INTRODUCTION
Allergic rhinitis (AR) is one of the most common chronic diseases worldwide, particularly prevalent among children and adolescents[1]. The core pathophysiological mechanism is an abnormal Th2 immune response in response to environmental allergens. Allergens are captured and processed by antigen-presenting cells in the nasal mucosa and presented to naïve CD4+ T cells, leading to their differentiation into Th2 cells. Activated Th2 cells secrete cytokines such as interleukin (IL)-4, IL-5, and IL-13, which in turn promote the production of specific immunoglobulin E (IgE) by B cells, triggering mast cell and basophil degranulation and eosinophil infiltration, resulting in typical clinical symptoms, including paroxysmal sneezing, clear nasal discharge, nasal congestion, and nasal itching. AR is not limited to the nasal cavity and often co-occurs with allergic conjunctivitis, asthma, sinusitis, and other diseases, severely impacting patients’ quality of life, sleep, and daytime activities, and imposing a heavy socioeconomic burden. The rising number of affected individuals imposes a substantial economic and social burden. AR is a chronic inflammatory condition of the nasal mucosa mediated by IgE[2]. Typical symptoms include sneezing, rhinorrhea, nasal congestion, nasal itching, and pale nasal mucosa[3]. Some patients also experience allergic conjunctivitis, presenting with itchy, red eyes and tearing[4]. In addition to conjunctivitis, AR frequently coexists with asthma and sinusitis, etc., reflecting the shared inflammatory characteristics summarized by the concept of “one airway, one disease”, which significantly impairs patients’ quality of life[5-7]. The pathogenesis of AR is a complex process triggered by environmental allergens and driven by immune dysregulation. Common airborne triggers are dust mites, pollen, animal dander, and mold[4]. In susceptible individuals, initial exposure to allergens of dust mites or pollen leads antigen-presenting cells, primarily dendritic cells (DCs), to capture and process the antigens, migrate to local lymph nodes, and present them to naive CD4+ T cells, thereby initiating an immune response[2,8,9]. The central mechanism involves Th1/Th2 immune imbalance and Th2 overactivation, resulting in the secretion of cytokines such as IL-4, IL-5, and IL-13, which facilitate IgE production and eosinophil infiltration, driving inflammation[10-13]. Furthermore, impaired regulatory T cell (Treg) function and damage to the nasal epithelial barrier can synergistically exacerbate inflammation, creating a vicious cycle. Chronic inflammation over time can also induce nasal mucosal tissue remodeling, including structural alterations such as epithelial barrier disruption[14,15].
Currently, drug therapy is the primary approach for managing AR, often involving oral antihistamines or topical corticosteroids to control symptoms. International guidelines recommend topical medications and nasal sprays as targeted treatments, which act directly at the lesion site, thereby reducing systemic side effects and improving therapeutic efficacy[16,17]. For patients who respond poorly to conventional drug therapy, allergen immunotherapy (AIT) offers an etiological treatment option[18,19]. AIT can be administered subcutaneously or sublingually and provides long-term benefits in improving disease outcomes[20]. However, current therapies have notable limitations. Pharmacological treatments are primarily symptomatic and do not correct underlying immune imbalances, and long-term use may carry side effects. AIT, while effective in some patients, involves prolonged treatment courses, carries a risk of allergic reactions, and may be ineffective for others[21]. Additionally, growing evidence highlights the role of nasal epithelial barrier dysfunction in AR pathogenesis, underscoring its importance in disease progression[22,23]. All these necessitate the exploration of novel therapies that can safely and effectively restore immune balance and promote tissue barrier repair. Cell-based regenerative medicine strategies, particularly those involving secreted extracellular vesicles (EVs) demonstrate potent immunomodulatory and tissue-repair capabilities, making them promising approaches for AR treatment.
EVs are a class of membrane-bound nanostructures secreted by cells. They excel in mediating intercellular and inter-organ communication, with their biological functions determined by the molecular cargo they carry, thereby participating in a wide range of physiological and pathological processes[24]. Current studies indicate that EVs can actively transport complex biomolecules, including proteins, lipids, and nucleic acids, which can modulate the functional activity and behavior of target cells. As natural carriers of non-secretory molecules, EVs are capable of crossing biological barriers to safely deliver their contents and regulate intercellular processes within barrier microenvironments. From modulating immune responses to promoting tissue repair and remodeling, as well as facilitating signal transduction in the nervous system, EVs essentially maintain cellular homeostasis and orchestrating disease progression and tissue repair[25]. EVs are naturally released by nearly all living cells and can be broadly classified based on their cellular origin into three main categories: EVs, microvesicles, and apoptotic bodies[26]. The clinical translation of EVs research faces a series of methodological challenges, the core of which lies in the standardization of isolation and characterization. Currently, EVs isolation relies on various techniques, such as ultracentrifugation, size exclusion chromatography, polymer precipitation, and immunoaffinity capture. Different methods yield EVs populations with significant differences in purity, yield, and subpopulation composition, directly impacting the comparability and reproducibility of research results. Regarding characterization, the International Society for Extracellular Vesicles has published minimum information standards, emphasizing the need for comprehensive identification using multiple techniques, including nanoparticle tracking analysis or dynamic light scattering to determine particle size and concentration, transmission electron microscopy to observe morphology, and western blotting or flow cytometry to detect specific marker proteins. However, in the specific research area of AR, many published studies have not yet fully standardized EVs isolation strategies, characterization depth, and data reporting, constituting a significant bottleneck in the transition from basic discovery to clinical application. Promoting and adhering to recognized standardized protocols is a crucial prerequisite for ensuring the reliability of EVs research results and enhancing their translational medical value[27-29].
Research on EVs began in 1984, when 50-nm vesicles released by cells were first observed using electron microscopy and subsequently named[30]. Studies on mesenchymal stem cells (MSCs) date back to the 1970s, when scientists such as Friedenstein et al[31] first discovered and isolated bone marrow-derived cells with osteogenic differentiation potential, although these cells had not yet been formally named MSCs. It was not until 1991 that Professor Caplan[32] officially designated these cells as “MSCs” and characterized their multi-lineage differentiation and tissue repair capabilities. The first therapeutic application of MSC-derived EVs can be traced to 1995, when Caplan’s team intravenously infused expanded bone marrow MSCs into patients with hematological diseases, demonstrating clinical feasibility and safety[33]. In rhinitis research, Qiu et al[34,35] were the first to detect EVs in patients with chronic atypical AR, revealing that these vesicles carry microbial products and antigens capable of modulating DC function and promoting antigen-specific CD8+ T cell responses. Subsequent studies have further elucidated the mechanisms of exosome-mediated immunomodulation in AR, including the regulatory roles of exosome-carried microRNAs (miRNAs)[36] and the potential of serum exosomal miRNAs (e.g., hsa-miR-4669) as biomarkers for predicting responses to immunotherapy[37]. Despite these advances, the application of EVs in rhinitis research has largely been limited to mechanistic studies and biomarker exploration, with no direct clinical applications reported to date. Nevertheless, a growing number of studies highlight the critical role of EVs in AR pathogenesis. This review aims to systematically examine the functions of EVs in AR, with particular emphasis on their roles in immune regulation and nasal epithelial barrier repair, and to explore the potential and challenges of exosome-based therapeutic strategies for clinical translation in AR. Figure 1 show the timeline of this field.
Figure 1 Timeline of this research field.
This figure illustrates the key developments of extracellular vesicles and mesenchymal stem cells in allergic rhinitis and related research. This timeline clearly presents the evolution of exosome research from basic discovery to precision diagnosis and translational applications of allergic rhinitis. sEVs: Small extracellular vesicles; AR: Allergic rhinitis; MSC: Mesenchymal stem cell; miRNA: MicroRNA.
THE IMMUNOMODULATORY MECHANISM OF EVS IN THE PATHOGENESIS OF AR
EVs derived from immune cells: Accomplices in exacerbating inflammation
Not all EVs play a protective or regulatory role. In the inflammatory microenvironment of AR, EVs released by activated immune cells can function as key inflammatory mediators, actively delivering pro-inflammatory molecules, amplifying and sustaining Th2 immune responses, and transforming from passive secretory products into “accomplices” that exacerbate disease progression. DCs, as central antigen-presenting cells bridging innate and adaptive immunity, pivotally drive AR pathogenesis[38]. During the initial sensitization phase, DCs capture and process allergens such as dust mites and pollen, presenting antigenic information to naive T cells and inducing their differentiation into Th2 or Th9 cells[39,40], thereby initiating the allergic response[2,8]. Follicular helper T (Tfh) cells, a specialized subset of CD4+ helper T cells, also contribute to AR pathogenesis via exosome-mediated signaling[41]. Tfh-derived EVs can induce DC maturation by modulating the miR-142-5p/cyclin-dependent kinase 5/signal transducer and activator of transcription 3 signaling pathway[42]. Specifically, the reduced expression of miR-142-5p in Tfh EVs relieves inhibition of the target gene cyclin-dependent kinase 5, activates the signal transducer and activator of transcription 3 pathway, promotes abnormal DC maturation, and enhances adaptive immune responses. Cao et al[43] using an AR mouse model, further demonstrated that Tfh-derived EVs regulate B cell function through the miR-149-5p/WNT3A/AXIN2 signaling axis, amplifying AR-related humoral immune responses. In this study, miR-149-5p expression was significantly downregulated in Tfh EVs from AR mice, and its expression was negatively correlated with B cell proliferation and maturation. Dual-luciferase reporter assays confirmed WNT3A as a direct target of miR-149-5p; low miR-149-5p levels relieved WNT3A inhibition, activated the WNT3A/AXIN2 pathway, and upregulated surface expression of CD69 and CD40 on B cells, thereby enhancing B cell proliferation and specific IgE secretion. The exosome inhibitor GW4869 effectively blocked these effects, confirming the mediating role of Tfh EVs. Furthermore, in vivo infusion of Tfh EVs from AR mice significantly exacerbated nasal mucosal inflammatory infiltration and B cell activation, highlighting their pathological role in amplifying humoral immune responses in AR. It is worth exploring further that these miRNAs, which play a key pathogenic role in AR, do not randomly enter EVs; their recruitment and enrichment in EVs may be precisely regulated. Existing research indicates that RNA-binding proteins and the specific RNA motifs they recognize play a central role in determining the sorting of RNA molecules to EVs[44]. For example, fragile X mental retardation protein has been shown to be involved in the selective sorting of miRNAs[45]. In the activated state of Tfh cells, specific intracellular RNA binding protein expression profiles or post-translational modifications may be altered. However, the specific RNA binding proteins and recognition sequences remain unclear. Elucidating this selective packaging mechanism is expected to reveal the specific programming of cell communication in disease states and provide potential molecular targets for future interventions in the generation or modification of pathogenic EV cargoes.
It is noteworthy that, in addition to EVs derived directly from local immune cells, circulating EVs, particularly plasma-derived EVs, serve as important mediators of intercellular communication throughout the body, involving immune cells, endothelial cells, platelets, and others. These EVs can provide a more systemic reflection of immune status and regulate immune responses in AR. At the level of innate immunity, upregulation of miR-150-5p in plasma EVs from AR patients promotes the differentiation of CD4+ T cells into Th2 subtypes, enhances activation of type 2 innate lymphoid cells (ILC2s), and facilitates secretion of inflammatory cytokines such as IL-4, IL-5, and IL-13, thereby exacerbating type 2 inflammation[46]. Furthermore, serum (plasma) EVs act as remote signaling messengers in immune regulation. For example, elevated levels of miR-146a-3p in serum EVs of AR patients can induce macrophage polarization toward the M2 phenotype by targeting the Vav guanine nucleotide exchange factor 3 gene and activating the phosphatidyl-inositol-3-kinase/protein kinase B/mammalian target of rapamycin pathway. This process contributes to immune tolerance imbalance and sustains the local inflammatory microenvironment[47]. Collectively, these studies demonstrate that immune cell-derived EVs are not passive byproducts but play a central pathogenic role in the initiation, maintenance, and exacerbation of AR through a complex network of miRNA-mediated signaling.
EVs derived from nasal mucosal barrier cells as biomarkers for disease diagnosis and prognosis
Given the central regulatory role of EVs in AR immune mechanisms, their expression profiles, along with those of their cargo molecules such as proteins and miRNAs, may reflect specific disease states. Consequently, isolating and analyzing EVs from patient body fluids promisingly help identify novel diagnostic and prognostic biomarkers for AR. Proteomics, a key tool in systems biology, enables comprehensive analysis of protein composition in complex biological systems and holds great potential for biomarker discovery, mechanistic studies, and drug target identification[48]. Data-independent acquisition combined with liquid chromatography-tandem mass spectrometry, offering high coverage and quantitative accuracy, provides a powerful method for in-depth profiling of disease-associated protein expression. In one AR study, researchers applied this technology to perform quantitative proteomic analysis of rat plasma EVs, identifying 92 differentially expressed proteins. Bioinformatics analysis revealed enrichment of these proteins in complement and coagulation cascade pathways, highlighting key proteins such as C4b, Clqa, Clqc, and Mbl1[49]. This study underscores the potential of exosome-carried proteins as novel biopsy markers or therapeutic targets. Similarly, investigations of EV-associated miRNAs have demonstrated their value in AR diagnosis. Analysis of nasal mucus from AR patients identified 21 significantly upregulated and 14 significantly downregulated miRNAs, which effectively distinguished patients from healthy controls. Independent validation confirmed six miRNAs, miR-30-5p, miR-199b-3p, miR-874, miR-28-3p, miR-203, and miR-875-5p, as potential diagnostic biomarkers[50]. These differentially expressed miRNAs are enriched in multiple immune and inflammatory pathways, including the B-cell receptor signaling pathway, T-cell receptor signaling pathway, natural killer cell-mediated cytotoxicity, RIG-I-like receptor signaling pathway, Wnt signaling pathway, and salivary secretion pathway. Dysregulation of these pathways is closely associated with AR pathogenesis, suggesting that EV-derived miRNAs not only serve as diagnostic biomarkers but may also participate in disease progression and immune regulation, thus possessing prognostic value. Importantly, miRNAs are highly stable within EVs and resistant to degradation, making them particularly suitable for non-invasive liquid biopsies[51,52]. Nasal mucus, being easily accessible, offers a practical medium for early diagnosis, disease subtyping, and monitoring of treatment response through analysis of EV miRNA profiles.
In addition to carrying host-derived molecular information, EVs can reflect the composition of the local microbiome, serving as a novel “ecological marker”[53,54]. Chiang et al[55] were the first to systematically characterize the microbiome of nasal EVs in AR patients. Relative to healthy controls, AR patients exhibited considerably reduced microbial diversity in nasal EVs and marked remodeling of the microbial community: The abundance of potentially beneficial genera, such as Zoogloea and Streptococcus, decreased, whereas genera such as Acetobacter and Mycoplasma were relatively enriched. These microbial signatures not only provide novel evidence for distinguishing AR disease states but also suggest that microbiome-mediated changes in metabolic pathways may influence the host immune microenvironment via EVs. Thus, profiling microbiome-encapsulated EVs opens new avenues for AR diagnosis and subtype classification from a microecological perspective[55]. Regarding treatment efficacy, serum-derived exosomal miRNAs, particularly hsa-miR-4669, have emerged as promising biomarkers for predicting responses to subcutaneous immunotherapy (SCIT). In pediatric AR patients, significant differences in serum exosomal miRNA profiles were detected between responders and non-responders following SCIT[37]. Validation across two independent cohorts confirmed that the expression levels of miRNAs, including hsa-miR-4669, correlated closely with clinical improvement indicators. According to bioinformatics analysis, the target genes of these miRNAs are enriched in pathways related to immune cell migration and immune regulation, such as cell adhesion molecules and the Wnt signaling pathway. These findings suggest that EVs may contribute functionally to SCIT-induced immune tolerance by transporting miRNAs like hsa-miR-4669, and that baseline levels of these miRNAs could serve as predictive biomarkers for personalized efficacy assessment in AR.
In the diagnosis and prognostic prediction of AR, the proteins, miRNAs, and microbiome information carried by EVs form a multi-dimensional and complementary biomarker system, offering a novel perspective for understanding the disease across different levels, including immune status, microecology, and therapeutic response[56,57]. Compared to traditional clinical biomarkers, EV-derived biomarkers exhibit unique added value. Traditional biomarkers primarily reflect the end effects of immune activation or inflammation levels, while EVs and their cargo molecules can more directly reflect the active communication activities and functional states of specific cell sources in disease states. However, translating these findings into reliable clinical tools faces substantial challenges. First, technical standardization remains a major bottleneck. Methods for EV isolation, purification, characterization, and downstream molecular analysis lack unified gold standards, resulting in poor comparability and reproducibility across studies. Second, most existing investigations are cross-sectional or involve limited sample sizes, and the reported biomarkers generally lack validation in large, multi-center, prospective cohorts. Their diagnostic specificity, such as distinguishing AR from other rhinitis subtypes or allergic diseases, as well as sensitivity and predictive robustness, require further confirmation. Moreover, the translation from biomarker discovery to the development of clinical diagnostic assays involves additional hurdles, including analytical performance validation, cost considerations, and regulatory approval. Therefore, while EVs and their molecular cargo from body fluids hold significant promise, their clinical application ultimately depends on achieving major advances in methodological standardization, large-scale clinical validation, and the clarification of translational pathways.
Potential of MSC-EVs in the treatment of rhinitis
Beyond their immunoregulatory functions, MSC-EVs can directly repair the damaged nasal epithelial barrier in allergic reactions, thereby addressing the allergy process at its source. Nasal epithelial barrier dysfunction critically dominates the development and progression of AR. According to recent studies, MSC-EVs can be internalized by nasal epithelial cells (NEpCs), delivering their cargo, particularly miR-143, to inhibit glycogen synthase kinase-3β activity. This inhibition restores the expression of the tight junction protein claudin-1, stabilizes the cytoskeleton, enhances cell migration, and ultimately reconstructs functional integrity of the nasal mucosal barrier[58]. This mechanism extends previous research, which largely focused on immune cells, by highlighting the direct tissue-repairing role of MSC-EVs in AR.
The immunomodulatory effects of MSCs are well established in immune-related diseases, including allergies[59-61]. Recent studies indicate that MSC-derived small EVs (sEVs) are key mediators of these therapeutic effects[62,63]. As natural carriers of bioactive molecules, MSC-EVs are enriched with proteins, lipids, mRNAs, long noncoding RNAs, and miRNAs[64,65], enabling direct regulation of varying immune cells, including T cells, B cells, and natural killer cells, and underpinning their broad immunomodulatory potential[66-68]. Regarding DC regulation, MSC-EVs exert effects opposite to those of immune cell-derived EVs. First, MSC-EVs modulate DC differentiation and function in an IL-10-dependent manner, suppressing Th2/Th9 responses and facilitating the expansion of Tregs. Second, MSC-EVs can mediate anti-inflammatory effects through the DC-ILC2 axis. Studies have shown that mature DCs treated with MSC-EVs significantly inhibit ILC2 activation, reduce IL-13 secretion, and downregulate the expression of the key transcription factor GATA binding protein 3. This effect relies on the prostaglandin E2 (PGE2) signaling pathway: DCs treated with MSC-EVs exhibit high expression of PGE2 synthase, promoting PGE2 synthesis and secretion, which in turn suppresses ILC2 function via the PGE2-EP2/EP4 receptor axis. Inhibition of PGE2 synthase with MF63 or blockade of EP2/EP4 receptors reverses this effect, indicating that MSC-EVs can enhance PGE2 production by modulating DC lipid metabolism, thereby suppressing ILC2-mediated type 2 inflammation[69] (Figure 2).
Figure 2 The role of extracellular vesicles in allergic rhinitis.
Extracellular vesicles (EVs) play a crucial role in allergic rhinitis (AR): The microRNAs (miRNAs) they carry (such as miR-30-5p, miR-199b-3p, miR-874, miR-149-5p, and miR-150-5p) promote Th2/type 2 innate lymphoid cell immune responses and B cell activation by regulating pathways such as Wnt/AXIN2, signal transducer and activator of transcription 3, and phosphatidyl-inositol-3-kinase/protein kinase B/mammalian target of rapamycin. Simultaneously, the microbiome in nasal EVs of AR patients is imbalanced, characterized by reduced Zoogloea and Streptococcus and enriched Acetobacter and Mycoplasma, reflecting local microecological dysbiosis. These characteristics collectively form the basis for EVs as multidimensional biomarkers of AR. EVs: Extracellular vesicles; miRNA: MicroRNA.
Notably, optimizing the stem cell culture microenvironment can significantly enhance the immunoregulatory effects of MSC-EVs on DCs. Wu et al[70] demonstrated that long-term hypoxia pretreatment of human MSCs at 1% O2 specifically upregulated vascular endothelial growth factor (VEGF) expression. VEGF-enriched hypoxia pretreatment of human MSCs more effectively inhibited the expression of DC maturation markers, including HLA-DR, CD80, CD40, and CD83. VEGF was identified as a key mediator of this inhibitory effect, capable of inducing immune tolerance during the initial stage of antigen presentation, suggesting a novel therapeutic target for AR[70]. These findings not only confirm the central role of MSC-EVs in DC regulation but also highlight the potential of optimizing culture conditions to enhance the immunomodulatory functions of EVs.
TREATMENT STRATEGIES BASED ON MSCS-EVS
MSC-EVs intervention for immune regulation
MSC-EVs have emerged as a promising alternative in regenerative medicine and disease therapy relying on their high biocompatibility, low immunogenicity, and ease of storage and transport[69,71]. However, their clinical efficacy is strongly influenced by administration strategies[72]. To evaluate the therapeutic value of MSC-EVs in AR, Yang et al[73] conducted a systematic study using human adipose-derived MSC-EVs (hADSC-EVs) in an ovalbumin (OVA)-induced mouse AR model. Tail vein injection of hADSC-EVs significantly alleviated nasal symptoms, reduced eosinophil infiltration and goblet cell proliferation in nasal tissue, and suppressed serum levels of OVA-specific IgE, IL-4, and interferon (IFN)-γ. Mechanistic analysis revealed that hADSC-EVs exert anti-inflammatory and immunomodulatory effects through regulating the Th1/Th2 balance in splenic lymphocytes, shifting the immune response toward a Th1-dominant state. To investigate the impact of administration routes, the researchers compared tail vein injection, intranasal administration, and a combination of both[74]. All three strategies alleviated AR symptoms, but intranasal and systemic administration appeared to act through distinct mechanisms, producing complementary immunomodulatory effects. Clinically, intranasal delivery may be preferable for mild AR due to ease of administration and minimal local side effects, whereas combined administration may offer synergistic, multi-target benefits in severe cases. To address the challenge of rapid systemic clearance, researchers have developed locally applied hydrogels as sustained-release delivery platforms. Zhao et al[75] engineered a temperature-sensitive chitosan hydrogel to encapsulate bone marrow MSC-EVs (Figure 3). This hydrogel remains liquid at room temperature for easy injection and gels in situ at nasal temperature, enabling sustained EV release. In animal models, bone marrow MSC-EVs delivered via this hydrogel effectively regulated Th1/Th2 balance and alleviated AR symptoms. Subsequently, the team developed an MXene-modified GelMA hydrogel to enhance EV stability and functionality[76]. MXene adsorption enables sustained EV release while the composite hydrogel provides antioxidant and antibacterial properties. In AR mouse models, intranasal implantation of this hydrogel significantly reduced behavioral symptoms, tissue inflammation, and oxidative stress, while modulating immune balance by decreasing OVA-specific IgE and IL-4 and increasing IL-10 and IFN-γ expression. Additionally, another study created a PLGA-based sustained-release system (PLGA-Exos) by encapsulating MSC-EVs in polylactic-co-glycolic acid nanoparticles[77]. This system prolonged EV retention in the nasal cavity and remodeled immune imbalance in AR mice by upregulating Th1 cytokines (IFN-γ, IL-2) and Tregs, while inhibiting Th2/Th17 responses and inflammatory cell infiltration. Collectively, these studies highlight promising material strategies for local MSC-EV delivery that integrate sustained release with immunomodulatory and therapeutic functions.
Figure 3 A thermosensitive injectable chitosan-based hydrogel used in allergic rhinitis.
A thermosensitive injectable chitosan-based hydrogel was intranasally delivered into allergic rhinitis mice models and proved to be effective in alleviating allergic rhinitis symptoms, reducing submucosal eosinophils infiltration and reversing Th1/Th2 imbalance in allergic rhinitis pathogenesis. Citation: Zhao C, Wei X, Kong W, Zhao Y, Yang J, Cheng J, Wang Z. Mesenchymal stem cell derived extracellular vesicles loaded thermosensitive chitosan-based hydrogel alleviates allergic rhinitis in mouse model. Mater Des 2023; 233: 112271. Copyright© The Authors 2023. Published by Elsevier Ltd. The authors have obtained the permission for figure using from the Elsevier Ltd (Supplementary material). BMSC: Bone marrow derived-mesenchymal stem cell; EVs: Extracellular vesicles; OVA: Ovalbumin.
NEpCs constitute the first line of defense against pathogen invasion in the respiratory tract, serving as both a physical and immune barrier and critically driving the development of nasal mucosal inflammation[78]. Recent research has highlighted the importance of repairing the damaged nasal epithelial barrier as a key step in AR pathogenesis, proposing a novel therapeutic approach using exosome-like nanovesicles (MSC-NVs)[79]. MSC-NVs have been shown to restore the epithelial barrier by upregulating tight junction proteins such as zonula occludens-1 and occludin, while modulating epithelial-derived cytokines, including thymic stromal lymphopoietin and IL-25/IL-33, thereby inhibiting the initiation and progression of Th2-type inflammation at its source. Compared to natural EVs, MSC-NVs offer advantages such as ease of preparation, high yield, and good homogeneity, enhancing their translational potential for clinical applications. This approach underscores a “barrier repair” perspective in AR treatment and confirms the crucial role of signaling pathways such as epidermal growth factor receptor/Wnt. The therapeutic efficacy of MSC-EVs is influenced by both administration route and delivery strategy. Hydrogel-based local sustained-release systems have emerged as a promising method to improve EV bioavailability and prolong their effects. Although such strategies have demonstrated encouraging outcomes in animal models, several challenges remain for clinical translation. Most preclinical studies have used acute or short-term AR models, whereas human AR is a chronic and relapsing disease; it remains unclear whether sustained-release systems can maintain stable barrier repair and immune regulation over long periods. Additionally, while the biocompatibility of hydrogel materials and their degradation products appears generally acceptable in nasal tissues, potential local immune responses or long-term effects in the complex human physiological environment require careful evaluation. These material-based strategies, while enhancing efficacy, may also introduce new complexities, necessitating careful assessment of feasibility and risk-benefit ratios for clinical application. Mechanistically, current research has primarily focused on classic immune axes, such as Th1/Th2 balance and Treg modulation. Nevertheless, corresponding molecular mechanisms by which specific active components within MSC-EVs regulate signaling pathways such as epidermal growth factor receptor/Wnt in epithelial cells to synergistically achieve barrier repair and immune homeostasis remain unclear. The lack of precise localization and characterization of core effector molecules hinders standardization and controllability, representing a major obstacle to translating these strategies into clinically viable therapeutics. Therefore, future research must simultaneously deepen mechanistic understanding and advance preclinical safety assessments to realize the full potential of MSC-EV-based therapies for AR.
EVs as antigen or drug carriers
In addition to their direct immunomodulatory effects, EVs serve as natural carriers of bioactive molecules, adding another important dimension to their therapeutic potential through their ability to transport and deliver these cargoes. AIT has been established as an effective treatment for AR[80,81]. Combining AIT with adjuvants can enhance therapeutic efficacy by increasing allergen immunogenicity and promoting targeted immune activation[82]. CpG DNA, a Toll-like receptor ligand, is a commonly used adjuvant; however, co-delivering CpG DNA with allergens presents a significant challenge[83]. EVs, as natural endogenous carriers, offer unique advantages for synergistic delivery[84]. For example, a composite system (CpG-OVA-sEVs) has been developed by simultaneously loading the model allergen OVA and the Th1-type adjuvant CpG DNA onto small sEVs. Following intranasal administration, these sEVs are efficiently targeted (NALT) and taken up by CD11c+ antigen-presenting cells. In a mouse AR model, this strategy synergistically modulates the Th1/Th2 balance, increases OVA-specific IgG antibody levels, suppresses IgE production, and alleviates allergic symptoms, thereby demonstrating a promising therapeutic effect.
Leveraging their unique roles in intercellular communication and immune regulation, EVs can function not only as natural nanoscale therapeutic carriers but also as platforms for enhanced functionality and precise delivery through advanced materials science approaches. Regarding their in vivo pharmacokinetics, particularly hydrogel and PLGA encapsulation systems for nasal delivery, existing research mainly focuses on local delivery and sustained-release effects. Photocurable hydrogels gel in the nasal cavity, extending the release time of MSC-EVs to several days, thereby maintaining effective drug concentrations locally and promoting sustained tissue repair[85]. PLGA encapsulation systems, through their slow degradation, also achieve sustained release of EVs, prolonging their retention time in the nasal mucosa and reducing rapid loss due to nasal cilia clearance[77]. However, detailed pharmacokinetic parameters of systemic absorption of these delivery systems in AR models are insufficient, limiting the assessment of their potential systemic effects or side effects. Whether nasal delivery can achieve brain targeting via the olfactory bulb generally depends on the size, surface properties, and nature of the molecules carried by the delivery carrier. Current research on AR therapy, whether using naked EVs or hydrogel/PLGA-encapsulated EVs, primarily aims, based on both design goals and experimental evidence, to limit their effects strictly to the nasal cavity and related nasal-associated lymphoid tissue, maximizing local immunomodulation and minimizing systemic exposure. While theoretically a very small fraction of nanoparticles could translocate into the brain via the olfactory nerve pathway, this is not the expected primary pathway in the current context of AR therapy research, and related evidence is extremely limited. Future research aiming to develop brain-targeted delivery will require specially designed carriers capable of specifically utilizing this pathway. Surface engineering strategies show great potential to further enhance the targeting and uptake efficiency of EVs or drug delivery systems on NEpCs. Ligand modification of EVs or nanocarriers can actively target specific receptors highly expressed on the surface of NEpCs. Modifying RVG peptides may target neurons, but a more relevant strategy might be modifying ligands, antibodies, or aptamers that recognize abundant receptors on NEpCs[86]. This active targeting strategy holds promise for overcoming the nasal mucosal barrier, significantly increasing the accumulation of therapeutic carriers at the lesion site, thereby achieving better efficacy with lower doses and potentially reducing off-target effects. Combining surface engineering with the aforementioned hydrogel or PLGA sustained-release systems is an important direction for developing efficient and precise AR therapies in the future. However, the clinical translation of these innovative strategies still faces key regulatory and manufacturing bottlenecks. In terms of manufacturing, large-scale, standardized, and GMP-compliant production of EVs and their composite systems is required, ensuring batch-to-batch consistency in activity, purity, and safety. In terms of regulation, EV-based products are generally classified as advanced therapies, and their review pathways are still immature, requiring comprehensive non-clinical safety and efficacy data, with even stricter requirements for engineered systems. To promote their clinical application, it is urgent to establish end-to-end standards from production to quality control and validate them through rigorous clinical trials. While actively envisioning MSC-EV-based treatment strategies, it is essential to be clearly aware of and deeply discuss their fundamental limitations and controversies, which are the core factors affecting the feasibility of their clinical translation. First, the preparation of MSC-EVs exhibits significant heterogeneity. Vesicle populations obtained using different isolation methods may differ in size, cargo composition, and biological function. However, current studies often discuss “MSC-EVs” as a relatively homogeneous entity, which may reduce the comparability and reproducibility of research results. Second, the long-term safety of MSC-EVs still requires rigorous evaluation. Although they have shown good tolerability in models such as AR, their potential pro-fibrotic effects, procoagulant activity, and the risk of transferring donor-derived genetic material to host cells have not been fully investigated, especially in the context of long-term, repeated administration. Finally, the differentiation of vesicle subsets is often ambiguous in existing studies. Many studies have failed to strictly distinguish vesicles from different biological pathways, which may carry different molecular cargoes and perform differentiated functions. In the pathological context of AR, vesicle subsets from different immune cell sources may play diametrically opposed roles. Therefore, future research urgently needs to standardize methodologies, conduct more systematic and comprehensive safety assessments, and perform more precise identification and functional analysis of vesicle subsets to solidify the scientific foundation for the clinical translation of MSC-EVs.
Researchers have successfully achieved sustained release and prolonged local retention of EVs using a PLGA-encapsulated exosome system prepared via a dual-emulsion method, demonstrating good biocompatibility and targeted delivery potential. This approach protects EVs from nasal mucosal ciliary clearance and enhances their uptake by NEpCs and immune cells, effectively regulating the Th1/Th2 balance, promoting Treg differentiation, and inhibiting inflammatory cell infiltration in AR models. Further refinement of this strategy could enable the construction of composite nanosystems, such as “PLGA-exo-PIO”, which co-load EVs with small-molecule drugs like peroxisome proliferator-activated receptor-gamma agonists to achieve synergistic delivery and multi-mechanism regulation. Such systems are applicable not only to upper respiratory tract diseases, including AR, but also hold promise for the combined treatment of lower respiratory tract conditions, such as asthma, via inhalation or systemic administration[87].
Treatment with EVs from other cell sources
In AR treatment strategies, in addition to MSC-derived EVs, EVs originating from local disease tissues also exhibit unique immunomodulatory potential[88]. For example, ephedra polysaccharide ESP-B4 is found to stimulate NEpCs to release EVs enriched with miR-146a-5p. These EVs are taken up by CD4+ T cells, where miR-146a-5p targets and suppresses Smad3 expression, thereby disrupting the formation of the Smad3/GATA binding protein 3 transcriptional complex. This mechanism promotes Th1 differentiation, inhibits Th2 responses, and restores immune homeostasis. This example highlights the directness and specificity of tissue-derived EVs in modulating the local immune microenvironment and introduces a novel paradigm for using natural bioactive compounds to regulate endogenous EV function, achieving “cell-vesicle-immune cell” cascade regulation. Looking forward, further exploration of the targeting, drug delivery potential, and integration of tissue-derived EVs with synthetic delivery systems could expand their therapeutic applications in respiratory immune diseases.
CONCLUSION
EVs play dual roles in AR as both participants in disease progression and as potential therapeutic agents. Immune cell-derived vesicles can transmit inflammatory signals, exacerbating disease, whereas vesicles from MSCs and other sources can exert therapeutic effects by modulating immune cell networks, correcting Th1/Th2 imbalances, and repairing the nasal epithelial barrier. In parallel, EVs and their cargo molecules have emerged as valuable biomarkers for disease diagnosis and treatment response prediction. Nevertheless, several challenges remain. First, standardized protocols for EV isolation, characterization, and quantification are lacking. Second, most mechanistic studies rely on animal models; the precise pathways in humans remain unclear, and differences in content and function among vesicles from various sources require detailed investigation. From a clinical translation perspective, issues such as large-scale production, quality control, stability, targeted delivery, and long-term safety continue to be major bottlenecks. Future research should focus on engineering vesicles to enhance efficacy, exploring their potential as drug delivery platforms in combination with existing therapies, and validating these approaches through rigorous clinical trials to facilitate their translation into clinical practice.
While acknowledging the potential of EVs, we must acknowledge several key limitations of current research and this review. Although many studies have characterized vesicles using standard methods, some cited works have not reported detailed characterization information or provided sufficient validation. This difference in characterization depth affects the comparability of different research results and the rigor of conclusions. Secondly, vesicle heterogeneity, challenges in large-scale production, targeted delivery in vivo and long-term safety, and the translational gap from animal models to complex human pathologies are all major bottlenecks hindering their clinical application. Furthermore, the precise mechanisms by which specific effector molecules in MSC-EVs synergistically repair the epithelial barrier and regulate immunity remain to be elucidated. Future development in this field depends on promoting the engineering of vesicles, in-depth multi-omics mechanism research, and rigorous clinical translation validation, all while strictly adhering to international guidelines.
