Application of extracellular vesicles in the diagnosis and treatment of chronic obstructive pulmonary disease (Review)

Introduction

Chronic obstructive pulmonary disease (COPD) is a progressive, largely irreversible lung disorder driven by genetic or environmental factors that is characterized by a complex inflammatory response and the destruction of lung parenchyma, leading to emphysema formation (1). From a genetic standpoint, a single amino acid substitution in the Z allele of the SERPINA1 gene results in reduced levels of α-1 antitrypsin (AAT) in the bloodstream of homozygous or loss-of-function homozygous individuals, which constitutes the primary pathogenesis of COPD (2). Environmental risk factors, particularly prolonged exposure to cigarette smoke and harmful particulate matter (PM), are key contributors, with cigarette smoke identified as a notable environmental risk factor for COPD (3).

The inhalation of carbon monoxide, nicotine and other components of cigarette smoke damages lung epithelial cells, thereby triggering the release of various inflammatory mediators. These mediators subsequently recruit circulating monocytes, mesophilic granulocytes and T cells into lung tissue, thereby initiating a complex inflammatory response (4). The recruited immune cells further release inflammatory mediators, which increase pulmonary vascular permeability, induce pulmonary edema and secrete elastases, which damage lung tissue and promote airway remodeling (5). As COPD advances, patients face an increased risk of cor pulmonale (also known as pulmonary heart disease) and respiratory failure (6). Current treatments are primarily symptomatic, comprising inhaled bronchodilators, glucocorticoids or oxygen therapy to alleviate dyspnea in individuals with persistent airflow limitation (7). Consequently, the molecular mechanisms underlying COPD and the development of higher-quality drug delivery systems require further research to improve the current the treatment of COPD.

Extracellular vesicles (EVs) are nanoscale lipid bilayer vesicles secreted by living cells, which are present in various body fluids, including blood, urine and ascites (8). Upon release into the extracellular space, EVs facilitate the efficient transfer of bioactive molecules, including proteins, microRNAs (miRNAs/miRs) and DNA, making them important mediators of intercellular communication (9). In previous years, EVs have been increasingly associated with the progression of various diseases, including malignant tumors, diabetes mellitus, stroke and lung inflammation (10,11). Furthermore, based on their widespread presence in biofluids, high biocompatibility, small particle size and other physiological properties, EVs have both been applied as a marker for disease diagnosis and developed as a platform for drug-targeted therapeutic carriers (12). The present review aims to explore the molecular mechanisms underlying COPD pathogenesis, with a focus on the potential role of EVs in COPD development. The present review also summarizes current approaches to using EVs as biomarkers for COPD prediction. Finally, the present review highlights previous advances that have been made in the development of EV-based therapies and their potential as drug delivery systems for COPD treatment, with the aim of offering novel research avenues for the diagnosis, treatment and prevention of COPD. Literature searches were conducted in PubMed (https://pubmed.ncbi.nlm.nih.gov/), Scopus (https://www.scopus.com/), and Web of Science (https://www.webofscience.com/) using the following search terms: ‘Chronic obstructive pulmonary disease’ AND ‘Extracellular vesicles’. After further filtering based on article content, 97 relevant articles were ultimately retained.

Molecular mechanisms of COPD pathogenesis

The pathogenesis of COPD is both complex and multifactorial, involving airway epithelial cell (AEC) damage from cigarette smoke inhalation, a dysregulated inflammatory response driven by an imbalance between oxidation and antioxidation, and the recruitment of immune cells to lung tissue that release various proteases, leading to protease/antiprotease imbalance and subsequent airway remodeling.

Imbalance of oxidation and anti-oxidation

An important factor in the pathogenesis of COPD is the imbalance between oxidant sources and sinks in the airways. Patients with COPD are exposed to exogenous oxidants, such as cigarette smoke and airborne PM, which trigger an endogenous inflammatory response (13). This response recruits neutrophils and mononuclear macrophages to the lungs, where they release superoxide anions, hydrogen peroxide and reactive oxygen species (ROS), thereby inducing oxidative stress in the airways (14). These ROS further amplify the inflammatory response by regulating redox-sensitive transcription factors, including nuclear factor κB (NF-κB), activator protein-1 and c-Jun N-terminal kinase (JNK) (15). Additionally, ROS promote mucous membrane metaplasia and the expression of the mucin genes MUC5AC and MUC5B, leading to increased mucus production (16). Notably, oxidative stress can damage DNA, and patients with COPD often fail to repair double-stranded DNA breaks promptly, which has the effect of raising their susceptibility to lung cancer (17).

Nuclear factor erythroid-2 counteracts oxidative reactions by regulating related factors, such as nuclear factor erythroid-2-related factor-2 (Nrf2), reducing cellular oxidative damage. In oxidative stress responses, Nrf2 undergoes phosphorylation and decouples from Kelch-like ECH-associated protein 1, subsequently entering the cell nucleus to participate in gene transcription, where it can activate the transcription of various antioxidant factors (including heme oxygenase 1, N-acetyl-L-tyrosine oxidoreductase and glutathione S-transferase) (18). However, in patients with COPD, reduced levels of this factor lead to an impairment of endogenous antioxidant production, thereby weakening the self-protective mechanisms of the body (19). Collectively, the residual effects of oxidative stress contribute to lung cell damage, excessive mucus secretion, protease inactivation and enhanced lung inflammation via activation of redox-sensitive transcription factors, thereby facilitating COPD progression. Additionally, cigarette smoke induces mitophagy in AECs in COPD, which both impairs oxidative phosphorylation, and leads to decreases in intracellular ATP levels and increases in mitochondrial ROS (mtROS) production. This mitochondrial dysfunction contributes to cellular senescence, further exacerbating the progression of COPD (20).

Chronic inflammation

COPD is characterized by chronic inflammation, with patients exhibiting distinctive inflammatory profiles in the lungs marked by increases in the numbers of macrophages, neutrophils, eosinophils and T lymphocytes (21). Inhalation of cigarette smoke, PM or other oxidants activates AECs and macrophages, stimulating them to release a range of inflammatory mediators, including leukotriene B4 (LTB4), interleukin (IL)-6, granulocyte-macrophage colony-stimulating factor, CXCL8, CXCL1, ROS and tumor necrosis factor α (TNF-α) (22). Even after smoking cessation, the inflammatory response persists, suggesting that COPD inflammation is maintained by self-sustaining mechanisms (21). The aforementioned inflammatory mediators not only exacerbate lung tissue damage and amplify the inflammatory response, but also serve to recruit monocytes, neutrophils and lymphocytes from the peripheral blood to the lungs (23). CXCL1 primarily recruits monocytes, whereas LTB4, CXCL1, CXCL5 and CXCL8 facilitate the recruitment of neutrophils. The recruited neutrophils and macrophages, activated by oxidative stress, contribute to the metaplasia of goblet cells and increased mucus secretion (24). The release of proteases, including matrix metalloproteinases (MMPs), tissue proteases and neutrophil elastase, degrades the extracellular matrix and alveolar elastin, leading to localized and generalized alveolar emphysema (25). Notably, collagen and elastin breakdown products act as chemotactic factors for inflammatory cells, further perpetuating the inflammatory cycle in patients with COPD (26).

Protease-antiprotease imbalance

In response to oxidative stress and inflammatory mediators, macrophages and neutrophils are recruited to the lungs, where they secrete various proteases (such as inherited deficiency of α1-antitrypsin, neutrophil elastase and MMPs) that degrade the structural components of the lungs, contributing to tissue remodeling (26). For example, macrophage secretions of MMP-2 and MMP-9 disrupt the elastic framework of the alveoli, leading to emphysema formation (27). MMP-12 is involved in pulmonary vascular remodeling through regulating the migration, proliferation and release of mitogens and growth factors from smooth muscle cells (28). In COPD, the endogenous MMP inhibitor, tissue inhibitor of metalloproteinases, is cleaved by neutrophil elastase and MMPs, resulting in elevated MMP-2 levels in lung tissue (29). Additionally, mutations in the AAT gene, particularly the Z allele, have been found to cause a marked deficiency in AAT, leading to the accumulation of the misfolded protein in hepatocytes (30). In addition, a deficiency of circulating AAT results in an imbalance between proteases and inhibitors in the lung tissue, promoting airway remodeling and airflow limitation (31).

EV involvement in COPD pathogenesis

General overview of EVs

EVs are non-replicating, nanoscale vesicles originally recognized for their role in maintaining homeostasis and eliminating waste, which has attracted notable research interest (31). During biogenesis, EVs effectively encapsulate nucleic acids, proteins, lipids and other cellular components from the donor cell (32). On the basis of their biogenesis pathways and particle sizes, EVs are classified into three categories: Exosomes (30–150 nm), microvesicles (MVs; 100 nm-1 µm) and apoptotic bodies (50 nm-5 µm) (33,34). Exosome biogenesis begins with the formation of plasma membrane indentations that encapsulate cell proteins and soluble proteins from the extracellular environment, creating a cup-shaped structure. These early endosomes undergo further sorting, development and maturation into late endosomes, which invaginate to form multivesicular bodies (MVBs). MVBs subsequently fuse with lysosomes or autophagosomes, releasing exosomes (35). The biogenesis of MVs involves localized accumulation of actin filaments and the flipping of phosphatidylserine from the inner to the outer leaflet of the membrane, altering membrane curvature and inducing vesicle budding (36). Apoptotic bodies are generated through the rupture of the plasma membrane in apoptotic cells, followed by continuous volume reduction and actinin-driven contraction, which causes the accumulation of cellular contents and an increase in hydrostatic pressure, ultimately leading to vesicle formation. The nucleus then fragments, resulting in the formation of apoptotic bodies (37). Once released into the extracellular space, EVs can be internalized by recipient cells via mechanisms such as granzyme-independent endocytosis, macropinocytosis and phagocytosis, where they release their cargo, including nucleic acids, proteins and other molecules, thereby facilitating intercellular communication (38) (Fig. 1).

Figure 1. Biogenesis, secretion and cell entry of EVs. EVs are categorized into exosomes, apoptotic bodies and MVs based on their biological origin and size. Exosomes produce ILVs through inward budding of early endosomes to form MVBs, which subsequently fuse with lysosomes or cell membranes and are released into the extracellular environment of the exosome. MVs are produced by outward budding from the plasma membrane. Apoptotic bodies are released during cell death through plasma membrane vesicles. EVs can deliver cargo to recipient cells through phagocytosis, megakaryocytosis, endocytosis, direct fusion and other means, and subsequently modulate biological behavior. Created with BioRender.com (https://app.biorender.com/illustrations/692ee7fb97717aa47f30567d?slideId=76ff231c-5a96-4f30-a8e7-0972073ada7c). EVs, extracellular vesicles; MVs, microvesicles; MVBs, multivesicular bodies; ILV, intraluminal vesicle.

Role of EVs in the pathogenesis of COPD

Prolonged exposure to irritants such as cigarette smoke leads to the destruction of the structural integrity of the lungs and their dysfunction (39). In response to such irritants, AECs secrete a variety of EVs, containing components such as miR-210, miR-21 and cellular communication network factor 1 (CCN1) (40). Among these, CCN1-containing EVs activate the Wnt signaling pathway, which induces the secretion of pro-inflammatory cytokines, such as IL-8 and monocyte chemoattractant protein 1, by epithelial cells. This activation promotes the recruitment of circulating T lymphocytes, neutrophils and monocytes to the lungs, further exacerbating the inflammatory response (41). Neutrophils and monocytes release matrix proteases that degrade the structural integrity of alveoli, contributing to the development of pulmonary emphysema. Furthermore, T lymphocytes secrete EVs that, through a positive feedback mechanism, stimulate epithelial cells to release IL-6 and inhibit the secretion of the anti-inflammatory cytokine IL-10, further damaging the airways (42).

Additionally, miR-210-containing EVs target the autophagy-related 7 gene in lung fibroblasts (LFs), inhibiting autophagosome formation (42). This impairment in autophagy leads to the increased expression of type I collagen and α-smooth muscle actin (α-SMA) in LFs, promoting myofibroblast differentiation and airway wall thickening (43). Similarly, miR-21-containing EVs promote myofibroblast differentiation by targeting von Hippel-Lindau protein (pVHL), stabilizing hypoxia-inducible factor 1α, thus facilitating airway remodeling (44). EVs from inhaled bacteria or viruses also serve a role in COPD pathogenesis. For example, bacterial outer-membrane vesicles (OMVs) from Moraxella catarrhalis contribute to pulmonary inflammation (45). OMVs derived from Pseudomonas aeruginosa inhibit chloride secretion mediated by cystic fibrosis transmembrane conductance regulator, reducing pathogen clearance through mucociliary action and amplifying the inflammatory response (46). Although the exact mechanisms via which EVs influence COPD have yet to be fully elucidated, their involvement in the pathogenesis of COPD is notable (Fig. 2).

Figure 2. Schematic overview of epithelial EV-induced airway remodeling. Epithelial cells secrete a large number of EVs after being stimulated by the external environment, and miR-210, miR-21, cellular communication network factor 1 and other components contained in EVs induce the increased expression of pro-inflammatory cytokines, such as IL-8 and monocyte chemoattractant protein-1, secreted by activated epithelial cells. This is achieved by activating the Wnt signaling pathway/hypoxia inducible factor-1α pathway, which enhances the differentiation of fibroblasts into myofibroblasts, the contraction of smooth muscle cells and the accumulation of extracellular matrix proteins, ultimately promoting airway remodeling. Created with BioRender.com (https://app.biorender.com/illustrations/692eed2c2bdb071cce996f3d?slideId=f9661b16-c75e-4ba9-a385-974bcfbc0a16). COPD, chronic obstructive pulmonary disease; EVs, extracellular vesicles; CCN1, cellular communication network factor 1; miRNA, microRNA; NK, natural killer; DC, dendritic cell; ROCK1, Rho-associated protein kinase 1; PVHL, von Hippel-Lindau tumor suppressor; HIF-1α, hypoxia inducible factor-1α; ATG7, autophagy related 7.

EVs in the diagnosis and treatment of COPD

Application of EVs in COPD diagnosis

Patients with COPD typically present with one or more symptoms such as exertional dyspnea, cough, sputum production, chest tightness or fatigue (47). Clinically, COPD diagnosis involves assessing lung function [using the ratio of forced expiratory volume in 1 sec (FEV1) to forced vital capacity (FVC) post-bronchodilator, with a ratio <0.70 indicating a positive COPD diagnosis], along with chest X-rays, chest CT scans, and serum markers such as soluble receptors for advanced glycation end products (48,49). The heterogeneity of EVs is associated with the type and state of the cells from which they originate. Analyzing the number or specific content of EVs in peripheral blood, bronchoalveolar lavage fluid (BALF) or sputum offers new diagnostic possibilities for COPD (50) (Fig. 3).

Figure 3. Schematic overview of EV-based biomarkers in COPD diagnosis. By detecting sputum, bronchoalveolar lavage fluid and EVs in blood samples as markers for the diagnosis of chronic obstructive pulmonary disease, the concentrations of CD31-MPs, CD66b-MPs, CD235ab-MPs and total protein in sputum sample-derived EVs are notably increased, the levels of miR-122 in bronchoalveolar lavage fluid are decreased, and the expression levels of EV miR-221, miR-23a and miR-574 in blood samples are also notably increased. Created with BioRender.com (https://app.biorender.com/illustrations/692eeccc235f309bfe04b81d?slideId=44a0ee63-27a9-433b-8188-676b7956b82a). COPD, chronic obstructive pulmonary disease; miR, microRNA; EVs, extracellular vesicles; MPs, microparticles.

EVs derived from sputum

Sputum, a secretion from the respiratory tract, is a vital diagnostic tool for lung diseases such as COPD, lung cancer, tuberculosis and chronic pneumonia. The analysis of sputum supernatant media or sputum cells can provide important diagnostic information (51). For example, miR-21, miR-155 and miR-210 levels are markedly elevated in sputum from patients with lung cancer, while detecting Mycobacterium tuberculosis in sputum is key for diagnosing pulmonary tuberculosis (52). However, spontaneous sputum samples often have poor quality, and hypertonic or isotonic saline containing salbutamol is typically used to induce sputum production (53). Sputum testing is less invasive and easier to perform compared with testing BALF (54). Recent research has successfully isolated and purified EVs from sputum samples of 20 patients with COPD, revealing a notable increase in the general protein concentration in COPD sputum samples (106.70±60.56 µg/ml) compared with the healthy control group (56.82±11.99 µg/ml) (55). Another study identified that EVs containing CD31, CD66b and CD235 in sputum are associated with COPD progression (56). Although few studies have been published on the physicochemical properties of sputum EVs, these findings suggest that analyzing EVs in sputum components could be a valuable diagnostic approach for COPD.

BALF

BALF is a standard clinical method for diagnosing infectious diseases, non-infectious immune diseases and malignant conditions in the lungs (57). The procedure involves introducing saline into the alveoli via a bronchoscope, and then aspirating alveolar cells and biochemical components under negative pressure (58). Compared with more invasive procedures, such as lung tissue or bronchial biopsies, BALF is less invasive and has fewer complications. BALF detects a variety of components, including cells, proteins, genes, microorganisms and EVs, making this procedure a more comprehensive reflection of lung pathology (59). Given the heterogeneity of EVs and their close association with the biology of their donor cells, analyzing EVs in BALF can provide a valuable basis for diagnosing COPD. For example, one study found that the expression of miR-122-5p in pulmonary EVs from patients with COPD was significantly downregulated compared with that in healthy smokers and non-smokers (57). A metabolomics database of BALF comprising 117 individuals, including non-smokers, smokers and patients diagnosed with COPD, revealed the presence of >11,000 lipids and ~650 water-soluble substances in BALF. Among these, one-tenth of the substances were present in all samples (60).

Blood-derived EVs

Peripheral blood contains millions of EVs, and the nucleic acids, proteins and other components within these vesicles can reflect the biological behavior of their donor cells (58). Consequently, analyzing the expression levels of nucleic acids and proteins in blood-derived EVs from patients with COPD compared with healthy individuals offers a promising foundation for diagnosing COPD. miRNAs are key regulators in gene expression networks, having roles in cell differentiation, apoptosis, inflammatory responses, tissue remodeling and angiogenesis. Profiling miRNA expression using miRNA microarrays or panel-based quantitative PCR technologies holds potential as a biomarker for COPD (61). A study comparing circulating miRNA and cytokine levels between 103 patients with COPD and 25 control patients found that miR-1 and miR-499 levels were 2.5 and 1.5 times higher, respectively, in the COPD group compared with in controls (62). However, one challenge of this detection method is that miRNAs in circulation are susceptible to degradation by endogenous RNA enzymes, which could lead to an inaccurate reflection of the miRNA expression levels in the donor cells (63). By contrast, EVs, as natural carriers of signal molecules, are encased in a phospholipid bilayer that protects miRNAs and proteins from degradation by proteases and nucleases in the blood. Therefore, analyzing miRNAs and proteins in blood-derived EVs offers a more reliable approach for COPD diagnosis (61). For example, a previous study found that the levels of miR-221, miR-23a and miR-574 in blood-derived EVs from 41 patients with COPD and 29 healthy individuals were negatively correlated with the FEV1/FVC ratio, with 95% confidence intervals of 0.776, 0.688 and 0.842, respectively (64). Another study identified a notable increase in miR-21 levels in blood-derived EVs from cigarette smoke-induced COPD, where miR-21 targets pVHL to regulate α-SMA gene expression, thereby contributing to airway remodeling. Furthermore, during acute COPD exacerbations, the number of plasma exosomes, along with the levels of plasma C-reactive protein and soluble TNF receptor-1, have been found to increase (65). Although differential detection of blood-derived EVs is not yet a routine diagnostic tool in clinical practice, their potential as a prospective biomarker for predicting COPD is increasingly evident (Table I) (55–57,61,62,64,65).

Table I. Potential markers for the diagnosis of EV-based chronic obstructive pulmonary disease.

Table I.

Potential markers for the diagnosis of EV-based chronic obstructive pulmonary disease.

First author, year	EV test sample source	Detection indicators	Trends in detection indicators	(Refs.)
Liu et al, 2025	Sputum	EVs containing CD31, CD66b and CD235	Upregulated	(56)
Kotsiou et al, 2024	Sputum	Total protein concentration	Upregulated	(55)
Kaur et al, 2021	Bronchoalveolar lavage fluid	miR-122-5p	Downregulated	(57)
Gon et al, 2020	Bronchoalveolar lavage fluid	Microvesicles containing CD14	Upregulated	(61)
Donaldson et al, 2013	Blood	miR-1 and miR-499	Upregulated	(62)
Shen et al, 2021	Blood	miR-221, miR-23a and miR-574	Upregulated	(64)
Tan et al, 2017	Blood	EV number and blood plasma C-reactive protein and soluble tumor necrosis factor receptor-1	Upregulated	(65)

[i] EV, extracellular vesicle; miR, microRNA.

Application of EVs in COPD treatment

In addition to the promising potential of liquid biopsy technology for diagnosing COPD, EVs also offer a new avenue for treatment. There are two current strategies for utilizing EVs in COPD treatment. Primarily, the removal of EVs containing nucleic acids or proteins associated with disease pathogenesis can be achieved by: i) Inhibiting the secretion of EVs that promote COPD progression; ii) blocking the effective binding of EVs to recipient cells; or iii) capturing circulating EVs that contribute to disease progression (66). For example, treatment with the EV-production inhibitor GW4869 has been shown to reduce miR-125a-5p expression in cigarette smoke-induced EV secretion from AECs. miR-125a-5p promotes M1 macrophage polarization by downregulating IL-1 receptor antagonist protein, thereby activating the Toll-like receptor (TLR)4/myeloid differentiation primary response 88/TNF receptor-associated factor 6/NF-κB signaling pathway and enhancing its pro-inflammatory effects (67). Alternatively, EVs can be used as therapeutic agents or as vehicles for delivering therapeutic agents to the lungs of patients with COPD. Mesenchymal stem cell (MSC)-derived EVs, in particular, have been explored for their therapeutic potential in COPD (Fig. 4) (68).

Figure 4. Therapeutic applications of engineered extracellular vesicles in COPD. EVs extracted from different cells, including mesenchymal stem cells, macrophages and DCs, by methods such as differential centrifugation, size exclusion chromatography, immunoaffinity capture, microfluidics or precipitation, can be used in the treatment of COPD. These EVs are loaded with drugs endogenously and/or exogenously, and their membrane surfaces are modified to improve lung tissue targeting. Created with BioRender.com (https://app.biorender.com/illustrations/692ee91969efa5da6646019a?slideId=15732dc8-116e-49fa-a545-6343ae7d7599). miR, microRNA; EVs, extracellular vesicles.

Application of MSC-derived EVs in therapy

MSCs are multipotent adult stem cells that can be isolated from various human tissues, including bone marrow (BM), adipose tissue, umbilical cord and placenta (69). MSCs secrete numerous anti-inflammatory cytokines, including IL-13, neurotrophin 3, ciliary neurotrophic factor and IL-10, and growth factors that aid tissue repair (70). Due to their anti-inflammatory and immunomodulatory properties, MSCs have been investigated for COPD treatment. A previous study has shown that BM-derived MSCs are able to inhibit p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase phosphorylation and activation, thereby reducing the production of prostaglandin E2 and cyclooxygenase 2 in macrophages and alleviating airway inflammation and emphysema in a COPD mouse model (71). Adipose-derived stem cells have been found to reduce chronic cigarette smoke-induced activation of JNK1 and protein kinase B (AKT) by eliminating p38 MAPK phosphorylation, which, in turn, mitigates lung inflammation and cysteine aspartate activity, protecting alveolar structural integrity in patients with COPD (72). A clinical trial assessing the systemic administration of allogeneic MSCs in patients with moderate-to-severe COPD evaluated inflammatory markers, including TNF-α, IFN-γ, IL-2 and TGF-β, pulmonary function and electrocardiographic parameters. The study confirmed the safety of systemic MSC infusion in individuals with impaired lung function, and demonstrated that MSCs may inhibit inflammation and reduce or reverse emphysematous changes (73). However, the clinical application of MSCs is limited by challenges such as the difficulty in obtaining high-quality MSCs, issues with proper storage, potential tumorigenicity and the ethical complexities of the infusion process. These factors constrain the broader therapeutic use of MSCs for patients with COPD (74). Consequently, there is a growing need to develop MSC-based cell-free therapies that overcome these limitations.

EVs derived from MSCs retain the anti-inflammatory, immunomodulatory and tissue-regenerative properties of their parent cells, effectively transporting proteins, lipids, DNA fragments, mRNA and other molecules to recipient cells, thereby facilitating intercellular communication and regulating the biological functions of these cells (75). Compared with the donor cells, MSC-derived EVs are enriched with functional cargo, including signaling molecules and growth factors, and offer advantages such as long-term stability for storage and transport, ethical acceptance for infusion, deep-tissue penetration and versatile administration methods (76). Furthermore, c-Myc gene transfection, hydrogen peroxide and lipopolysaccharide pretreatment can further enhance EV secretion from MSCs (77,78). Over time, MSC-derived EVs have emerged as a promising cell-free therapeutic alternative.

The bioactive substances contained within EVs contribute towards COPD treatment through promoting lung cell proliferation and reducing systemic inflammatory-mediator production (79). Chronic exposure to harmful particles or gases triggers a complex inflammatory response, driving the characteristic pathological features of COPD, including emphysematous destruction, alveolar remodeling and narrowing of small airways (80). A key event in this process is endothelial cell apoptosis in the lung parenchyma, which is closely associated with emphysema development. Notably, patients with COPD exhibit reduced expression levels of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) in their bodies, and this leads to extensive endothelial cell death and microvascular damage upon exposure to cigarette smoke extract, accelerating emphysema progression (81). MSC-derived EVs retain ~70% of the beneficial effects of MSCs and can effectively deliver signaling molecules and growth factors to lung tissue, reducing inflammation and promoting the restoration of damaged lung function (82). Human umbilical cord MSC exosomes (hUCMSC-Exos) contain hsa-miR-10a-5p and hsa-miR-146a-5p, which inhibit endothelial cell apoptosis by facilitating the specific binding of VEGF to VEGFR2 on endothelial cells, thereby activating the phosphoinositide 3-kinase/AKT pathway (83). Additionally, hUCMSC-Exo treatment can reduce the lung tissue inflammation score in COPD model mice, decreasing the influx of peripheral blood neutrophils, eosinophils, lymphocytes and macrophages into the lungs, while also reducing the number of mucus-secreting goblet cells. Mechanistically, hUCMSC-Exos exert their therapeutic effects by inhibiting the phosphorylation of the pro-inflammatory transcription factor NF-κB subunit p65 (84).

Mesenchymal stem cells, potent mitochondrial donors, exhibit elevated levels of the mitochondrial fission protein dynamin-related protein 1 (DRP1) upon exposure to cigarette smoke, leading to impaired mitochondrial function. Prolonged exposure reduces the expression of fusion proteins such as mitofusin (MFN)1, MFN2 and optic atrophy 1 (OPA1), causing mitochondrial damage, and ultimately triggers mitophagy (85). Disrupted mitophagy results in the accumulation of perinuclear mitochondria in lung epithelial cells and fibroblasts, impaired oxidative phosphorylation and an increase in mtROS, all of which exacerbate COPD progression (86). To address COPD induced by mitochondrial damage, a previous study employed BM-derived MSCs and exosomes (MSC + EXO) in a COPD mitochondrial Keima mouse model induced by acute cigarette smoke exposure over a period of 10 days (87). The combination therapy markedly upregulated the expression of MFN1, MFN2 and OPA1, whereas the levels of damage-associated molecular pattern (DAMP) markers, including MMP9 and high mobility group box 1, were markedly increased, supporting the hypothesis that MSC + EXO therapy protects against lung mitochondrial dysfunction. By contrast, MSC or EXO treatments alone exhibited limited effects (87). These results highlight MSC-derived EVs as promising cell-free therapeutic agents capable of addressing COPD through multiple mechanisms.

Treatment of COPD with EVs as a drug delivery vehicle

The current standard of care for COPD involves triple therapy, combining inhaled corticosteroids, long-acting β-2 agonists and long-acting muscarinic receptor antagonists (88). However, steroid resistance in a number of patients necessitates the administration of higher doses, which increase the risk of pneumonia and a range of side effects that negatively impact the function of normal cells, including osteoporosis, metabolic disorders, cardiac arrhythmias and hyperglycemia (89). This underscores the notable need for the development of a high-quality drug-delivery platform that targets lung tissue to enhance COPD treatment efficacy.

EVs have been engineered to deliver a wide range of drugs for the treatment of various diseases (90). For example, EVs loaded with miR-451a can target MMP10 to inhibit the proliferation, migration and invasion of hepatocellular carcinoma (91). EVs carrying gemcitabine (GEM) enhance the therapeutic effect of GEM treatment on pancreatic ductal carcinoma, while reducing GEM-induced toxicity to the liver and kidneys (92). Curcumin-loaded EVs mitigate ischemic stroke by suppressing the expression of phosphorylated-p65 and caspase-3 (93). Similarly, EVs as a drug delivery platform show notable potential in COPD treatment. Drug-loaded EVs can regulate key signaling pathways involved in COPD pathogenesis to inhibit disease progression. For example, CD24-overexpressing EVs can block the activation of the NF-κB pathway by binding to DAMPs, preventing their interaction with pattern recognition receptors, such as TLRs and NOD-like receptors, thereby dampening the excessive inflammatory response in COPD (94). Furthermore, nebulized human platelet-derived exosome products have been shown to reduce smoke-induced emphysema in mice by inhibiting NF-κB activation, inflammatory cytokine production and apoptotic protein expression, while promoting an increase in CD4+/forkhead box P3+ regulatory T cells in lung tissue, thereby alleviating oxidative lung injury, inflammation and apoptotic alveolar epithelial cell death (95). In addition, artificial nanovesicles derived from adipose-derived stem cells expressing fibroblast growth factor 2 have been shown to enhance the differentiation potential of type II alveolar cells into type I alveolar cells, thereby inhibiting emphysema formation (96). Some practical applications of EV-based treatments for COPD are summarized in Table II (83,84,87,95,96).

Table II. Practical application of EV-based treatment of chronic obstructive pulmonary disease.

Table II.

Practical application of EV-based treatment of chronic obstructive pulmonary disease.

First author, year	Sources of EVs	Cargo	Mechanism of action	Effect of action	(Refs.)
Chen et al, 2022	Human umbilical cord mesenchymal stem cells	miR-10a and miR-146a	Promotes specific binding of VEGF to VEGF receptor 2 of endothelial cells to activate the phosphoinositide 3-kinase/protein kinase B pathway	Inhibition of endothelial cell apoptosis	(83)
Ridzuan et al, 2021	Human umbilical cord mesenchymal stem cells	Not applicable	Inhibits phosphorylation of the pro-inflammatory transcription factor NF-κB subunit p65	Reduces influx of neutrophils, eosinophils, lymphocytes and macrophages into lung tissue	(84)
Maremanda et al, 2019	Bone marrow mesenchymal stem cells	Not applicable	Increases the expression levels of MFN1, MFN2 and optic atrophy 1	Strengthens protection against pulmonary mitochondrial dysfunction	(87)
Xuan et al, 2024	Platelets	Not applicable	Inhibits NF-κB activation, inflammatory cytokine production and apoptotic protein expression	Inhibits oxidative lung injury, inflammation and apoptotic alveolar epithelial cell death	(95)
Kim et al, 2017	Adipose-derived stem cells	Fibroblast growth factor 2	Enhances type II alveolar cell proliferation and differentiation potential to type I alveolar cells	Inhibits the formation of emphysema	(96)

[i] EV, extracellular vesicle; VEGF, vascular endothelial growth factor; MFN, mitofusin; NF-κB, nuclear factor κB.

Achieving the therapeutic effects of drug-loaded EVs targeting lung tissue involves several important steps: i) Isolation and purification of EVs; ii) loading of therapeutic agents into EVs; and iii) artificial modification of EVs to enhance targeting efficiency. The first step, the isolation and purification of EVs, primarily utilizes methods based on the physicochemical properties of EVs such as size, density and surface marker proteins (97). Techniques used include differential centrifugation, size-exclusion chromatography, immunoadsorption capture, microfluidics and precipitation (98). Among these, differential centrifugation is considered the ‘gold standard’ for EV isolation in laboratories, and remains the predominant method for extracting EVs from biofluids. The second step is the effective loading of therapeutic drugs into EVs, which can occur either before or after EV isolation (98,99). The third important step is modifying EVs to improve their targeting capability. Natural EVs typically exhibit poor targeting properties and are rapidly cleared from circulation, primarily accumulating in the liver and kidneys following intravenous administration (100). To address this limitation, artificial modification of EVs is necessary to enhance their tissue- or cell-specific delivery. A key approach involves using genetic engineering to transfect plasmids encoding targeting peptides or proteins, such as lysosomal-associated membrane protein 2B (LAMP2B) and tetraspanins, into EVs. These targeting peptides are either incorporated into the biogenesis pathway of the EVs or directly loaded onto the EV membrane surface, improving the ability of the EVs to target and deliver cargo to specific tissues or cells (101). For example, the fusion of neuron-specific rabies virus glycoprotein with the N-terminus of LAMP2B enables the targeted delivery of GAPDH small interfering RNA (siRNA) to neurons, microglia and oligodendrocytes in the brain. Furthermore, other tissues do not absorb GAPDH siRNA in this treatment (102).

The route of administration for EVs targeting lung tissue has garnered notable interest from researchers. Currently, the primary methods of EV-based COPD treatment involve intravenous administration and intratracheal delivery. Intravenous administration is the most widely used method (103), with therapeutic EVs reaching the lungs through the circulatory system to exert their effects. In studies performed in isolated cells, typical injection doses of EV therapeutics range from 2–40 µg/ml (104). However, intravenous administration carries a risk of microcirculation aggregation, potentially leading to mutagenicity and carcinogenicity (105). Intratracheal delivery, which directly targets the airways, reduces systemic side effects compared with intravenous administration, although the complexity of the procedure markedly increases (106).

Previous review summaries and novelty of the present review

A recent review summarized the current understanding of EV-miRNAs in conditions such as COPD, asthma, lung cancer and COVID-19, highlighting their potential as both biomarkers and therapeutic targets in these respiratory diseases (107). The previous work emphasized the crucial role of EV-miRNAs in regulating immune cell function, chronic airway inflammation and disease progression. Additionally, the previous work largely focused on EVs as biomarkers and therapeutic targets, whereas the present review provides a more detailed analysis of bacteria-derived OMVs and their impact on COPD, linking impaired mucociliary clearance and inflammation. Furthermore, the current review uniquely addresses EV engineering strategies for targeted drug delivery in COPD, including pre- and post-isolation drug-loading methods and genetic modifications to enhance targeting efficiency, topics not as extensively covered in previous reviews (1,8). A previous study also emphasized the importance of EVs in regulating immune cell function, chronic inflammation and disease progression (108). However, the present review introduces novel insights by exploring mitochondrial dysfunction as a key mechanism in COPD progression, a topic not deeply covered in the prior study. Furthermore, while the previous review discussed EVs as biomarkers detected in biological fluids, such as blood and sputum, the current review offers a more detailed quantitative analysis of miRNA expression in various fluids. Additionally, this review expands on EV engineering strategies for targeted drug delivery in COPD, including both pre- and post-isolation drug-loading methods and genetic modifications, which is a more focused exploration compared with the broader discussions in the previous review.

Furthermore, another review highlighted the involvement of EVs in COPD pathogenesis, particularly their influence on inflammation, immune responses and airway remodeling (109). However, the present review introduces novel insights by exploring mitochondrial dysfunction as a key mechanism in COPD progression, specifically through mitochondrial fission/fusion proteins, which were not extensively covered in the referenced review. Furthermore, while both reviews have discussed EVs as biomarkers, the current review provides a more detailed quantitative analysis of miRNAs in various biological fluids, offering deeper insights into their diagnostic potential. Additionally, this review expands on EV engineering strategies for targeted drug delivery in COPD, which is more thoroughly explored than in the previous review, which provided a broader discussion of EVs as biomarkers and therapeutic targets.

The present review has provided novel insights into EV-mediated mitochondrial dysfunction, characterized by imbalances in key regulators such as DRP1/MFN/OPA1 and increased mtROS production, as a key driver of COPD progression, a mechanism frequently overlooked in previous reviews (107–109). Furthermore, the present review has broadened the perspective of current EV-based research by linking bacteria-derived OMVs, for example from Moraxella and Pseudomonas, to impaired mucociliary clearance and inflammatory responses, thereby establishing a notable association between microbiology and EV biology.

In terms of diagnostic value, the present review has systematically compared and evaluated potential EV biomarkers in blood, BALF and sputum, supplemented with quantitative data. For example: i) The general protein concentration of sputum EVs in patients with COPD is higher compared with that in healthy controls (55); ii) the expression levels of miR-122-5p in EVs from the lungs of patients with COPD are significantly downregulated, and a structured dataset of BALF-derived EV particles, encompassing particle size distribution and concentration, has been established and analyzed (57,60); and iii) the expression levels of miR-1 and miR-499 are higher in the blood of patients with COPD, and the diagnostic confidence intervals of miR-23a, miR-221 and miR-574-5p have been analyzed (62,64). These have filled in several gaps in previous studies.

Regarding therapeutic applications, the present review has detailed EV engineering strategies for targeted drug delivery in COPD, including both pre-isolation and post-isolation drug-loading methods, modification technologies such as genetically engineered fusion targeting peptides, and their specific processes. The present review has also objectively analyzed the limitations and challenges of using EVs as drug delivery systems, and discussed the therapeutic potential of stem cell-derived EVs, such as the effects of hUCMSC-Exos and combined therapy with BM-derived MSC + EXO treatment.

Conclusion

In conclusion, EVs represent a ‘double-edged sword’ in COPD treatment. On one hand, the substances they carry can contribute to the progression of COPD by regulating key signaling pathways. On the other hand, liquid biopsy technologies that detect EVs in sputum, blood and alveolar lavage fluid, along with their associated cargo, may offer valuable diagnostic markers for early COPD detection. Additionally, EVs derived from MSCs or used as drug delivery vehicles for COPD treatment present a promising new cell-free therapeutic strategy. Therefore, further research into the role of EVs in COPD progression is important for advancing preventative measures. Simultaneously, extensive preclinical studies and clinical trials are required to validate the therapeutic applications of EVs or therapeutic vectors in COPD.

Acknowledgements

Not applicable.

Funding

The present work was supported by the National Natural Science Foundation of China (grant no. 8180150490).

Availability of data and materials

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Authors' contributions

YZ, ZG and CF conceived and designed the review. TR, JX and YY retrieved the relevant literature, acquired relevant references, screened data, and analyzed and organized reference data. XZ, XH and WY critically revised and edited the manuscript. YZ, ZG and CF created figures and reviewed the article. Data authentication is not applicable. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

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References