Extracellular Vesicles for Therapeutic Applications

Abstract

Extracellular vesicles (EVs) are cell-derived nanoparticles containing endogenous bioactivators or loading exogenously therapeutics, which serve as “messengers” in intercellular and inter-organismal communication, in both normal and pathological processes. EVs are reshaping our perspective on life science and public health. They are tools for mediating information exchange between cells and are unique in protecting and delivering their internal cargo to target cells through ligand-receptor interactions. Therefore, EVs are one of the most potential delivery systems for treating various diseases. This chapter summarizes the recent progress made in EV-based delivery systems applications, including cancer, cardiovascular diseases, liver, kidney, nervous system diseases, and COVID-19.

1. Introduction

Cells participate in the exchange of information between cells through a variety of biomolecules, which can be cytokines, chemokines, and metabolites. It has been found that cell-cell communication happens through extracellular vesicles (EVs). EVs are lipid bilayer vesicles with a diameter of 30–150 nm secreted by most cells, which carry a variety of biological molecules, including nucleic acids, proteins, lipids, and metabolites. When EVs are ingested by other cells, these goods are transferred and affect the biological behavior of receptor cells. Recent studies have shown that EVs carry many important signaling molecules and are the “messengers” between intercellular communications [1]. EVs are widely distributed in all body fluids, such as blood, brain effusion, saliva, amniotic fluid, and urine. When the EVs bind to the recipient cells, they transmit the “cargo” into the recipient cells, thereby mediating the signal communication and substance exchange between the cells to adjust or change the function of the recipient cells [2]. In recent years, many studies have shown that EVs are involved in the development and metastasis of tumors and play an important role in immune response and inflammation [3]. In addition, compared to normal cell-derived EVs, “cargo” packaged in diseased cell-derived EVs is different in abundance and variety and could be a biomarker for diagnosis. In particular, EVs have been used as nanocarriers for drug delivery and have great potential in the field of disease treatment [4].

EVs have some advantages as a nanocarrier for precision therapy due to their low immunogenicity and biocompatibility, among other features. As its biological function in the human body, the membrane of EVs can protect its “cargo”, and the inherent or artificially modified biomacromolecules expressed on the surface of EVs can help to recognize targeting cells. Moreover, natural EVs are safer than artificial nanocarriers such as liposomes. Therefore, EVs provide a yet source of delivery systems, and even treatment means to a great extent [5].

2. EVs biology

EVs are divided into three types, including exosomes, microvesicles, and apoptotic vesicles, which originates from the endosomal system and whose formation is associated with multivesicular bodies (MVBs) and intraluminal vesicles (ILVs). The ILVs are generated from the inward budding of the endosomal membrane during the maturation of MVBs. Ultimately, ILVs are secreted as exosomes with a diameter range of 40–160 nm when MVBs fuse with the endosomal membrane [6] (Figure 1).

Figure 1.

The biogenesis of EVs originates from the endocytic pathway, where the invagination of the inner membrane leads to the formation of luminal vesicles (ILVs) contained within the inner body. The resulting compartment is called multivesicular vesicles (MVB), which fuse with the plasma membrane and release in the form of EVs outside the cell.

EVs were first identified in the reticulocytes of sheep and were considered involved in removing unwanted proteins [7, 8]. Subsequent studies have, in turn, shown that EVs have an important regulatory role in the immune system [9, 10, 11]. At the beginning of the twenty-first century, researchers also discovered that EVs could transmit biological information [12] between cells depending on RNA [13, 14], lipids, and proteins [15, 16]. In the past few years, the significance of EVs in endo-environmental homeostasis [17], inflammation regulation, and tumor metastasis [18] have been elucidated. Recent studies suggest that EVs also play an increasingly irreplaceable role in the biomedical field as nano-vesicular carrier systems.

Almost all living cells can produce EVs, which on the one hand, can be modified by molecular engineering techniques to load exogenous cargo into EVs for exogenous loading. On the other hand, they can also be loaded by endogenous means, using cell sorting mechanisms to sort cargo into EVs. EVs can be loaded with therapeutic drugs, RNAs, and proteins for delivery to the interior of recipient cells. At the same time, therapeutic EVs’ lipid bilayer membrane structure can also be modified to express specific surface molecules and thus mediate specific biological functions or target specific recipient cell types. In this chapter, we will focus on the recent years of EVs as carriers for the delivery of endogenous or exogenous cargoes for the treatment of relevant diseases, as well as discuss the latest relevant methods for EV isolation, loading, and storage, and provide thoughts on their breakthrough in the medical field.

3. EVs as potential delivery vehicles

EVs are stable in biological fluids and organisms, and they can circulate/travel over short and long distances and even penetrate biological barriers [19]. The unique feature of EVs is that they can protect their internal cargo and transport it to target cells through ligand-receptor interactions. Previous studies have shown that proteins on the surface of EVs promote the delivery of goods by promoting membrane fusion with target cells, inhibiting CD47-mediated phagocytosis clearance, and increasing the half-life in circulation, thereby enhancing the pharmacological properties of EVs. The uptake of EVs depends on their surface ligands, such as HSPG, or receptor cell surface receptors, such as SR-B1. Recent studies have found that EVs are vulnerable to the influence of specific organs. Based on this, we can load goods on EVs and deliver them to targeted receptor cells. Other issues need to be considered; for example, the size of the engineered EVs should be small enough to avoid uptake by the reticuloendothelial system (RES) and large enough to avoid rapid renal clearance [20]. As nano-sized particles, EVs can be easily transported through body fluids and biological barriers. If this specific method can be efficiently and accurately controlled, EVs will be an effective tool for transferring therapeutic components. Some biological molecules, such as miRNA, siRNA, and complex recombinant proteins or molecules, are challenging to deliver within cells without carriers, but engineered EVs can load specific goods into target cells and induce gene modification through endocytosis [21].

When developing EV-based therapeutics, the primary consideration is to select donor cells for the exocrine body. According to GMP principles, to ensure that EVs secreted by donor cells do not cause serious adverse effects such as proinflammatory, teratogenic, and carcinogenic effects in humans, the choice should then be made in relation to the therapeutic target, as EVs from different sources generally retain the characteristics of the donor cells. Currently, immature DC-derived EVs, mesenchymal stem cell-derived EVs, or HEK293-derived EVs are used as nanocarriers for drug delivery. Among them, the EVs from immature DC sources have lower toxicity and a weaker ability to induce immune responses, but the disadvantage is that the amount of EVs that can be collected is relatively small. Furthermore, compared to EVs derived from tumors, EVs derived from DC can induce more effective anti-tumor immunity. Mesenchymal stem cells (MSCs) and HEK293 cells secrete a large number of EVs. MSCs have a wide range of cell sources and exhibit good stability and sustainability in human plasma at −20°C. Human bone marrow MHCs incubated with drugs have anti-tumor effects. In addition, the immortalization of cells does not affect the quantity and quality of EVs produced, thus ensuring the possibility of a continuous supply of EVs. However, it has been found that tumor-derived EVs may cause malignant changes in target cells due to related miRNAs. In order to overcome the problems related to mammalian EVs, many researchers have also started loading cargosinto EVs from plants [22], bacteria [23], and milk [24, 25] to study the regulatory treatment effect. For example, milk-derived EVs have been found to promote the healing of ulcer wounds in diabetes patients and can also be used to load nucleic acid drugs such as siRNA and miRNA. In addition, milk-derived EVs have been proven to exist under the degradation conditions of strong acidic gastric juice and the presence of digestive enzymes in the intestine. As oral drugs, they can greatly reduce costs and related inconveniences for intravenous treatment. Although milk-derived EVs are safe, stable, and cost-effective, there is a lack of data comparing their effectiveness with other mammalian cell-derived EVs.

4. Therapeutic applications of EVs as delivery systems

4.1 The treatment of cancer

Cancer is considered a significant threat to human health and ranks as the second leading cause of death globally. Intercellular communication can also contribute to changes in the tumor microenvironment, influencing the occurrence, development, and metastasis of cancer cells [26]. This signaling can occur through EVs. EVs play diverse roles in cancer progression, possibly due to the heterogeneity in their origin and composition. The release of EVs is crucial for the maintenance of pluripotency in embryonic stem cells (ESCs) activated by focal adhesion kinase (FAK), which may be one of the conserved mechanisms maintaining the stemness of both ESCs and tumor stem cells. Meanwhile, the tumor microenvironment (TME) plays a significant role in promoting or inhibiting cancer progression and treatment of resistance. The contribution of EVs from different cell subpopulations within the TME remains unclear and requires further investigation to elucidate the roles of distinct EVs in cancer progression.

During cancer metastasis, locally invasive cancer cell-derived EVs exhibit increased secretion, promoting cancer cell adhesion and directional migration. This is because EVs from cancer cells exchange mRNA with neighboring tissues, potentially leading to the transfer of cancer cells. Live imaging of zebrafish embryos demonstrates that EVs released into circulation by cancer cell sources can enter endothelial cells and macrophages, activating the latter to promote metastatic growth. At future metastatic sites, EVs can reshape the microenvironment, creating a new TME that supports tumor growth and metastasis. EVs not only influence the angiogenesis of bone marrow-derived cells (BMDCs) to facilitate vascular metastasis but also promote lymphatic metastasis to sentinel lymph nodes by enhancing extracellular matrix (ECM) deposition and vascular generation. Therefore, bidirectional communication between cancer cells and the microenvironment is likely a key factor in promoting cancer metastasis [26, 27] (Figure 2).

Figure 2.

Comparison of exosomes from normal cell and tumor cell. Compared with normal cells, the expression of rho-ROCK, RalB, YKT6, EARK 1/2, MAPK, and PI3K/AKT signaling pathway in tumor cells is increased, which leads to angiogenesis and over-expression of ESCRT complexes, syntenin, and heparinase in the tumor, thereby secreting more exosomes.

Due to the significant role of EVs in the occurrence, development, and metastasis of cancer, they also hold great potential in cancer treatment [28]. In addition to utilizing endogenous components of EVs from different sources, EVs are also considered as delivery vehicles capable of packaging therapeutic molecules such as small molecules, nucleic acids, and proteins and protecting cargo inside the membrane, thereby prolonging the circulation time of the therapeutic molecules in the body [29]. It has been demonstrated that chemotherapeutic drugs such as doxorubicin, paclitaxel, and gemcitabine when packaged in EVs, exhibit significant inhibition of tumor growth [28]. Furthermore, for tumors with different gene mutation types, siRNA can be loaded into EVs for specific gene therapy. Research has also found that a combination of EVs and synthetic materials can further enhance the drug-loading capacity and biocompatibility of drug-delivery systems [30]. Currently, there are new therapies targeting cancer cells, including anticancer drugs and immunotherapy drugs. As excellent nanocarriers, EVs have attracted attention due to their efficient and selective drug delivery, providing promising possibilities for modern drug administration. Zhu’s team designed an EV system with two positive charges, which can rapidly enter the lipid bilayer. Simultaneously, the loading of AIEgens and PPI, combined with glutamine inhibition therapy and photodynamic therapy, effectively inhibits tumor growth in a subcutaneous cancer model.

Cancer cachexia is a multifactorial syndrome characterized by significant skeletal muscle loss, which significantly negatively impacts the patient’s quality of life. The negative regulator of muscle growth, myostatin (Mstn), is potentially effective in preventing muscle wasting in cancer cachexia. However, existing administration methods have low delivery efficiency and high toxicity, leading to repeated failures of Mstn inhibitors in clinical trials. Li discovered that red blood cell-derived EVs (RBCEVs) could target and deliver Mstn siRNA to skeletal muscles and validated it in a mouse model of cancer cachexia [31]. Through muscle injection, RBCEVs-Mstn siRNA specifically and efficiently inhibited the expression of Mstn, alleviated skeletal muscle loss in cachexia mice, prolonged their survival, and had no significant systemic toxicity, reducing the mortality rate of cancer cachexia patients and providing a safe and efficient potential treatment method for muscle degenerative diseases.

In colorectal cancer patients, microsomal triglyceride transfer protein (MTTP) expression in plasma EVs is increased, acting as an inhibitor of ferroptosis. In response to this new mechanism, EVs loaded with oxaliplatin may reverse chemoresistance in chemotherapy. Index enrichment systems have been developed, generating ligands with high affinity (Kd = 3.41 nM) for EVs derived from colorectal cancer (CRC). The data show that the ligands and CRC-EVs exhibit high affinity, with a detection limit of 1.0*103 particles/μL, and the ligands significantly inhibit the EV-induced transfer process [32].

Gu et al. overcame the poor solubility and high liver and kidney toxicity of triptolide (TPL) by using hUCMSCs-derived EVs loaded with TPL and engineered with cyclic peptides, establishing a biomimetic targeted drug delivery system. This system demonstrated good tumor targeting, prolonged the half-life of TPL, significantly inhibited tumor growth through caspase cascade and mitochondrial pathways, and prolonged the survival time of a malignant melanoma mouse model.

To address the problem of heterogeneity in hepatocellular carcinoma (HCC) that makes the identification of new antigens difficult, Zhou and his colleagues designed a specific vaccine called DEXP&A2&N by combining the functional domains of DC-derived EVs (DEX), high mobility group nucleosome-binding protein 1 (HMGN1), and an immune adjuvant. The vaccine promoted the recruitment and activation of DCs by targeting A2 and HCC-specific peptide P47 (P) and α-fetoprotein epitope (AFP212-A2). Significant tumor delay was observed in HCC mice through the recruitment and activation of cross-presenting CD103⁺CD11⁺ and CD8α⁺CD11c⁺CD cells in the tumor upon intravenous injection of CD11c⁺CD cells.

In head and neck squamous cell carcinoma (HNSCC), EVs expressing CD73 were found to promote malignant progression and activate the NF-κB pathway in tumor-associated macrophages (TAMs), leading to immune evasion and increased secretion of cytokines such as IL-6, IL-10, TNF-α, and TGF-β1, thereby inhibiting the immune system. Additionally, previous studies have shown a correlation between EVs enriched with HAX1 and metastasis in nasopharyngeal carcinoma (NPC), which can now be explained by the increased presence of ITGB6 in HAX1-enriched EVs.

In a xenograft model of chronic myeloid leukemia (CML), tumors in mice treated with EVs from CML cells were larger compared to the control group treated with PBS. It was found that anti-apoptotic molecules such as BCL-w and BCL-xl increased, while pro-apoptotic molecules BAD, BAX, and PUMA decreased in both in vitro and in vivo samples. Furthermore, TGF-β1 was enriched in CML cell-derived EVs, and it stimulated CML cell proliferation by activating the ERK, AKT, and anti-apoptotic pathways. EVs play a role in tumor initiation, development, immunity, and drug resistance, providing new targets for improving chemotherapy efficacy and offering new insights into precision therapy.

4.2 Treatment of kidney diseases

There is increasing evidence confirming the central role of EVs in kidney physiology and pathology. EVs present in urine or circulation may participate in the regulation of renal function and communication between glomeruli and renal tubules. Potential biomarkers related to kidney diseases can be detected in EVs isolated from urine, such as AQP1, for assessing water balance in acute kidney injury. The therapeutic potential of EVs has been demonstrated, particularly those derived from mesenchymal stem cells (MSCs), which have therapeutic properties that accelerate kidney recovery. This may be attributed to the high C-C motif chemokine receptor-2 (CCR2) expression that can bind with its ligand CCL2. Studies have shown using a mouse model that CCR2-overexpressing EVs reduce CCL2 concentration, inhibit macrophage activation, and provide superior rescue of renal function in early-stage acute kidney injury (AKI). Tang reported a method using EVs to deliver interleukin-10 (IL-10) for alleviating AKI by engineering macrophages. This approach not only enhances the stability of IL-10 but also enhances its targeting to the kidneys, significantly improving tubular injury, and effectively preventing progression to chronic kidney disease. MSC-derived EVs have also been considered as a promising cell-free therapeutic approach for AKI. MSC-derived EVs have been regarded as a promising cell-free therapeutic approach for acute kidney injury (AKI). A supramolecular hydrogel containing Arg-Gly-Asp (RGD) peptides has been developed to enhance the therapeutic efficacy of MSC-derived EVs in AKI treatment. Data has shown that RGD EV hydrogel, through the interaction between RGD and integrins, provides superior rescue of renal function in the early stage of AKI, reduces tubular injury, and promotes cell proliferation.

Chronic kidney disease (CKD) is a global public health issue, and tubulointerstitial inflammation (TII) is a common pathological feature of CKD, leading to progressive renal fibrosis and driving CKD progression. The interplay between renal tubular epithelial cells and macrophages mediated by EVs is considered an important mechanism in the development of inflammation, with heat shock proteins (Hsp) 70 and Hsp90 playing crucial regulatory roles in increasing EV release. Thus, previous studies have demonstrated that the administration of the Hsp inhibitor quercetin can reduce EV release in a TII mouse model, thereby alleviating tubulointerstitial inflammation and fibrosis. Additionally, it has been found that EVs derived from HK-2 cells induced by an epidermal growth factor receptor (EGFR) mimetic peptide significantly inhibit macrophage viability and promote macrophage apoptosis, with a potential molecular mechanism involving an elevation in MHC-1B concentration. Further experimental evidence has confirmed that the injection of EVs derived from EGFR-induced HK-2 cells can significantly alleviate collagen deposition and macrophage infiltration in renal tissue, thereby ameliorating kidney fibrosis [33].

4.3 Treatment of liver diseases

In pathological conditions of liver cells, cellular stress leads to the activation of various signaling pathways, resulting in diverse biological effects. EVs have been widely accepted as carriers of signals and cargo, making them a subject of significant interest in the treatment of liver diseases. Liver cell-derived EVs directly fuse with target liver cells and transfer neutral sphingomyelinase and sphingosine kinase 2 (SK2), leading to increased synthesis of sphingosine-1-phosphate (S1P) in the target liver cells, thereby mediating liver repair and regeneration. Furthermore, systemically administered EVs exhibit significant accumulation in the liver with minimal reduction in renal clearance, making them a suitable therapeutic approach for liver diseases. Studies on hepatitis, liver failure, or cancer have found that EVs play an important role in their treatment. Mice treated with conditioned medium from mesenchymal stem cells (MSCs), which primarily consists of EVs, showed lower levels of serum INF-γ, IL-1β, and IL-6 and higher levels of serum IL-10 in an acute liver failure (ALF) model after 48 hours. Additionally, Zhao et al. demonstrated that treatment with bone marrow mesenchymal stem cell (BMSC)-derived EVs reduced the expression levels of pro-apoptotic proteins Bax and caspase-3 and increased the expression level of anti-apoptotic protein Bcl-2 in ALF mice. Therefore, it is hypothesized that BMSC-derived EVs prevent liver cell apoptosis through autophagy in ALF. EVs derived from the livers of mice infected with Schistosoma japonicum deliver miR-142a-3p to target WASL, inducing the release of neutrophil extracellular traps (NETs) and inhibiting the development of Schistosoma japonicum. miR-142a-3p and NETs upregulate the expression of CCL2, which activates the immune system and recruits macrophages to suppress the development of Schistosoma japonicum. Furthermore, knocking out WASL accelerates the formation of NETs, suggesting that WASL may be a potential therapeutic target and that the delivery of miR-142a-3p via EVs can effectively treat Schistosoma japonicum. With increasing research on EVs, it is believed that their application in liver diseases will have a higher significance in the future.

4.4 Treatment of orthopedic diseases

With the global aging population, orthopedic diseases are becoming an increasingly significant social issue that threatens human health. Bone undergoes constant remodeling through the balance of osteoblast-mediated bone formation and osteoclast-mediated bone resorption. In the bone metabolism microenvironment, EVs also participate in the regulation of bone formation, bone resorption, and bone remodeling. Both endothelial cell-derived EVs and mesenchymal stem cell-derived EVs have been shown to promote bone regeneration. Cartilage, a dense avascular connective tissue, presents a major challenge in delivering drugs to treat osteoarthritis. It has been reported [34] that cartilage-affinity peptide (CAP) and lysosome-associated membrane protein 2b (Lamp-2b) can be incorporated on the surface of EVs and loaded with miR-140 to target chondrocytes, effectively alleviating the progression of osteoarthritis in rats. Osteoporosis is a systemic disease characterized by progressive loss of bone mass. Xu’s team [35] developed a drug delivery system targeting osteoclasts called OT-RBCEVs, which can deliver miRNA therapeutics to osteoclasts, thereby inhibiting osteoclast activity, improving bone density, and treating osteoporosis. In another study, Luo et al. conjugated the surface of BMSCs-EVs with BMSC-specific ligands and found that intravenous injection of these EVs in ovariectomized mice increased their bone content. Rheumatoid arthritis is a chronic autoimmune disease characterized by dysregulated macrophage activity, leading to joint inflammation, destruction of bone and cartilage, and loss of function. Drug-loaded EVs can reduce the systemic toxicity of drugs and effectively modulate the activity of pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages. Research has shown that MSC-EVs can inhibit rheumatoid arthritis, which may be related to the ability of MSC-EVs to inhibit the proliferation and maturation of T cells and B cells in a dose-dependent manner. These findings demonstrate the tremendous potential of EVs in the treatment of orthopedic diseases, and their underlying mechanisms are being extensively explored.

4.5 Treatment of pulmonary diseases

Acute respiratory distress syndrome (ARDS) is the most severe form of acute lung injury caused by direct or indirect lung damage, requiring intensive care and prolonged hospitalization. Once the lungs are damaged, pro-inflammatory signaling pathways are upregulated, leading to the recruitment of large numbers of neutrophils and the secretion of pro-inflammatory cytokines. Excessive inflammatory damage to the pulmonary capillaries further disrupts the alveoli, impairing gas exchange, reducing lung compliance, and ultimately resulting in respiratory failure. Research has reported that EVs derived from dermal fibroblasts loaded with anti-inflammatory cytokines interleukin-4 and -10 (IL-4 and IL-10) genes, mRNA, and proteins can be targeted to mouse lung tissue, significantly reducing the secretion of pro-inflammatory cytokines, infiltration of neutrophils, and tissue damage. EVs loaded with miR-671-sp can also alleviate lung inflammation and injury through the NF-κB axis [36].

In 2019, COVID-19 emerged as a highly infectious respiratory illness with severe sequelae. Evidence from plasma lipidomics and metabolomics analysis suggests that EVs enriched in monosialoganglioside GM3 are associated with the pathogenesis of COVID-19, and GM3 levels increase with disease severity. Some severe COVID-19 patients develop a severe cytokine storm syndrome (CSS), a severe inflammatory immune response that threatens multiple organs. Recent clinical trials have shown that MSC-derived EVs are safe and effective for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related pneumonia [37]. MSC-derived EV therapy can significantly improve patient oxygenation and immune reconstitution within 72 hours without any side effects. mRNA lipid nanoparticle (LNP) vaccines for COVID-19 have been successfully developed [38] but their administration via intramuscular injection limits their pulmonary bioavailability. Inhalation therapy, on the other hand, can achieve effective drug concentrations in the lungs and has greater patient compliance, making it a favorable route for local administration. Pulmonary EVs are excellent candidates for inhaled therapy with nanoparticles, showing superior mRNA and protein distribution compared to commercial standards of bioengineered EVs and LNPs, thereby enhancing pulmonary bioavailability and therapeutic efficacy. Additionally, an inhalable COVID-19 vaccine has been designed that remains stable at room temperature for 3 months.

4.6 Treatment of other diseases

In addition to the diseases mentioned above, EV therapy also holds great potential in many other common diseases. For example, in patients with immune thrombocytopenia (ITP), mesenchymal stem cells (MSCs) in the bone marrow are damaged to varying degrees, resulting in abnormal immune regulation and immune tolerance and imbalance. MSC-derived EVs have been shown to regulate the secretion of antiplatelet antibodies by splenic cells, promote megakaryocyte generation of platelets, and alleviate bleeding symptoms in an ITP mouse model [39]. In stroke patients, ischemia/reperfusion injury further induces brain cell death in ischemic stroke patients. Ferroptosis, a novel form of cell death, is also present in ischemic stroke patients. Wang et al. found that intranasal administration of ADSC-derived EVs in mice can deliver miR-760-3p to cells undergoing ferroptosis, inhibit ferroptosis, improve motor and coordination abilities, and promote neural functional recovery [40].

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the targeted destruction of β-cells by self-reactive T cells. Recent studies have found that MSC-derived EVs can promote β-cell proliferation and have anti-apoptotic effects, improving T1D, possibly due to the high expression of PD-L1 in MSC-derived EVs. In the high glucose microenvironment of diabetic wounds, neutrophils are abnormally increased. Research has found that these abnormal neutrophils can induce apoptosis of human skin fibroblasts, leading to delayed wound healing. Conductive hydrogels loaded with EVs and metformin can promote angiogenesis and wound healing in chronic diabetic wounds [41]. Additionally, researchers have discovered that miR-17-5p can target the MAPK pathway to prevent the formation of abnormal neutrophils. Under hypoxic conditions, the expression of miR-17-5p in EVs secreted by mesenchymal stem cells increases, as does EV production. Therefore, EVs from hypoxic mesenchymal stem cells can inhibit the progression of diabetic wounds, improve patients’ quality of life, and provide a better understanding of diseases associated with abnormal neutrophils. The hypothalamus is the source of secretion for various hormones, and small extracellular vesicles (sEVs) derived from the hypothalamus can mediate hypothalamic AMP-activated protein kinase (AMPK) and target the central nervous system to treat obesity. Currently, research on many refractory diseases is also progressing, and with continued exploration of EV mechanisms, we believe that there will be more treatment options and better therapeutic outcomes in the future.

5. Conclusion

As drug delivery vehicles, EVs have shown tremendous potential, and clinical trials evaluating the efficacy and safety of EV-based therapies are gradually progressing. EV-based clinical drugs may be approved and enter clinical practice in the future. We believe that the production scale of EVs should be determined based on dosage, demand, and shelf life. Therefore, the production scale should be evaluated early to optimize the production process and avoid costly production expenses. Basic research on EVs should also gradually align with industrialization to lay the foundation for future applications based on EVs. We believe that by scientifically and reasonably combining these methods, the yield of EVs can be significantly improved, while also exploring and discovering new ways to increase production.

This chapter provides a brief overview of the generation, functions, and therapeutic applications of EVs in diseases. Due to their ability to achieve targeted delivery, high drug loading capacity, and controlled release, EVs are a promising drug delivery system in the field of disease treatment. However, the clinical application of EVs still faces many challenges, and the engineering and clinical translations of EVs remain major issues that need to be addressed. While increasingly complex therapeutic approaches expand our treatment options, they may also introduce additional regulatory barriers. Therefore, a careful balance between benefits and obstacles is required for the successful clinical translation of EV therapy.

In the future, what we can do is ① strive to solve the difficulties encountered in the engineering of EVs and complete the transformation of EVs research into clinical applications. ② Standardize EVs therapy and develop relevant standards. In addition, utilize the advantages of multiple disciplines to achieve mass production of EVs.