consistently addressed [139]. The ideal GMP-grade EVs manufacturing technique should be individually determined by the EVs donors and the type of material loaded [140, 105].

Lastly, maintaining the sterility of clinical-grade EVs is essential to prevent any contamination and ensure the safety of patients who receive them. Proper storage and transportation temperatures should be maintained, and sterilisation methods such as filtration and irradiation can be employed to ensure the stability and quality of clinical-grade EVs. To ensure the long-term preservation of clinical-grade EVs, freezing or lyophilization may be employed. Before clinical administration, preclinical and clinical assessments should be conducted to evaluate the safety and effectiveness of the clinical-grade EVs. The production of clinical-grade EVs requires a multidisciplinary approach, which involves experts in cell biology, biochemistry and clinical research [141, 142]. To guarantee the safety and effectiveness of the final product, careful attention must be paid to quality control and standardisation.

Conclusion

EVs hold tremendous potential for revolutionizing clinical treatments and enhancing patients’ quality of life. As a cell-free therapeutic source and an innovative delivery vehicle for biotherapeutics, EVs are increasingly recognized as an efficient and reliable form of therapy. Their unique attributes, such as the ability to overcome physical barriers, intrinsic targeting capabilities, utilization of natural intracellular trafficking routes, and superior biocompatibility, surpass current treatment strategies [9]. Additionally, EVs exhibit lower immunogenicity, inherent cell targeting properties, and improved circulatory stability, contributing to enhanced safety and stability in clinical applications [131, 143]. However, despite these promising characteristics, the intricate molecular mechanisms underlying EVs’ intracellular biogenesis, transportation, and absorption processes remain incompletely understood. This includes the role of key signaling molecules between MSCs and damaged tissue, which may introduce complexities in interpreting their therapeutic effects.

Nonetheless, it is essential to acknowledge that the clinical use of EVs faces certain challenges, including the need for large-scale production, effective and scalable isolation methods, precise biodistribution and tissue targeting, as well as robust storage and preservation techniques. Although EVs demonstrate the capacity to function similarly to MSCs, offering unique therapeutic opportunities, future research efforts should focus on mitigating these challenges, enabling high-throughput production of clinical-grade EVs, and developing stable long-term storage solutions. This will be crucial in addressing the rising prevalence of clinical diseases and meeting unmet medical needs. EVs play a critical role in mediating communication between MSCs and injured tissues, with the advantage of specifically targeting injured tissues even when MSCs are distributed in other organs or tissues. However, a comprehensive understanding of MSC–EVs' targeting and biodistribution mechanisms is essential to clarify their effects, which can be multifaceted. While large-scale production of MSC–EVs presents a challenge, their ease of use and numerous advantages have sparked significant research interest in exploring their therapeutic potential.

In conclusion, EVs are small lipid bilayer-bound vesicles released by various cell types, playing a vital role in intercellular communication. Their diverse cargo, containing proteins, lipids, and nucleic acids, makes them a promising source for diagnostic and therapeutic applications across different disorders. However, several significant challenges hinder the clinical implementation of EV-based therapies. Firstly, the inherent heterogeneity of EVs, reflected in variations in size, shape, and composition, complicates the identification and isolation of specific subpopulations suitable for clinical use. Secondly, the current time-consuming and costly methods for EV isolation and purification limit their scalability for therapeutic production. Thirdly, environmental factors like temperature and pH can affect EV stability and biological activity during storage and transportation. Moreover, the lack of standardized protocols for EV isolation and characterization hinders result comparison and reproducibility across different laboratories and clinical contexts. Furthermore, the regulatory framework for EV-based therapies is still evolving, lacking clear guidelines for their clinical application. Lastly, achieving targeted delivery of EVs to specific cells or tissues presents a major challenge, given the rapid clearance from circulation and the potential uptake by non-target cells in EV-based therapies. Addressing these obstacles will be crucial for unlocking the full potential of EVs in clinical settings and advancing their transformative impact on medical treatments.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors declare that they have no conflict of interests.

Ethical statement

There are no animal experiments carried out for this article.