From bench to bedside: the research status and application opportunity of extracellular vesicles and their engineering strategies in the treatment of skin defects

Abstract

Engineered extracellular vesicles (EVs), which are EVs modified to enhance certain biological properties, offer a promising therapeutic strategy for the treatment of skin defects. Conventional nanomaterials often encounter clinical translation challenges due to potential toxicity and limited targeting. Engineered EVs, utilizing inherent biocompatibility and effective physiological barrier traversal, can ameliorate the limitations of conventional EV therapies to some extent, including detection, isolation, purification, and therapeutic validation. Recent advances in EV engineering, such as genetic modification of production cells to control cargo, surface engineering for targeted delivery, and pre-treatment of parental cells to optimize production and bioactivity, have improved therapeutic efficacy in laboratory studies through enhanced targeting, prolonged retention time, and increased yield. Many studies have suggested the potential ability of engineered EVs to treat a variety of skin defects, including diabetic wounds, burns, and hypertrophic scars, providing a promising avenue for their clinical translation in this area. This paper reviews the therapeutic potential of engineered EVs in skin regeneration, highlighting their role in promoting cell migration and angiogenesis, modulating inflammation and reducing scar formation during wound healing. In addition, given the investment in this rapidly evolving field and the growing clinical trial activity, this review also explores recent global advances and provides an outlook on future application opportunities for EVs in the treatment of skin defects.

Graphical abstract

Introduction

In recent decades, advancements in nanoscale technologies and tools have facilitated the development of a diverse array of nanomaterials. Within the field of biomedicine, a variety of engineered nanoplatforms have been employed for the diagnosis and treatment of various diseases [1]. Nevertheless, the potential for adverse systemic effects and immunogenicity of foreign synthetic nanomaterials may act as a barrier to their progression towards clinical application and commercial viability [2, 3]. Extracellular vesicles (EVs) represent naturally occurring nanocarriers that encapsulate functional cargoes, presenting an intriguing option from a materials standpoint. These entities have attracted significant attention in the biomedical field owing to their potential to surmount the limitations associated with alternative nanoparticle-based approaches [4].

Exosomes (Exos) are a type of EVs that have a bilayer lipid membrane structure and are ubiquitously distributed throughout various bodily fluids such as serum, saliva, and cerebrospinal fluid [5], with sizes ranging from 30–150 nm [6]. The structural properties of exosomes enable them to shield their internal components from degradation by the external environment [7]. Since their initial discovery in the 1980s, exosomes had remained largely unrecognized until their function as mediators of intercellular communication was recognized in the early twenty-first century [8–13]. Numerous studies have demonstrated the therapeutic potential and safety of exosomes for a variety of diseases in the context of cellular and cell-free therapeutic methods [14–17].

EVs, especially exosomes, exhibit excellent biocompatibility and biological action potential due to their nanoscale size structure, the presence of ligands on the membrane surface, and their contents [18]. This is evidenced by their ability to transcend physiological barriers and penetrate recipient cells through direct fusion, phagocytosis, and endocytosis [19]. Once internalization, they discharge their cargo, thereby initiating a cascade of downstream cellular responses [20]. In contrast to stem cells, exosomes are safer and have a reduced risk of embolization and tumorigenesis [21]. Additionally, their inherent stability and ease of storage render them increasingly favored in the realms of research and practical applications, particularly in the context of tissue repair and regeneration [22].

In specific cases, such as burns, mechanical injuries, diabetic ulcers, and conditions of prolonged stress, the endogenous capacity of human skin to self-repair falls short of being effective [23, 24]. This inadequacy results in a compromised skin barrier that is difficult to repair and makes it challenging to maintain the body’s homeostasis [25]. In such scenarios, interventions such as skin grafts may be necessary [26]. Difficult-to-heal skin injuries can lead to prolonged hospitalization, thereby exerting a substantial burden on the healthcare infrastructure through the consumption of surgical and wound care supplies, as well as the demand for physician and nursing resources [27]. Moreover, the healthcare system is currently under considerable stress due to the ongoing challenges posed by other health crises, such as the Corona Virus Disease-2019 (COVID-19) pandemic, emphasizing the urgent need for low-risk wound management strategies [28, 29].

EV therapy is thought to have the potential to ameliorate the problem of delayed healing to some extent. However, the practical and widespread use of EVs in clinical settings has been hampered by several challenges, including extraction and isolation, purification and identification [30, 31], and low yields of production [17, 32]. To overcome these obstacles, researchers have investigated EV engineering strategies as new approaches to modify them. Modified EVs have the potential to improve yield, therapeutic efficiency, and targeting ability, making them applicable across a broad spectrum of prospective clinical interventions in disease management [33].

This review aims to elucidate the biological characteristics of exosomes, the most representative entities among EVs, along with their biogenesis processes and isolation methodologies. It will also discuss the strategies employed for EV engineering and highlight the various biological effects of both conventional EVs and those modified through engineering techniques in wound healing. Furthermore, the current state of research in this field and the potential clinical applications of EVs will be analyzed. It is noteworthy that some studies do not rigorously distinguish between “exosomes” and “EVs”. Therefore, unless explicitly specified, the term “extracellular vesicles” will be used throughout this article.

Exosome biogenesis, secretion, cellular up-taking and in vitro production

Biogenesis, secretion, and cellular up-taking of exosomes

Exosomes, identified as nanoscale lipid bilayer vesicles of biological origin [34], can be secreted by a broad spectrum of cell types and are prevalently present in various bodily fluids as well as cell culture supernatants [35]. Being a representative vesicle of cellular origin, the exosome’s architecture and surface-associated molecules possess unique properties. Figure 1a illustrates the diverse array of molecules localized on the surface of exosomes, including lipids, cell-specific proteins, adhesion molecules, tetraspanins, and some glycoproteins. These molecules are pivotal in the biogenesis, transportation, and functionality of exosomes. The internal composition of exosomes includes an assortment of enzymes, cytokines, nucleic acids, and other bioactive molecules, which enable exosomes to mediate a wide array of cellular functions such as targeting, adhesion, metabolic processes, and the modulation of inflammation.

Fig. 1.

Overview of exosome characteristics. a Illustrative representation of exosomes, depicting surface molecules and internal constituents. b Depiction of the processes involved in the intracellular biogenesis and outward secretion of exosomes. The content within the illustrations is based on the current state of scientific understanding

The fundamental principles of exosome biogenesis are relatively well-established. Exosomes originate from endosomes through a series of processes including generation, circulation, degradation, and eventual release [36]. As depicted in Fig. 1b, this process initiates with the inward budding of the cytomembrane, culminating in the creation of early endosomes. Extracellular substances undergo fusion with early sorting endosomes via the processes of plasma membrane invagination and endocytosis. Subsequently, these endosomes evolve into late sorting endosomes, within which intraluminal vesicles are formed through further invagination. These vesicles are integral to the formation of multivesicular bodies (MVBs) [36, 37] or act as a part of the endomembrane system [38]. MVBs may either be directed toward lysosomes for degradation or proceed to exocytosis, merging with the plasma membrane to discharge exosomes into extracellular space [39]. Once in the extracellular milieu, exosomes serve as facilitators of intercellular communication, transferring bioactive molecules to recipient cells via mechanisms such as direct fusion, endocytosis, and receptor-ligand interactions [40, 41]. This exchange triggers downstream cascade reactions that affect cellular operations and participate in the physiological and dysfunctional activities of the organic living system [42].

Studies have demonstrated that the endosomal sorting complex required for transport (ESCRT) family is critical in the process of exosomes’ biogenesis [43–45]. Besides, heat shock protein 90 (HSP90) and the mechanistic target of rapamycin complex 1 (mTORC1) can also exert an impact on the secretion of exosomes [46, 47]. In addition to the ESCRT-dependent exosome pathway, investigations have also revealed the ESCRT-independent pathway that is involved in exosome biogenesis and can also load substances into exosomes with the assistance of exosomal proteins [48].

Methods for obtaining exosomes

The distinctive features of exosomal architecture and composition suggest specific facets of their physiological impact. Achieving high yield and purity in exosome isolation is a critical foundational step for both research and practical applications. Figure 2 detailing exosome processing and characterization is presented below. A multitude of techniques has been employed to isolate exosomes, including centrifugation, ultrafiltration, size-exclusion chromatography (SEC), immunoaffinity separation, polymer precipitation, and microfluidic technology. The pros and cons of commonly used isolation techniques for exosomes are listed in Table 1.

Fig. 2.

Flowchart depicting exosome processing and characterization. Exosomes can be extracted from various sources through many techniques. The section on exosome characterization includes quantitative and qualitative assessments, and other examinations, such as their structures, sizes, topology and so on

Table 1.

Comparison of commonly used exosome isolation techniques and their benefits and limitations

Techniques	Principles	Advantages	Limitations
Differential ultracentrifugation	Separate by density, size, and shape	Low cost High productivity Easy to apply	Large sample volume Time consuming Relatively low purity
Density-gradient centrifugation	Separate by buoyant density	More purified	Contamination of extracellular microvesicles Negatively affects structures and biological functions of isolated exosomes
Ultrafiltration	Molecular size or molecular weight recognition by ultrafine nanomembranes	Quick Easy to operate	Low yield Co-existence of nanoparticles of comparable sizes to exosomes
Size-exclusion chromatography	Separate by different molecular weights	Preservation of the natural bioactivity of exosomes High-throughput preparation	Contaminants of protein aggregates and lipoproteins
Immunoaffinity isolation	Interaction of exosomal antigen and antibody	High yield More purified Low sample volume	High cost Potential disruption of the structural and functional integrity
Polymer precipitation	Solubility and isoelectric point	Easy to apply High yield	Contamination of nucleic acids, lipoproteins, proteins, and viruses
Microfluidic technique	Physical and biochemical properties	Quick Small volume samples Automatic operation	Relatively complex equipment No standardized protocol

Centrifugation

Centrifugation is an extensively utilized approach for the fractionation of small particles, like microorganisms and cellular organelles, based on the application of centrifugal forces. For the isolation of exosomes, forces typically within the range of 100,000 to 150,000 × g are employed, offering a cost-effective means of exosome extraction [49, 50]. In general, centrifugation methods can be categorized into two main types: differential ultracentrifugation and density gradient centrifugation.

Differential ultracentrifugation, alternatively known as simple ultracentrifugation or pelleting technique, is a centrifugal-force-based method used to separate components of fluid samples according to their density, size, and shape [51, 52]. Initially reported by Johnston in 1989 [53] and afterwards refined by Théry and colleagues [54] in 2006, this methodology has been a favored approach for the extraction of exosomes from diverse origins over several decades [55–57]. This is due to its straightforwardness and ability to produce large quantities. However, the method is based on a principle that allows for the co-separation of all components sharing similar buoyant density, size, and mass by precipitation under a chosen centrifugal force [58]. Exosome samples isolated by differential ultracentrifugation are of suboptimal purity, which may adversely affect downstream both quantitative and functional analytical studies related to exosomes [59, 60].

To enhance the effectiveness of the classical centrifugal-force-based separation technique, density gradient centrifugation was introduced and widely adopted as a representative improvement [61, 62]. This method has proven effective for obtaining purer exosome samples by exploiting the density disparities among various extracellular constituents [63, 64]. Although the method was effective in separating exosomes from protein aggregates, the method has not been entirely successful in eliminating contamination by extracellular microvesicles, attributed to the buoyancy density factor [65].

Ultrafiltration

Ultrafiltration is a method for separating extracellular vesicles from collected specimens or culture media, utilizing ultrafine nanomembranes with varying molecular weight cut-offs to differentiate EVs based on size [66]. Remarkably, this process can be completed in a mere 16 min and does not necessitate specialized equipment, presenting itself as a preferable substitute for ultracentrifugation methods [67]. It is noteworthy that by adjusting the pore size of the filtration membrane, ultrafiltration enables the classification of extracellular vesicles based on specific size criteria [68].

However, the employment of ultrafine nanomembranes during the separation process may result in the entrapment of larger vesicles within the membrane pores, leading to the aggregation of exosomes and clogging of the pores. This phenomenon can significantly reduce the yield of isolated exosomes and contribute to the deterioration of the ultrafiltration membranes [49, 69]. In addition, the concurrent presence of nanoparticles of a size similar to that of exosomes, coupled with the potential for exosome deformation induced by transmembrane pressure, represent additional limitations of the ultrafiltration technique [49].

Size-exclusion chromatography

SEC, first invented by Grant and Colin in the 1950s, represents a separation technique predicated on the differential molecular weights of substances as they traverse a chromatographic column [70]. Following the introduction of a variety of finely porous materials such as dextran polymers, agarose, and polyacrylamide, the utility of this technique has seen a gradual expansion [71]. In the context of exosome research applications, SEC facilitates the separation of exosomes through passive gravity flow, offering the distinct advantage of precise isolation of the target exosomes while maintaining the vesicles’ structural and functional integrity [72, 73]. Importantly, the method is applicable not only for the isolation of trace samples but also supports scalability and automation for high-throughput exosome production.

Notwithstanding its advantages, the SEC method also encounters some challenges, including the presence of contaminants like protein aggregates and lipoproteins within the smaller size fraction. It has been documented that amalgamating SEC with the immunoaffinity capture isolation technique presents a viable solution to mitigate this challenge [72, 74].

Other techniques

In addition to the methods mentioned above, techniques such as immunocapture, polymer precipitation and microfluidics have also been applied and investigated for obtaining exosomes.

Theoretically, any protein or cellular membrane component that is either exclusively or predominantly located on the exosome membrane, and absent in the extracellular fluid, could serve as a target for immunoaffinity-based exosome capture. Several exosome markers like cluster of differentiation 9 (CD9), CD63, and CD81 have been widely utilized for the targeted isolation of exosomes [75, 76]. While immunoaffinity enables the attainment of a high yield of more pure exosomes from comparatively minimal sample volumes, this technique’s drawbacks include the elevated cost associated with isolation and the possible compromise of exosome integrity and biological functionality [49, 50].

Polymer-induced precipitation capitalizes on the interaction between highly hydrophilic polymer molecules and the water molecules enveloping the exosomes, thereby generating a hydrophobic environment that induces the precipitation of exosomes. Typically, researchers have used hydrophilic polyethylene glycol (PEG) of a certain molecular weight to interact with the cell culture medium of cells or body fluids, subsequently utilizing centrifugation to isolate exosomes. The polymer precipitation technique is straightforward, does not demand elaborate apparatus, and can be readily scaled up to generate large volumes of exosomes. However, polymers do not exclusively precipitate exosomes but also co-precipitate a variety of water-soluble entities, thereby elevating the risk of contamination with other extracellular materials in comparison to the methods previously described [77, 78].

Microfluidic separation technology enables the rapid extraction of exosomes from minimal volumes of liquid samples and enables the analysis of exosomal properties at a microscopic scale, thereby permitting the real-time characterization and potential on-site diagnostic evaluation of exosomes. This technique was first introduced for the isolation of exosomes by Chen and colleagues using anti-CD63 antibodies [79]. Fundamentally, microfluidics has revolutionized the conventional biphasic process—comprising exosome isolation followed by characterization—into a streamlined, singular procedure [80, 81]. However, some critical parameters of the analytical system, like selectivity and affinity of the deployed antibodies, need to be meticulously assessed when designing microfluidic devices for real-time analysis.

Methodologies for EV engineering

As previously mentioned, traditional EV therapy is encumbered by issues such as diminished effectiveness, low production yields, limited local persistence, and inadequate targeting capabilities. These limitations have catalyzed numerous investigations, leading to the development of the emerging field of EV engineering [82].

Engineered EVs are EVs that have undergone modifications and optimizations through bioengineering methods or physical or chemical interventions, which are designed to fulfill particular functions or augment their inherent characteristics to satisfy clinical or other specific requirements. Typically, these engineered EVs preserve the fundamental structural and physicochemical attributes of natural EVs, including nanoscale dimensions, lipid bilayer configuration, and distinct protein profiles. Besides, engineered EVs exhibit enhanced properties in comparison to natural EVs including functional contents, ligands, and receptors [83, 84]. Broadly speaking, methodologies within EV engineering can be categorized into three principal strategies: adapting the culture environment of the cells, directly treating the parent cells, and modifying the isolated EVs (Fig. 3). A comprehensive understanding of these methods is essential for comprehending the engineering pathway of EVs, thereby establishing the technical foundation for their application in skin regeneration.

Fig. 3.

Methodologies for extracellular vesicle engineering. Diverse approaches can be utilized to produce engineered EVs during different stages of the extraction procedure. These techniques can be applied to the cell culture environment, parental cells, or directly on the EVs. The figure depicts an array of technical approaches for EV engineering, grounded in the current knowledge

Adjustment of the culture environment

Hypoxia microenvironment

It is well known that oxygen is essential for life. As oxygen is involved in many biochemical reactions such as glycolysis, it is necessary to maintain the oxygen balance in the cell culture environment [85]. The level of oxygen, or oxygen tension is regarded as a crucial factor that impacts the biological characteristics of stem cells in vitro culturing. Intriguingly, research by Cooper et al. in 1958 revealed that hypoxic conditions could augment the proliferation rates of certain cell types [86]. This proliferative enhancement under hypoxic conditions has similarly been observed in mesenchymal stem cells (MSCs) [87]. It has been suggested that oxygen levels can potentially induce DNA damage within cells [88]. As a result, the cultivation of MSCs in a hypoxic environment is believed to potentially improve their condition and, in certain instances, lead to more efficacious therapeutic outcomes.

In conditions of hypoxia, cells trigger a series of complex reactions, including those involved in autophagy, cellular stress responses, and pathways related to energy metabolism [89]. For example, hypoxia-inducible factors (HIFs) play a crucial role in the transcriptional regulation of genes that are pivotal in various biological processes, like cell proliferation, apoptosis, metabolic regulation, immune responses, and the mechanisms of tumorigenesis and metastasis [90]. When exposed to low oxygen levels, MSCs exhibit an upregulation of HIF-1α [91]. Additionally, evidence suggests it has been shown that pre-exposure to hypoxic conditions can amplify the secretion capabilities of exosomes [92]. It is important to note that hypoxia is one of the pathological microenvironment characteristics of diabetic ulcer [93]. Therefore, from this standpoint, the engineered EVs obtained by this method have the potential to be applied to diabetic wounds.

However, in such culture conditions, evidence suggests that hypoxia exerts a limited influence on the miRNA composition of EVs from bone marrow derived mesenchymal stem cells (BMSC-EVs) [94]. The discrepancy in findings raises questions regarding the potential impact of variability in cell sources or the techniques employed for EV engineering. Furthermore, the feasibility of scaling up hypoxia, a culture condition that may not be conducive to cell viability, warrants further investigation.

Stereoscopic culture environment

To date, the majority of traditional cell culture methodologies have been conducted within a two-dimensional setting, typically utilizing static adherent wall cultures. However, it is essential to recognize that there are significant differences that exist between the two-dimensional (2D) culture conditions and the three-dimensional (3D) microenvironment encountered in vivo, particularly concerning biological functionality and the structure of the medium. Within a two-dimensional culture environment, there is a tendency for cells to progressively deviate from their native morphology, structural integrity, and functional capacities as observed in vivo. Such alterations may lead to disruptions in a range of cellular biogenesis activities, which, in turn, may impact the EV produced [95, 96]. Table 2 compares the advantages and drawbacks of planar versus spatial cell culture techniques.

Table 2.

Comparison of 2D and 3D cell culture techniques

	2D cell culture	3D cell culture
Cellular morphology	Elongated and flat, growing in a single layer	Maintain natural shape and growth pattern, forming 3D aggregates or spheres, or growing stereoscopic along 3D scaffolds
Cell attachment	Rare to be seen	Common and easily generated communication
Cell differentiation	Relatively poor	Relatively well
Cell proliferation	Unnaturally rapid rate	Closer to the real situation, depending on the techniques and cell types
Influences on EV production	Lower yields Lower bioactivity Potentially leading to discrepancies in the compositions and functions	Relatively higher yields More effective bioactivity Better represent in vivo conditions
Evaluation	Lower cost, suitable for long-term, large-scale studies	More costly, more time-consuming, and relatively difficult to reproduce
	Does not accurately reflect mechanical stimulation of cells and the effects of gravity	Can accurately reflect cellular responses to mechanical stimuli, and gravity

Currently, three-dimensional culture techniques can be broadly divided into two directions: those that do not rely on materials and those that do. Material-independent three-dimensional culture methods mainly include stirred culture conditions [97], scaffold-free suspension culture [98], and so on. Material-reliant cell culture techniques have garnered extensive investigation in recent times, particularly within the realm of tissue engineering. This includes cell scaffolds [99], bioreactors [100], quantum cell expansion systems [101], etc. Biomaterials, recognized for their superior biocompatibility, are a distinct category of materials whose properties and biodegradation characteristics can be tuned with minimal alterations to their chemical structure [102]. This adaptability facilitates the development of materials that support cell culture, offering porosity, bioactivity, and mechanical robustness to satisfy the requirements of diverse cellular types. 3D culture systems have been developed to expedite the generation of mesenchymal stem cells [96, 103] and their exosomes [100, 104], achieving greater yields within shorter time spans or utilizing lesser volumes. Recent evidence indicates that hollow bioreactor-based 3D culture systems have enhanced the overall yield of MSC-exosomes, achieving an approximate increase of 19.4-fold in comparison to traditional 2D cultures [100]. Moreover, it has been observed that 3D-exos from space-based culture systems confer unique benefits in terms of enhancing the ability of proliferation and migration of endothelial cells, angiogenic capabilities, and the repair of various forms of damage compared with 2D-exos [105]. Therefore, these findings offer a preliminary foundation for the utilization of this method in producing engineered EVs to promote wound healing.

Treatment of EV-producing cells

Pretreatment of source cells represents an alternative strategic approach capable of modifying cellular characteristics. Studies have shown that these techniques are effective in augmenting the efficiency and output of EVs [106]. In this section, we will discuss treatment strategies encompassing physical stimulation, pre-application of chemicals or cytokines, and the implementation of genetic engineering.

Physical stimulation

Investigations have revealed that low-intensity ultrasound stimulation enhances the proliferation of MSCs, modifies their physiological activity responses, and directs their developmental pathways [107, 108]. In a study by Zheng et al. [109], an approach utilizing low-intensity ultrasound stimulation (US) on adipose-derived stem cells (ADSCs) was devised to increase the secretion of EVs for potential clinical application. In this investigation, an appropriate ultrasound setting was determined, which in turn increased the production of ADSC-EVs. In addition, US-EVs have been demonstrated to enhance the biological functions of fibroblasts, keratinocytes, and endothelial cells in vitro experiments, and to facilitate the healing of diabetic wound animal model in vivo through mechanisms including supporting re-epithelialization, enhancing collagen production, promoting cell proliferation, facilitating keratinocyte differentiation and migration, and inducing angiogenesis [109].

Chemical treatment

Many cells, including MSCs, are capable of reacting to a diversity of chemical agents, such as metformin, and melatonin, among others, which can profoundly modify their phenotypic characteristics. These modifications consequently influence both the volume and composition of EVs released, offering a method for EV engineering.

N, N-dimethylbiguanide, serves as an oral antihyperglycemic agent prevalently employed in diabetes therapy [110]. It is known to activate adenosine 5’-monophosphate-activated protein kinase (AMPK) and has been observed to influence the secretion of EVs from MSCs [111]. Detailed mechanistic studies have shown that metformin induces the integration of the α-trypsin inhibitor heavy chain H4 (ITIH4) into MVBs, leading to an enrichment of ITIH4 in the exosomes. The MSC-derived exosomes were demonstrated to mitigate senescence in nucleus pulposus cells, thus enhancing their therapeutic application value intervertebral disc degeneration (IDD) [112]. However, although metformin is a first-line treatment for type 2 diabetes [113], there has been no exploration of preconditioning with metformin to obtain engineered EVs suitable for the treatment of diabetic ulcers as the time of writing.

Melatonin (MT), a free radical scavenger and broad-spectrum antioxidant, was originally isolated from the pineal gland and is widely distributed throughout the body. It has been shown to promote M2-like polarization of macrophages [114] and has been effectively employed in the therapy of diverse illnesses [115–118]. Recent studies have revealed that MT-pretreated extracellular vesicles exert advantageous impacts on conditions such as renal ischemia–reperfusion injury and cerebral ischemia, among others. Furthermore, in the context of diabetic wound healing, MT has been indicated to exhibit beneficial outcomes [119–122].

Treatment with other chemical factors was also investigated, such as glycyrrhizin acid [123], kartogenin [124], thrombin [125], oridonin [126], and atorvastatin [127]. The collective evidence from these studies indicates that different chemical compounds can augment the restorative or immunosuppressive capacity of EVs by altering the secretome of mesenchymal stem cells. However, further research is required to ascertain the types of chemicals utilized, in addition to the optimal concentrations and pretreatment times, with a view to producing engineered EVs that facilitate wound healing.

Cytokine preconditioning

The approach of cytokine pretreatment is similar to that of chemical interventions, with the distinct characteristic being that the biological effects of cytokines can typically be detected. EVs mediate paracrine secretion, serving as a mechanism for cellular communication [128]. Cytokine-induced stimulation has been recognized for its ability to increase the efficiency of paracrine communication and control the expression and release of various therapeutic substances within EVs [87]. Studies have also demonstrated that cytokine pretreatment of MSC-exos can modulate immune responses. This adjustment involves nudging MSCs toward a less inflammatory phenotype and aiding in the shift of macrophages from the inflammatory M1 type to the anti- inflammatory M2 type [129–132]. These M2 macrophages, noted for their anti-inflammatory functions, are crucial in aiding the healing process during the inflammation phase of wound recovery [133, 134].

Inflammatory stimuli, for example, like tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interferon-γ (IFN-γ), have been shown to enhance the regenerative potential and anti-inflammatory ability of MSC-exos. TNF-α has been found to elevate miRNAs linked to the suppression of inflammation, thereby increasing the release of anti-inflammatory exos from MSCs, leading to the secretion of more anti-inflammatory MSC exosomes [129, 131]. IL-1β promotes the anti-inflammatory functions of MSC exosomes in osteoarthritis by upregulating miRNA-147b expression [135]. Additionally, Yang et al. [136] reported that IFN-γ also improved the ability of MSC-exos to suppress Th17 cell differentiation and alleviate colitis. Li et al. [137] used engineered macrophage-derived extracellular vesicles to create a bioactive hydrogel ink, aimed at enhancing wound healing. The EVs were obtained from M2 macrophages stimulated with interleukin-4 (IL-4), which promotes their anti-inflammatory properties. These EVs were incorporated into sodium alginate hydrogel precursors, resulting in a hydrogel that exhibits angiogenic effects, promotes endothelial cell migration, and facilitates the polarization of macrophages towards the M2 phenotype, providing a promising strategy for effective and personalized wound management.

Genetic engineering

For targeted transportation

EVs possess the ability to traverse the bloodstream and penetrate tissues, including crossing the blood–brain barrier (BBB). Therefore, enhancing their tissue-specific targeting capabilities is crucial for improving their therapeutic outcomes. Strategies have been employed to improve their targeting ability. Surface modifications directly alter EV surface molecules, augmenting their therapeutic effects and enhancing their targeting capabilities [138]. Genetic engineering of the source cells allows for the incorporation of targeted signaling molecules, enabling the precise engineering of EVs for optimized delivery to targeted tissues and lesions.

Genetic engineering techniques, including molecular cloning and lentiviral vector assembly, can be utilized to produce engineered EVs with tailored characteristics [139]. This procedure entails linking the genetical sequence of a guide protein or peptide to the genetical sequence of a chosen EV membrane protein, leading to the assembly of specific signaling proteins on the EV surface. Then, the parental donor cells are transfected with a plasmid that encodes the fusion protein, resulting in the displaying of the desired ligand on their surface. For example, Zhang et al. [140] induced the differentiation of human induced pluripotent stem cells (iPSCs) into endothelial cells (iPS-ECs) utilizing genetic engineering methodologies. These iPS-ECs inherently exhibit elevated expression of C-X-C chemokine receptor type 4 (CXCR4), a chemokine receptor instrumental in enabling precise delivery to endothelial cell populations. Employing a sequential extrusion protocol, the membrane-associated proteins of iPS-ECs, notably CXCR4, were effectively retained within the resultant nanovesicles (NVs). These engineered NVs emulate the intrinsic characteristics of natural EVs and were subsequently modified to incorporate dapagliflozin (DA) as a therapeutic payload. The strategic delivery of DA via CXCR4-enriched NVs markedly enhanced angiogenesis and expedited the healing process in diabetic wounds. This innovative strategy underscores the potential of genetic engineering in crafting EVs with tailored targeting and therapeutic properties, thereby advancing tissue regeneration in chronic wounds.

For cargo packaging

EVs can be loaded with many diverse cargo molecules to elicit different biological functions, including DNAs, RNAs, lipids, metabolites, and proteins. As of May 2024, the ExoCarta online database (http://www.exocarta.org) has cataloged 9769 proteins, 3408 mRNAs, 2838 miRNAs, and 1116 lipids identified in exosomes from different types of cells and organisms. Cargo loading into EVs can be categorized into endogenous and exogenous pathways [106]. This section will focus on endogenous pathways, while exogenous methods will be discussed subsequently.

Endogenous carriers, like viral vectors and plasmids [106], are genetically engineered to modify the source cells. These carriers are commonly employed to load endogenous molecules with therapeutic effects [138]. MiRNAs have important functions in many disease models and have been extensively studied [141–145]. Mounting evidence suggests that packaging endogenous proteins can augment the therapeutic efficacy of EVs in a variety of diseases [146–148].

For example, You et al. [149] found that encapsulated mesenchymal stem cells-EVs (EVs-L-PGDS) inhibited the progression of gastric cancer through the delivery of lipocalin-type prostaglandin D2 synthase (L-PGDS). For the therapy of chronic wounds, Huang et al. [150] investigated the construction of engineered small extracellular vesicles (sEVs) derived from MSCs that overexpress circular RNA derived from cyclin-dependent kinase 13 gene (circCDK13), which is downregulated in diabetic wounds. These engineered vesicles demonstrated a significant ability to promote the proliferation and migration of human dermal fibroblasts and human epidermal keratinocytes, thereby accelerating wound healing in diabetic models.

Modification of isolated-EVs

A more direct approach to modifying EVs involves processing them immediately following isolation, extraction, and purification to alter their contents (i.e. the exogenous pathways mentioned previously) to improve their biological functionality or confer them the ability to target transport. Overall, such approaches generally include incubation, physical or chemical treatment, and membrane fusion techniques.

Incubation

Co-incubation is a straightforward and facile way to load EVs with substances that have therapeutic effects. When EVs are co-incubated with a drug, the concentration gradient between the interior and exterior of the EV membrane drives the diffusion of the therapeutic molecules into the lumen [151, 152]. EVs loaded with the drug will then be obtained by purification and isolation [153]. For example, Salarpour et al. conducted a study wherein paclitaxel (PTX) was successfully loaded into exosomes derived from human glioblastoma U-87 cells for drug delivery purposes through the application of incubation techniques [154].

However, drug loading efficiency is often constrained when employing this technique for drug encapsulation, attributable to the lipid bilayer membrane of EVs and the physicochemical properties of the drug [155]. The interaction of the lipid membrane surface of the EV with the loading molecule, the hydrophobic properties of the target cargo, and the concentration gradient within and outside the EV will significantly affect the loading efficiency [156]. Therefore, it is challenging to control the loading inside the actual EV for cargo loading performed under this method.

Physical treatment

Extrusion

Extrusion represents another widely used technique [157–160]. This method involves isolating and assembling EV-like nanoparticles from the cytomembrane of the parental cell via a compression process utilizing polycarbonate membranes with sequentially decreasing pore sizes. Such EV-like nanovesicles exhibit similar properties as cell-secreted extracellular vesicles, such as size, shape, biology functions, and so on [161], thus circumventing the limitation of low yield and providing a method for relatively substantial scale production of extracellular vesicles.

For example, Yu et al. [162] utilized the extrusion technique to generate exosome mimetics (EM) from polymorphonuclear neutrophils (PMNs). This method markedly improved both the production and purity of these mimetics, with a notable focus on activated neutrophil-derived exosome mimetics (aPMNEM) that preserved the antimicrobial properties of PMNs. By integrating aPMNEM into an extracellular matrix hydrogel, they achieved a substantial enhancement in wound healing for diabetic rats, driven by increased collagen deposition, angiogenesis, and a reduction in bacterial infections. Recently, Liu et al. [163] employed the extrusion method to prepare biomimetic NVs from endothelial progenitor cells (EPCs), which exhibited high yield, rapid production, and inheritance of parental cell phenotypes. Furthermore, a multifunctional hydrogel was developed to enable sustained release of cyclic arginine-glycine-aspartic acid (RGD) peptide modified NVs while providing antioxidant and antibacterial properties. The hydrogel demonstrated excellent biocompatibility and effectively promoted diabetic wound healing by enhancing angiogenesis, scavenging reactive oxygen species (ROS), and inhibiting bacterial infection in a synergistic manner.

Electroporation

Electroporation is an extensively applied method for introducing exogenous cargoes into EVs [164]. By applying an electric field, it is possible to induce the formation of tiny pores within the phospholipid bilayer of nanovesicles, facilitating the entry of various small molecule substances into EVs through the force from the electric field, thereby enabling drug encapsulation. Additionally, these small pores are reparable, mitigating the risk of drug leakage or premature release. For example, a recent study employed electroporation to load small interfering RNA (si-TNF-α) into EVs, thereby generating engineered EVs (si-TNF-α-EVs). Following in vitro and in vivo experiments demonstrated that si-TNF-α-EVs significantly promoted macrophage polarization toward the M2 phenotype, markedly suppressed TNF-α secretion in wound exudates, and accelerated wound closure [165].

Acoustical method

Sonication, an acoustic-based technique, momentarily disrupts the structural stability of the EV membrane, thereby permitting therapeutic drugs to permeate into the vesicle interior [166, 167]. This method can restore membrane structure, and the shear forces generated by sound waves do not permanently affect the membrane contents [155]. Compared to simple incubation methods, utilizing this method for the delivery of stable molecules into EVs demonstrates increased loading efficiency [153]. However, it may not be optimal for the conveyance of specific therapeutic RNAs due to sonication-induced aggregation and degradation. Additionally, the drug may stick to the exterior surface EV or be incorporated into its interior, resulting in uneven loading and a biphasic drug release pattern. The drug is first released in rapid bursts from the surface, succeeded by a gradual release from the interior core [155].

Guo et al. [168] innovatively utilized ultrasound irradiation to controllably remove PEG by cleaving thioketal (TK) through ROS generated by Chlorin e6 (Ce6). They developed a variable stealth coating, CD63-penetrating 05 peptide-TK- methoxy PEG (CP05-TK-mPEG), which was tethered to exosomes via the binding of the CP05 peptide to the exosomal surface marker CD63. Under sonication, ROS produced by Ce6 enabled the controlled removal of PEG, followed by the encapsulation of bone morphogenetic protein 7 (Bmp7) mRNA into exosomes for targeted delivery to the omental adipose tissue (OAT) of obese C57BL/6 mice, successfully inducing browning and demonstrating therapeutic efficacy. This study provides significant technical insights into the development of an intelligent exosome-based drug delivery system. Its capabilities in controlled release and targeted delivery hold substantial potential for applications in skin wound healing, such as the precise regulation of the wound microenvironment through the delivery of pro-healing factors (e.g., growth factors or anti-inflammatory molecules), thereby accelerating tissue repair and regeneration.

Freeze–thaw cycles

In earlier studies, rapid freeze–thaw cycles were used as a liposome-forming reconstitution procedure [169, 170]. However, subsequent research has identified this approach as a mechanical strategy for the incorporation of cargo into EVs [171]. This procedure involves the cyclical heating of a blend containing EVs and the intended cargo from ambient temperature or 37 °C to conditions achieved with liquid nitrogen. A study by Hettich et al. investigated several drug-loading techniques targeting extracellular vesicles. Their findings indicated that the freeze–thaw technique was efficacious, yielding a satisfactory rate of drug encapsulation and concurrently maintaining the structural integrity status and biological functions of the small vesicles [172].

Hajipour et al. [173] loaded human chorionic gonadotropin (hCG) into exosomes using freeze–thaw cycles and sonication methods. The results showed that hCG-loaded exosomes had a greater influence on endometrial tolerance than hCG or exosomes used alone. Their findings imply that uterine fluid-derived exosomes could serve as efficient carriers for the therapeutic substances to the target cite. Nevertheless, despite its simplicity of execution, the freeze–thaw technique exhibits lower efficacy in drug encapsulation compared to sonication or extrusion methods [153, 155]. Additionally, it has been noticed that the freeze–thaw cycle may induce protein denaturation and EV aggregation, culminating in an increased heterogeneity in particle size distribution [174].

Chemical modification

Chemical modifications utilize various methods to display an array of native or artificial ligand receptors on the EV exterior. The chemical composition of EVs allows for the direct and rapid formation of covalent chemical bonds with different functional groups due to the existence of amine groups and carboxyl-terminated phospholipids on their surface [175].

The copper-catalyzed azidoalkane cycloaddition, commonly known as click chemistry, is an efficacious reaction for forming triazole linkages between alkanes and azides [176]. The presence of a catalyst markedly enhances the reaction rate of click chemistry, enabling its execution in diverse solvents such as water, alcohol, and dimethyl sulfoxide (DMSO) [177]. Zeng et al. [178] developed a novel nanoreagent for spinal cord injury (SCI) therapy by attaching biologically active Ile-Lys-Val-Ala-Val (IKVAV) peptides to the surface of M2-type macrophage-derived exosomes via click chemistry. These engineered exosomes specifically target the SCI site in vivo and enhance motor function recovery. Hao and colleagues [179] employed click chemistry to attach the collagen-binding peptide (SILY) to EVs derived from MSCs, substantially improving the retention and therapeutic effectiveness of EVs in injured tissue. The resulting SILY-EVs exhibited enhanced binding to collagen, effectively reduced inflammation, and stimulated muscle regeneration and blood vessel formation, underscoring their promise in tissue repair applications.

Apart from covalent bonding strategies, noncovalent modification approaches, including ligand-receptor binding [180], hydrophobic insertion [181], electrostatic binding [182], and anchoring peptides [183], have also been applied to the customization of EV membrane surfaces.

Membrane fusion technique

Membrane fusion technology leverages the inherent characteristic whereby the lipid bilayer membrane of EVs is capable of spontaneously merging with other membrane structures. This process allows for the alteration of the contents or structural arrangement of EVs, providing new possibilities for improving their cell-targeted therapeutic efficiency. As targeted drug delivery vehicles, liposomes are the most extensively employed nanoparticulate systems. They are vesicles with a bilayer membrane structure formed spontaneously from phospholipids dispersed in an aqueous medium [184]. The development of embrane Fusion Hybrid Exosomes (MFHE) introduces a novel potential for drug delivery systems by amalgamating liposomes and exosomes through diverse membrane fusion techniques [185].

Liang et al. [186] constructed a hybrid CAP-exo by fusing chondrocyte affinity peptide (CAP) with an exosome surface protein lamp2b terminal gene. They then fused CAP-exo with the liposome membrane to encapsulate the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) plasmid. The expression of matrix metalloproteinase 13 (MMP-13) in chondrocytes decreased significantly after the intervention of hybrid CAP-exo, which alleviated the damage to cartilage. In addition, Yang et al. [187] innovatively introduced a membrane editing technique that uses virus-analogous fusion exosomes to transfer membrane proteins with special functions directly into cell membranes. These studies, based on fusion EV-mediated delivery, broaden the application scope of membrane fusion technology, promote the targeting of EVs to different tissue types, and demonstrate its application potential in promoting tissue repair.

Therapeutic potential of unmodified and engineered EVs for wound healing

The skin, as the human body’s outermost protective barrier, serves a crucial role in defending against external pathogens and contributes to metabolic functions essential for homeostasis [25]. The skin is known to be anatomically organized into three primary layers: the epidermis, the dermis, and the subcutaneous layer. Wound healing is a multifaceted process that necessitates the coordinated interaction of a variety of factors, including diverse cells, proteases, growth factors, and extracellular matrix (ECM) [188]. In skin defect therapy, EVs hold promise for facilitating wound repair in different phases. This section presents a discussion of the therapeutic potential of unmodified and engineered EVs in the context of skin wound treatment.

Biological functions of unmodified EVs in different phases of wound healing

In general, the progression of wound healing is segmented into four phases: hemostasis phase, inflammatory phase, proliferation phase, and remodeling phase [189, 190]. The restoration of skin integrity involves the coordinated action of multiple cell types across a complex and sequentially organized process. The phases of wound healing are closely intertwined and the boundaries between them are not always clear [191]. As a result, skin repair is one of the most complex biological processes in the body. Figure 4 shows the main physiological events in the various stages of wound healing with the cells involved. It is worth noting that the tissue repair process in chronic wounds deviates significantly from the conventional “linear” model of sequential and overlapping stages. The different stages of wound repair may alternate and recur, while various sections of the same wound could concurrently be undergoing different stages of the healing process [192–194]. Therapeutic applications of exosomes have demonstrated superior efficacy compared to traditional interventions [35, 195] and influence all phases of the wound healing process [35]. They commonly participate in governing cell viability and differentiation, modulating immunological and inflammatory responses, and facilitating angiogenesis and tissue regeneration [196], including wound healing [197].

Fig. 4.

The main physiological events and key cellular participants involved in the process of wound healing. The edges of different stages within normal wound healing often lack clarity. In chronic wounds, the healing process’s stages do not adhere to a defined timeline, which means there may be the possibility of phase recurrence or extension, and some parts of the wound may be in different stages of healing, resulting in a non-linear healing process. The images’ contents are based on currently available knowledge

Hemostasis phase

Upon reaching a certain depth, skin injuries expose intravascular platelets to subendothelial collagen, initiating thrombin formation [198]. These resultant blood clots composed of platelets, erythrocytes, and ECM molecules, serve to safeguard the wound site [199]. Platelets release growth factors like platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), and TGF-β, among others [191]. These substances spread into the neighboring tissues, attracting monocytes by chemotaxis [200], which subsequently differentiate into macrophages. These macrophages then orchestrate a cascade of processes essential for wound healing [201].

EVs have been implicated in facilitating hemostasis during this phase. A study conducted by Hu et al. aimed to evaluate the effectiveness of gelatin sponges loaded with exos derived from human umbilical cord mesenchymal stem cells (hUC-MSCs) in promoting wound healing and stopping bleeding. The coagulation index test demonstrated its ability to promote coagulation. Additionally, the outcomes from full-thickness skin wound healing assays in Sprague–Dawley (SD) rats revealed improved healing in wounds treated with the exosome-infused gelatin sponges compared to controls. Thus, employing gelatin sponges containing exosomes derived from hUC-MSCs proves to be a safe and effective strategy for promoting hemostasis and accelerating wound healing [202]. It should be noted that there is currently limited research on EVs during this period, and researchers may need to invest more in the EV therapy of early-stage injuries.

Inflammatory phase

A series of myeloid cells, such as neutrophils, monocytes, and macrophages, play a major role in this period. Initially, neutrophils secrete chemokines that not only recruit cytokines from macrophages but also prompt fibroblasts to migrate to the injured site [199]. In particular, macrophages are indispensable in the wound healing continuum, and at the end of this stage, they change from M1 to M2, which has anti-inflammatory properties that promote wound repair [133, 134]. Moreover, the duration of this stage has an impact on the wound-healing effect. long-term inflammation is not conducive to effective wound healing, which may lead to scar hyperplasia, mainly in chronic or burn wounds [189, 203]. Therefore, effectively managing the transition of wounds from the inflammatory stage to the proliferative phase through various strategies is crucial for optimal healing [204], which is also the focus of many studies.

To date, EVs have been demonstrated to suppress of T-lymphocyte differentiation, activation, and proliferation, while concomitantly diminishing IFN-γ production [205]. Besides, they have been found to lower the levels of pro-inflammatory cytokines, including TNF-α, inducible nitric oxide synthase (iNOS), IL-1β, and IL-6 [206] and concurrently elevate the synthesis of the immunosuppressive cytokine IL-10 [207, 208]. Additionally, exosomes from adipose mesenchymal stem cells (AMSCs) also have been shown to decrease the production of inflammatory markers such as IL-6, TNF-α, CD14, CD19, and CD68, thus promoting the healing of wounds [209]. In addition, MSC-exosomes have the capability to adjust pknox1 protein expression through the transport of miR-223 contained within the exosomes, which triggers the polarization of macrophages to the M2-type, thereby aiding in wound recovery [52]. The intradermal injection of exos from menstrual blood-derived MSCs (MenSC-Exos) has also been shown to encourage macrophage transition from the M1 to the M2 phenotype [210]. Notably, A marked reduction in M1 macrophages and an increase in M2 type macrophages were observed in two different animal models treated with BMSC-Exos. Moreover, BMSC-Exos stimulated with melatonin (MT-Exos) activated the phosphatase and tensin homolog/phosphoinositide 3-kinase/protein kinase B (PTEN/PI3K/AKT) signal transduction pathway, leading to M2 polarization of macrophages, angiogenesis, and collagen formation, which in turn improved wound healing, mitigated persistent inflammation, and supported the progression from the inflammatory stage to the proliferative phase of tissue restoration [208]. Hao et al. [211] developed a gelatin-alginate hydrogel (GelAlg) integrated with reduced graphene oxide (rGO) and platelet-derived extracellular vesicles (pEVs). In a diabetic rat model, the GelAlg@rGO-pEV hydrogel significantly reduced the expression of inflammatory biomarkers and modulated immune responses thereby accelerating wound healing. Their findings suggested that the GelAlg@rGO-pEV hydrogel has the potential to serve as a novel wound dressing that effectively improves the healing process of diabetic wounds.

Proliferative phase

Throughout the proliferative stage, fibroblasts transition into myofibroblasts and synthesize a matrix rich in collagen, contributing to the tensile strength of the wound. Meanwhile, these cells release growth factors that not only stimulate the migration and proliferation of keratinocytes but also inhibit cellular migration via contact inhibition, thereby facilitating re-epithelialization. Additionally, the processes of angiogenesis during this phase ensure an adequate oxygen supply, ensuring essential nutritional support for the proliferation of migrating cells [199, 212, 213].

Exosomes have been demonstrated to facilitate angiogenesis through the positive modulation of vascular endothelial cell (EC) functions. Furthermore, MSC-exos exert a straightforward influence on the proliferative phase of wound healing, enhancing angiogenesis at the injury locus [214]. Exos derived from MSCs pretreated with pioglitazone (PGZ-Exos) have been shown to augment the angiogenic capacity of human umbilical vein endothelial cells (HUVECs) by triggering the PI3K/AKT/ endothelial nitric oxide synthase (eNOS) pathway [215]. Shabbir et al. also reported a notable increase in cell proliferation following the administration of exos from mesenchymal stem cells [216]. In the context of diabetic wounds receiving treatment with exosomes from human MSCs, there was a notable enhancement in angiogenesis, as well as in the proliferation, migration, and differentiation of fibroblasts, coupled with an improved reduction in fibroblast senescence, decreased scar width, and the formation of larger, more structured collagen deposits [217].

Adipose mesenchymal stem cell-exos (AMSC-Exos) can improve the migration and proliferation ability of keratinizing cells by activating the AKT/HIF-1α and Wingless/Integrated (Wnt)/β-catenin signal pathways [218, 219]. This action not only promotes angiogenesis but also contributes to the restoration of skin barrier functions and stimulates the production of large, regularly aligned, and densely distributed neo-collagen [209]. In addition, MSC-exos derived from the human umbilical cord Wharton’s jelly have been observed to modulate the function of human immortalized keratinocytes (HaCaT cells) by preventing the nuclear translocation of apoptosis-inducing factor (AIF) and the hyperactivation of poly(ADP-ribose) polymerase-1 (PARP-1), which contributes to the amelioration of full-thickness skin injury through improved re-epithelialization and angiogenesis [220]. Fetal dermal-derived MSC-Exos have also been reported to expedite wound closure in an animal model of full-thickness skin wounds, by boosting fibroblast motility and secretory functions via the activation of the Notch signaling pathway [221].

Remodeling phase

The remodeling phase, marking the conclusive stage of wound healing, extends over a period ranging from several months to years. This phase is distinguished by the programmed cell death of fibroblasts, myofibroblasts, and additional cellular populations, as well as the breakdown of the extracellular matrix [199]. The systematic and sufficient accumulation of collagen is crucial for effective wound healing [222]. Enzymes such as MMPs play an irreplaceable role throughout all phases of skin wound healing, with particular significance in the remodeling phase [223]. Fibroblasts, epidermal cells, endothelial cells, and macrophages degrade most type III collagen fibers by modulating MMP release [224]. Similarly, uncontrolled proliferation of myofibroblasts at the wound site during this stage frequently results in scarring. Therefore, managing the appropriate assembly of fibroblasts is also pivotal for achieving optimal wound healing outcomes.

BMSC-Exos have been evidenced to reinstate typical skin architecture in rats afflicted with full-thickness skin tissue injury models. Human umbilical cord MSC (hucMSC-Exos) also promotes phosphorylation of Yes-associated protein (YAP) in the Hippo pathway, thereby negatively regulating the Wnt4/β-catenin pathway and balancing the relationship between tissue regeneration and repair, and curtailing excessive cellular growth and collagen build-up during the remodeling phase [225]. Some studies have revealed that intravenous administration of ADSC-Exos can increase the type-III/type-I collagen ratio and TGF-β3/TGF-β1 ratio, inhibit the differentiation of fibroblasts into myofibroblasts, and lessen scarring at the position of incision [226]. These insights collectively highlight the indispensable function of MSC-derived exos in the extracellular matrix remodeling stage of wound repair.

Therapeutic effects of engineered EVs for skin defects

Unmodified EVs play a pivotal role in all stages of wound healing, demonstrating significant therapeutic potential through diverse mechanisms such as promoting cell proliferation, angiogenesis, and anti-inflammatory effects. However, their therapeutic efficacy is often limited by issues such as lack of target specificity, rapid clearance in vivo, and inconsistent biological activity. In contrast, engineered EVs offer significant advantages in wound treatment. Through modifications, engineered EVs can achieve targeted delivery to wound sites, prolonged circulation time, and optimized therapeutic outcomes. Additionally, engineered EVs can be tailored to address specific pathological mechanisms, such as chronic inflammation or abnormal scar formation, making them a more versatile and effective tool for advanced wound healing applications. Table 3 provides examples of research conducted in this field in recent years.

Table 3.

Research examples about tailored exosomes in the management of skin defects recently

References	Source of exosomes	Engineering methods	Disease indication	Biological functions
[285]	Rat adipose-derived stem cells	Genetic engineering	Diabetic wound	Promoted the proliferation and migration of fibroblasts, and the angiogenesis of endothelial cells
[229]	Human placenta-derived mesenchymal stem cells	Genetic engineering	Diabetic wound	Anti-inflammatory. Promoted collagen deposition and neovascularization
[248]	Adipose stem cells	Genetic engineering	Diabetic wound	Reduced inflammation and promoted angiogenesis
[232]	Adipose-derived stem cells	Hypoxia pretreatment	Diabetic wound	Increased the migratory, proliferative, and blood vessel regeneration potential of vascular endothelial cells
[286]	Umbilical cord mesenchymal stem cells	Blue light irradiation	Diabetic wound	Reduced the expression of inflammatory factors and apoptosis. Enhanced vascular neogenesis and matrix remodeling
[239]	Bone marrow mesenchymal stem cells	Electroporation	Normal wound	Promoted cell proliferation, collagen deposition, and neovascularization
[247]	Adipose mesenchymal stem cells	Genetic engineering	UV skin injury	Induced M2 type macrophage activation and inhibited inflammatory responses
[287]	Adipose-derived stem cells	Genetic engineering	Normal wound	Promoted collagen formation and angiogenesis
[246]	Mesenchymal stem cells	TNF-alpha-treated under hypoxia and encapsulated cationic antimicrobial carbon dots	Infected diabetic wound	Suppressed oxidative injury and tissue inflammation. Promoted angiogenesis. Eradicated bacterial infection
[237]	Milk-derived	Electroporation	Diabetic wound	Promoted angiogenesis
[288]	Mesenchymal stem cells	Selenium stimulation	Normal wound	Inhibited inflammation and pro-angiogenesis
[289]	Human adipose-derived stem cells	Genetic engineering	Skin explant	Promoted skin epidermal stem cell proliferation
[290]	Human embryonic kidney 293 cells	Genetic engineering	Diabetic wound	Promoted angiogenesis, fibrogenesis, and re-epithelization
[291]	Umbilical cord-derived mesenchymal stem cells	Hypoxia pretreatment	Normal wound	Reduced hypoxia-induced cell apoptosis

Promoting tissue regeneration

Angiogenesis is a critical and far-reaching restorative mechanism in the comprehensive tissue regeneration cascade at the wound location. A deficiency in the genes or proteins essential for this process, like vascular endothelial growth factor (VEGF), could hinder neovascularization, leading to delayed healing of wounds [227]. Genetic engineering techniques enable the modification of parental source cells to secrete EVs with enhanced pro-angiogenic properties. For example, targeted genetic modifications can amplify the presence of specific miRNAs and proteins within exosomes. A study by Tao et al. [228] illustrated that MSC exosomes with overexpressed miR126-3p can stimulate the proliferation, migration, and angiogenesis of human dermal microvascular endothelial cells. In a recent study [229], the investigators utilized phage display to screen for a silk fibronectin-binding peptide (SFBP) and subsequently generated SFBP-Gluc-MS2 (SGM) and pac-miR146a-pac fusion proteins. Engineered exos named SGM-Exos, miR146a-Exos, and SGM-miR146a-Exos were then isolated from genetically modified placental mesenchymal stem cells (PMSCs) expressing SGM, pac-miR146a-pac, or both. Subsequent studies revealed that SGM-miR146a-Exo@SFP exhibited the capacity to enhance wound healing, collagen deposition, and neovascularization.

Additionally, modifications to EV compositions through physical or chemical means can enhance their function in angiogenesis. A study found that blue light treatment increased the expression levels of miR-135b-5p and miR-499a-3p in exos derived from hUC-MSCs, thereby promoting the proliferation, migration, and angiogenesis of HUVECs [230]. Wu et al. utilized iron oxide nanoparticles and a magnetic field to stimulate exos from BMSCs. Their research revealed that this activation could enhance angiogenesis by increasing the expression of miR-21-5p, targeting SPRY2 gene, and initiating the PI3K/AKT and ERK1/2 pathways in wound repair [231]. In a recent study [232], a hypoxic environment was utilized to precondition ADSC-derived exosomes for the delivery of circ-Snhg11. The findings suggest that circ-Snhg11-modified ADSC-exosomes enhanced the migration, proliferation, and vascular regeneration of vascular endothelial cells compared to standard exosomes. Subsequent mechanistic investigations revealed that hypoxia-pretreated ADSC-Exo-embedded gelatin methacryloyl hydrogels (GelMA-HExo) facilitated the delivery of circ-Snhg11, potentially improving cell survival and preserving endothelial cell function through the activation of the miR-144-3p/NFE2L2/HIF-1α signal transduction pathway. This engineered stem cell exosome showed promise for therapeutic applications in enhancing wound healing by modulating angiogenic potential.

EVs, with their unique structure, can afford a protective milieu for biomacromolecules against enzymatic or other biological degradation [233–235]. This makes them an effective delivery platform for therapeutic substances that can reach the target cells, eliciting desired biological responses [155, 233]. Shi et al. have developed a clinical-grade exosome product, enriched with TGF-β. Such exosomes could promote cell proliferation and migration, skin organogenesis, and exhibit enhanced healing in a full-thickness ischemic wound model [236]. Yan et al. utilized milk-derived exos to transport miR-31-5p mimics for diabetic wound therapy. This delivery system stabilized miR-31-5p from degradation and improved angiogenesis at the wound site, thereby promoting wound recovery [237]. Electroporation has been used to prepare engineered exos of with miR-21-5p and has been shown to have a role in promoting the healing of diabetic wounds by promoting collagen deposition, angiogenesis, and re-epithelialization, thereby promoting the healing of diabetic wounds. Additionally, the expression level of CD31 and alpha smooth muscle actin (α-SMA) was increased with exosomes containing miR-21 compared to those without miR-21 [238]. In a recent study [239], electroporation was employed to encapsulate miRNA-542-3p into BMSCs-Exo. The results demonstrate that miRNA-542-3p-Exos can be uptaken by skin fibroblasts (HSFs) and human dermal microvascular endothelial cells (HMECs), stimulating their functionality and enhancing the proliferation, migration, and vasculogenesis of HSFs/HMECs both in vitro and in vivo. These findings suggest that miRNA-542-3p-Exo holds promise as a therapeutic strategy for enhancing cutaneous wound repair.

Low molecular weight pharmaceuticals can also be loaded into EVs presenting pro-angiogenesis capabilities. Pioglitazone (PZG), a drug commonly used to treat type 2 diabetes mellitus [240], was employed in a study by Hu et al. Pioglitazone-pretreated bone marrow MSCs were used to obtain PGZ-Exos. Cellular experiments showed that PGZ-Exos enhanced the survival and proliferative capacity of HUVECs under high glucose injury. Furthermore, PGZ-Exos improved in vitro migration, angiogenesis, wound repair, and VEGF expression of HUVECs. Mechanistic studies demonstrated that PGZ-Exos promoted the expression of p-AKT, p-PI3K, and p-eNOS proteins, and inhibited the expression of PTEN proteins. In vivo investigations employing a diabetic rat wound model revealed that pretreatment with pioglitazone enhanced the therapeutic potency of exos derived from MSCs, facilitating accelerated angiogenesis and diabetic wound closure by promoting collagen synthesis, ECM reconstitution, and upregulation of VEGF and CD31 expression. These findings indicate that this approach offers a promising new cell-free strategy for treating diabetic wounds [215].

Metallic nanoparticles have garnered significant attention for their application in defective skin regeneration, attributed to their roles in enhancing wound repair and suppressing harmful bacterial growth [241–243]. Li and colleagues investigated the effects of exos from MSCs with iron oxide nanoparticles on the wound healing process. They found that exosomes equipped with iron oxide nanoparticles promoted cellular proliferation, migration, and angiogenesis. Additionally, the application of magnetically directed navigation has been shown to improve their accumulation at the injury site, thereby expediting the wound-healing process [244]. Qian et al. employed ultrasound techniques to encapsulate silver nanoparticles (AgNPs) within hUCMSC-exos. The dressing based on Ag-Exo has comprehensive antimicrobial activity, moisturizing, and electrolyte-balancing properties, thus facilitating the repair of contaminated injuries [245].

Anti-inflammation

As described in previous sections, chronic inflammation significantly hinders the wound-healing cascade, and persistent localized inflammatory responses may lead to abnormal wound repair. Scholars have developed many ways to modify EVs aimed at modulating inflammatory responses, either through alterations in the parental cell culture conditions or by modifying the constituent contents of the EVs.

Sun and colleagues created an engineered exosome by pre-treating the parent cells in a hypoxic environment. The researchers used a two-dimensional reduced covalent organic framework coated with an antimicrobial immuno-engineered exosome to stabilize HIF-1α, resulting in anti-inflammatory M2 macrophage polarization, and exhibit bactericidal activity against Gram⁻, Gram⁺, and antibiotic-resistant bacteria. This approach enables an effective and holistic integrated treatment for diabetic wound management [246]. Lu et al. engineered lentivirus vectors carrying siRNA targeting nuclear factor kappa-B (NF-κB), a crucial gene associated with skin damage, to establish stable cell lines capable of producing abundant siRNA exosomes (si-ADMSC-EXOs) through lentiviral transduction of adipose-derived mesenchymal stem cells (ADMSCs). The following findings showed that si-ADMSC-EXOs effectively suppressed the expression of genes linked to skin damage, promoted the healing of injured skin tissue, and attenuated the production of inflammatory cytokines [247]. In a recent study, scientists isolated engineered exosomes derived from adipose stem cells with elevated expression of miR-132 (miR-132-exo). The research illustrated that miR-132-exo notably improved flap viability and expedited the recovery of diabetic wounds by mitigating inflammation, fostering angiogenesis, and inducing M2-type macrophage polarization via the NF-κB pathway. These results propose that miR-132-exo holds potential as a promising therapeutic intervention for managing diabetic wounds and disorders associated with inflammation [248].

For drug delivery applications, EVs can optimize the delivery of small molecules by elevating drug concentration in targeted cells and augmenting drug stability throughout circulation in the bloodstream and the healing continuum [235, 249]. Fan et al. recently embedded milk-derived extracellular vesicles containing polydopamine (PDA) into Schiff base cross-linked hydrogels through an extrusion process. The resultant hydrogel, imbued with engineered extracellular vesicles containing antioxidants, manifests potential as a prospective therapeutic strategy for wound repair and regeneration, fostering the proliferation, migration, and anti-inflammatory responses of 3T3 cells via the activation of the PI3K-AKT pathway [250]. In addition, Gondaliya et al. introduced miR-155 inhibitors into exos derived from MSCs in diabetic wounds. The combined application of the miR-155 inhibitor and MSC-Exos exhibits a synergistic enhancement in the migration of keratinocytes and anti-inflammatory effects [251].

Decreasing scar formation

Wound healing is an intricately orchestrated process that transcends the mere restoration of skin integrity. With the progression of human evolution into more sophisticated entities, the capacity for healing via regenerative modalities diminished. Instead, humans have adopted strategies aimed at arresting hemorrhage post-trauma and expediting the closure of acute wounds, albeit at the expense of scar formation [252]. Scarring ensues as a consequence of the healing of damaged skin, occasionally accompanied by alterations in skin pigmentation [253]. Persistent inflammation and the accumulation of myofibroblasts can culminate in the pathological hypertrophy of scars within the wound site. Such an overproduction of scar tissue may precipitate a fibrotic condition, characterized by the development of proliferative scars and keloid genesis. Hypertrophic scarring (HTS), a fibroproliferative disorder, commonly occurs following full-thickness skin defects or severe burns. It is characterized by the transdifferentiating of fibroblasts into myofibroblasts, which then produce an abundance of α-SMA, collagen type-I, and type-III, facilitated by the TGF-β/Smad3 signal transduction pathway [254]. Unlike HTS, which remains confined to the boundaries of the original injury, keloids proliferate beyond the initial wound margins. Therefore, the ultimate aim is to diminish scar formation or to achieve healing without scarring, ensuring the wound heals in a manner that is both functionally and aesthetically satisfactory.

Meng et al. designed an engineered exo encapsulating miR-141-3p, which was derived from adipose mesenchymal stem cells transfected with Lv-miR-141-3p. The study showed that this engineered exosome improved the distribution of fibroblasts and the orientation of collagen fibers, thereby diminishing the thickness of hypertrophic scars [255]. Similarly, another study by Yuan et al. showed that exosomes derived from human adipose-derived MSCs with overexpressed miR-29a suppress scar hyperplasia following burn injury by targeting the TGF-β2/Smad3 signaling pathway [256]. Furthermore, exosomes derived from mesenchymal stem cells that overexpress TNF-stimulated gene-6 (TSG-6) were found to attenuate inflammatory responses, and decrease scar formation [257]. All these results collectively suggest that engineered exosomes can effectively enhance their biological activities, improve the reparative outcomes, and mitigate scar formation.

Status and perspectives

A wound is delineated as a tissue trauma that disrupts the skin’s integrity and continuity, potentially leading to severe complications and even mortality [258–260]. Chronic wounds, characterized by failing to proceed through the natural healing process and remaining unhealed for an excess of one month [261], notably compromise the quality of life to an extent comparable with cardiac and renal diseases. Moreover, it is worth noting that the mortality rates among patients suffering from chronic wounds are akin to those observed in cancer patients. The etiology of chronic wounds includes aging, obesity, diabetes, sensory neuropathy, autoimmune disorders, and cardiovascular system diseases [262, 263]. By the year of 2025, approximately 1% to 2% of individuals in developed countries experience chronic wounds that remain unhealed, notwithstanding advancements in healthcare [264]. This issue presents a significant financial burden, with yearly costs in the United States reaching around $25 billion [264].

Meanwhile, the global market for wound dressing products is witnessing expansion [265]. According to Verified Market Research, the worldwide wound care market is anticipated to experience a compound annual growth rate (CAGR) of 4.61% from 2023 to 2030 [266]. This growth trajectory is primarily attributed to the rising incidence of wound healing complications among patients, which are linked to aging and the prevalence of chronic diseases [266]. Furthermore, healthcare systems are under strain due to the spread of COVID-19 and other infectious diseases, which has necessitated a re-evaluation of the existing wound care paradigms [28, 29]. Several studies have highlighted the risks faced by patients with chronic wounds who are infected with COVID-19, particularly those who are older or have pre-existing metabolic and physiological conditions like chronic obstructive pulmonary disease or renal dysfunction [267–269].

Therefore, chronic wounds are a global health challenge. Traditionally, primary care was responsible for wound healing [270]. Approximately 2500 years ago, Hippocrates introduced seminal concepts such as pus, a binary classification of wound types, and an early form of surgical intervention aimed at facilitating wound healing. These concepts, despite their simplicity, retain a degree of relevance today and underpin the therapeutic strategies currently employed. With the evolution of specialized wound care practices, there has been an emphasis on rigorous clinical training, research, and evidence-based methodologies to enhance the prognosis of wound healing in patients [271]. Regional application of diverse modalities is the principal treatment for dermal injuries due to the substantial exposed region of prolonged ulcers and the detrimental impacts of systemic pharmaceuticals. For example, debridement a critical technique in local wound management, entails the removal of necrotic tissue and foreign materials from the wound bed. Additionally, compression therapy, which encompasses the implementation of graded external pressure via customized dressings or pre-manufactured stratified compression bandage apparatuses, stands as the cornerstone treatment modality for venous leg ulcers [272–274].

Notwithstanding progressive improvements in the clinical management of chronic wounds, certain challenges remain intractable, including limited therapeutic efficacy and incongruent residence times [275–277]. EV interventions have garnered significant research attention as a substitute for cell therapy, which emerged concurrently with the advancement of nanotechnology. At present, the procedure to obtain high-quality EVs relies on assessing their size, density, and immunoaffinity through various methods, including ultracentrifugation, density gradient centrifugation, size-exclusion chromatography, polymer-based precipitation, microfluidic separation, and immunoaffinity capture. Among them, ultracentrifugation is considered the benchmark, widely adopted by researchers for the separation of EVs. However, an optimal method for extracting EVs that combines high yield, rapid production speed, high product purity, and cost-effectiveness has not yet been established. To compensate for the differences between different techniques, researchers need to use a combination of modalities according to their investigational needs.

The route of administration for isolated EVs can vary, encompassing intravenous, intraperitoneal, or subcutaneous injections, with the selected delivery method substantially impacting their in vivo bio-distribution [278]. However, upon entry into the bloodstream, EVs are swiftly cleared from the circulatory system, with a subsequent rapid elimination from blood vessels and distribution into parenchymal organs [279]. The plasma half-life is notably brief, ranging merely from 2 to 4 min [280]. Additionally, EVs are expeditiously cleared from tissue sites. Enhancing the persistence of EVs at the target location is pivotal for attaining the therapeutic dosage required for efficacy. As previously outlined, the intrinsic limitations of unmodified EVs can be reduced by employing an EV engineering strategy that can moderately enhance efficacy, yield and targeting precision. This provides a foundation for the clinical application of EV-based therapies.

Figure 5a shows the trend in the number of publications on the topic of exosomes in wound healing, alongside their geographical dissemination (Fig. 5b) up to the year 2023, utilizing data sourced from the Web of Science Core Collection database as of March 28th, 2024. Clearly, there has been a marked escalation in scholarly pursuits on this topic, with the leading sources of publication being mainland China and the United States. The propitious applications of exosomes in regenerative medicine [17] have also concurrently prompted a progressive rise in clinical trials involving exosome-based therapeutics [281]. The International Society for Extracellular Vesicles (ISEV) documented that the expansive intellectual property domain has incited a considerable influx of both interest and financial support for the clinical translation of the research in this area [282]. As of the year 2018, research related to exosomes has secured investments amounting to $250 million (USD) [283]. Currently, there are 235 clinical trials related to EV treatment registered on Clinicaltrials.gov, covering the treatment and diagnosis of a broad spectrum of pathologies. Table 4 presents a summary of clinical trials of EV therapies currently available on Clinicaltrials.gov for skin injury as of the time of writing. However, the widespread use of EV products in clinical practice remains uncommon [284] and there are no exosome-based therapies that have been approved for the treatment of skin defects in the United States or in China. Only Becaplermin gel (biologic) and two generally effective cellular therapies (Dermagraft and Apligraf; Organogenesis, Massachusetts) are approved by the Food and Drug Administration (FDA) of the U.S. for the treatment of refractory wounds. No other therapies have been approved by the FDA for the marketing applications of non-healing chronic wounds yet [261].

Fig. 5.

Worldwide publication overview on exosome research in wound healing. a The trajectories in the quantity of scholarly works from 2000 to 2023. b Geographic distribution of relative publications. The data was extracted from the Web of Science Core Collection Database on March 28th, 2024. The world map with publication data was created using the website www.mapchart.net

Table 4.

Clinical trials of extracellular vesicle therapies for skin injuries

Study title	NCT number	Status	Conditions	Interventions	Study type	Phase
Effects of Mesenchymal Stem Cell Supernatant on Prevention and Treatment of Skin/Mucosal Injury in Hematology Patients	NCT06599346	Recruiting	Mucositis; Hematopoietic Stem Cell Transplantation; Chemotherapy-Induced Mucositis; Radiation-Induced Mucositis	Other: Standard Care; Biological: MSC Supernatant + Standard Care	Interventional	Not Applicable
Phase 2a Multi-Center Prospective, Randomized Trial to Evaluate the Safety & Efficacy of Topical PEP-TISSEEL for Diabetic Foot Ulcers (DFU)	NCT06319287	Recruiting	Diabetic Foot Ulcer	Biological: PEP (Purified Exosome Product) / TISSEEL	Interventional	Phase 2
Pilot Study of Human Adipose Tissue Derived Exosomes Promoting Wound Healing	NCT05475418	Completed	Wounds and Injuries	Procedure: Adipose tissue derived exosomes	Interventional	Not Applicable
PEP on a Skin Graft Donor Site Wound	NCT04664738	Active, not recruiting	Skin Graft	Biological: 10% PEP; Drug: TISSEEL; Biological: 20% PEP	Interventional	Phase 1
Effect of Plasma Derived Exosomes on Cutaneous Wound Healing	NCT02565264	Unknown status	Ulcer	Other: plasma-derived exosomes	Interventional	Early Phase 1
Clinical Observation of Exosomes in Patients After Q-switched Laser Surgery	NCT06279039	Recruiting	Exosome; Skin Regeneration; Laser	Drug: Exosome liquid dressing	Interventional	Not Applicable
Randomized, Controlled, Multicenter Study of Extracellular Vesicles from Human Adipose Tissue Promoting Wound Healing	NCT06253975	Recruiting	Wound Heal	Biological: adipose tissue derived extracellular vesicles (AT-EVs); Drug: Hyaluronic acid	Interventional	Not Applicable
Safety of Extracellular Vesicles for Burn Wounds	NCT05078385	Recruiting	Burns	Drug: AGLE-102	Interventional	Phase 1
Autologous Serum-derived EV for Venous Trophic Lesions Not Responsive to Conventional Treatments	NCT04652531	Recruiting	Ulcer Venous	Other: Autologous extracellular vesicles from serum	Interventional	Not Applicable

Conclusion

Advancements in nanotechnology are driving innovative developments in the future generation of nanotherapies. EV-based investigations in the past decades have enhanced nanomedicine research by leveraging the diverse and versatile properties of EVs. Engineered EVs offer an innovative approach to improve the limitations associated with the low-scale production, reduced purity, and constrained efficacy observed in the application of traditionally sourced EVs. This objective may be realized through the modification of the cells responsible for EV production and their cultivation conditions, or by directly altering the EVs. Translating the most recent advances and technologies into clinical practice necessitates the dissemination of clinical trial findings, optimization of cost-effectiveness for market viability, and strict adherence to regulatory guidelines. In other words, the primary hurdle in advancing EV-based therapies lies in the transition from laboratory practices to clinically approved methodologies that emphasize safety, high fidelity, and operational effectiveness.

Author contributions

LC and YS organized and wrote the manuscript, designed and prepared illustrations. ZH guided and edited the manuscript. LY, SG, and CW revised the paper and provided financial support. LC and YS contributed equally to this article. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by National Key Research and Development Program of China (grant No. 2024YFA1107805), and Project of Liaoning Xingliao Talents Plan (grant No. XLYC2002103), and Joint Project of Science and Technology Plan of Liaoning Province (grant No. 2024-MSLH-5512024), and the Natural Science Foundation of Liaoning Province (grant No. 2024JH2/102600340), and the Support Program for Excellent Young Scholars of China Medical University.

Data availability

No datasets were generated or analysed during the current study.

Declarations.

Ethics approval and consent to participate

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Not applicable.

Competing interests

The authors declare no competing interests.