Abstract
Chronic wounds, including diabetic foot ulcers, are notoriously difficult to heal due to their complex pathological microenvironments, which are marked by persistent inflammation and impaired angiogenesis. These challenges place a significant burden on both patients and healthcare systems. Extracellular vesicles (EVs), as natural carriers, can modulate the wound microenvironment and facilitate tissue regeneration by delivering bioactive molecules such as proteins and nucleic acids. EVs offer advantages including low immunogenicity and excellent biocompatibility. However, unmodified EVs face limitations such as low therapeutic payloads, limited targeting capability, and vulnerability to degradation. Building on this foundation, this review provides a detailed overview of the specific roles and mechanisms of EVs in wound healing. It focuses particularly on condition-specific strategies, engineering approaches, and the use of biomaterials to further enhance the therapeutic efficacy of EVs in treating chronic wounds. Finally, we highlight the current challenges faced in the clinical translation of EV-based therapies and propose emerging strategies to address these obstacles, offering new directions for chronic wound management.
Keywords: Extracellular vesicles, Chronic wound healing, Advanced delivery systems, Engineered EVs, Biomaterials
Graphical abstract
1. Introduction
The skin, as the largest organ of the human body, performs essential physiological functions such as protection against external insults, regulation of body temperature, and sensory perception, while serving as the primary physical barrier for maintaining internal homeostasis [1]. When this barrier is compromised, the body activates intricate repair mechanisms to restore tissue integrity [2]. However, adverse conditions like hyperglycemia, infection, and ischemia disrupt the healing process [3]. Persistent inflammation and impaired angiogenesis further impede tissue repair, ultimately leading to chronic wounds [4,5]. Common chronic wound types include diabetic foot ulcers, venous ulcers, arterial ulcers, pressure ulcers, and non-healing postoperative wounds, with diabetic foot ulcers being the leading cause of non-traumatic limb amputations [6]. Recent data indicate that over 828 million individuals worldwide were affected by diabetes in 2022 [7]. Among diabetic patients, 19 %–34 % develop diabetic foot ulcers (DFUs), with recurrence rates ranging from 7.7 % to 44 %.Notably, 80 % of patients undergoing amputation have a documented history of DFUs [8,9]. Chronic wounds impose prolonged treatment demands and substantial economic burdens, underscoring their status as a critical public health challenge [10]. These findings emphasize the urgent need for innovative therapeutic strategies specifically designed for chronic wound management.
Extracellular vesicles (EVs) are naturally occurring, spherical structures with diameters spanning 50 nm to 5 μm [11]. Secreted by nearly all cell types, they have been isolated from diverse biological sources, including breast milk, urine, dissociated tissues, and cell culture supernatants [[12], [13], [14]]. Based on biogenesis and physical characteristics, EVs are categorized into three primary subtypes: apoptotic bodies (ABs) derived from programmed cell death; exosomes originating from multivesicular bodies; and microvesicles formed through outward budding of the plasma membrane—all recognized as endogenous EV forms (Fig. 1) [15]. Their ability to traverse complex physiological barriers via receptor-mediated endocytosis, macropinocytosis, or phagocytosis enables targeted delivery of therapeutic cargos to recipient cells [16]. A As natural drug delivery systems, EVs overcome the immunogenicity limitations of synthetic carriers and demonstrate enhanced tissue penetration and pathological site targeting due to their intrinsic homing capabilities [17,18]. Compared with conventional strategies, EVs provide multi-phase and synergistic support for wound healing, encompassing hemostasis, inflammation resolution, tissue regeneration, and scar remodeling. Surface modification further enables targeted delivery and precise regulation of key signaling pathways while minimizing systemic side effects. As cell-free carriers, EVs possess intrinsic immunomodulatory activity, which reduces excessive inflammation and avoids immune rejection, resulting in a favorable safety profile relative to cell-based transplantation. Notably, ExoFlo, the most clinically advanced EV-based product candidate, has entered phase III clinical trials and demonstrated significant potential for DFU treatment [19]. This progress highlights the translational promise of EV-based therapies in chronic wound management.
Fig. 1.
Classification of EVs and composition of exosomes. The left panel depicts the three primary types of EVs secreted by cells: exosomes (30–150 nm), derived from the endosomal pathway and released via exocytosis; microvesicles (100–1000 nm), produced by plasma membrane budding; and apoptotic bodies (50–5000 nm), released during programmed cell death. The right panel shows the molecular composition of exosomes, containing lipids, proteins, enzymes, and nucleic acids. Their membranes contain tetraspanins, transporters, transferrin receptors, MHC-I/II molecules, lipid rafts, and adhesion molecules.
Despite their excellent biocompatibility, intrinsic targeting capabilities, and demonstrated therapeutic potential in tissue repair, the clinical application of EVs in wound healing is impeded by several critical limitations. Native EVs carry limited quantities of therapeutic microRNAs and regenerative factors, often insufficient to address the complex pathological microenvironment [20,21]. The absence of tissue-specific targeting leads to suboptimal accumulation at wound sites [22]. Additionally, elevated levels of matrix metalloproteinases (MMPs) and reactive oxygen species (ROS) in lesion areas accelerate EV degradation, while rapid clearance by the reticuloendothelial system further compromises their local stability and retention [23,24]. The inherently low production yields of natural EVs also present a substantial barrier to large-scale manufacturing and clinical translation [25]. To overcome these challenges, condition-specific and engineered EVs have been developed to enhance therapeutic efficacy. Parental cells dynamically respond to biochemical and biophysical cues in their microenvironment through adaptive regulatory mechanisms. Condition-specific EVs secreted under these tailored conditions inherit the functional profiles of their source cells, thereby exhibiting amplified biological activities such as enhanced proangiogenic or anti-inflammatory effects [26]. Engineered EVs not only preserve the inherent advantages of native vesicles but also confer improved functionalities. Through therapeutic cargo loading, they enable multi-targeted combinatorial therapies [27,28]. Moreover, surface modification with targeting ligands and integration with biomaterials enhance EV stability, bioavailability, and controlled release, facilitating the development of next-generation wound dressings [29,30]. Notably, artificial cell-derived vesicles (ACDVs) emerge as a promising solution to the technical bottleneck of large-scale EV production, offering scalable alternatives that retain biological activity while addressing manufacturing constraints [31]. Collectively, these strategies significantly broaden the therapeutic potential of EVs in chronic wound healing.
Chronic wounds remain a significant clinical challenge due to their prolonged and dysregulated healing process, characterized by persistent inflammation, impaired angiogenesis, and deficient tissue remodeling. Conventional therapies often fail to deliver satisfactory outcomes. In this context, EVs have emerged as a promising cell-free therapeutic modality. This review discusses a range of advanced strategies for EV-based therapy, including the generation of functionally enhanced EVs under specific conditions, engineering modifications to improve tissue targeting and drug loading efficiency, and the development of intelligent delivery platforms by integrating EVs with hydrogels, microneedles, and other biomaterials. Additionally, ACDVs offer a scalable alternative that retains biological activity while addressing production limitations. Through a comprehensive analysis of the therapeutic mechanisms and engineering strategies of EVs, this review provides insights to accelerate their clinical translation and integration into chronic wound management.
2. Wound healing mechanisms and dysregulation in chronic wounds
2.1. General wound healing mechanisms
Comprehensive understanding of the molecular mechanisms underlying wound healing is critical to overcoming current therapeutic limitations. Wound healing is a tightly regulated biological process involving four overlapping phases: hemostasis, inflammation, granulation tissue formation, and tissue remodeling [32]. During the hemostasis phase, platelets adhere to exposed collagen via integrin αIIbβ3 receptors, forming thrombi and initiating the release of fibrinogen and coagulation factors to activate the coagulation cascade, thereby achieving rapid hemostasis [33]. The inflammatory phase overlaps with hemostasis. Release of damage- and pathogen-associated molecular patterns (DAMPs/PAMPs) from the injured tissue rapidly triggers neutrophil infiltration, which clears pathogens by releasing ROS and neutrophil extracellular traps (NETs) [34]. Neutrophils also secrete interleukin (IL)-8 to recruit monocytes and promote macrophage polarization [35]. M1-type macrophages release proinflammatory factors such as tumor necrosis factor-α (TNF-α) and IL-6 to combat infection early on [36], and subsequently transition to M2-type macrophages, secreting IL-10 and transforming growth factor-β (TGF-β) to inhibit inflammation while releasing pro-repair factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) to promote tissue regeneration [37]. Notably, sustained activation of the nuclear factor kappa-B (NF-κB) pathway disrupts the inflammation–repair balance, exacerbating the chronic wound microenvironment [38].
During the granulation phase, fibroblasts and endothelial cells are recruited to the injury site. The TGF-β/SMAD signaling pathway activates, mediating fibroblast-driven collagen synthesis [39]. VEGF promotes angiogenesis via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. Concurrently, stimulation by epidermal growth factor (EGF) and PDGF initiates a kinase cascade through the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway, promoting keratinocyte proliferation and directional migration to drive re-epithelialization [40,41]. In the remodeling phase, granulation tissue is gradually replaced by mature connective tissue. Keratinocytes continue to migrate and reconstruct the epidermal barrier [42]. MMPs participate in the degradation of the extracellular matrix (ECM), maintaining a balance between collagen synthesis and degradation. Type III collagen is progressively replaced by type I collagen, ultimately resulting in the formation of scar tissue with complete biomechanical properties [43].
2.2. Chronic wound healing mechanisms
Chronic wounds are characterized by a triad of persistent inflammation, impaired angiogenesis, and abnormal ECM remodeling [44]. Especially in DFUs, these mechanisms converge to impede the healing process, with the persistent inflammatory microenvironment representing a central obstacle in chronic wounds.In the diabetic state, macrophage polarization undergoes significant disruption, resulting in an M1/M2 phenotypic imbalance. M1 macrophages undergo abnormal activation via the TLR4/NF-κB signaling pathway, resulting in sustained production of pro-inflammatory mediators, including TNF-α, IL-1β, and IL-6, that perpetuate chronic inflammation [45,46]. Concurrently, impairment of the IL-4/signal transducer and activator of transcription (STAT) 6 pathway hinders the transition to M2 macrophages, suppressing the production of immunomodulatory cytokines such as IL-10 and TGF-β, and thereby impeding inflammation resolution [47]. Impaired angiogenesis arises from suppression of the PI3K/AKT signaling pathway. Under hyperglycemic conditions, VEGF expression undergoes downregulation, and ROS activate the phosphatase and tensin homolog (PTEN), which in turn inhibits the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway. This cascade compromises endothelial cell migration and tube formation [48]. Additionally, dysfunction of nitric oxide synthase (NOS) impairs vasodilation, further exacerbating local hypoxia and nutrient deficiency, thereby perpetuating a vicious cycle [49].
ECM homeostasis is disrupted by dysregulation of both fibroblasts and the protease system. In diabetic wounds, excessive activation of the TGF-β/Smad pathway in fibroblasts drives aberrant type I collagen deposition. Simultaneously, diminished MMP activity compromises collagen degradation, resulting in an imbalance between ECM synthesis and degradation [50]. This dysregulation not only hinders granulation tissue remodeling but also results in the formation of dense fibrotic scars. Inadequate ECM support further leads to recurrent ulceration of newly formed epithelium. Despite significant progress in elucidating the pathophysiological mechanisms underlying chronic wound pathophysiology, translating this knowledge into effective therapies remains challenging. Traditional interventions often target isolated aspects of wound repair and fall short of addressing the dynamic and multifaceted interactions within the wound microenvironment. Recent advances in regenerative medicine have shifted the focus toward holistic, microenvironment-modulating strategies. Among these, EVs have garnered increasing attention as next-generation therapeutic agents capable of orchestrating complex intercellular communication and restoring homeostatic balance across multiple wound healing pathways.
3. Mechanisms underlying EV-based therapeutic strategies in chronic wound healing
Although traditional cell-based therapies have made advancements in tissue repair, their clinical application is limited by low cell survival rates, the risk of immune rejection, and potential tumorigenicity [20,51]. In contrast, cell-free therapies based on EVs have emerged as a promising alternative, demonstrating notable benefits in accelerating wound repair. Owing to their nanoscale dimensions and lipid bilayer architecture, EVs can be efficiently internalized by recipient cells, enabling targeted modulation of cellular processes with a precision and versatility beyond that of conventional cell-based therapies. EVs carry and safeguard diverse bioactive molecules, including proteins, mRNA, and miRNAs [11,41,52]. These molecules can be delivered directly to the wound microenvironment with low immunogenicity. As research into the composition and function of EVs advances, their biomedical applications have garnered increasing attention. Several EV-specific databases, such as Vesiclepedia (https://www.microvesicles.org/) and ExoCarta (https://www.exocarta.org/), have been established based on accumulated data. These databases provide comprehensive information about the proteins, nucleic acids, and lipids in EVs, offering crucial support for EV analysis and the design of engineered therapeutic vectors. Beyond delivering bioactive molecules, EVs influence chronic wound healing by transferring their cargo to recipient cells, thereby modulating gene expression, protein interaction networks, and cellular metabolism.
3.1. Regulation of gene expression
The persistent inflammatory state of chronic wounds is strongly linked to the dysregulated expression of key genes. EVs regulate gene transcription by delivering functional miRNAs that target inflammation-related pathways. For instance, exosomes derived from stem cells modulate macrophage polarization and inflammatory homeostasis via miR-20a-5p-mediated regulation of the JAK/STAT pathway (Fig. 2) [53]. Additionally, EVs carrying anti-inflammatory miRNAs, such as miR-146a, suppress the NF-κB signaling pathway, thereby alleviating chronic inflammation and promoting the transition of the wound microenvironment from a pro-inflammatory state to the tissue remodeling phase [54]. EVs can also promote angiogenesis. Mesenchymal stem cell (MSC)-secreted exosomes carry a characteristic miRNA cargo, such as miR-21-5p [55] and miR-145-5p [56], which stimulate angiogenesis by modulating the PI3K/AKT signaling pathway. Meanwhile, angiogenesis-associated growth factors enriched in EVs, such as VEGF and EGF, can trigger the PI3K/AKT cascade and the MAPK signaling axis, which in turn promotes endothelial cell proliferation and neovascularization, ultimately enhancing oxygen supply within the wound microenvironment [57]. These changes contribute to increased fibroblast activity and upregulated collagen deposition, facilitating more efficient wound tissue regeneration. Beyond modulating inflammation and stimulating angiogenesis, EVs play vital roles in regulating cell proliferation and migration. Exosomes derived from bone marrow mesenchymal stem cells (BMSCs) are rich in miR-136-5p, which facilitates osteoblast proliferation via the Wnt/β-catenin signaling pathway, thereby expediting tissue repair [58]. In the later stages of wound healing, EVs effectively suppress excessive fibroblast activation and reduce scar formation. For example, miR-125b and miR-145 target the TGF-β/Smad signaling pathway to inhibit fibroblast activation, downregulate the expression of α-smooth muscle actin (α-SMA), and reduce abnormal collagen deposition, thereby modulating scar formation [59].
Fig. 2.
Therapeutic mechanisms of EVs in chronic wound healing. Schematic illustration of key signaling pathways involved in EV-mediated skin repair. EVs suppress inflammation via miR-20a-mediated inhibition of the JAK/STAT pathway and miR-146a-induced suppression of the NF-κB pathway. VEGF and EGF carried by EVs activate the PI3K/AKT/mTOR and Ras/MEK/ERK pathways, respectively. miR-21 and miR-145 further enhance angiogenesis by targeting both pathways. Wnt proteins in EVs activate the β-catenin pathway to promote cell proliferation. Anti-fibrotic effects are achieved through 14-3-3ζ, which binds to phosphorylated LATS and subsequently to phosphorylated YAP, resulting in cytoplasmic retention of YAP and preventing its translocation into the nucleus, thereby reducing abnormal cell proliferation and scar formation. miR-145 and miR-125b suppress the TGF-β/Smad2/3 pathway, reducing fibrosis.
3.2. Protein regulatory networks
EVs regulate the dynamic balance of cellular signaling pathways by delivering key proteins. For instance, fibroblast-derived EVs deliver heat shock protein 90 alpha (HSP90α), leading to the phosphorylation of STAT3 and inducing macrophage polarization toward the M2 phenotype, thereby modulating the inflammatory microenvironment [41]. In addition, EVs contribute to angiogenesis by delivering bioactive proteins [60]. EVs derived from adipose-derived mesenchymal stem cells (ADSCs) deliver Wnt4, which activates the Wnt/β-catenin signaling pathway. This activation leads to the proliferation of keratinocytes, endothelial cells, and fibroblasts, and accelerates re-epithelialization, angiogenesis, and tissue regeneration [61]. VEGF and EGF enriched in MSC-derived EVs (MSC-EVs) directly activate endothelial cell surface receptors, which activate the PI3K/AKT and MAPK signaling pathways through phosphorylation cascades, thereby enhancing endothelial cell migration and angiogenesis. Moreover, EVs derived from ADSCs enhance macrophage secretion of TGF-β1, which subsequently activates the TGF-β/SMAD3 signaling pathway, thereby enhancing fibroblast proliferation [62]. Conversely, EVs derived from umbilical cord mesenchymal stem cells deliver the regulatory protein 14-3-3ζ, which modulates YAP phosphorylation via p-LATS signaling. This leads to YAP cytoplasmic retention and degradation, thereby facilitating the inhibition of the Wnt/β-catenin pathway, suppressing fibroblast proliferation and collagen deposition, and ultimately reducing scar formation [63].
3.3. Metabolic reprogramming
Metabolic reprogramming serves as a core mechanism through which cells flexibly regulate glucose, lipid, and amino acid metabolism in response to microenvironmental changes [64]. With EVs functioning as natural carriers of metabolic products and regulatory molecules, they reshape recipient cell metabolism by delivering bioactive components such as metabolic enzymes, non-coding RNA, and signaling proteins [65]. Traditionally, endothelial cells have been considered highly glycolytic; however, recent studies demonstrate that fatty acid metabolism is also essential for nucleotide biosynthesis in these cells, and its inhibition significantly impairs endothelial cell proliferation [66]. These findings highlight the complex metabolic regulation underlying angiogenesis. It has been demonstrated that miR-122-5p enhances fatty acid utilization in endothelial cells and promotes vascular neogenesis by targeting AGPAT1, which leads to increased expression of CPT1A and CD36—two key regulators of fatty acid oxidation [67]. Other studies further reveal the diversity of metabolic regulation mediated by EVs. For instance, skeletal muscle-derived EVs deliver the glycolytic enzyme lactate dehydrogenase A (LDHA), inducing glycolysis in BMSCs and promoting osteogenesis [68]. Importantly, EV-mediated metabolic modulation exhibits bidirectional plasticity. A recent study has identified and characterized a glucose metabolism regulatory protein (GMRSP), encoded by the long non-coding RNA H19. The delivery of GMRSP via exosomes reduces the expression of pyruvate kinase M2 (PKM2), thereby attenuating glycolytic activity [69].
4. Condition-specific EVs
EVs are secreted by nearly all cell types and are found in various body fluids, where they mediate intercellular communication. However, conventional EV studies often treat EVs as homogeneous entities, thereby overlooking their dynamic nature and how their composition changes under different physiological or pathological conditions. This oversimplification limits their diagnostic and therapeutic potential, as EVs from the same cell type can exhibit distinct biological properties depending on their microenvironment [26]. The cargo composition of EVs varies in response to the state of their parental cells and reflects their adaptive responses to biochemical and biophysical cues within the microenvironment. By precisely modulating the cellular microenvironment or mimicking disease-relevant conditions, condition-specific EVs with tailored composition and functions can be generated (Fig. 3) [70]. This strategy not only enhances the therapeutic potential of EVs in chronic wound healing but also offers valuable insights into disease mechanisms and the development of targeted therapies.
Fig. 3.
Strategies for generating condition-specific EVs through microenvironmental and pathophysiological cues. Condition-specific EVs generated through modulation of the cellular microenvironment exhibit enhanced therapeutic efficacy in chronic wound healing. Physical stimuli, chemical inducers, and biological factors can condition cells to adaptively respond to biochemical and biophysical cues, resulting in the release of EVs with improved biological functions. In vivo, physiological conditions such as exercise, and pathological states such as aging and diabetes, also lead to the production of EVs with distinct functional properties.
4.1. Physical stimuli
Physical stimuli modulate the metabolic status and gene expression of parental cells by altering biophysical cues such as oxygen tension, mechanical forces, and culture dimensionality, which in turn modifies the composition and functionality of EVs. Common physical strategies to achieve this include hypoxic preconditioning, three-dimensional (3D) culture, mechanical stimulation, and light stimulation.
4.1.1. Hypoxic preconditioning
Hypoxic preconditioning represents an effective strategy to enhance the pro-angiogenic potential of EVs. For instance, culturing MSCs and human umbilical vein endothelial cells (HUVECs) under hypoxic conditions activates the hypoxia-inducible factor-1α (HIF-1α) signaling pathway, upregulates genes such as VEGF, and alters the protein and RNA cargo of EVs, thereby enhancing their functionality [71,72]. Furthermore, hypoxia has been shown to enrich EVs with specific miRNAs, including miR-486-5p [73], miR-4645-5p [74] and miR-17-5p [75]. EVs enriched in miR-486-5p, derived from hypoxia-treated urine-derived stem cells, specifically target and suppress SERPINE1, thereby activating the HIF-1α signaling pathway and promoting angiogenesis [73]. Similarly, EVs enriched in miR-4645-5p, generated under hypoxic conditions, suppress MAPK-activated protein kinase 2 (MK2) expression by binding to the 3’ untranslated region of its mRNA. This modulation affects the AKT-mTOR signaling pathway and induces autophagy in keratinocytes, ultimately facilitating diabetic wound healing [74]. In addition, EVs enriched in miR-17-5p, obtained from hypoxia-preconditioned cells, inhibit excessive formation of neutrophil extracellular traps (NETs) by targeting the TLR4/ROS/MAPK pathway, which contributes to improved diabetic wound healing [75]. EVs derived from stem cells of human exfoliated deciduous teeth, when subjected to hypoxic preconditioning, also exhibit outstanding potential in promoting osteogenesis [76]. Beyond oxygen tension, nitric oxide (NO) has also been implicated in regulating EV bioactivity. By upregulating VEGF and miR-126 expression within EVs, NO significantly enhances their angiogenic capacity [77].
Hypoxic preconditioning has emerged as a promising strategy for enhancing cell-derived products, owing to its simplicity, cost-effectiveness, scalability, and potential for widespread application in large-scale manufacturing. Despite these advantages, several challenges must be addressed before it can be advanced toward clinical application. A key consideration is the optimization of oxygen concentration during hypoxic activation, as different cell types exhibit distinct oxygen requirements based on their tissue-specific physiological niches [78]. Moreover, the optimal duration of hypoxic exposure remains unclear and requires further investigation to balance therapeutic efficacy with cellular safety.
4.1.2. Optimization via three-dimensional (3D) culture
Culture conditions significantly influence cellular behavior, which in turn affects the production and composition of EVs. For example, compared to two-dimensional (2D) culture, the rotary cell culture system (RCCS) has been shown to increase EV secretion from MSCs [79]. Studies demonstrate that cells cultured in three-dimensional (3D) environments not only produce a higher quantity of EVs but also exhibit improved therapeutic efficacy [80,81]. EVs derived from 3D cultures (3D-derived EVs) promote collagen deposition and are more effective in addressing challenges such as impaired angiogenesis, persistent inflammation, and oxidative stress in diabetic wounds [82,83]. Evidence suggests that MSC-derived 3D EVs are produced in significantly higher yields (approximately 6.7-fold) and exhibit superior pro-angiogenic and wound healing properties compared to 2D-derived EVs. Additionally, 3D-derived EVs demonstrate enhanced anti-inflammatory, anti-apoptotic, and anti-fibrotic capacities [84,85]. Three-dimensional culture maximizes EV secretion and enhances their associated bioactivity. Scaffold-based techniques, including hollow fiber bioreactors and microcarrier-based bioreactors, have been widely used to support 3D culture systems [86,87]. Beyond dimensionality, cell adhesion plays a crucial role in EV production. In 2D-cultured BMSCs, EV yield decreases with increasing cell density. In contrast, 3D spheroidal culture of BMSCs enhances EV secretion as the cells transition to a non-adherent, rounded morphology [88]. This suggests that non-adherent states may favor EV release. Therefore, selecting appropriate cell culture strategies is essential for optimizing EV production to facilitate wound healing. However, variations in 3D culture duration and reproducibility may affect the consistency of these differences and ultimately impact cell function. The formation of hypoxic gradients within 3D spheroids may result in batch-to-batch variability in EV functionality. This reflects the inherent heterogeneity of three-dimensional culture systems. Future studies should integrate single-cell secretomic analyses to delineate the functional contributions of distinct cellular subpopulations to EV production under 3D conditions. The development of intelligent bioreactor systems holds great promise for enabling dynamic microenvironmental regulation and maximizing the therapeutic potential of EVs.
4.1.3. Other physical stimuli
Cells can sense mechanical stimuli, including stretch, acoustic waves, and light irradiation, and transduce them into biochemical signals that initiate intracellular changes and ultimately modulate the properties and functions of EVs. For example, static mechanical stretching enhances fibroblast activity and significantly increases the secretion of EVs with improved abilities to promote cell proliferation, migration, and angiogenesis. Proteomic analyses of EVs from stretched cells have identified 12 upregulated and 12 downregulated candidate miRNAs, suggesting that mechanical cues may alter EV function through epigenetic modifications [89]. Moreover, mechanical stimulation has also been shown to enhance EV yield [79]. Physical stimuli-based interventions can synergistically enhance the therapeutic efficacy of EVs. Low-intensity pulsed ultrasound (LIPUS), a noninvasive mechanical stimulus, has demonstrated remarkable benefits in treating diabetic wounds. A recent study reported that LIPUS pretreatment enhances the ability of EVs to promote endothelial cell proliferation, migration, and tube formation. LIPUS also increases EV uptake by human umbilical vein endothelial cells (HUVECs) by nearly 11-fold [90]. In addition, low-level ultrasound stimulation has been shown to promote EV production [91]. Light-based stimulation can similarly modulate EV function. Blue light irradiation (455 nm) enhances the pro-angiogenic activity of mesenchymal stem cell-derived EVs (MSC-EVs) by upregulating miR-135b-5p and miR-499a-3p [92]. Likewise, low-level laser irradiation promotes EV secretion from human endothelial cells by activating the Wnt signaling pathway [93]. Mechanical stimulation, as a noninvasive and controllable physical strategy, offers advantages in biocompatibility and potential for synergistic regulation. However, excessive stimulation may induce cellular stress responses, including disruption of mitochondrial membrane potential, thereby affecting cellular homeostasis.
4.2. Chemical induction
Chemical induction strategies enhance the therapeutic potential of EVs by pharmacologically modulating cellular metabolism or signaling pathways. For example, although metformin is widely used to stimulate autophagy and restore mitochondrial function, its direct systemic delivery is limited by rapid clearance and low bioavailability. Engineering EVs to carry metformin thus presents a promising alternative [94]. In one study, ADSCs preconditioned with metformin produced mitochondria-rich EVs that ameliorate mitochondrial dysfunction by restoring membrane potential, increasing ATP production, and reducing ROS levels. These EVs also promote macrophage polarization toward the anti-inflammatory M2 phenotype, thereby accelerating skin wound healing [95]. Furthermore, mitochondria-enriched EVs have been shown to enhance the regenerative capacity of aged tissues by reprogramming cellular metabolism [94]. Regarding angiogenesis, condition-specific EVs exhibit synergistic effects. For instance, EVs derived from pioglitazone-preconditioned cells demonstrate superior pro-angiogenic capabilities compared to untreated EVs [96]. Similarly, EVs generated from MSCs pretreated with atorvastatin [97], melatonin [98], or quercetin [99] show enhanced wound-healing efficacy in diabetic wound models. Additionally, EVs from copper ion–activated macrophages stimulate vascularization both in vitro and in vivo [100]. Exosomes derived from hydrogen sulfide (H2S)-preconditioned M2 macrophages are highly enriched in moesin protein, which facilitates their uptake by MSCs and significantly enhances therapeutic outcomes [101]. Fibroblasts treated with chitosan oligosaccharides (COS) produce exosomes enriched in transcription factor AP-2 gamma (TFAP2C), effectively promoting peripheral nerve axon regeneration [102]. These findings indicate that preconditioning cells with specific biological or chemical cues allows precise modulation of EV composition and function, thereby improving their potential for tissue repair and regeneration. Intriguingly, such preconditioning approaches not only enhance EV biological activity but also increase their yield. For example, preconditioning MSCs with the Wnt signaling agonist CHIR99021 boosts EV production by 1.5-fold without inducing MSC differentiation or altering EV particle size. Animal experiments further confirm that CHIR99021-modified MSC-EVs significantly accelerate wound closure in diabetic models by promoting collagen matrix accumulation, stimulating neovascularization, and alleviating chronic inflammatory responses compared to unmodified EVs [103].
While these studies highlight the broad applicability and precise controllability of chemical induction strategies in enhancing EV therapeutic potential, the efficacy of this approach often depends on the type, concentration, and duration of the inducing agents. Potential risks, such as cytotoxicity or increased heterogeneity, must also be carefully evaluated. Future research should focus on systematically elucidating the underlying mechanisms and establishing clear dose–response relationships to facilitate the translational application of this strategy in tissue repair.
4.3. Stimulation by biological factors
Beyond chemical induction, biological factors also powerfully modulate EV function. In recent years, multiple studies have demonstrated that pretreating cells with stimulatory factors and biological agents is an effective strategy to modulate both the function and cargo of EVs, thereby enhancing their bioactivity and regenerative capacity. To date, researchers have employed various biological factors to engineer tissue-specific EV contents. For example, PDGF stimulation of ADSCs enhances exosome secretion and increases the expression of pro-angiogenic molecules such as c-KIT and stem cell factor within exosomes, thereby improving their angiogenic potential [104]. Similarly, MSCs pretreated with transforming growth factor-beta 1 (TGF-β1) exhibit elevated levels of miR-135b in their exosomes, which promotes chondrocyte proliferation [105]. In another study, EVs enriched in programmed death-ligand 1 (PD-L1) were isolated from cells stimulated with interferon-gamma (IFN-γ) or from genetically engineered cells overexpressing PD-L1.These EVs suppress the production of inflammatory cytokines by CD8+ T cells and reduce the population of CD8+ T cells in the spleen and peripheral lymph nodes, offering a novel paradigm for immunometabolic reprogramming strategies [106]. Another investigation revealed that IFN-γ-primed MSCs attenuate murine colitis by enriching exosomal cargo with miR-125a and miR-125b, thereby modulating inflammatory responses.As an inflammatory cytokine, IL-1β has also been shown to modulate MSC-derived exosomes [107]. These exosomes activate the nuclear factor erythroid 2–related factor 2 (Nrf2) signaling pathway, thereby suppressing inflammation in hippocampal astrocytes [108]. Collectively, these findings suggest that both growth factors and inflammatory mediators can significantly enhance the regenerative and anti-inflammatory properties of exosomes.
4.4. Regulated by physiological and pathological conditions
Cellular physiological and pathological states, such as physical exercise, disease, and aging, directly regulate the biogenesis of EVs, conferring functional plasticity by specifically enriching their cargo with components like proteins, lipids, metabolites, and nucleic acids [109]. During exercise, activation of metabolic stress signals promotes the release of EVs enriched with regenerative proteins. For instance, a study reported that 1 h of high-intensity cycling results in a significant increase in more than 300 EV-associated proteins in human circulation, many of which are involved in energy metabolism and tissue repair [110]. This suggests that exercise-induced exosomes may promote wound healing by transmitting regenerative signals. Additionally, a single bout of exercise markedly elevates the expression of superoxide dismutase 3 (SOD3) in plasma-derived exosomes (Exos), and this SOD3-dependent mechanism enhances the pro-angiogenic potential of circulating Exos in a type 2 diabetic mouse model [111]. This dynamic relationship provides a theoretical basis for precisely modulating EVs based on physiological conditions. Furthermore, exercise has been shown to significantly increase circulating levels of miR-122-5p, a potent pro-angiogenic factor capable of activating VEGF signaling and promoting angiogenesis [67].
However, under pathological conditions, EVs may exacerbate imbalances in the wound microenvironment. For example, proteomic analysis revealed that mitochondrial-derived vesicles (MDVs) isolated from skin tissues and culture supernatants of DFU patients exhibit elevated levels of components associated with oxidative stress and mitochondrial dysfunction. These MDVs share substantial similarity with high-glucose (HG)-conditioned MDVs, which induce fibroblast apoptosis through mechanisms involving oxidative stress, mitochondrial dysfunction, and suppression of aerobic metabolic pathways [112]. Similarly, plasma-derived EVs from DFU patients show elevated expression of miR-181b-5p compared to non-diabetic individuals. This miRNA induces senescence in human umbilical vein endothelial cells (HUVECs), impairs diabetic wound healing, and inhibits neovascularization via suppression of the Nrf2/heme oxygenase 1 (HO-1) signaling pathway [113]. In addition, EVs derived from the liver tissue of aged mice are enriched with miR-23b-3p, which promotes cellular senescence by targeting TNFα-induced protein 3 (TNFAIP3) [114]. The increased expression of miR-23b-3p in EVs has been implicated as a candidate biomarker for age-related changes and metabolic disturbances.
Compared with conventional cell-based therapies, EVs generated under defined culture conditions or physiological states exhibit functional adaptability. This allows for the selective enrichment of therapeutic cargo, such as regenerative proteins, functional mitochondria, and anti-inflammatory miRNA, while avoiding risks like immune rejection and ethical concerns associated with live cell transplantation. Such EVs represent a promising strategy for chronic wound repair.
5. Drug loading and targeting strategies for EVs
5.1. Enhancing EVs function via active ingredient loading
EVs exhibit excellent biocompatibility, low immunogenicity, and the capacity to cross physiological barriers. Additionally, they are amenable to modification and possess intrinsic targeting potential, which collectively establish them as promising platforms in the biomedical field [23]. The therapeutic efficacy of EVs can be enhanced by incorporating a variety of bioactive agents, such as nucleic acids, small molecules, proteins, and peptides, as well as nanoparticles. EVs loaded with these agents not only act as drug carriers but also function as active therapeutic platforms.
5.1.1. Nucleic acids
Among the active ingredients loaded into EVs, nucleic acids represent the most popular category, particularly microRNAs (miRNAs) and small interfering RNAs (siRNAs), which are widely regarded as holding significant potential for the treatment of wound healing. However, these molecules face inherent challenges, including susceptibility to enzymatic degradation, limited biological stability, and the potential to trigger immune responses, which collectively restrict their clinical translation [115]. Utilizing EVs as delivery vehicles effectively overcomes these limitations, thereby promoting the translational potential of nucleic acid therapeutics.
MiRNAs are a class of endogenous, non-coding RNA molecules that play a critical role in post-transcriptional gene regulation [116]. Despite their regulatory importance, clinical translation is hampered by their rapid enzymatic degradation in physiological environments and inefficient cellular internalization due to unfavorable physicochemical properties [117]. EVs, owing to their intrinsic bilayer membrane structure, serve as ideal carriers for nucleic acid delivery systems. MSC-EVs can deliver key regulatory molecules, such as miR-146a-5p [118] and miR-155-5p [119], which collaboratively promote angiogenesis, suppress apoptosis, and modulate the inflammatory microenvironment. For instance, exosomes derived from BMSCs and loaded with miR-146a-5p have been shown to suppress TRAF6 expression in vitro, thereby enhancing the proliferation, migration, and angiogenic capacity of HUVECs under hyperglycemic conditions. Additionally, treatment with exosomes containing miR-146a promotes macrophage polarization toward the anti-inflammatory M2 phenotype while inhibiting the pro-inflammatory M1 phenotype [118]. Notably, exosomes derived from M1 macrophages (M1-Exos) exhibit high expression of miR-155-5p, which significantly impairs angiogenesis in HUVECs [120]. Based on this regulatory mechanism, a study targeting diabetic wound healing demonstrated that engineering MSC-derived exosomes to deliver miR-155-5p inhibitors facilitates keratinocyte migration, restores fibroblast growth factor 7 (FGF7) levels, and enhances anti-inflammatory responses, ultimately improving collagen deposition, neovascularization, and epithelial regeneration [119].
Beyond miRNAs, other nucleic acid species also show therapeutic promise. The delivery of siRNA to target cells for gene silencing represents a key strategy in gene therapy. Exosomes derived from BMSCs, loaded with siRNA targeting ferredoxin 1 (FDX1) via water bath sonication, successfully silence FDX1 expression, thereby inhibiting goblet cell apoptosis and promoting wound healing [121]. Additionally, studies focusing on cuproptosis, a form of metal-dependent cell death, in the wound microenvironment have shown that engineered exosomes modified with copper ion chelators can efficiently sequester intracellular copper ions, providing a novel approach for regulating this process [121].
Circular RNAs (circRNAs), a newly recognized family of highly conserved, endogenous non-coding RNAs, have attracted increasing attention for their regulatory potential [122]. For example, engineered MSC-derived small EVs (MSC-sEVs) overexpressing circCDK13 have been shown to markedly promote cellular growth and migration in both human dermal fibroblasts (HDFs) and epidermal keratinocytes (HEKs). These engineered vesicles demonstrate superior efficacy in promoting diabetic wound healing compared to native MSC-sEVs [123].
Improving the nucleic acid loading efficiency in EVs remains a major research focus. An emerging technique, cellular nanoporation (CNP), transiently perturbs the plasma membrane of donor cells, enabling the efficient encapsulation of fully transcribed mRNA into EVs during their biogenesis [124]. In a study involving the delivery of collagen type I alpha 1 chain (COL1A1) mRNA, CNP treatment led to a tenfold increase in EV production compared to conventional electroporation or untreated cells [125]. Moreover, the CNP technique has been applied to load VEGF-A mRNA into fibroblast-derived EVs, resulting in vesicles with high mRNA content. These VEGF-A-loaded EVs effectively deliver and express VEGF-A mRNA and protein, significantly promoting neovascularization in ischemic tissues [126]. Engineered EVs are increasingly recognized as efficient vectors for nucleic acid delivery, owing to their ability to overcome multiple extracellular and intracellular barriers, including immunogenic responses, molecular instability, limited systemic availability, and inefficient cellular internalization.
5.1.2. Small molecules
The precise delivery of small-molecule drugs remains a critical challenge in the field of regenerative medicine. EVs demonstrate versatility in encapsulating and delivering small molecules. By integrating their intrinsic delivery capacity with functional modifications involving small-molecule cargo, therapeutic efficacy is significantly enhanced. A notable example involves the use of ADSC-derived exosomes to encapsulate oxygen nanobubbles formed from bovine serum albumin, resulting in oxygen-loaded exosomes (Fig. 4). This system effectively reduces excess ROS within wound tissues while enabling oxygen release and promoting intracellular delivery of the EVs [127]. HIF-1α, a transcription factor that regulates genes associated with angiogenesis, cell proliferation, and metabolic adaptation, has been a focus of therapeutic strategies [128]. However, HIF-1α-based therapies are limited by its inherent instability and rapid degradation. To address this, researchers have successfully employed EV-based delivery systems to encapsulate and stabilize biologically active HIF-1α, thereby enabling targeted delivery to wound sites [129]. Similarly, biomimetic nanovesicles derived from polymorphonuclear neutrophils (PMNs) have been engineered to deliver VEGF therapeutically. These vesicles retain the intrinsic antimicrobial properties of PMNs while simultaneously promoting angiogenesis in chronic diabetic wounds through VEGF delivery [130]. ABs offer a unique advantage in attracting macrophages via “eat-me” signaling molecules, such as phosphatidylserine and apoptosis-related proteins C1QC and C3b.Leveraging this mechanism, ADSC-derived ABs have been utilized to load β-hydroxybutyrate (BHB) for the treatment of chronic diabetic wounds. Experimental results revealed that BHB-loaded ABs effectively delivered NOD-like receptor protein 3 (NLRP3) inflammasome inhibitors through a dual strategy of macrophage targeting and lysosomal escape facilitation [131].
Fig. 4.
Exosome-encapsulated oxygen nanobubbles incorporated into a hydrogel accelerate wound repair by enhancing neovascularization, improving exosomal transport efficiency, mitigating hypoxic conditions, and suppressing inflammation. This system provides an effective strategy for delivering exosome-coated nanoparticles under hypoxic conditions. Copyright © 2024, The Author(s).
By leveraging the natural tropism, biocompatibility, and modularity of EVs and their mimetics, researchers have developed highly adaptable delivery vehicles capable of stabilizing labile molecules, enhancing cellular uptake, and achieving targeted therapeutic effects. By leveraging the natural tropism, biocompatibility, and modularity of EVs and their mimetics, researchers have developed highly adaptable delivery vehicles capable of stabilizing labile molecules, enhancing cellular uptake, and achieving targeted therapeutic effects.
5.1.3. Proteins
The therapeutic delivery of functional proteins to address genetic deficiencies, pathological mutations, or insufficient endogenous expression has emerged as a transformative approach in contemporary pharmacotherapeutics [132]. With the rapid advancement of protein-based therapeutics, EVs have become natural carriers capable of shuttling functional proteins between cells, thereby enabling more precise and targeted treatments. For example, myeloid differentiation factor 88 (MyD88), a central intracellular signaling molecule downstream of most toll-like receptors (TLRs), plays a pivotal role in inflammatory signaling pathways. EVs loaded with anti-inflammatory peptides that specifically target MyD88 can synergistically suppress the production of inflammatory cytokines [133]. Compared to traditional viral vectors such as adenovirus or lentivirus, EVs offer superior biocompatibility and significantly reduced immunogenic risk [134]. Their bilayer membrane structure not only protects encapsulated proteins from extracellular proteolytic degradation but also facilitates targeted delivery to endothelial cells at injury sites through surface molecules like the E-selectin ligand sialyl Lewis X (sLeX) [135].
Despite these advantages, the application of EVs for protein delivery in wound healing and chronic ulcer therapy remains limited. This is primarily due to the molecular weight and structural complexity of proteins, which often exceed the inherent loading capacity of EVs and pose significant challenges in maintaining protein stability and activity [136]. One such strategy employs transmembrane domain fusion to enhance protein incorporation into EVs by directing them toward the plasma membrane and promoting their enrichment within lipid raft microdomains. Additionally, the incorporation of lipid tags to modulate membrane-protein interactions further facilitates efficient protein incorporation into EVs, significantly improving the loading efficiency of large molecules such as VEGF [137]. Traditional loading strategies often rely on fusing the protein of interest (POI) with scaffold proteins intrinsic to EVs, such as tetraspanins (CD63, CD9, CD81), membrane-associated proteins (MFGE8), and type I transmembrane proteins (Lamp2b, PTGFRN) [138] However, although this method allows for scalable production and spatial programmability of protein incorporation, it is often associated with reduced protein stability and activity. To overcome these limitations, an innovative strategy called IDEA (EV-based Intracellular Delivery of Exogenous Proteins) has been proposed. IDEA utilizes EVs as delivery vehicles and leverages the co-expression of VSV-G to achieve effective encapsulation and targeted delivery of non-fused therapeutic proteins, thereby bypassing the dependence on scaffold proteins and their associated drawbacks [132].
Currently, the direct application of EVs for therapeutic protein delivery in wound healing remains in its infancy. Most existing research in this field continues to focus on the delivery of nucleic acids (such as miRNA and siRNA) or small molecule drugs, as summarized in Table 1.
Table 1.
Applications of EVs as drug delivery systems.
| Donor cell type | Types of EVs | Active ingredient | Loading strategy | Mechanism of action | Reference |
|---|---|---|---|---|---|
| BMSC | Exosomes | miR-146a-5p | Electroporation | Suppresses TRAF6 expression and promotes proliferation, migration, and angiogenesis of HUVECs | [118] |
| BMSC | Exosomes | miR-155 Inhibitor | Transfection | Enhances keratinocyte migration, restores FGF-7 levels, and exerts anti-inflammatory effects | [119] |
| BMSC | Exosomes | lncRNA H19 | Transfection | Inhibits miR-152-3p and promotes PTEN expression to enhance fibroblast proliferation and migration and inhibit apoptosis | [139] |
| BMSC | Exosomes | siRNA-FDX1 | Sonication | Inhibits FDX1 expression to prevent goblet cell apoptosis | [121] |
| BMSC | EVs | Peptides targeting MyD88 | Sonication | Targets MyD88 to suppress pro-inflammatory cytokine production and reduce inflammation | [133] |
| ADSC | ABs | DFO | Sonication | Acts on endothelial cells, upregulates VEGF expression, and promotes angiogenesis | [140] |
| ADSC | Exosomes | O2 | Sonication | Supplies oxygen to promote angiogenesis and suppress inflammatory responses | [127] |
| ADSC | Exosomes | mmu_circ_0001052 | Transfection | Inhibition of apoptosis and miR-106a-5p expression and activation of the FGF4/p38MAPK pathway promote angiogenesis | [141] |
| ADSC | Exosomes | circ-Snhg11 | Transfection | Inhibits HG-induced endothelial cell injury and induces M2-like macrophage polarization via miR-144-3p/HIF-1α axis | [142] |
| ADSC | Exosomes | IRF1 | Transfection | Induction of miR-16-5p expression targets and inhibits SP5 | [143] |
| ADSC | Exosomes | linc00511 | Transfection | Inhibition of PAQR3-induced Twist1 ubiquitin degradation accelerates angiogenesis | [144] |
| ADSC | Exosomes | miR-132 | Transfection | Inhibits NF-κB signaling pathway, induces M2 macrophage polarization, promotes angiogenesis | [145] |
| ADSC | Exosomes | YARA-miR-21-5p | Co-incubation | Significantly enhances proliferation, migration, and invasion of human and murine fibroblasts | [146] |
| ADSC | Exosomes | miR-21-5p | Electroporation | Stimulates keratinocyte expansion and motility through activation of the Wnt/β-catenin pathway, thus accelerating re-epithelialization, matrix remodeling, and neovascularization | [21] |
| ADSC | ABs | BHB | Extrusion | ABs with lysosomal escape capability target M1 macrophages to deliver NLRP3 inflammasome inhibitors | [131] |
| HDF | EVs | COL1A1 mRNA | Transfection | Induces COL1A1 mRNA expression to enhance collagen synthesis | [125] |
| HDF | EVs | VEGF-A mRNA | Transfection | Stimulates angiogenesis and improves tissue regeneration | [126] |
| HEK293T | EVs | HIF-1α | Transfection | Activates the HIF signaling pathway, facilitating angiogenesis and tissue repair | [129] |
| UCMSC | Exosomes | eNOS | Transfection | Reduces oxidative stress-induced inflammatory factor expression and apoptosis, enhances vascularization and matrix remodeling | [49] |
| PMN | Exosomes | VEGF | Extrusion | Promotes proliferation and migration of endothelial cells, thereby facilitating angiogenesis | [130] |
5.2. Drug loading strategies
Researchers have developed two major strategies for EV-based drug delivery systems: endogenous loading and exogenous loading [147] (Fig. 5). These strategies differ in their principles, advantages, and applications.
Fig. 5.
Strategies for loading active ingredient into EVs. EVs can be loaded with active ingredient through endogenous or exogenous strategies to enhance their therapeutic efficacy. Endogenous loading preconditions donor cells via transfection or co-incubation, leading to intracellular active ingredient expression and subsequent incorporation into secreted EVs. Exogenous loading is applied post-isolation, using techniques such as sonication, electroporation, or extrusion to directly introduce drugs or nucleic acids into EVs.
Endogenous loading strategies operate during EV biogenesis by harnessing the biosynthetic machinery of donor cells to incorporate therapeutic molecules into the EV lumen or membrane [23,25]. This approach primarily depends on two technical methods: (1) Co-incubation, which refers to the process of culturing donor cells together with drug molecules, which allows passive uptake through dynamic membrane remodeling. This method requires careful consideration of the biocompatibility between the drug and the donor cells, as well as the drug encapsulation efficiency. (2) Transfection refers to the delivery of plasmids or viral vectors encoding specific genes into donor cells, enabling them to actively carry specific proteins or RNA molecules during the biogenesis of EVs. The primary advantage of endogenous loading is the preservation of the native EV membrane structure. However, it presents challenges, including the need for high compatibility between the drug and donor cells, difficulty in precisely modulating drug loading efficiency, and limited encapsulation capacity for macromolecules like antibodies. In contrast, exogenous loading strategies target isolated and purified EVs, using physical methods to load therapeutic agents. Mainstream techniques include electroporation, sonication, extrusion, and freeze-thaw cycles [23]. Compared to endogenous loading, exogenous methods offer greater versatility, faster processing times, and scalability for large-scale production. Nevertheless, the harsh physicochemical conditions employed may compromise EV membrane integrity, leading to vesicle aggregation, cargo leakage, and diminished biological activity [[148], [149], [150]]. Both strategies exhibit distinct advantages and limitations regarding loading efficiency, EV structural preservation, and application scenarios (Table 2). The establishment of standardized loading protocols is critical for advancing EV engineering, optimizing therapeutic efficacy, and accelerating scalability and clinical translation.
Table 2.
Comparison of drug loading strategies.
| Strategy type | Loading Method | Principle | Advantages | Limitations | Example | Reference |
|---|---|---|---|---|---|---|
| Endogenous | Co-incubation (with cells) | Passive diffusion of drug into cells | Preserves membrane integrity, simple procedure | Low loading efficiency, potential cytotoxicity | Exosomes loaded with Adriamycin | [151,152] |
| Transfection | Gene engineering for expression | Enables protein modification, stable expression | Low loading efficiency, technically complex, difficult to quantify | Exosomes loaded with miR-155 Inhibitor | [13,119] | |
| Exogenous | Electroporation | Electric field-induced changes in membrane permeability | Triggers aggregation of EVs, loss of functional proteins | EVs aggregation risk, possible protein loss | Exosomes loaded with miR-146a-5p | [15,118] |
| Sonication | Creation of diffused micropores by mechanical shear forces | High loading efficiency | Preserves membrane integrity | Exosomes loaded with siRNA-FDX1 | [121,153] | |
| Extrusion | Repeated extrusion induced membrane recombination and encapsulation of drug | High loading efficiency; uniform exosome size | EVs membrane damage | ABs loaded with BHB | [25,131] | |
| Freeze-thaw cycle | Mechanical disruption of cell membranes by ice crystals induces membrane remodeling | No complex equipment required, suitable for small laboratory scale | Exosome aggregation risk, potential loss of protein activity | Exosomes loaded with catalase | [67,154] | |
| Co-incubation (with EVs) | Passive diffusion into EVs | Simple operation | Low loading efficiency | Evs loaded with kaempferol | [152,155] |
5.3. Modifications to enhance the targeting ability of EVs
The therapeutic efficacy of EVs is closely associated with their accumulation at disease sites and their subsequent uptake by target cells. The intrinsic targeting capability of native EVs primarily depends on membrane proteins derived from their donor cells, such as integrins and CD47 [20]. However, due to cellular heterogeneity and the complexity of the disease microenvironment, this natural targeting is often limited. Therefore, enhancing the tissue-specific delivery of EVs and prolonging their retention at target sites are critical for achieving optimal therapeutic outcomes. To enhance EV targeting, researchers have proposed surface engineering strategies, where ligands are artificially introduced to the EV membrane [29]. Such strategies can be broadly divided into genetic and chemical modifications. Other approaches, including electroporation, extrusion, sonication, or membrane fusion, focus on cargo delivery rather than precise targeting and were discussed earlier, so they are not covered here.
Genetic engineering is a cornerstone approach for enhancing EV functionality. This method involves introducing gene expression vectors—such as plasmids or viral vectors—into donor cells, enabling the expression of target molecules on the EV surface or within their cargo, thereby enhancing their biological function. For instance, EVs engineered to overexpress HOX transcript antisense RNA (HOTAIR) through plasmid transfection significantly promoted angiogenesis and accelerated wound healing in diabetic mice [156]. In addition, the A3 domain of von Willebrand factor (vWF) exhibits high affinity and specificity for type I and type III collagen [157]. Based on this, researchers have designed a strategy to express the collagen-binding domain derived from vWF on the EVs membrane via plasmid transfection, thereby enhancing the binding capacity of EVs to collagen at wound sites and improving their retention efficiency [129]. Viral vectors, including retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses, are widely used for EVs engineering due to their high gene delivery efficiency. For example, lentiviral transfection was used to modify MSCs, thereby enhancing the expression of sLeX on the surface of MSC-EVs. Genetically modified EVs specifically target HUVECs and accelerate angiogenesis [135]. Furthermore, by transducing MSCs with lentivirus, researchers utilized the specific binding between MS2 bacteriophage coat protein and the pac site to encapsulate anti-inflammatory miR-146a into EVs. The results showed that EVs derived from lentivirus-transfected MSCs exhibited a tenfold increase in miR-146a expression compared to the non-transfected group [54]. The application of CRISPR-Cas9 technology enables the knockout of specific gene sequences in cells. It has been demonstrated that knocking out β2-microglobulin, the light chain of HLA, in umbilical cord-derived MSCs significantly reduces the immune rejection response induced by allogeneic transplantation of their EVs [158]. Although the integration of CRISPR-Cas9 into EV-based therapeutic approaches for chronic wounds is still in its infancy, its accuracy in gene editing offers considerable potential for targeted intervention. It should be noted, that several limitations remain, including variable transfection efficiency and the complexity and high cost of production processes, which continue to pose major challenges.
Apart from employing genetic modification approaches, chemical conjugation strategies have also been utilized to improve the targeting efficiency of EVs. Covalent conjugation reactions allow for stable modification of EVs surface proteins. For instance, REDV peptides, which specifically bind to α4β1 integrin on endothelial cells, were conjugated onto ginseng-derived exosomes to enable precise targeting of diseased endothelial cells. This strategy effectively reversed endothelial dysfunction under high-glucose conditions and promoted vascular regeneration [159]. Furthermore, leveraging the overexpression of folate receptors on M1 macrophages, folate was conjugated to the membrane surface of artificial vesicles to target M1 macrophages, thereby modulating the inflammatory response and promoting wound healing [131]. These findings prompt us to identify cell-specific surface molecules expressed on macrophages, endothelial cells, and fibroblasts in chronic wounds, which may facilitate the development of engineered EVs with enhanced targeting specificity. In contrast, chemical conjugation provides a relatively straightforward and broadly applicable route, since it bypasses the need for donor cell modification. Such methods can improve targeting specificity in a versatile manner. Nevertheless, the structural complexity of EV surface proteins can limit modification efficiency, and chemical conjugation may also alter protein conformation, potentially impairing biological activity.
In summary, the functional enhancement of EV-based therapies primarily relies on two strategies: drug loading and targeting modification. Endogenous loading integrates therapeutic agents during the donor cell's biosynthesis (e.g., via plasmid transfection or co-culture), preserving the EVs' native structure yet limited by compatibility and low efficiency in packaging large molecules; exogenous loading (e.g., electroporation or sonication) offers simplicity and scalability for production, while potentially compromising membrane integrity. In terms of improved targeting, genetic engineering achieves precise functionalization by modifying donor cells, however, it faces challenges in process complexity and high costs; chemical conjugation directly modifies the EV surface, eliminating the need for cell engineering but requiring a balance between targeting efficiency and protein conformational stability. Overall, endogenous and exogenous loading strategies represent distinct approaches to functional enhancement—endogenous loading prioritizes structural preservation, while exogenous loading emphasizes production scalability. Similarly, genetic and chemical modifications complement each other in improving targeting capabilities: genetic engineering enables precise functional control through donor cell manipulation, whereas chemical conjugation offers operational flexibility by directly modifying the EV surface. However, both strategies must overcome bottlenecks in efficiency, cost, and standardization to accelerate the translation of EV-based therapies from bench to bedside.
6. Production strategies of EVs for clinical translation
6.1. Scalable production and translational challenges of natural EVs
As EVs research gradually moves towards clinical practice, how to achieve large-scale preparation while ensuring quality and functional activity has become a bottleneck restricting its wide application in regenerative medicine fields such as wound repair [160]. For large-scale production, commonly used platforms comprise ultracentrifugation, density-gradient centrifugation, ultrafiltration, size-exclusion chromatography (SEC), anion-exchange chromatography (AIEX) and polymer precipitation (Table 3). Although widely adopted in discovery-stage studies, each method exhibits constraints at clinical scale. Ultracentrifugation, the conventional “gold standard,” is operationally mature yet hampered by low throughput, lengthy processing times, and potential vesicle damage. Density-gradient centrifugation affords high purity but is limited by low throughput and high energy/time demand, rendering continuous production and lot-to-lot consistency difficult [13,161]. By contrast, SEC, AIEX and ultrafiltration are increasingly considered GMP-amenable options owing to their gentle handling and modular scalability [[162], [163], [164]]. Polymer precipitation offers simplicity and high recovery, but often introduces co-precipitated protein contaminants, compromising purity and comparability [165]. Where applicable, immunoaffinity capture enables subtype-specific enrichment but remains challenged by cost and limited scalability. In sum, achieving standardized, large-scale manufacturing while preserving EVs functional integrity remains the pivotal hurdle for clinical deployment in wound healing.
Table 3.
Comparative analysis of production strategies for natural EVs and ACDVs.
| Vesicle type | Technique | Principle | Advantages | Limitations | Applicable scenarios |
|---|---|---|---|---|---|
| Natural EVs | Ultracentrifugation | Separation based on particle size and density under high centrifugal force | Well-established, widely used | Low throughput, time-consuming, potential structural damage to EVs | Small-scale laboratory research |
| Density Gradient Centrifugation | Migration of particles in media with different density gradients | High purity | Not scalable for industrial production | High-purity analysis | |
| Size-Exclusion Chromatography (SEC) | Separation according to molecular size through porous gel matrix | Gentle to EVs, scalable | Limited separation efficiency | GMP-compliant, controlled production | |
| Anion-Exchange Chromatography (AIEX) | Separation based on surface charge differences between EVs and stationary phase | High purity, applicable to large sample volumes | High cost, buffer-dependent | GMP production | |
| Ultrafiltration | Physical separation and concentration based on membrane pore size | Simple, scalable | Membrane fouling, partial EV loss | Large-scale sample concentration | |
| Microfluidic Platforms | Physical stimulation within microchannels to enhance EV secretion and automated collection | Automated, high-throughput, minimal damage | Not fully industrialized | Clinical-grade production exploration | |
| Bioreactors | High-density cell culture and continuous EV harvesting in controlled bioreactors | Continuous collection, high-density yield | High cost, complex release criteria | GMP production systems | |
| Immunoaffinity Capture | Utilize antibodies or ligands to specifically bind to EV surface markers | High specificity; enables isolation of specific EV subpopulations | High cost; limited scalability | Precision research, biomarker detection | |
| Artificial cell-derived vesicles (ACDVs) | Nanoporous extrusion | Membrane reassembly induced by mechanical shear | High yield; simple operation | Incomplete protein preservation; batch-to-batch variability | Engineering application |
| Microfluidic technology | Hydrodynamically controlled self-assembly | Uniform particle size; suitable for continuous production | High equipment requirements; limited throughput | Alternative delivery system | |
| Ultrasonic fragmentation | Membrane reassembly triggered by ultrasonic shear | Rapid preparation; scalable for mass production | Risk of protein denaturation; broad size distribution | Drug delivery and basic research | |
| Hypotonic treatment | Membrane disruption and reassembly due to osmotic pressure | Simple operation; low cost | Low yield; insufficient membrane protein preservation | Basic research | |
| Repeated freeze–thaw | Physical stress-induced membrane disruption and reassembly | Equipment-independent; minimal chemical intervention | Poor size uniformity; low vesicle integrity; unsuitable for sensitive cargos | Basic research |
To overcome these limitations, clinical-grade EV production is shifting from conventional two-dimensional culture toward three-dimensional and perfusion-based systems. Representative platforms include hollow-fiber bioreactors (HFBs) and stirred-tank bioreactors with three-dimensional microcarriers. These systems support cell expansion under closed, low-shear, and high-density conditions while enabling continuous EV harvesting, and have demonstrated significant advantages in MSC-based models. For instance, one report showed that HFBs increased the yield of small EVs by approximately 7.5-fold compared with 2D culture, while preserving pro-proliferative and pro-migratory regenerative activities [87]. In parallel, process-intensification strategies, such as hypoxia, temperature modulation, and mechanical or chemical stimulation, have also been validated to enhance per-cell EV secretion [166]. Furthermore, the “Small Extracellular vEsicles Developer (SEED)” microfluidic platform, which applies high-throughput and non-destructive physical stimulation to cells, markedly increased EV output, with reports indicating a four-fold improvement in secretion [167]. Collectively, these advances are establishing a scalable manufacturing paradigm that better supports tissue regeneration and clinical translation. The absence of standardized definitions and detection methods also presents a significant barrier [168]: the classification of EVs, such as exosomes, microvesicles, and apoptotic bodies, as well as their functional evaluation, lacks universally accepted standards (e.g., detection thresholds for surface markers like CD63 and CD81, or quantitative metrics for biological activity), resulting in inconsistent and non-comparable results across studies, which hinders the translation from preclinical research to clinical application. Additionally, the incomplete regulatory framework increases the uncertainty in clinical development [169]: there is currently no dedicated regulatory framework for EV-based therapies, and safety (such as potential tumorigenicity and immunogenicity) and efficacy assessment criteria must still be collaboratively established with regulatory authorities (e.g., the FDA) to ensure the scientific validity and regulatory compliance of clinical trial designs.
6.2. Production strategies and clinical prospects of ACDVs
In addition to optimizing the production of natural EVs, ACDVs are emerging as a promising alternative to address the challenges of low yield and batch-to-batch variability. ACDVs are generated through physical approaches, such as nanopore extrusion, microfluidics, hypotonic treatment, and ultrasonic disruption (Table 3), which enable the reconstruction and self-assembly of cell membranes. These processes allow ACDVs to retain membrane structures and partially mimic the functional attributes of natural EVs, while achieving yields that can exceed those of natural EVs by more than two orders of magnitude [170,171].
The nanoporous extrusion technique is widely used to produce nanoscale ACDVs. In this method, cell suspensions are repeatedly passed through polycarbonate membranes under controlled pressure. Mechanical shear disrupts the plasma membrane, which subsequently self-assembles into homogeneous vesicles [172]. By optimizing parameters such as pore size, pressure, and extrusion cycles, vesicle size (typically 50–200 nm) and drug encapsulation efficiency can be precisely controlled, enhancing their potential for drug delivery applications. Nanovesicles derived from ADSCs (ANVs), prepared via a liposome-based nanoporous extrusion method, address the low yield of natural exosomes. ANVs average ∼177 nm in diameter, resemble exosomes in structure, and share a similar protein profile. In vitro, they modulate inflammation and enhance cell proliferation, migration, and neovascularization [173]. This technique has been extended beyond stem cells to include neutrophils [130], macrophages [174], and tumor cells [175]. However, vesicle heterogeneity remains an issue due to random assembly of membrane and organelle components during extrusion. To address this, modified protocols have been developed, involving ionic stress-induced separation of cellular membranes from cytoplasmic contents followed by ultrasonic purification, yielding vesicles with improved purity [133]. As a frontier advancement in biomanufacturing, microfluidic technology enables the engineering of ACDVs through precise control of hydrodynamic parameters within micron-scale channels. This approach induces membrane shearing and reassembly within the channels, offering advantages such as miniaturization, high efficiency, automation, high purity, and scalability [176]. In addition, techniques for preparing ACDVs include hypotonic treatment, ultrasonic fragmentation, and repeated freeze-thawing [176]. These methods are often followed by gradient extrusion to purify the resulting vesicles, or combined with nanoparticle co-extrusion to generate functionalized nanocarriers with biological membrane coatings. Such modifications markedly enhance the biocompatibility and systemic stability of nanomaterials in vivo [177,178].
It is noteworthy that natural EVs and ACDVs are not entirely equivalent in their biological properties. Natural EVs carry cell type–specific cargos, whereas ACDVs are predominantly membrane self-assembled products and may lack certain endogenous functions. Consequently, ACDVs are better suited as efficient carriers for drugs or bioactive molecules, while natural EVs may be superior in retaining the intrinsic “regenerative instructions” of their parent cells. In the context of standardized production and quality control, although the synthetic process is tunable, minor batch-to-batch variations in critical parameters such as particle size, surface charge, and drug loading efficiency may significantly impact therapeutic efficacy, thus highlighting the urgent need to establish a comprehensive quality control system that spans the entire process from material synthesis, functional modification, to biological activity validation. Furthermore, compared to natural vesicles, the challenges posed by the lack of a regulatory framework and the inherent complexity of preclinical studies are even more pronounced for ACDVs. As a novel biomaterial, ACDVs currently lack a unified preclinical research paradigm for the assessment of safety (e.g., long-term toxicity, immunogenicity) and the validation of efficacy (e.g., efficacy differences across various wound types). It is therefore imperative to collaborate with regulatory authorities to establish specific approval standards tailored for synthetic vesicles, thereby accelerating their translation from the laboratory to the clinic. Overcoming these challenges will require progressive interdisciplinary innovation at the interface of materials science, bioengineering, and clinical medicine, ultimately ensuring the reproducibility and clinical reliability of the therapy. In future clinical applications, these two modalities may become complementary: natural EVs serving as “signal carriers for tissue repair” and ACDVs acting as “high-efficiency delivery vehicles,” together advancing the therapeutic landscape of wound healing.
7. Biomaterial-assisted cell-free therapies
As a cutting-edge therapeutic strategy, cell-free therapy has attracted increasing attention in regenerative medicine and wound repair. Although EVs exhibit greater structural stability and resistance to enzymatic degradation compared with free RNA or proteins, they still face several in vivo challenges, including limited tissue retention time, low local accumulation efficiency, and rapid clearance [[22], [23], [24]]. To address these limitations, the development of suitable biomaterials as delivery carriers for EVs has become one of the key strategies. In the field of wound healing, commonly used biomaterials include hydrogels, microneedles, electrospun scaffolds, three-dimensional porous scaffolds, and nanoparticles [13,[179], [180], [181]]. These biomaterials can protect EVs membranes from damage caused by pH fluctuations, enzymatic degradation, and temperature changes, thereby improving their in vivo stability [182]. They also enable sustained and localized release of EVs, prolonging their bioactivity and enhancing local bioavailability. Moreover, certain biomaterials possess intrinsic properties such as pro-healing or anti-inflammatory effects, which may synergize with EVs to further promote tissue repair within the wound microenvironment.
7.1. Hydrogels
Hydrogels have been widely utilized for the loading and delivery of EVs due to their excellent biocompatibility, biodegradability, and three-dimensional network structure that resembles the natural extracellular matrix. For instance (Fig. 6a), one study developed a thermosensitive chitosan-based hydrogel modified with hyaluronic acid oligosaccharide to enable the stable release of EVs derived from hemangioma stem cells. This delivery system significantly improved the in vivo retention and stability of EVs, while in vitro release kinetics confirmed its capacity for sustained and steady EVs release over 36 h. Additionally, the hydrogel was soft, highly elastic, deformable, and biocompatible, which not only preserved the morphology and structure of EVs but also significantly improved their therapeutic efficacy [183]. Similarly, another research group developed a multifunctional hydrogel composed of gallic acid (GA)-modified chitosan and oxidized hyaluronic acid (OHA) as a carrier for hypoxia-preconditioned BMSC-derived exosomes (hyBMSC-Exos) to address macrophage dysfunction in diabetic wound healing. The GA and OHA components covalently interacted with amino groups on the transmembrane proteins of EVs, enhancing their stability and reducing their release rate in diabetic wounds, while significantly improving the uptake of EVs by macrophages [184]. Notably, the biophysical properties of hydrogels can substantially influence cell behavior. Through single-cell RNA sequencing (scRNA-seq) analysis, it was demonstrated that highly crosslinked GelMA hydrogels elicited more pronounced inflammatory and foreign body reactions, with macrophages showing a tendency toward pro-inflammatory activation. In contrast, low-crosslinked GelMA was more readily internalized and integrated into tissue by macrophages [185]. This study underscores the critical role of hydrogel crosslinking and cell–material interactions, offering valuable perspectives to guide the systematic design and refinement of hydrogel systems.
Fig. 6.
(a) Biomimetic thermosensitive hydrogel encapsulating hemangioma stem cell-derived extracellular vesicles promotes microcirculatory reconstruction in diabetic wounds. Copyright © 2023 Wiley‐VCH GmbH. (b) Multifunctional self-repairing hydrogels with smart response properties based on reversible Schiff base covalent bonds was developed for controlled exosome release. Copyright © 2024 Wiley‐VCH GmbH. (c) Wireless, biocompatible thermoelectric hydrogel (HFN) loaded with targeted ginseng-derived exosomes (RGE) enables directional stimulation by endogenous electric fields (EFs), thereby promoting angiogenesis and re-epithelialization. Copyright © 2025 Wiley‐VCH GmbH.
Biomimetic thermosensitive hydrogel encapsulating hemangioma stem cell-derived EVs promotes microcirculatory reconstruction in diabetic wounds. The functional scope of hydrogels has gradually expanded to include responsiveness to microenvironmental stimuli such as pH, temperature, ROS, light, heat, and glucose. These stimuli-responsive properties have endowed hydrogels with multifunctional capabilities such as antibacterial, anti-inflammatory, and intelligent monitoring functions, driving their development toward enhanced versatility and intelligent performance. For instance (Fig. 6b), a pH-responsive, self-healing hydrogel based on Schiff base linkages was developed for smart EVs release [186]. More advanced designs have incorporated ROS/glucose dual-responsive conductive hydrogels, enabling on-demand exosome release and offering a more efficient therapeutic strategy for chronic wound healing [187]. In addition, a pioneering study (Fig. 6c) introduced a biodegradable, wireless smart thermoelectric hydrogel, overcoming the non-degradability limitations of traditional conductive materials. This system integrates high biocompatibility with electrostimulation and enables electrofield-exosome synergistic effects. The resulting cationic trap drives the directional migration of epithelial and fibroblast cells and facilitates the targeted release of ginseng-derived exosomes for vascular repair [159]. The advancement of 3D printing technology has further enhanced the application value of hydrogels in tissue engineering and drug delivery. Its precise and controllable structural fabrication enables the optimization of hydrogel microporosity, thereby improving the loading capacity and release efficiency of therapeutic agents or EVs. For example, a cryo-extruded 3D printing technology that can be used to deliver exosomes [188]. Compared to conventional extrusion printing, the low-temperature process freezes water within the bioink to induce phase separation and rapid solidification. Subsequent freeze-drying removes ice crystals, forming abundant micropores within the scaffold. This increased porosity and surface roughness facilitates sustained exosome release and promotes cell proliferation, thereby enhancing wound healing.
7.2. Microneedles
Microneedles (MN), representing an innovative transdermal delivery modality, have shown considerable promise in therapeutic agent delivery. By penetrating the stratum corneum with micron-scale projections, MNs create microchannels in the skin, substantially enhancing drug permeability [189]. This approach improves bioavailability while reducing drug dosage and concentration, offering an novel approach for the treatment of skin disorders and the development of transdermal delivery systems [190]. A variety of MN-based therapeutic strategies have been developed to promote wound healing. For instance, an adhesive MN patch has been designed to combine MSC-Exos with antibacterial silver nanoparticles (AgNPs). Through the skin-penetrating capability of MNs, the encapsulated MSC-Exos can be locally and continuously delivered to the wound site, thereby inducing angiogenesis and suppressing inflammation [191]. Similarly, MNs have been employed to deliver HUVEC-derived exosomes to enhance neovascularization [192].
Achieving uniform EV loading remains a key challenge in optimizing MN-based delivery systems. Spray-coating and lyophilization techniques have proven effective, maintaining consistent drug distribution and preserving the bioactivity of EVs under ultralow temperatures. Based on this, researchers (Fig. 7) have developed a microneedle patch utilizing a spray–freeze–drying cycle to coat exosomes derived from young fibroblasts for deep transdermal delivery in the treatment of aging-related skin wounds [193]. In parallel, advancements in materials science have further expanded the functionality of MNs. For example, dissolvable microneedles fabricated from methacrylated decellularized dermal matrix hydrogels have been developed to deliver platelet-derived exosomes (PLT-Exos) into the dermis. These dissolvable MNs can be fully absorbed by the skin while enabling sustained drug release [194]. Despite their promising applications in wound healing, MNs still face several limitations. The small tip volume of MNs restricts drug loading capacity, posing a significant challenge, particularly for sustained-release applications. The penetration depth of microneedles may not be sufficient to treat deep or complex wounds [195,196]. In addition, regarding degradation byproducts, dissolving microneedles typically employ biodegradable materials such as poly(lactic-co-glycolic acid), where the biocompatibility of degradation products (e.g., lactic acid and glycolic acid) is critical. Imbalanced degradation rates could alter the local pH environment, potentially disrupting the wound healing process. Furthermore, in terms of potential immune responses, although exosomes derived from mesenchymal stem cells exhibit low immunogenicity due to the absence of nuclei and DNA, the microneedles themselves may induce transient inflammatory responses upon skin penetration, and their matrix materials could be recognized as foreign bodies [197]. This necessitates a delicate balance between physical stimulation and immune modulation functions to ensure optimal therapeutic outcomes. Therefore, further technological and regulatory advancements are required to facilitate broader clinical translation. In terms of long-term safety, a systematic evaluation of the complete metabolic clearance and potential chronic toxicity of microneedle degradation products in tissues is essential. Equally important is addressing the potential for delayed immune responses or long-term effects on the chronic wound microenvironment that may arise from the prolonged release of exosomes, to avoid introducing new inflammatory risks while accelerating the healing process. In terms of standardization, unified quality standards must be established that encompass the source cell donor of exosomes, isolation and purification protocols, particle size, concentration, marker expression (e.g., CD63, CD81), and biological activity (e.g., miRNA content) [198]. Additionally, the entire production process of the MN-exosome composite product, including matrix material selection, loading technology, sterilization processes, and stability studies, must be standardized through standard operating procedures to ensure batch-to-batch consistency.
Fig. 7.
Exosomes were coated onto the tips of microneedles via spray and freeze-drying cycles, preserving their bioactivity and effectively promoting tissue regeneration. Copyright © 2024 Wiley‐VCH GmbH.
7.3. Electrospinning
Electrospinning offers distinct advantages as a biomaterial platform for tissue regeneration, featuring nanofibrous topography with ECM-mimetic properties, optimal porosity for nutrient exchange, and tunable hydrophobicity while maintaining breathability [199]. Based on these properties, electrospinning systems have also been adapted for the delivery of EVs. For instance, electrospun scaffolds composed of polycaprolactone (PCL), a polymer known for its outstanding biocompatibility and mechanical strength, have been employed to deliver ABs derived from MSCs. Research indicates PCL scaffold systems facilitate sustained release of MSC-ABs containing mmu-miR-21a-5p, thereby regulating CCL1 mediated signaling pathways that drive macrophage functional reprogramming towards immunoregulatory functions. This modulation of the immune response and enhancement of angiogenesis synergistically promote wound healing [200]. In another study, to improve the stability of EVs loading, researchers fabricated electrospun fiber networks from hyaluronic acid (HA) modified with a polydopamine (PDA) coating. Milk-derived exosomes (mEXOs) were immobilized onto the PDA-coated meshes using a dip-coating technique. The results demonstrated that mEXOs were gradually released over a 14-day period without an initial burst effect [201]. This sustained release profile effectively promoted in vitro cell proliferation and significantly accelerated wound closure in vivo.
Nonetheless, electrospinning alone suffers from limitations in complex wound environments. For instance, in complex wound environments, electrospun scaffolds tend to lose structural integrity, exhibit insufficient mechanical strength, and show difficulties in precisely controlling degradation rates. To improve wound healing and address the constraints of conventional mono-strategy methods, the integration of electrospinning with hydrogels and 3D printing has been increasingly investigated [202]. For example, one study developed an anisotropic nanofibrous hydrogel via electrospinning, which exhibited well-aligned fiber orientation and remarkable multicellular modulation capabilities. By incorporating a VEGF-mimicking peptide, this system facilitated immunomodulation, angiogenesis, and neurogenesis, synergistically promoting diabetic wound healing [203]. Moreover, nanoparticles and microspheres have also been employed for EVs delivery to improve their stability and targeting efficiency. For instance, a bioinspired polydopamine-coated microsphere was designed to adsorb exosomes, enabling sustained exosome release for up to 21 days [76]. With the continuous advancement of biomaterials science, the construction of multifunctional composite delivery systems has emerged as a promising direction in EV-based therapy for chronic wounds. Future efforts may focus on optimizing material design and integrating cutting-edge engineering strategies to achieve more efficient and precise EVs delivery, ultimately improving wound healing outcomes.
8. Challenges and prospects
As research on EVs continues to advance, the field has witnessed rapid and substantial breakthroughs. EVs exhibit notable advantages in safety, controllability, and translational applicability. For instance, EVs derived from sources such as mesenchymal stem cells have demonstrated therapeutic potential in both preclinical and clinical studies, particularly in the treatment of inflammatory diseases, neurological disorders, cancer, and inflammation caused by coronavirus disease [[204], [205], [206], [207]]. These studies may further elucidate the safety, efficacy, and underlying mechanisms of EV-based therapies. At present, several studies investigating the application of EVs in wound healing have progressed to the clinical trial stage. Representative clinical trials employing EVs as therapeutic agents are summarized in Table 4.
Table 4.
Clinical trials using extracellular vesicle-based therapies for wound healing.
| Start year | Type of EVs | Type of wound | Phase | Country | Sponsor | Status | Study design | Patients number | NCT number |
|---|---|---|---|---|---|---|---|---|---|
| 2024 | PLT-Exos | Diabetic Foot Ulcer | Phase 2 | United States | Rion Inc. | Recruiting | Randomized; Parallel Assignment; Open Label | 40 | NCT06319287 |
| 2019 | EVs in MSC conditioned medium | Chronic Ulcer | Phase 1 | Indonesia | Sukma Skin Treatment | Completed | Single Group Assignment; Open Label | 38 | NCT04134676 |
| 2015 | Plasma-derived Exos | Chronic Ulcer | Early Phase 1 | Japan | Kumamoto University | Enrolling | Single Group Assignment; Open Label | 5 | NCT02565264 |
| 2023 | BMSC-EVs | Burn Wounds | Phase 1 | United States | Aegle Therapeutics | Completed | Single Group Assignment; Open Label | 1 | NCT05078385 |
| 2022 | Adipose tissue derived exosomes | Wounds and Injuries | Not Applicable | China | Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University | Completed | Single Group Assignment; Open Label | 5 | NCT05475418 |
| 2021 | PLT-Exos | Skin Graft | Phase 1 | United States | University of Miami | Active, not recruiting | Non-Randomized; Sequential Assignment; Single (Outcomes Assessor) | 8 | NCT04664738 |
| 2020 | ADSC-EVs | Periodontitis (Periodontal regeneration) | Early Phase 1 | Egypt | Beni-Suef University | Recruiting | Single Group Assignment; Open Label | 10 | NCT04270006 |
| 2017 | MSC-Exos | Macular Holes | Early Phase 1 | China | Tianjin Medical University | Active, not recruiting | Randomized; Parallel Assignment; Single (Participant) | 44 | NCT03437759 |
| 2020 | Exosomes | COVID-19 (Reduce lung inflammation and pathological impairment.) | Phase 1/2 | Russian Federation | State-Financed Health Facility "Samara Regional Medical Center Dinasty" | Completed | Randomized; Parallel Assignment; Double (Participant, Care Provider) | 30 | NCT04491240 |
From the perspective of clinical translation, MSC-EVs remain the most advanced and clinically relevant source. Multiple recent in vitro and in vivo studies, as well as systematic reviews, have demonstrated that MSC-EVs promote wound closure, angiogenesis, and collagen remodeling by regulating macrophage polarization and activating key miRNAs and signaling pathways such as miR-146a-5p, Wnt/β-catenin, and PI3K/AKT [12,208]. Notably, ADSC-EVs combined with fractional CO2 laser therapy have already shown promising outcomes in the treatment of acne scars [209]. Among non-MSC sources, platelet-derived EVs, enriched with diverse growth factors, exhibit inherent advantages in hemostasis, granulation, and chemotaxis [210]. Their safety profile has been further validated in a phase I clinical trial, where subcutaneous administration was well tolerated without significant adverse effects [211]. Macrophage-derived EVs have also emerged as promising candidates for chronic wounds dominated by excessive inflammation, as they suppress inflammatory responses, induce phenotypic reprogramming, and promote the transition to a pro-repair microenvironment [212]. In addition, skin-derived EVs, such as those from keratinocytes and fibroblasts, contribute to re-epithelialization, vascular reconstruction, collagen deposition, and ECM remodeling. Endothelial cell-derived EVs play a distinct role in revascularization, particularly in ischemia-related wounds [213,214].
Although MSC-EVs and other mammalian-derived EVs have demonstrated significant therapeutic efficacy in preclinical studies, large-scale expansion, manufacturing compliance, and ethical oversight remain critical barriers to clinical translation. These limitations have prompted increasing interest in non-mammalian alternatives. Plant-derived EVs can be isolated from organs, sap, root hairs, seeds, and pollen, offering abundant yield and scalability [215]. Their low immunogenicity and superior stability make them attractive platforms for drug delivery and therapeutic interventions [216]. For example, ginger-derived EVs have been reported to reprogram macrophages and alleviate colitis [217], and an oral clinical trial of ginger-derived exosomes for inflammatory bowel disease has been registered (NCT04879810). In parallel, bacterial EVs have shown therapeutic potential in chronic bacterial infections [218]. Leveraging established fermentation and bioprocessing technologies, bacteria can be rapidly expanded at high density in inexpensive media, enabling scalable and sustainable EV production. However, their potential toxicity and immunological side effects still require rigorous validation [219]. Regardless of their source (mammalian, plant, or bacterial), EVs face common translational barriers: the absence of standardized production and isolation protocols; and a strong dependence of yield and functionality on source and processing methods [220]. A widely adopted standardized framework for EV characterization, established by the International Society for Extracellular Vesicles (ISEV), is the MISEV guidelines. Unlike a single experimental protocol, these guidelines constitute a set of ‘minimum information’ standards established to ensure the reproducibility and reliability of EV research findings [221]. The core content of the MISEV guidelines can be summarized into three main aspects: biochemical characterization (detection of specific markers such as CD9, CD63, and CD81, along with purity assessment), biophysical characterization (determination of particle size and concentration, typically using techniques like nanoparticle tracking analysis, dynamic light scatterin, or transmission electron microscop, and functional characterization (verification of the biological activity of EVs, for example, whether they can be taken up by target cells or deliver specific molecules). Since their initial release in 2014, the MISEV guidelines have undergone multiple revisions and have become the global benchmark for EV research, although their effective implementation remains critically dependent on the standardization of EV isolation methods.
The long-term stability and biosafety of exosomes are pivotal determinants for their successful clinical translation. Various storage conditions can affect particle size, sample purity, zeta potential, and concentration, potentially causing protein leakage from EVs [222]. Studies have shown that at 4 °C and room temperature, the diameter of EVs tends to decrease within two days. Currently, the primary storage method for EVs is at −80 °C, often with the addition of cryoprotectants such as trehalose during freezing and thawing to minimize structural damage and aggregation caused by freeze-thaw cycles [223]. The in vivo behavior and long-term biosafety of EVs remain to be fully investigated. While significant progress has been made in understanding EVs biodistribution and uptake, the molecular mechanisms governing their cellular entry and subsequent cargo release remain elusive. Fluorescent labeling using fluorophores or tracking dyes is commonly employed in EVs imaging studies; however, this approach has limitations. It has been reported that fluorescent probes may dissociate from EVs, leading to signals that do not accurately reflect the true distribution of EVs, a phenomenon previously observed in liposome studies [23]. Allogeneic or xenogeneic EVs may still pose risks of immunogenicity and potential tumorigenicity [224]. Most current studies employ xenogeneic or allogeneic EVs, with only a minority utilizing autologous sources [225]. Evidence suggests that autologous EVs demonstrate superior viability in tissue repair [226]. Whether of allogeneic or xenogeneic origin, immunogenicity and potential tumorigenicity remain central concerns for EV-based therapies. For allogeneic EVs, including those from MSCs or tumor cells, overall immunogenicity is generally lower than that of intact cells, yet donor EVs may still present antigens such as MHC class I/II that can be recognized by the host immune system, leading to rejection and reduced efficacy. EVs derived from cell sources with inherent tumorigenic potential, such as embryonic or induced pluripotent stem cells, may also retain undifferentiated components or oncogenic signals in their cargo, thereby elevating the risk of tumor formation [227]. These risks highlight the importance of strict donor selection, standardized purification, and comprehensive functional validation. For xenogeneic EVs, interspecies antigenic differences and residual pathogens pose additional challenges, as they may provoke chronic inflammation or immune dysregulation that hinders clinical application. Translational efforts are further complicated by the heterogeneity of outcomes across animal models, which reflect species-specific differences, wound types, and modeling strategies [228]. For example, wound closure in mice is driven mainly by contraction due to the panniculus carnosus, limiting their relevance to human chronic wounds, whereas large-animal models such as pigs or dogs more closely reproduce human skin architecture and repair mechanisms but remain restricted by ethical and financial constraints [229]. Together, these challenges emphasize the need for source-specific risk assessment frameworks, advanced engineering approaches to mitigate immunogenicity and tumorigenicity, and more predictive preclinical models to ensure the safety and reliability of EV-based wound therapies.
With the progressive elucidation of EV biogenesis, cargo sorting mechanisms, and intercellular communication pathways, EVs demonstrate unique therapeutic advantages in areas where conventional treatments fall short, such as chronic wounds, autoimmune disorders, and inflammatory diseases. Innovations at the interface of biomaterials science, nanotechnology, and molecular biology are expected to drive the engineering of tailored EVs with optimized cargo-loading capacity, surface functionalization for enhanced targeting, and prolonged systemic circulation. Such progress will not only accelerate the clinical translation of EV-based therapeutics but also redefine existing treatment paradigms, unlocking new frontiers in regenerative and personalized medicine.
9. Conclusions
Chronic wounds remain a persistent clinical challenge, marked by delayed healing, high recurrence, and vulnerability to infection. As a cell-free therapeutic modality, EVs offer distinctive advantages through their endogenous bioactivity, low immunogenicity, and ability to mediate multifaceted intercellular communication. Refinement of EV functionality, achieved through microenvironmental preconditioning and precision bioengineering strategies such as targeted drug encapsulation and surface modification, have significantly enhanced their therapeutic efficacy, stability, and capacity to traverse physiological barriers. Concurrently, the incorporation of intelligent biomaterials, including hydrogels, microneedles, and electrospun scaffolds, provides a dynamic platform for controlled, localized, and sustained EV delivery. While the clinical translation of EV-based therapies is still constrained by challenges in large-scale manufacturing, standardization, and in vivo consistency, these limitations are being progressively addressed through improvements in isolation techniques, delivery strategies, and quality control frameworks. With these developments, EVs are expected to achieve broader and more reliable clinical application in the management of chronic wounds.
Abbreviations
| ABs | Apoptotic bodies |
| ADSC | Adipose-derived mesenchymal stem cell |
| ADSC-Exos | ADSC-derived exosomes |
| ACDVs | Artificial cell-derived vesicles |
| AgNPs | Silver nanoparticles |
| AKT | Protein kinase B |
| ANVs | Nanovesicles derived from ADSCs |
| AIEX | Anion-exchange chromatography |
| BHB | β-hydroxybutyrate |
| BMSCs | Bone marrow mesenchymal stem cells |
| circRNA | Circular RNA |
| CNP | Cellular nanoporation |
| COL1A1 | Collagen type I alpha 1 chain |
| COS | Chitosan oligosaccharides |
| DAMPs | Damage-associated molecular patterns |
| DFUs | Diabetic foot ulcer |
| EVs | Extracellular vesicles |
| ECM | Extracellular matrix |
| EGF | Epidermal growth factor |
| ERK | Extracellular signal-regulated kinase |
| FDX1 | Ferredoxin 1 |
| FGF7 | Fibroblast growth factor 7 |
| GA | Gallic acid |
| GMRSP | Glucose metabolism regulatory protein |
| HA | Hyaluronic acid |
| HDFs | Human dermal fibroblasts |
| HEKs | Epidermal keratinocytes |
| HG | High-glucose |
| HIF-1α | Hypoxia-inducible factor-1α |
| HO-1 | Heme oxygenase 1 |
| HSP90α | Heat shock protein 90 alpha |
| HUVECs | Human umbilical vein endothelial cells |
| HFBs | Hollow-fiber bioreactors |
| hyBMSC-Exos | Hypoxia-preconditioned BMSC-derived exosomes |
| IFN-γ | Interferon-gamma |
| IL | Interleukin |
| ISEV | International Society for Extracellular Vesicles |
| JAK | Janus tyrosine kinase |
| LDHA | Lactate dehydrogenase A |
| LIPUS | Low-intensity pulsed ultrasound |
| MAPK | Mitogen-activated protein kinase |
| MDVs | Mitochondrial-derived vesicles |
| mEXOs | Milk-derived exosomes |
| MMPs | Matrix metalloproteinases |
| MN | Microneedles |
| mRNA | Messenger RNA |
| MSC | Mesenchymal stem cell |
| MSC-EVs | MSC-derived EVs |
| MSC-sEVs | MSC-derived small EVs |
| mTOR | Mammalian target of rapamycin |
| MyD88 | Myeloid differentiation factor 88 |
| NETs | Neutrophil extracellular traps |
| NF-κB | Nuclear factor kappa-B |
| NLRP3 | NOD-like receptor protein 3 |
| NO | Nitric oxide |
| NOS | Nitric oxide synthase |
| Nrf2 | Nuclear factor erythroid 2–related factor 2 |
| OHA | Oxidized hyaluronic acid |
| PAMPs | Pathogen-associated molecular patterns |
| PCL | Polycaprolactone |
| PDA | Polydopamine |
| PDGF | Platelet-derived growth factor |
| PD-L1 | Programmed death-ligand 1 |
| PI3K | Phosphatidylinositol 3-kinase |
| PKM2 | Pyruvate kinase M2 |
| PLT-Exos | Platelet-derived exosomes |
| PMNs | Polymorphonuclear neutrophils |
| POI | Protein of interest |
| PTEN | Phosphatase and tensin homolog |
| RCCS | Rotary cell culture system |
| ROS | Reactive oxygen species |
| scRNA-seq | Single-cell RNA sequencing |
| SEED | Small extracellular vesicles developer |
| siRNA | Small interfering RNA |
| sLeX | Sialyl Lewis X |
| SOD3 | Superoxide dismutase 3 |
| STAT | Signal transducer and activator of transcription |
| SEC | Size-exclusion chromatography |
| TFAP2C | Transcription factor AP-2 gamma |
| TGF-β | Transforming growth factor-β |
| Tnfaip3 | TNF alpha-induced Protein 3 |
| TNF-α | Tumor necrosis factor-α |
| VEGF | Vascular endothelial growth factor |
| Vwf | Von Willebrand factor |
| YAP | Yes-associated protein |
| 3D | Three-dimensional |
| 3D-EVs | EVs derived from 3D cultures |
| 2D | Two-dimensional |
| α-SMA | α-smooth muscle actin |
CRediT authorship contribution statement
Le Ding: Writing – original draft, Visualization, Project administration, Conceptualization. Tingrui Zhang: Writing – review & editing, Visualization, Software, Conceptualization. Yixiao Pan: Writing – original draft, Visualization, Software, Methodology. Jun Liu: Writing – original draft, Visualization, Data curation, Conceptualization. Tianyou Ma: Writing – original draft, Data curation. Hanxue Zhou: Software, Data curation. Quangang Zhu: Writing – review & editing, Validation. Zongguang Tai: Writing – review & editing, Validation, Supervision, Funding acquisition, Conceptualization. Zhongjian Chen: Writing – review & editing, Validation, Resources, Project administration, Funding acquisition, Conceptualization.
Ethical statement
There are no animal experiments carried out in this article.
Consent for publication
All authors consent for publication of this review.
Funding
This manuscript was supported by the National Natural Science Foundation of China (No. 82172706 and 82373274) and the Fundamental Research Funds for the Central Universities (No. 22120240325).
Declaration of competing interest
The authors declare no competing interests.
Acknowledgements
We are grateful to BioRender (https://app.biorender.com/) for facilitating the mapping.
Footnotes
This article is part of a special issue entitled: Low-dimensional biomaterials published in Materials Today Bio.
Contributor Information
Quangang Zhu, Email: qgzhu@126.com.
Zongguang Tai, Email: taizongguang@126.com.
Zhongjian Chen, Email: aajian818@163.com.
Data availability
No data was used for the research described in the article.
References
- 1.Gravitz L. Skin, Nature. 2018;563(7732) doi: 10.1038/d41586-018-07428-4. S83-S83. [DOI] [PubMed] [Google Scholar]
- 2.Gurtner G.C., Werner S., Barrandon Y., Longaker M.T. Wound repair and regeneration. Nature. 2008;453(7193):314–321. doi: 10.1038/nature07039. [DOI] [PubMed] [Google Scholar]
- 3.Yu J.R., Navarro J., Coburn J.C., Mahadik B., Molnar J., Holmes J.H.t., et al. Current and future perspectives on skin tissue engineering: key features of biomedical research, translational assessment, and clinical application. Adv. Healthcare Mater. 2019;8(5) doi: 10.1002/adhm.201801471. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Marchete R., Oliveira S., Bagne L., Silva J.I.S., Valverde A.P., Aro A.A., et al. Anti-inflammatory and antioxidant properties of Alternanthera brasiliana improve cutaneous wound healing in rats. Inflammopharmacology. 2021;29(5):1443–1458. doi: 10.1007/s10787-021-00862-3. [DOI] [PubMed] [Google Scholar]
- 5.Lou D., Luo Y., Pang Q., Tan W.Q., Ma L. Gene-activated dermal equivalents to accelerate healing of diabetic chronic wounds by regulating inflammation and promoting angiogenesis. Bioact. Mater. 2020;5(3):667–679. doi: 10.1016/j.bioactmat.2020.04.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Morabbi A., Karimian M. Therapeutic potential of exosomal lncRNAs derived from stem cells in wound healing: focusing on mesenchymal stem cells. Stem Cell Res. Ther. 2025;16(1):62. doi: 10.1186/s13287-025-04200-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Worldwide trends in diabetes prevalence and treatment from 1990 to 2022: a pooled analysis of 1108 population-representative studies with 141 million participants. Lancet. 2024;404(10467):2077–2093. doi: 10.1016/s0140-6736(24)02317-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Jeffcoate W., Boyko E.J., Game F., Cowled P., Senneville E., Fitridge R. Causes, prevention, and management of diabetes-related foot ulcers. Lancet Diabetes Endocrinol. 2024;12(7):472–482. doi: 10.1016/s2213-8587(24)00110-4. [DOI] [PubMed] [Google Scholar]
- 9.Graves N., Phillips C.J., Harding K. A narrative review of the epidemiology and economics of chronic wounds. Br. J. Dermatol. 2022;187(2):141–148. doi: 10.1111/bjd.20692. [DOI] [PubMed] [Google Scholar]
- 10.Olsson M., Järbrink K., Divakar U., Bajpai R., Upton Z., Schmidtchen A., et al. The humanistic and economic burden of chronic wounds: a systematic review. Wound Repair Regen. 2019;27(1):114–125. doi: 10.1111/wrr.12683. [DOI] [PubMed] [Google Scholar]
- 11.Manno M., Bongiovanni A., Margolis L., Bergese P., Arosio P. The physico-chemical landscape of extracellular vesicles. Nature Reviews Bioengineering. 2024;3(1):68–82. doi: 10.1038/s44222-024-00255-5. [DOI] [Google Scholar]
- 12.Li Y., Zhu Z., Li S., Xie X., Qin L., Zhang Q., et al. Exosomes: compositions, biogenesis, and mechanisms in diabetic wound healing. J. Nanobiotechnol. 2024;22(1):398. doi: 10.1186/s12951-024-02684-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Yang C., Xue Y., Duan Y., Mao C., Wan M. Extracellular vesicles and their engineering strategies, delivery systems, and biomedical applications. J. Contr. Release. 2024;365:1089–1123. doi: 10.1016/j.jconrel.2023.11.057. [DOI] [PubMed] [Google Scholar]
- 14.Ding J.Y., Chen M.J., Wu L.F., Shu G.F., Fang S.J., Li Z.Y., et al. Mesenchymal stem cell-derived extracellular vesicles in skin wound healing: roles, opportunities and challenges. Mil Med Res. 2023;10(1):36. doi: 10.1186/s40779-023-00472-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Liao W., Du Y., Zhang C., Pan F., Yao Y., Zhang T., et al. Exosomes: the next generation of endogenous nanomaterials for advanced drug delivery and therapy. Acta Biomater. 2019;86:1–14. doi: 10.1016/j.actbio.2018.12.045. [DOI] [PubMed] [Google Scholar]
- 16.Xie Q., Hao Y., Li N., Song H., Chen X., Zhou Z., et al. Cellular uptake of engineered extracellular vesicles: Biomechanisms, engineered strategies, and disease treatment. Adv. Healthcare Mater. 2024;13(2) doi: 10.1002/adhm.202302280. [DOI] [PubMed] [Google Scholar]
- 17.Song H., Chen X., Hao Y., Wang J., Xie Q., Wang X. Nanoengineering facilitating the target mission: targeted extracellular vesicles delivery systems design. J. Nanobiotechnol. 2022;20(1):431. doi: 10.1186/s12951-022-01638-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Kalluri R., LeBleu V.S. The biology, function, and biomedical applications of exosomes. Science. 2020;367(6478) doi: 10.1126/science.aau6977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Lightner A.L., Sengupta V., Qian S., Ransom J.T., Suzuki S., Park D.J., et al. Bone marrow mesenchymal stem cell-derived extracellular vesicle Infusion for the treatment of respiratory failure from COVID-19: a randomized, Placebo-controlled dosing clinical trial. Chest. 2023;164(6):1444–1453. doi: 10.1016/j.chest.2023.06.024. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Liu L., Liu D. Bioengineered mesenchymal stem cell-derived exosomes: emerging strategies for diabetic wound healing. Burns Trauma. 2024;12 doi: 10.1093/burnst/tkae030. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Lv Q., Deng J., Chen Y., Wang Y., Liu B., Liu J. Engineered human adipose stem-cell-derived exosomes loaded with miR-21-5p to promote diabetic cutaneous wound healing. Mol. Pharm. 2020;17(5):1723–1733. doi: 10.1021/acs.molpharmaceut.0c00177. [DOI] [PubMed] [Google Scholar]
- 22.Li Q., Song Y., Wang Q., Chen J., Gao J., Tan H., et al. Engineering extracellular vesicles with platelet membranes fusion enhanced targeted therapeutic angiogenesis in a mouse model of myocardial ischemia reperfusion. Theranostics. 2021;11(8):3916–3931. doi: 10.7150/thno.52496. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Wang L., Wang D., Ye Z., Xu J. Engineering extracellular vesicles as delivery systems in therapeutic applications. Adv. Sci. (Weinh.) 2023;10(17) doi: 10.1002/advs.202300552. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Wang L., Yang H., Zhang C., Zhang Y., He Y., Liu Y., et al. A blood glucose fluctuation-responsive delivery system promotes bone regeneration and the repair function of Smpd3-reprogrammed BMSC-derived exosomes. Int. J. Oral Sci. 2024;16(1):65. doi: 10.1038/s41368-024-00328-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Kimiz-Gebologlu I., Oncel S.S. Exosomes: large-scale production, isolation, drug loading efficiency, and biodistribution and uptake. J. Contr. Release. 2022;347:533–543. doi: 10.1016/j.jconrel.2022.05.027. [DOI] [PubMed] [Google Scholar]
- 26.Dai W., Dong Y., Han T., Wang J., Gao B., Guo H., et al. Microenvironmental cue-regulated exosomes as therapeutic strategies for improving chronic wound healing. NPG Asia Mater. 2022;14(1) doi: 10.1038/s41427-022-00419-y. [DOI] [Google Scholar]
- 27.Brennan M., Layrolle P., Mooney D.J. Biomaterials functionalized with MSC secreted extracellular vesicles and soluble factors for tissue regeneration. Adv. Funct. Mater. 2020;30(37) doi: 10.1002/adfm.201909125. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Zeng H., Guo S., Ren X., Wu Z., Liu S., Yao X. Current strategies for exosome cargo loading and targeting delivery. Cells. 2023;12(10) doi: 10.3390/cells12101416. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Liu Q., Li D., Pan X., Liang Y. Targeted therapy using engineered extracellular vesicles: principles and strategies for membrane modification. J. Nanobiotechnol. 2023;21(1):334. doi: 10.1186/s12951-023-02081-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Ma Y., Brocchini S., Williams G.R. Extracellular vesicle-embedded materials. J. Contr. Release. 2023;361:280–296. doi: 10.1016/j.jconrel.2023.07.059. [DOI] [PubMed] [Google Scholar]
- 31.Pang L., Jin H., Lu Z., Xie F., Shen H., Li X., et al. Treatment with mesenchymal stem cell-derived nanovesicle-containing gelatin methacryloyl hydrogels alleviates osteoarthritis by modulating chondrogenesis and macrophage polarization. Adv. Healthcare Mater. 2023;12(17) doi: 10.1002/adhm.202300315. [DOI] [PubMed] [Google Scholar]
- 32.Peña O.A., Martin P. Cellular and molecular mechanisms of skin wound healing. Nat. Rev. Mol. Cell Biol. 2024;25(8):599–616. doi: 10.1038/s41580-024-00715-1. [DOI] [PubMed] [Google Scholar]
- 33.Huang C., Xu X., Fu J., Yu D.G., Liu Y. Recent progress in electrospun Polyacrylonitrile nanofiber-based wound dressing. Polymers. 2022;14(16) doi: 10.3390/polym14163266. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Aloysius A., Saxena S., Seifert A.W. Metabolic regulation of innate immune cell phenotypes during wound repair and regeneration. Curr. Opin. Immunol. 2021;68:72–82. doi: 10.1016/j.coi.2020.10.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Driskell R.R., Lichtenberger B.M., Hoste E., Kretzschmar K., Simons B.D., Charalambous M., et al. Distinct fibroblast lineages determine dermal architecture in skin development and repair. Nature. 2013;504(7479):277–281. doi: 10.1038/nature12783. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Xu D., Zhu W., Ding C., Mei J., Zhou J., Cheng T., et al. Self-homeostasis immunoregulatory strategy for Implant-related infections through remodeling redox balance. ACS Nano. 2023;17(5):4574–4590. doi: 10.1021/acsnano.2c10660. [DOI] [PubMed] [Google Scholar]
- 37.Lee J., Byun H., Madhurakkat Perikamana S.K., Lee S., Shin H. Current advances in immunomodulatory biomaterials for bone regeneration. Adv. Healthcare Mater. 2019;8(4) doi: 10.1002/adhm.201801106. [DOI] [PubMed] [Google Scholar]
- 38.Guo Q., Jin Y., Chen X., Ye X., Shen X., Lin M., et al. NF-κB in biology and targeted therapy: new insights and translational implications. Signal Transduct. Targeted Ther. 2024;9(1):53. doi: 10.1038/s41392-024-01757-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Zhang T., Wang X.F., Wang Z.C., Lou D., Fang Q.Q., Hu Y.Y., et al. Current potential therapeutic strategies targeting the TGF-β/Smad signaling pathway to attenuate keloid and hypertrophic scar formation. Biomed. Pharmacother. 2020;129 doi: 10.1016/j.biopha.2020.110287. [DOI] [PubMed] [Google Scholar]
- 40.Sarate R.M., Hochstetter J., Valet M., Hallou A., Song Y., Bansaccal N., et al. Dynamic regulation of tissue fluidity controls skin repair during wound healing. Cell. 2024;187(19):5298–5315.e5219. doi: 10.1016/j.cell.2024.07.031. [DOI] [PubMed] [Google Scholar]
- 41.Yang B., Lin Y., Huang Y., Zhu N., Shen Y.Q. Extracellular vesicles modulate key signalling pathways in refractory wound healing. Burns Trauma. 2023;11 doi: 10.1093/burnst/tkad039. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Yang J., Wang Z., Liang X., Wang W., Wang S. Multifunctional polypeptide-based hydrogel bio-adhesives with pro-healing activities and their working principles. Adv. Colloid Interface Sci. 2024;327 doi: 10.1016/j.cis.2024.103155. [DOI] [PubMed] [Google Scholar]
- 43.Liang Y., He J., Guo B. Functional hydrogels as wound dressing to enhance wound healing. ACS Nano. 2021;15(8):12687–12722. doi: 10.1021/acsnano.1c04206. [DOI] [PubMed] [Google Scholar]
- 44.Chen D., Tan G., Tian S., Han L., Li Y., Tan Y., et al. Advancements in nanozymes research for the management of chronic wounds. Chem. Eng. J. 2024;500 doi: 10.1016/j.cej.2024.157299. [DOI] [Google Scholar]
- 45.Teh H.X., Phang S.J., Looi M.L., Kuppusamy U.R., Arumugam B. Molecular pathways of NF-ĸB and NLRP3 inflammasome as potential targets in the treatment of inflammation in diabetic wounds: a review. Life Sci. 2023;334 doi: 10.1016/j.lfs.2023.122228. [DOI] [PubMed] [Google Scholar]
- 46.Meng W., Chen X., Chen Y., Li M., Zhang L., Luo Q., et al. Self-cascade of ROS/Glucose-Scavenging immunomodulatory hydrogels for programmed therapeutics of infected diabetic ulcers via Nrf2/NF-κB pathway. Small. 2025;21(7) doi: 10.1002/smll.202411189. [DOI] [PubMed] [Google Scholar]
- 47.Audu C.O., Melvin W.J., Joshi A.D., Wolf S.J., Moon J.Y., Davis F.M., et al. Macrophage-specific inhibition of the histone demethylase JMJD3 decreases STING and pathologic inflammation in diabetic wound repair. Cell. Mol. Immunol. 2022;19(11):1251–1262. doi: 10.1038/s41423-022-00919-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.Jere S.W., Houreld N.N., Abrahamse H. Role of the PI3K/AKT (mTOR and GSK3β) signalling pathway and photobiomodulation in diabetic wound healing. Cytokine Growth Factor Rev. 2019;50:52–59. doi: 10.1016/j.cytogfr.2019.03.001. [DOI] [PubMed] [Google Scholar]
- 49.Zhao X., Fu L., Zou H., He Y., Pan Y., Ye L., et al. Optogenetic engineered umbilical cord MSC-derived exosomes for remodeling of the immune microenvironment in diabetic wounds and the promotion of tissue repair. J. Nanobiotechnol. 2023;21(1):176. doi: 10.1186/s12951-023-01886-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.Liu Y., Liu Y., He W., Mu X., Wu X., Deng J., et al. Fibroblasts: immunomodulatory factors in refractory diabetic wound healing. Front. Immunol. 2022;13 doi: 10.3389/fimmu.2022.918223. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 51.Jeong J.O., Han J.W., Kim J.M., Cho H.J., Park C., Lee N., et al. Malignant tumor formation after transplantation of short-term cultured bone marrow mesenchymal stem cells in experimental myocardial infarction and diabetic neuropathy. Circ. Res. 2011;108(11):1340–1347. doi: 10.1161/circresaha.110.239848. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52.Liang Y., Duan L., Lu J., Xia J. Engineering exosomes for targeted drug delivery. Theranostics. 2021;11(7):3183–3195. doi: 10.7150/thno.52570. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 53.Mao J., Qian S., Zhao Q., Zhao B., Lu B., Zhang L., et al. Balancing macrophage polarization via stem cell-derived apoptotic bodies for diabetic wound healing. Méd. Sur. 2024;5(2):148–168.e148. doi: 10.1016/j.medj.2024.01.006. [DOI] [PubMed] [Google Scholar]
- 54.Li Q., Hu W., Huang Q., Yang J., Li B., Ma K., et al. MiR146a-loaded engineered exosomes released from silk fibroin patch promote diabetic wound healing by targeting IRAK1. Signal Transduct. Targeted Ther. 2023;8(1):62. doi: 10.1038/s41392-022-01263-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55.Yang Y., Zhang J., Wu S., Deng Y., Wang S., Xie L., et al. Exosome/antimicrobial peptide laden hydrogel wound dressings promote scarless wound healing through miR-21-5p-mediated multiple functions. Biomaterials. 2024;308 doi: 10.1016/j.biomaterials.2024.122558. [DOI] [PubMed] [Google Scholar]
- 56.Su J., Wei Q., Ma K., Wang Y., Hu W., Meng H., et al. P-MSC-derived extracellular vesicles facilitate diabetic wound healing via miR-145-5p/CDKN1A-mediated functional improvements of high glucose-induced senescent fibroblasts. Burns Trauma. 2023;11 doi: 10.1093/burnst/tkad010. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 57.Chen R., Wang P., Xie J., Tang Z., Fu J., Ning Y., et al. A multifunctional injectable, self-healing, and adhesive hydrogel-based wound dressing stimulated diabetic wound healing with combined reactive oxygen species scavenging, hyperglycemia reducing, and bacteria-killing abilities. J. Nanobiotechnol. 2024;22(1):444. doi: 10.1186/s12951-024-02687-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 58.Tan F., Li X., Wang Z., Li J., Shahzad K., Zheng J. Clinical applications of stem cell-derived exosomes. Signal Transduct. Targeted Ther. 2024;9(1):17. doi: 10.1038/s41392-023-01704-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 59.Shi H., Wang M., Sun Y., Yang D., Xu W., Qian H. Exosomes: emerging cell-free based therapeutics in Dermatologic diseases. Front. Cell Dev. Biol. 2021;9 doi: 10.3389/fcell.2021.736022. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 60.Zhang Y., Wu D., Zhou C., Bai M., Wan Y., Zheng Q., et al. Engineered extracellular vesicles for tissue repair and regeneration. Burns Trauma. 2024;12 doi: 10.1093/burnst/tkae062. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 61.Li C., An Y., Sun Y., Yang F., Xu Q., Wang Z. Adipose mesenchymal stem cell-derived exosomes promote wound healing through the WNT/β-catenin signaling pathway in dermal fibroblasts. Stem Cell Rev Rep. 2022;18(6):2059–2073. doi: 10.1007/s12015-022-10378-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 62.Hsu H.H., Wang A.Y.L., Loh C.Y.Y., Pai A.A., Kao H.K. Therapeutic potential of exosomes derived from diabetic adipose stem cells in cutaneous wound healing of db/db mice. Pharmaceutics. 2022;14(6) doi: 10.3390/pharmaceutics14061206. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 63.Zhang B., Shi Y., Gong A., Pan Z., Shi H., Yang H., et al. HucMSC exosome-delivered 14-3-3ζ Orchestrates self-control of the Wnt response via modulation of YAP during cutaneous regeneration. Stem Cell. 2016;34(10):2485–2500. doi: 10.1002/stem.2432. [DOI] [PubMed] [Google Scholar]
- 64.Wang Z., Zhao F., Xu C., Zhang Q., Ren H., Huang X., et al. Metabolic reprogramming in skin wound healing. Burns Trauma. 2024;12 doi: 10.1093/burnst/tkad047. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 65.Kuang L., Wu L., Li Y. Extracellular vesicles in tumor immunity: mechanisms and novel insights. Mol. Cancer. 2025;24(1):45. doi: 10.1186/s12943-025-02233-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 66.Bruning U., Morales-Rodriguez F., Kalucka J., Goveia J., Taverna F., Queiroz K.C.S., et al. Impairment of angiogenesis by fatty acid synthase inhibition involves mTOR Malonylation. Cell Metab. 2018;28(6):866–880.e815. doi: 10.1016/j.cmet.2018.07.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 67.Lou J., Wu J., Feng M., Dang X., Wu G., Yang H., et al. Exercise promotes angiogenesis by enhancing endothelial cell fatty acid utilization via liver-derived extracellular vesicle miR-122-5p. J Sport Health Sci. 2022;11(4):495–508. doi: 10.1016/j.jshs.2021.09.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 68.Ma S., Xing X., Huang H., Gao X., Xu X., Yang J., et al. Skeletal muscle-derived extracellular vesicles transport glycolytic enzymes to mediate muscle-to-bone crosstalk. Cell Metab. 2023;35(11):2028–2043.e2027. doi: 10.1016/j.cmet.2023.10.013. [DOI] [PubMed] [Google Scholar]
- 69.Wang J., Liu J., Yang F., Sun Y., Chen J., Liu J., et al. GMRSP encoded by lncRNA H19 regulates metabolic reprogramming and alleviates aortic dissection. Nat. Commun. 2025;16(1):1719. doi: 10.1038/s41467-025-57011-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 70.Li M., Jiang Y., Hou Q., Zhao Y., Zhong L., Fu X. Potential pre-activation strategies for improving therapeutic efficacy of mesenchymal stem cells: current status and future prospects. Stem Cell Res. Ther. 2022;13(1):146. doi: 10.1186/s13287-022-02822-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 71.Hu N., Cai Z., Jiang X., Wang C., Tang T., Xu T., et al. Hypoxia-pretreated ADSC-derived exosome-embedded hydrogels promote angiogenesis and accelerate diabetic wound healing. Acta Biomater. 2023;157:175–186. doi: 10.1016/j.actbio.2022.11.057. [DOI] [PubMed] [Google Scholar]
- 72.Li Z., Bai Y., Wu H., Feng Y., Wang X., Zhao C., et al. PTEN/PI3K/AKT pathway activation with hypoxia-induced human umbilical vein endothelial cell exosome for angiogenesis-based diabetic skin reconstruction. Mater. Today Bio. 2025;32 doi: 10.1016/j.mtbio.2025.101651. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 73.Fan M.H., Zhang X.Z., Jiang Y.L., Pi J.K., Zhang J.Y., Zhang Y.Q., et al. Exosomes from hypoxic urine-derived stem cells facilitate healing of diabetic wound by targeting SERPINE1 through miR-486-5p. Biomaterials. 2025;314 doi: 10.1016/j.biomaterials.2024.122893. [DOI] [PubMed] [Google Scholar]
- 74.Shi Y., Wang S., Liu D., Wang Z., Zhu Y., Li J., et al. Exosomal miR-4645-5p from hypoxic bone marrow mesenchymal stem cells facilitates diabetic wound healing by restoring keratinocyte autophagy. Burns Trauma. 2024;12 doi: 10.1093/burnst/tkad058. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 75.Chu Z., Huang Q., Ma K., Liu X., Zhang W., Cui S., et al. Novel neutrophil extracellular trap-related mechanisms in diabetic wounds inspire a promising treatment strategy with hypoxia-challenged small extracellular vesicles. Bioact. Mater. 2023;27:257–270. doi: 10.1016/j.bioactmat.2023.04.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 76.Gao Y., Yuan Z., Yuan X., Wan Z., Yu Y., Zhan Q., et al. Bioinspired porous microspheres for sustained hypoxic exosomes release and vascularized bone regeneration. Bioact. Mater. 2022;14:377–388. doi: 10.1016/j.bioactmat.2022.01.041. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 77.Du W., Zhang K., Zhang S., Wang R., Nie Y., Tao H., et al. Enhanced proangiogenic potential of mesenchymal stem cell-derived exosomes stimulated by a nitric oxide releasing polymer. Biomaterials. 2017;133:70–81. doi: 10.1016/j.biomaterials.2017.04.030. [DOI] [PubMed] [Google Scholar]
- 78.Silva L.H.A., Antunes M.A., Dos Santos C.C., Weiss D.J., Cruz F.F., Rocco P.R.M. Strategies to improve the therapeutic effects of mesenchymal stromal cells in respiratory diseases. Stem Cell Res. Ther. 2018;9(1):45. doi: 10.1186/s13287-018-0802-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 79.Yan L., Liu G., Wu X. Exosomes derived from umbilical cord mesenchymal stem cells in mechanical environment show improved osteochondral activity via upregulation of LncRNA H19. J Orthop Translat. 2021;26:111–120. doi: 10.1016/j.jot.2020.03.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 80.Yu W., Li S., Guan X., Zhang N., Xie X., Zhang K., et al. Higher yield and enhanced therapeutic effects of exosomes derived from MSCs in hydrogel-assisted 3D culture system for bone regeneration. Biomater. Adv. 2022;133 doi: 10.1016/j.msec.2022.112646. [DOI] [PubMed] [Google Scholar]
- 81.Haraszti R.A., Miller R., Stoppato M., Sere Y.Y., Coles A., Didiot M.C., et al. Exosomes produced from 3D cultures of MSCs by tangential Flow filtration show higher yield and improved activity. Mol. Ther. 2018;26(12):2838–2847. doi: 10.1016/j.ymthe.2018.09.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 82.Zhu D., Hu Y., Kong X., Luo Y., Zhang Y., Wu Y., et al. Enhanced burn wound healing by controlled-release 3D ADMSC-derived exosome-loaded hyaluronan hydrogel. Regen. Biomater. 2024;11 doi: 10.1093/rb/rbae035. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 83.Peng H., Li H., Zhang X., Tang J., Liang Y., Qiao L., et al. 3D-exosomes laden multifunctional hydrogel enhances diabetic wound healing via accelerated angiogenesis. Chem. Eng. J. 2023;475 doi: 10.1016/j.cej.2023.146238. [DOI] [Google Scholar]
- 84.Kim J.Y., Rhim W.K., Cha S.G., Woo J., Lee J.Y., Park C.G., et al. Bolstering the secretion and bioactivities of umbilical cord MSC-derived extracellular vesicles with 3D culture and priming in chemically defined media. Nano Converg. 2022;9(1):57. doi: 10.1186/s40580-022-00349-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 85.Yuan X., Sun L., Jeske R., Nkosi D., York S.B., Liu Y., et al. Engineering extracellular vesicles by three-dimensional dynamic culture of human mesenchymal stem cells. J. Extracell. Vesicles. 2022;11(6) doi: 10.1002/jev2.12235. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 86.Chen W., Wu P., Jin C., Chen Y., Li C., Qian H. Advances in the application of extracellular vesicles derived from three-dimensional culture of stem cells. J. Nanobiotechnol. 2024;22(1):215. doi: 10.1186/s12951-024-02455-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 87.Yan L., Wu X. Exosomes produced from 3D cultures of umbilical cord mesenchymal stem cells in a hollow-fiber bioreactor show improved osteochondral regeneration activity. Cell Biol. Toxicol. 2020;36(2):165–178. doi: 10.1007/s10565-019-09504-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 88.Kim M., Yun H.W., Park D.Y., Choi B.H., Min B.H. Three-dimensional spheroid culture increases exosome secretion from mesenchymal stem cells. Tissue Eng Regen Med. 2018;15(4):427–436. doi: 10.1007/s13770-018-0139-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 89.Xie F., Wen G., Sun W., Jiang K., Chen T., Chen S., et al. Mechanical stress promotes angiogenesis through fibroblast exosomes. Biochem. Biophys. Res. Commun. 2020;533(3):346–353. doi: 10.1016/j.bbrc.2020.04.159. [DOI] [PubMed] [Google Scholar]
- 90.Zhong F., Cao S., Yang L., Liu J., Gui B., Wang H., et al. Low-intensity pulsed ultrasound accelerates diabetic wound healing by ADSC-derived exosomes via promoting the uptake of exosomes and enhancing angiogenesis. Int. J. Mol. Med. 2024;53(3) doi: 10.3892/ijmm.2024.5347. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 91.Zhao Z., Qu L., Shuang T., Wu S., Su Y., Lu F., et al. Low-intensity ultrasound radiation increases exosome yield for efficient drug delivery. J. Drug Deliv. Sci. Technol. 2020;57 doi: 10.1016/j.jddst.2020.101713. [DOI] [Google Scholar]
- 92.Yang K., Li D., Wang M., Xu Z., Chen X., Liu Q., et al. Exposure to blue light stimulates the proangiogenic capability of exosomes derived from human umbilical cord mesenchymal stem cells. Stem Cell Res. Ther. 2019;10(1):358. doi: 10.1186/s13287-019-1472-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 93.Bagheri H.S., Mousavi M., Rezabakhsh A., Rezaie J., Rasta S.H., Nourazarian A., et al. Low-level laser irradiation at a high power intensity increased human endothelial cell exosome secretion via Wnt signaling. Laser Med. Sci. 2018;33(5):1131–1145. doi: 10.1007/s10103-018-2495-8. [DOI] [PubMed] [Google Scholar]
- 94.Zhuang Y., Jiang S., Deng X., Lao A., Hua X., Xie Y., et al. Energy metabolism as therapeutic target for aged wound repair by engineered extracellular vesicle. Sci. Adv. 2024;10(15) doi: 10.1126/sciadv.adl0372. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 95.Yao W.D., Zhou J.N., Tang C., Zhang J.L., Chen Z.Y., Li Y., et al. Hydrogel microneedle Patches loaded with stem cell mitochondria-enriched microvesicles boost the chronic wound healing. ACS Nano. 2024;18(39):26733–26750. doi: 10.1021/acsnano.4c06921. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 96.Hu Y., Tao R., Chen L., Xiong Y., Xue H., Hu L., et al. Exosomes derived from pioglitazone-pretreated MSCs accelerate diabetic wound healing through enhancing angiogenesis. J. Nanobiotechnol. 2021;19(1):150. doi: 10.1186/s12951-021-00894-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 97.Yu M., Liu W., Li J., Lu J., Lu H., Jia W., et al. Exosomes derived from atorvastatin-pretreated MSC accelerate diabetic wound repair by enhancing angiogenesis via AKT/eNOS pathway. Stem Cell Res. Ther. 2020;11(1):350. doi: 10.1186/s13287-020-01824-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 98.Liu W., Yu M., Xie D., Wang L., Ye C., Zhu Q., et al. Melatonin-stimulated MSC-derived exosomes improve diabetic wound healing through regulating macrophage M1 and M2 polarization by targeting the PTEN/AKT pathway. Stem Cell Res. Ther. 2020;11(1):259. doi: 10.1186/s13287-020-01756-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 99.Wu S., Zhou Z., Li Y., Wu R., Jiang J. Pretreatment of human umbilical cord mesenchymal stem cell-derived exosomes with quercetin enhances the healing of diabetic skin wounds by modulating host-Microbiota interactions. Int. J. Nanomed. 2024;19:12557–12581. doi: 10.2147/ijn.S491471. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 100.Wang Z., Zhao Y., Zhao Y., Zhang Y., Yao X., Hang R. Exosomes secreted by macrophages upon copper ion stimulation can promote angiogenesis. Mater. Sci. Eng., C. 2021;123 doi: 10.1016/j.msec.2021.111981. [DOI] [PubMed] [Google Scholar]
- 101.Zhou Y.K., Han C.S., Zhu Z.L., Chen P., Wang Y.M., Lin S., et al. M2 exosomes modified by hydrogen sulfide promoted bone regeneration by moesin mediated endocytosis. Bioact. Mater. 2024;31:192–205. doi: 10.1016/j.bioactmat.2023.08.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 102.Zhao Y., Liu J., Liu S., Yang P., Liang Y., Ma J., et al. Fibroblast exosomal TFAP2C induced by chitosan oligosaccharides promotes peripheral axon regeneration via the miR-132-5p/CAMKK1 axis. Bioact. Mater. 2023;26:249–263. doi: 10.1016/j.bioactmat.2023.03.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 103.Wang L., Chen J., Song J., Xiang Y., Yang M., Xia L., et al. Activation of the Wnt/β-catenin signalling pathway enhances exosome production by hucMSCs and improves their capability to promote diabetic wound healing. J. Nanobiotechnol. 2024;22(1):373. doi: 10.1186/s12951-024-02650-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 104.Lopatina T., Bruno S., Tetta C., Kalinina N., Porta M., Camussi G. Platelet-derived growth factor regulates the secretion of extracellular vesicles by adipose mesenchymal stem cells and enhances their angiogenic potential. Cell Commun. Signal. 2014;12:26. doi: 10.1186/1478-811x-12-26. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 105.Wang R., Xu B., Xu H. TGF-β1 promoted chondrocyte proliferation by regulating Sp1 through MSC-exosomes derived miR-135b. Cell Cycle. 2018;17(24):2756–2765. doi: 10.1080/15384101.2018.1556063. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 106.Su D., Tsai H.I., Xu Z., Yan F., Wu Y., Xiao Y., et al. Exosomal PD-L1 functions as an immunosuppressant to promote wound healing. J. Extracell. Vesicles. 2019;9(1) doi: 10.1080/20013078.2019.1709262. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 107.Yang R., Huang H., Cui S., Zhou Y., Zhang T., Zhou Y. IFN-γ promoted exosomes from mesenchymal stem cells to attenuate colitis via miR-125a and miR-125b. Cell Death Dis. 2020;11(7):603. doi: 10.1038/s41419-020-02788-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 108.Liu K., Cai G.L., Zhuang Z., Pei S.Y., Xu S.N., Wang Y.N., et al. Interleukin-1β-Treated mesenchymal stem cells inhibit inflammation in hippocampal astrocytes through exosome-activated Nrf-2 signaling. Int. J. Nanomed. 2021;16:1423–1434. doi: 10.2147/ijn.S289914. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 109.van Niel G., D'Angelo G., Raposo G. Shedding light on the cell biology of extracellular vesicles. Nat. Rev. Mol. Cell Biol. 2018;19(4):213–228. doi: 10.1038/nrm.2017.125. [DOI] [PubMed] [Google Scholar]
- 110.Whitham M., Parker B.L., Friedrichsen M., Hingst J.R., Hjorth M., Hughes W.E., et al. Extracellular vesicles provide a Means for tissue crosstalk during exercise. Cell Metab. 2018;27(1):237–251.e234. doi: 10.1016/j.cmet.2017.12.001. [DOI] [PubMed] [Google Scholar]
- 111.Abdelsaid K., Sudhahar V., Harris R.A., Das A., Youn S.W., Liu Y., et al. Exercise improves angiogenic function of circulating exosomes in type 2 diabetes: role of exosomal SOD3. FASEB J. 2022;36(3) doi: 10.1096/fj.202101323R. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 112.Zhang H., Yan Z., Zhu J., Li Z., Chen L., Zheng W., et al. Extracellular mitochondrial-derived vesicles affect the progression of diabetic foot ulcer by regulating oxidative stress and mitochondrial dysfunction. Adv. Sci. (Weinh.) 2025;12(10) doi: 10.1002/advs.202407574. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 113.Wang S., Shi M., Zhou J., Wang W., Zhang Y., Li Y. Circulating exosomal miR-181b-5p promoted cell senescence and inhibited angiogenesis to impair diabetic foot ulcer via the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway. Front. Cardiovasc. Med. 2022;9 doi: 10.3389/fcvm.2022.844047. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 114.Jin Y., Sun G., Chen B., Feng S., Tang M., Wang H., et al. Delivering miR-23b-3p by small extracellular vesicles to promote cell senescence and aberrant lipid metabolism. BMC Biol. 2025;23(1):41. doi: 10.1186/s12915-025-02143-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 115.Tian Y., Tirrell M.V., LaBelle J.L. Harnessing the therapeutic potential of Biomacromolecules through intracellular delivery of nucleic acids, peptides, and proteins. Adv. Healthcare Mater. 2022;11(12) doi: 10.1002/adhm.202102600. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 116.Krol J., Loedige I., Filipowicz W. The widespread regulation of microRNA biogenesis, function and decay. Nat. Rev. Genet. 2010;11(9):597–610. doi: 10.1038/nrg2843. [DOI] [PubMed] [Google Scholar]
- 117.Han X., Zhang H., Butowska K., Swingle K.L., Alameh M.G., Weissman D., et al. An ionizable lipid toolbox for RNA delivery. Nat. Commun. 2021;12(1):7233. doi: 10.1038/s41467-021-27493-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 118.Zhou X., Ye C., Jiang L., Zhu X., Zhou F., Xia M., et al. The bone mesenchymal stem cell-derived exosomal miR-146a-5p promotes diabetic wound healing in mice via macrophage M1/M2 polarization. Mol. Cell. Endocrinol. 2024;579 doi: 10.1016/j.mce.2023.112089. [DOI] [PubMed] [Google Scholar]
- 119.Gondaliya P., Sayyed A.A., Bhat P., Mali M., Arya N., Khairnar A., et al. Mesenchymal stem cell-derived exosomes loaded with miR-155 inhibitor ameliorate diabetic wound healing. Mol. Pharm. 2022;19(5):1294–1308. doi: 10.1021/acs.molpharmaceut.1c00669. [DOI] [PubMed] [Google Scholar]
- 120.Lou R., Chen J., Zhou F., Zhang T., Chen X., Wang C., et al. Exosomal miRNA-155-5p from M1-polarized macrophages suppresses angiogenesis by targeting GDF6 to interrupt diabetic wound healing. Mol. Ther. Nucleic Acids. 2023;34 doi: 10.1016/j.omtn.2023.102074. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 121.Sun H., Zou Y., Chen Z., He Y., Ye K., Liu H., et al. Nanodrug-engineered exosomes achieve a Jointly dual-pathway inhibition on cuproptosis. Adv. Sci. (Weinh.) 2025;12(4) doi: 10.1002/advs.202413408. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 122.Chen L.L. The expanding regulatory mechanisms and cellular functions of circular RNAs. Nat. Rev. Mol. Cell Biol. 2020;21(8):475–490. doi: 10.1038/s41580-020-0243-y. [DOI] [PubMed] [Google Scholar]
- 123.Huang Q., Chu Z., Wang Z., Li Q., Meng S., Lu Y., et al. circCDK13-loaded small extracellular vesicles accelerate healing in preclinical diabetic wound models. Nat. Commun. 2024;15(1):3904. doi: 10.1038/s41467-024-48284-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 124.Yang Z., Shi J., Xie J., Wang Y., Sun J., Liu T., et al. Large-scale generation of functional mRNA-encapsulating exosomes via cellular nanoporation. Nat. Biomed. Eng. 2020;4(1):69–83. doi: 10.1038/s41551-019-0485-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 125.You Y., Tian Y., Yang Z., Shi J., Kwak K.J., Tong Y., et al. Intradermally delivered mRNA-encapsulating extracellular vesicles for collagen-replacement therapy. Nat. Biomed. Eng. 2023;7(7):887–900. doi: 10.1038/s41551-022-00989-w. [DOI] [PubMed] [Google Scholar]
- 126.You Y., Tian Y., Guo R., Shi J., Kwak K.J., Tong Y., et al. Extracellular vesicle-mediated VEGF-A mRNA delivery rescues ischaemic injury with low immunogenicity. Eur. Heart J. 2025 doi: 10.1093/eurheartj/ehae883. [DOI] [PubMed] [Google Scholar]
- 127.Han X., Saengow C., Ju L., Ren W., Ewoldt R.H., Irudayaraj J. Exosome-coated oxygen nanobubble-laden hydrogel augments intracellular delivery of exosomes for enhanced wound healing. Nat. Commun. 2024;15(1):3435. doi: 10.1038/s41467-024-47696-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 128.Jeon S., Jeon M., Choi S., Yoo S., Park S., Lee M., et al. Hypoxia in skin cancer: molecular basis and clinical implications. Int. J. Mol. Sci. 2023;24(5) doi: 10.3390/ijms24054430. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 129.Jeon S., Cho S., Yoo S., Lee Y., Goo J., Jeong Y.J., et al. Controlled delivery of HIF-1α via extracellular vesicles with collagen-binding activity for enhanced wound healing. J. Contr. Release. 2025;380:330–347. doi: 10.1016/j.jconrel.2025.02.010. [DOI] [PubMed] [Google Scholar]
- 130.Yu Y., Jin H., Li L., Zhang X., Zheng C., Gao X., et al. An injectable, activated neutrophil-derived exosome mimetics/extracellular matrix hybrid hydrogel with antibacterial activity and wound healing promotion effect for diabetic wound therapy. J. Nanobiotechnol. 2023;21(1):308. doi: 10.1186/s12951-023-02073-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 131.Mao J., Xia W., Wu Y., Li M., Zhao Y., Zhai P., et al. vol. 8. Research (Wash D C); 2025. p. 581. (Biosynthesis of Lysosomally Escaped Apoptotic Bodies Inhibits Inflammasome Synthesis in Macrophages). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 132.Ma D., Xie A., Lv J., Min X., Zhang X., Zhou Q., et al. Engineered extracellular vesicles enable high-efficient delivery of intracellular therapeutic proteins. Protein Cell. 2024;15(10):724–743. doi: 10.1093/procel/pwae015. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 133.Park K.S., Bergqvist M., Lässer C., Lötvall J. Targeting Myd88 using peptide-loaded mesenchymal stem cell membrane-derived synthetic vesicles to treat systemic inflammation. J. Nanobiotechnol. 2022;20(1):451. doi: 10.1186/s12951-022-01660-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 134.Huang R., Jia B., Su D., Li M., Xu Z., He C., et al. Plant exosomes fused with engineered mesenchymal stem cell-derived nanovesicles for synergistic therapy of autoimmune skin disorders. J. Extracell. Vesicles. 2023;12(10) doi: 10.1002/jev2.12361. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 135.Wang K., He Q., Yang M., Qiao Q., Chen J., Song J., et al. Glycoengineered extracellular vesicles released from antibacterial hydrogel facilitate diabetic wound healing by promoting angiogenesis. J. Extracell. Vesicles. 2024;13(11) doi: 10.1002/jev2.70013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 136.Liu W., He H., Qi J. Special issue "delivery systems of peptides and proteins: challenges, status Quo and future perspectives". Pharmaceuticals. 2023;16(9) doi: 10.3390/ph16091285. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 137.Peruzzi J.A., Gunnels T.F., Edelstein H.I., Lu P., Baker D., Leonard J.N., et al. Enhancing extracellular vesicle cargo loading and functional delivery by engineering protein-lipid interactions. Nat. Commun. 2024;15(1):5618. doi: 10.1038/s41467-024-49678-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 138.Zhao H., Li Z., Liu D., Zhang J., You Z., Shao Y., et al. PlexinA1 (PLXNA1) as a novel scaffold protein for the engineering of extracellular vesicles. J. Extracell. Vesicles. 2024;13(11) doi: 10.1002/jev2.70012. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 139.Li B., Luan S., Chen J., Zhou Y., Wang T., Li Z., et al. The MSC-derived exosomal lncRNA H19 promotes wound healing in diabetic foot ulcers by upregulating PTEN via MicroRNA-152-3p. Mol. Ther. Nucleic Acids. 2020;19:814–826. doi: 10.1016/j.omtn.2019.11.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 140.Qian S., Mao J., Zhao Q., Zhao B., Liu Z., Lu B., et al. "Find-eat" strategy targeting endothelial cells via receptor functionalized apoptotic body nanovesicle. Sci. Bull. 2023;68(8):826–837. doi: 10.1016/j.scib.2023.03.030. [DOI] [PubMed] [Google Scholar]
- 141.Liang Z.H., Pan N.F., Lin S.S., Qiu Z.Y., Liang P., Wang J., et al. Exosomes from mmu_circ_0001052-modified adipose-derived stem cells promote angiogenesis of DFU via miR-106a-5p and FGF4/p38MAPK pathway. Stem Cell Res. Ther. 2022;13(1):336. doi: 10.1186/s13287-022-03015-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 142.Shi R., Jin Y., Zhao S., Yuan H., Shi J., Zhao H. Hypoxic ADSC-derived exosomes enhance wound healing in diabetic mice via delivery of circ-Snhg11 and induction of M2-like macrophage polarization. Biomed. Pharmacother. 2022;153 doi: 10.1016/j.biopha.2022.113463. [DOI] [PubMed] [Google Scholar]
- 143.Wu M., Tu J., Huang J., Wen H., Zeng Y., Lu Y. Exosomal IRF1-loaded rat adipose-derived stem cell sheet contributes to wound healing in the diabetic foot ulcers. Mol Med. 2023;29(1):60. doi: 10.1186/s10020-023-00617-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 144.Qiu J., Shu C., Li X., Ye C., Zhang W.C. Exosomes from linc00511-overexpressing ADSCs accelerates angiogenesis in diabetic foot ulcers healing by suppressing PAQR3-induced Twist1 degradation. Diabetes Res. Clin. Pract. 2021;180 doi: 10.1016/j.diabres.2021.109032. [DOI] [PubMed] [Google Scholar]
- 145.Ge L., Wang K., Lin H., Tao E., Xia W., Wang F., et al. Engineered exosomes derived from miR-132-overexpresssing adipose stem cells promoted diabetic wound healing and skin reconstruction. Front. Bioeng. Biotechnol. 2023;11 doi: 10.3389/fbioe.2023.1129538. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 146.Hade M.D., Suire C.N., Suo Z. An effective peptide-based platform for efficient exosomal loading and cellular delivery of a microRNA. ACS Appl. Mater. Interfaces. 2023;15(3):3851–3866. doi: 10.1021/acsami.2c20728. [DOI] [PubMed] [Google Scholar]
- 147.Escudé Martinez de Castilla P., Tong L., Huang C., Sofias A.M., Pastorin G., Chen X., et al. Extracellular vesicles as a drug delivery system: a systematic review of preclinical studies. Adv. Drug Deliv. Rev. 2021;175 doi: 10.1016/j.addr.2021.05.011. [DOI] [PubMed] [Google Scholar]
- 148.Tréton G., Sayer C., Schürz M., Jaritsch M., Müller A., Matea C.T., et al. Quantitative and functional characterisation of extracellular vesicles after passive loading with hydrophobic or cholesterol-tagged small molecules. J. Contr. Release. 2023;361:694–716. doi: 10.1016/j.jconrel.2023.08.010. [DOI] [PubMed] [Google Scholar]
- 149.de Abreu R.C., Ramos C.V., Becher C., Lino M., Jesus C., da Costa Martins P.A., et al. Exogenous loading of miRNAs into small extracellular vesicles. J. Extracell. Vesicles. 2021;10(10) doi: 10.1002/jev2.12111. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 150.Gui H., Wang W., Xue Y., Zhao W., Wang M., Shi L., et al. Extracellular vesicles as nanomaterial carriers: Endowing superior targeting capability and biosafety. Coord. Chem. Rev. 2025;541 doi: 10.1016/j.ccr.2025.216784. [DOI] [Google Scholar]
- 151.Kanchanapally R., Deshmukh S.K., Chavva S.R., Tyagi N., Srivastava S.K., Patel G.K., et al. Drug-loaded exosomal preparations from different cell types exhibit distinctive loading capability, yield, and antitumor efficacies: a comparative analysis. Int. J. Nanomed. 2019;14:531–541. doi: 10.2147/ijn.S191313. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 152.Fu S., Wang Y., Xia X., Zheng J.C. Exosome engineering: current progress in cargo loading and targeted delivery. NanoImpact. 2020;20 doi: 10.1016/j.impact.2020.100261. [DOI] [Google Scholar]
- 153.Sridharan B., Lim H.G. Exosomes and ultrasound: the future of theranostic applications. Mater. Today Bio. 2023;19 doi: 10.1016/j.mtbio.2023.100556. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 154.Haney M.J., Klyachko N.L., Zhao Y., Gupta R., Plotnikova E.G., He Z., et al. Exosomes as drug delivery vehicles for Parkinson's disease therapy. J. Contr. Release. 2015;207:18–30. doi: 10.1016/j.jconrel.2015.03.033. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 155.Liu G.S., Chen H.A., Chang C.Y., Chen Y.J., Wu Y.Y., Widhibrata A., et al. Platelet-derived extracellular vesicle drug delivery system loaded with kaempferol for treating corneal neovascularization. Biomaterials. 2025;319 doi: 10.1016/j.biomaterials.2025.123205. [DOI] [PubMed] [Google Scholar]
- 156.Born L.J., Chang K.H., Shoureshi P., Lay F., Bengali S., Hsu A.T.W., et al. HOTAIR-loaded mesenchymal stem/stromal cell extracellular vesicles enhance angiogenesis and wound healing. Adv. Healthcare Mater. 2022;11(5) doi: 10.1002/adhm.202002070. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 157.Addi C., Murschel F., De Crescenzo G. Design and Use of Chimeric proteins containing a collagen-binding domain for wound healing and bone regeneration. Tissue Eng., Part B. 2017;23(2):163–182. doi: 10.1089/ten.TEB.2016.0280. [DOI] [PubMed] [Google Scholar]
- 158.Shao L., Zhang Y., Pan X., Liu B., Liang C., Zhang Y., et al. Knockout of beta-2 microglobulin enhances cardiac repair by modulating exosome imprinting and inhibiting stem cell-induced immune rejection. Cell. Mol. Life Sci. 2020;77(5):937–952. doi: 10.1007/s00018-019-03220-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 159.Tan M., Liu Y., Wang Y., Li Y., Wu C., Jiang Z., et al. Wireless thermoelectric hydrogel Recreates biomimetic electric field and angiogenic signal accelerating diabetic ulcer repair. Adv. Funct. Mater. 2025 doi: 10.1002/adfm.202425610. [DOI] [Google Scholar]
- 160.Duong A., Parmar G., Kirkham A.M., Burger D., Allan D.S. Registered clinical trials investigating treatment with cell-derived extracellular vesicles: a scoping review. Cytotherapy. 2023;25(9):939–945. doi: 10.1016/j.jcyt.2023.04.007. [DOI] [PubMed] [Google Scholar]
- 161.Uddin M.J., Mohite P., Munde S., Ade N., Oladosu T.A., Chidrawar V.R., et al. Extracellular vesicles: the future of therapeutics and drug delivery systems. Intelligent Pharmacy. 2024;2(3):312–328. doi: 10.1016/j.ipha.2024.02.004. [DOI] [Google Scholar]
- 162.Paganini C., Capasso Palmiero U., Pocsfalvi G., Touzet N., Bongiovanni A., Arosio P. Scalable production and isolation of extracellular vesicles: Available sources and Lessons from current Industrial Bioprocesses. Biotechnol. J. 2019;14(10) doi: 10.1002/biot.201800528. [DOI] [PubMed] [Google Scholar]
- 163.Guo J., Wu C., Lin X., Zhou J., Zhang J., Zheng W., et al. Establishment of a simplified dichotomic size-exclusion chromatography for isolating extracellular vesicles toward clinical applications. J. Extracell. Vesicles. 2021;10(11) doi: 10.1002/jev2.12145. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 164.Yang D., Zhang W., Zhang H., Zhang F., Chen L., Ma L., et al. Progress, opportunity, and perspective on exosome isolation - efforts for efficient exosome-based theranostics. Theranostics. 2020;10(8):3684–3707. doi: 10.7150/thno.41580. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 165.de Almeida Fuzeta M., Gonçalves P.P., Fernandes-Platzgummer A., Cabral J.M.S., Bernardes N., da Silva C.L. From promise to Reality: bioengineering strategies to enhance the therapeutic potential of extracellular vesicles. Bioengineering (Basel) 2022;9(11) doi: 10.3390/bioengineering9110675. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 166.Debbi L., Guo S., Safina D., Levenberg S. Boosting extracellular vesicle secretion. Biotechnol. Adv. 2022;59 doi: 10.1016/j.biotechadv.2022.107983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 167.Hao R., Hu S., Zhang H., Chen X., Yu Z., Ren J., et al. Mechanical stimulation on a microfluidic device to highly enhance small extracellular vesicle secretion of mesenchymal stem cells. Mater. Today Bio. 2023;18 doi: 10.1016/j.mtbio.2022.100527. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 168.Xu G., Jin J., Fu Z., Wang G., Lei X., Xu J., et al. Extracellular vesicle-based drug overview: research landscape, quality control and nonclinical evaluation strategies. Signal Transduct. Targeted Ther. 2025;10(1):255. doi: 10.1038/s41392-025-02312-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 169.Rai A., Claridge B., Lozano J., Greening D.W. The discovery of extracellular vesicles and their emergence as a next-generation therapy. Circ. Res. 2024;135(1):198–221. doi: 10.1161/circresaha.123.323054. [DOI] [PubMed] [Google Scholar]
- 170.Hahm J., Kim J., Park J. Strategies to enhance extracellular vesicle production. Tissue Eng Regen Med. 2021;18(4):513–524. doi: 10.1007/s13770-021-00364-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 171.Jang S.C., Kim O.Y., Yoon C.M., Choi D.S., Roh T.Y., Park J., et al. Bioinspired exosome-mimetic nanovesicles for targeted delivery of chemotherapeutics to malignant tumors. ACS Nano. 2013;7(9):7698–7710. doi: 10.1021/nn402232g. [DOI] [PubMed] [Google Scholar]
- 172.Rodrigues C.F., Fernandes N., de Melo-Diogo D., Correia I.J., Moreira A.F. Cell-derived vesicles for nanoparticles' coating: biomimetic approaches for enhanced blood circulation and cancer therapy. Adv. Healthcare Mater. 2022;11(23) doi: 10.1002/adhm.202201214. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 173.Zhao M., Kang M., Wang J., Yang R., Zhong X., Xie Q., et al. Stem cell-derived nanovesicles embedded in dual-Layered hydrogel for programmed ROS regulation and comprehensive tissue regeneration in burn wound healing. Adv. Mater. 2024;36(32) doi: 10.1002/adma.202401369. [DOI] [PubMed] [Google Scholar]
- 174.Choo Y.W., Kang M., Kim H.Y., Han J., Kang S., Lee J.R., et al. M1 macrophage-derived nanovesicles potentiate the Anticancer efficacy of immune Checkpoint inhibitors. ACS Nano. 2018;12(9):8977–8993. doi: 10.1021/acsnano.8b02446. [DOI] [PubMed] [Google Scholar]
- 175.Hu M., Zhang J., Kong L., Yu Y., Hu Q., Yang T., et al. Immunogenic hybrid nanovesicles of liposomes and tumor-derived nanovesicles for cancer Immunochemotherapy. ACS Nano. 2021;15(2):3123–3138. doi: 10.1021/acsnano.0c09681. [DOI] [PubMed] [Google Scholar]
- 176.Xu X., Xu L., Wen C., Xia J., Zhang Y., Liang Y. Programming assembly of biomimetic exosomes: an emerging theranostic nanomedicine platform. Mater. Today Bio. 2023;22 doi: 10.1016/j.mtbio.2023.100760. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 177.Wang Z., Zhang C. Nanomaterials for targeted therapy of kidney diseases: strategies and advances. Mater. Today Bio. 2025;31 doi: 10.1016/j.mtbio.2025.101534. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 178.Chen S., Sun F., Qian H., Xu W., Jiang J. Preconditioning and engineering strategies for improving the efficacy of mesenchymal stem cell-derived exosomes in cell-free therapy. Stem Cell. Int. 2022;2022 doi: 10.1155/2022/1779346. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 179.Wang W., Cui Y., Wei X., Zang Y., Chen X., Cheng L., et al. CuCo(2)O(4) Nanoflowers with multiple enzyme activities for treating Bacterium-Infected wounds via cuproptosis-like death. ACS Nano. 2024;18(24):15845–15863. doi: 10.1021/acsnano.4c02825. [DOI] [PubMed] [Google Scholar]
- 180.Hu Z., Shan J., Jin X., Sun W., Cheng L., Chen X.L., et al. Nanoarchitectonics of in Situ Antibiotic-releasing Acicular nanozymes for targeting and inducing cuproptosis-like death to eliminate drug-Resistant bacteria. ACS Nano. 2024;18(35):24327–24349. doi: 10.1021/acsnano.4c06565. [DOI] [PubMed] [Google Scholar]
- 181.Dan X., Li S., Chen H., Xue P., Liu B., Ju Y., et al. Tailoring biomaterials for skin anti-aging. Mater. Today Bio. 2024;28 doi: 10.1016/j.mtbio.2024.101210. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 182.Ghasempour A., Dehghan H., Mahmoudi M., Lavi Arab F. Biomimetic scaffolds loaded with mesenchymal stem cells (MSCs) or MSC-derived exosomes for enhanced wound healing. Stem Cell Res. Ther. 2024;15(1):406. doi: 10.1186/s13287-024-04012-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 183.Lu E., Yang X., Wang T., Huang X., Chen Y., Wang R., et al. Biomimetic Thermo‐sensitive hydrogel encapsulating hemangiomas stem cell derived extracellular vesicles promotes microcirculation reconstruction in diabetic wounds. Adv. Funct. Mater. 2023;33(45) doi: 10.1002/adfm.202304250. [DOI] [Google Scholar]
- 184.Shi Y., Wang S., Wang K., Yang R., Liu D., Liao H., et al. Relieving macrophage dysfunction by inhibiting SREBP2 activity: a hypoxic mesenchymal stem cells-derived exosomes loaded multifunctional hydrogel for accelerated diabetic wound healing. Small. 2024;20(25) doi: 10.1002/smll.202309276. [DOI] [PubMed] [Google Scholar]
- 185.Butenko S., Nagalla R.R., Guerrero-Juarez C.F., Palomba F., David L.M., Nguyen R.Q., et al. Hydrogel crosslinking modulates macrophages, fibroblasts, and their communication, during wound healing. Nat. Commun. 2024;15(1):6820. doi: 10.1038/s41467-024-50072-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 186.Yang P., Ju Y., Shen N., Zhu S., He J., Yang L., et al. Exos-loaded Gox-modified smart-response self-healing hydrogel improves the microenvironment and promotes wound healing in diabetic wounds. Adv. Healthcare Mater. 2025;14(7) doi: 10.1002/adhm.202403304. [DOI] [PubMed] [Google Scholar]
- 187.Jing Y., Huang T., Zhao B., Zhao L., Zhang N., Zhang K., et al. A ROS/glucose stimulated-responsive ADSCs-derived exosomes-release hydrogel system for diabetic wound healing. Chem. Eng. J. 2024;487 doi: 10.1016/j.cej.2024.150561. [DOI] [Google Scholar]
- 188.Hu Y., Wu B., Xiong Y., Tao R., Panayi A.C., Chen L., et al. Cryogenic 3D printed hydrogel scaffolds loading exosomes accelerate diabetic wound healing. Chem. Eng. J. 2021;426 doi: 10.1016/j.cej.2021.130634. [DOI] [Google Scholar]
- 189.Li H., Cui J., Zhang T., Lin F., Zhang G., Feng Z. Research progress on chitosan microneedle Arrays in transdermal drug delivery. Int. J. Nanomed. 2024;19:12957–12973. doi: 10.2147/ijn.S487313. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 190.Zheng B., Li Q., Fang L., Cai X., Liu Y., Duo Y., et al. Microorganism microneedle micro-engine depth drug delivery. Nat. Commun. 2024;15(1):8947. doi: 10.1038/s41467-024-53280-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 191.Gan J., Zhang X., Ma W., Zhao Y., Sun L. Antibacterial, adhesive, and MSC exosomes encapsulated microneedles with spatio-temporal variation functions for diabetic wound healing. Nano Today. 2022;47 doi: 10.1016/j.nantod.2022.101630. [DOI] [Google Scholar]
- 192.Yuan M., Liu K., Jiang T., Li S., Chen J., Wu Z., et al. GelMA/PEGDA microneedles patch loaded with HUVECs-derived exosomes and Tazarotene promote diabetic wound healing. J. Nanobiotechnol. 2022;20(1):147. doi: 10.1186/s12951-022-01354-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 193.Xu J., Lin S., Chen H., Yang G., Zhou M., Liu Y., et al. Highly active Frozen nanovesicles microneedles for senile wound healing via Antibacteria, Immunotherapy, and skin regeneration. Adv. Healthcare Mater. 2024;13(12) doi: 10.1002/adhm.202304315. [DOI] [PubMed] [Google Scholar]
- 194.Cao Y., Chen B., Liu Q., Mao Y., He Y., Liu X., et al. Dissolvable microneedle-based wound dressing transdermally and continuously delivers anti-inflammatory and pro-angiogenic exosomes for diabetic wound treatment. Bioact. Mater. 2024;42:32–51. doi: 10.1016/j.bioactmat.2024.08.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 195.Zhang Y., Wang S., Yang Y., Zhao S., You J., Wang J., et al. Scarless wound healing programmed by core-shell microneedles. Nat. Commun. 2023;14(1):3431. doi: 10.1038/s41467-023-39129-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 196.Lyu S., Dong Z., Xu X., Bei H.P., Yuen H.Y., James Cheung C.W., et al. Going below and beyond the surface: microneedle structure, materials, drugs, fabrication, and applications for wound healing and tissue regeneration. Bioact. Mater. 2023;27:303–326. doi: 10.1016/j.bioactmat.2023.04.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 197.Liu L., Wang F., Chen X., Liu L., Wang Y., Bei J., et al. Designing multifunctional microneedles in biomedical engineering: materials, methods, and applications. Int. J. Nanomed. 2025;20:8693–8728. doi: 10.2147/ijn.S531898. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 198.Lei L., Zhou S., Zeng L., Gu Q., Xue H., Wang F., et al. Exosome-based therapeutics in Dermatology. Biomater. Res. 2025;29:148. doi: 10.34133/bmr.0148. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 199.Cheng F., Song D., Li H., Ravi S.K., Tan S.C. Recent progress in biomedical scaffold fabricated via electrospinning: design, fabrication and tissue engineering application. Adv. Funct. Mater. 2025;35(1) doi: 10.1002/adfm.202406950. [DOI] [Google Scholar]
- 200.Su X., Yang J., Xu Z., Wei L., Yang S., Li F., et al. Fibrous scaffolds loaded with BMSC-derived apoptotic vesicles promote wound healing by inducing macrophage polarization. Genes Dis. 2025;12(2) doi: 10.1016/j.gendis.2024.101388. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 201.Bui H.D., You G., Lee M., Mao W., So C., Byeon C., et al. Milk exosome-infused fibrous matrix for treatment of acute wound. J. Contr. Release. 2024;376:79–93. doi: 10.1016/j.jconrel.2024.09.053. [DOI] [PubMed] [Google Scholar]
- 202.Wang Y., Yue G., Bai T., Liu F., Wang N., Bai J., et al. Hierarchical assembly of electrospun nanofibers for the next generation tissue repairing materials. Sustain. Mater. Technol. 2025;44 doi: 10.1016/j.susmat.2025.e01355. [DOI] [Google Scholar]
- 203.Kim K., Yang J., Li C., Yang C.Y., Hu P., Liu Y., et al. Anisotropic structure of nanofiber hydrogel accelerates diabetic wound healing via triadic synergy of immune-angiogenic-neurogenic microenvironments. Bioact. Mater. 2025;47:64–82. doi: 10.1016/j.bioactmat.2025.01.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 204.Semchenko E.A., Tan A., Borrow R., Seib K.L. The Serogroup B Meningococcal Vaccine bexsero Elicits Antibodies to Neisseria gonorrhoeae. Clin. Infect. Dis. 2019;69(7):1101–1111. doi: 10.1093/cid/ciy1061. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 205.Shi M.M., Yang Q.Y., Monsel A., Yan J.Y., Dai C.X., Zhao J.Y., et al. Preclinical efficacy and clinical safety of clinical-grade nebulized allogenic adipose mesenchymal stromal cells-derived extracellular vesicles. J. Extracell. Vesicles. 2021;10(10) doi: 10.1002/jev2.12134. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 206.Lotfy A., AboQuella N.M., Wang H. Mesenchymal stromal/stem cell (MSC)-derived exosomes in clinical trials. Stem Cell Res. Ther. 2023;14(1):66. doi: 10.1186/s13287-023-03287-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 207.Zarrabi M., Shahrbaf M.A., Nouri M., Shekari F., Hosseini S.E., Hashemian S.R., et al. Allogenic mesenchymal stromal cells and their extracellular vesicles in COVID-19 induced ARDS: a randomized controlled trial. Stem Cell Res. Ther. 2023;14(1):169. doi: 10.1186/s13287-023-03402-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 208.Feng H., Gong S., Liu J., Aghayants S., Liu Y., Wu M., et al. Adipose-derived stem cell exosomes: mechanisms and therapeutic potentials in wound healing. Biomark. Res. 2025;13(1):88. doi: 10.1186/s40364-025-00801-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 209.Kwon H.H., Yang S.H., Lee J., Park B.C., Park K.Y., Jung J.Y., et al. Combination treatment with human adipose tissue stem cell-derived exosomes and fractional CO2 laser for acne scars: a 12-week Prospective, Double-blind, randomized, Split-face study. Acta Derm. Venereol. 2020;100(18) doi: 10.2340/00015555-3666. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 210.Hao P.C., Burnouf T., Chiang C.W., Jheng P.R., Szunerits S., Yang J.C., et al. Enhanced diabetic wound healing using platelet-derived extracellular vesicles and reduced graphene oxide in polymer-coordinated hydrogels. J. Nanobiotechnol. 2023;21(1):318. doi: 10.1186/s12951-023-02068-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 211.Johnson J., Law S.Q.K., Shojaee M., Hall A.S., Bhuiyan S., Lim M.B.L., et al. First-in-human clinical trial of allogeneic, platelet-derived extracellular vesicles as a potential therapeutic for delayed wound healing. J. Extracell. Vesicles. 2023;12(7) doi: 10.1002/jev2.12332. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 212.Zhao X., Chen X., Deng Y., Wu C., Ruan Z., Li C., et al. A novel adhesive dual-sensitive hydrogel for sustained release of exosomes derived from M2 macrophages promotes repair of bone defects. Mater. Today Bio. 2023;23 doi: 10.1016/j.mtbio.2023.100840. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 213.Chen K., Li Y., Xu L., Qian Y., Liu N., Zhou C., et al. Comprehensive insight into endothelial progenitor cell-derived extracellular vesicles as a promising candidate for disease treatment. Stem Cell Res. Ther. 2022;13(1):238. doi: 10.1186/s13287-022-02921-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 214.Cheng P., Xie X., Hu L., Zhou W., Mi B., Xiong Y., et al. Hypoxia endothelial cells-derived exosomes facilitate diabetic wound healing through improving endothelial cell function and promoting M2 macrophages polarization. Bioact. Mater. 2024;33:157–173. doi: 10.1016/j.bioactmat.2023.10.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 215.Hao S., Yang H., Hu J., Luo L., Yuan Y., Liu L. Bioactive compounds and biological functions of medicinal plant-derived extracellular vesicles. Pharmacol. Res. 2024;200 doi: 10.1016/j.phrs.2024.107062. [DOI] [PubMed] [Google Scholar]
- 216.Huang J., Chen H., Li N., Liu P., Yang J., Zhao Y. Emerging technologies towards extracellular vesicles large-scale production. Bioact. Mater. 2025;52:338–365. doi: 10.1016/j.bioactmat.2025.06.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 217.Yan L., Cao Y., Hou L., Luo T., Li M., Gao S., et al. Ginger exosome-like nanoparticle-derived miRNA therapeutics: a strategic inhibitor of intestinal inflammation. J. Adv. Res. 2025;69:1–15. doi: 10.1016/j.jare.2024.04.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 218.Li Y., He W., Piao Y., Wang Y., Peng M., Li H., et al. Bacterial membrane nanovesicles encapsulating prodrug assemblies combine chemical and immunological therapies for chronic bacterial infection. Nat. Commun. 2025;16(1):5246. doi: 10.1038/s41467-025-60570-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 219.Jeong G.J., Khan F., Tabassum N., Cho K.J., Kim Y.M. Bacterial extracellular vesicles: modulation of biofilm and virulence properties. Acta Biomater. 2024;178:13–23. doi: 10.1016/j.actbio.2024.02.029. [DOI] [PubMed] [Google Scholar]
- 220.Witwer K.W., Van Balkom B.W.M., Bruno S., Choo A., Dominici M., Gimona M., et al. Defining mesenchymal stromal cell (MSC)‐derived small extracellular vesicles for therapeutic applications. J. Extracell. Vesicles. 2019;8(1) doi: 10.1080/20013078.2019.1609206. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 221.Welsh J.A., Goberdhan D.C.I., O'Driscoll L., Buzas E.I., Blenkiron C., Bussolati B., et al. Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches. J. Extracell. Vesicles. 2024;13(2) doi: 10.1002/jev2.12404. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 222.Maroto R., Zhao Y., Jamaluddin M., Popov V.L., Wang H., Kalubowilage M., et al. Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses. J. Extracell. Vesicles. 2017;6(1) doi: 10.1080/20013078.2017.1359478. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 223.Bosch S., de Beaurepaire L., Allard M., Mosser M., Heichette C., Chrétien D., et al. Trehalose prevents aggregation of exosomes and cryodamage. Sci. Rep. 2016;6 doi: 10.1038/srep36162. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 224.Wang M., Zhao X., Cui Y., Gui H., Wang S., Liu Z., et al. Extracellular vesicles in burn injury: roles, mechanisms, and applications. Burns Trauma. 2025;13 doi: 10.1093/burnst/tkaf006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 225.Al-Masawa M.E., Alshawsh M.A., Ng C.Y., Ng A.M.H., Foo J.B., Vijakumaran U., et al. Efficacy and safety of small extracellular vesicle interventions in wound healing and skin regeneration: a systematic review and meta-analysis of animal studies. Theranostics. 2022;12(15):6455–6508. doi: 10.7150/thno.73436. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 226.Lu M., Peng L., Ming X., Wang X., Cui A., Li Y., et al. Enhanced wound healing promotion by immune response-free monkey autologous iPSCs and exosomes vs. their allogeneic counterparts. EBioMedicine. 2019;42:443–457. doi: 10.1016/j.ebiom.2019.03.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 227.Moretti E.H., Lin A.L.Y., Peruzzotti-Jametti L., Pluchino S., Mozafari S. Neural stem cell-derived extracellular vesicles for advanced neural repair. J. Neurochem. 2025;169(8) doi: 10.1111/jnc.70170. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 228.Cirka H., Nguyen T.T. Comparative analysis of animal models in wound healing research and the utility for Humanized mice models. Adv. Wound Care. 2025 doi: 10.1089/wound.2024.0082. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 229.Pignet A.L., Schellnegger M., Hecker A., Kamolz L.P., Kotzbeck P. Modeling wound Chronicity in vivo: the translational challenge to capture the complexity of chronic wounds. J. Invest. Dermatol. 2024;144(7):1454–1470. doi: 10.1016/j.jid.2023.11.024. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
No data was used for the research described in the article.