Insights into optimizing exosome therapies for acute skin wound healing and other tissue repair

Abstract

Exosome therapy holds great promise as a novel approach to improve acute skin wound healing. This review provides a comprehensive overview of the current understanding of exosome biology and its potential applications in acute skin wound healing and beyond. Exosomes, small extracellular vesicles secreted by various stem cells, have emerged as potent mediators of intercellular communication and tissue repair. One advantage of exosome therapy is its ability to avoid potential risks associated with stem cell therapy, such as immune rejection or stem cells differentiating into unwanted cell types. However, further research is necessary to optimize exosome therapy, not only in the areas of exosome isolation, characterization, and engineering, but also in determining the optimal dose, timing, administration, and frequency of exosome therapy. Thus, optimization of exosome therapy is critical for the development of more effective and safer exosome-based therapies for acute skin wound healing and other diseases induced by cancer, ischemia, or inflammation. This review provides valuable insights into the potential of exosome therapy and highlights the need for further research to optimize exosome therapy for clinical use.

Introduction

Acute skin wounds generally result from surgery, traumatic wounds, superficial burns, and abrasions, and can lead to complications such as wound infection, delayed healing or non-healing, and scar formation. These complications not only require significant medical resources but can also affect the mental health of some patients [1,2]. The severity of acute skin wounds is rising rapidly, making it a global public health concern. Therefore, the therapy and care of acute skin wounds is an urgent health issue.

Skin is the largest organ in the human body and it is exposed to various types of damage, such as burns, cuts, abrasions, and infections. There are several types of therapies that can help repair or replace damaged skin, including cell therapy and tissue-engineered skin. Cell therapy involves using stem cells or other types of cells to promote skin regeneration. Stem cells have the ability to differentiate into different types of cells, such as skin cells, endothelial cells, and stromal cells [3-6]. These adult stem cells can be obtained from various sources, such as bone marrow or adipose tissue. Cell therapy can also involve the use of growth factors or cytokines to stimulate cell growth and differentiation. Recent studies have demonstrated the effect of growth factors and cytokines, including platelet-derived growth factors, epidermal growth factor, fibroblast growth factors, vascular endothelial growth factor and transforming growth factor-β (TGF-β), and keratinocyte growth factors on therapeutic strategies in wound healing, which can significantly increase the level of neovascularization and the number of capillaries to promote angiogenesis and reduce scar formation at the wound site [7,8]. Tissue-engineered skin, on the other hand, involves creating a skin substitute using a combination of cells and biomaterials. The process typically involves seeding skin cells onto a scaffold made of biodegradable materials, such as collagen or hyaluronic acid. The scaffold provides a framework for the cells to grow and organize into tissue that can be transplanted onto the patient’s skin. Both cell therapy and tissue-engineered skin have shown promise in treating various skin conditions and injuries [9,10]. Our preliminary research also indicates that stem cells derived from mesenchymal tissues and kidney present in the urine can promote skin wound healing [11-13].

While stem cells have great potential for skin regeneration, they also have limitations and risks. One of the potential risks associated with stem cell therapy is the formation of tumors during the differentiation process, especially embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) [14-17]. What’s more, Min et al. discovered that dysplastic stem cells can drive the neoplastic transformation of precancerous gastric mucosa [18]. In addition, stem cell therapy may be limited by immune rejection [19-21], which occurs when the patient’s immune system attacks the transplanted cells as foreign [22]. A recent study explored the immunogenicity of iPSCs derivatives in allogeneic recipients. While iPSCs themselves are considered less immunogenic, the study reveals that their derivatives may still trigger an immune response, particularly if they are not properly modulated for immune evasion [19]. This can lead to the failure of the treatment and potential harm to the patient [23]. Furthermore, stem cell therapy can be time-consuming and expensive, as it requires the growth and differentiation of large numbers of cells in culture. This can limit the practicality and affordability of the therapy, making it less accessible to patients [24].

The extracellular matrix (ECM) plays a critical role in the repair of skin tissue. It is a complex network of proteins and other molecules that provide structural support and help regulate cellular behavior. Our previous studies have shown that skin ECM can better maintain skin-derived cells, and promote cell proliferation and differentiation, but their phenotype and function may change during ECM culture [25]. In skin tissue repair, xenogenous skin decellularized matrix serves as a scaffold for the migration and proliferation of skin cells, such as keratinocytes and fibroblasts. Skin ECM also contains various growth factors and cytokines that help regulate cell behavior and promote tissue regeneration [26].

The use of stem cell-derived exosomes as a novel therapy for skin wound healing is becoming increasingly important [27]. Exosomes are tiny vesicles ranging in size from 30 to 150 nm, which are released by cells and contain various proteins, lipids, and nucleic acids that can affect the behavior of other cells [28,29]. In various tissue-repairing processes, implanted exosomes regulate necrotic apoptosis, reduce renal tubular epithelial cell damage and enhance repair, enhance the function of the pancreas and provide immune isolation for pancreatic transplantation, and promote endogenous sperm recovery in non-obstructive azoospermic mice [30-32]. Recent studies have shown that exosome therapy has the potential to promote skin regeneration and reduce inflammation in various types of skin injury [33,34].

Stem cell-derived exosome therapy is suitable for several types of skin injuries, such as wounds, burns, scars, dermatitis, and aging-related skin changes. For example, exosome therapy promotes wound healing by stimulating the growth of new blood vessels, promoting cell proliferation and differentiation, and reducing inflammation [33]. Exosome therapy can also reduce the inflammatory response and improves tissue repair and regeneration in burn injuries [33]. For scars, exosome therapy helps reduce scar formation by promoting the remodeling of collagen fibers and reducing inflammation [35]. Moreover, exosome therapy reduces inflammation and promote skin regeneration in various types of dermatitis, such as atopic dermatitis and allergic contact dermatitis [36,37]. Finally, exosome therapy helps to reduce the appearance of wrinkles and fine lines, improve skin texture and tone, and promote the production of collagen and elastin in aging-related skin changes [38].

Although exosomes hold great potential in the treatment of skin injuries, their application is currently limited due to changes in isolation and purification, loading efficiency, low yield, and biological distribution in vivo. In addition, it is important to note that exosome therapy is still in the early stages of development for skin injury. Therefore, further research is needed to optimize exosome therapy, and its safety and efficacy for skin injury therapy needs to be thoroughly evaluate.

Exosome biology and potential applications in skin acute wound healing

Biomarkers of exosomes

Exosomes are nano-sized vesicles (with a diameter of 30–100 nm) with a bilayer lipid membrane structure. They are originated from the intima, stored in polyvesicles, and released outside the cell through plasma membrane fusion [39]. Exosomes carry a variety of bioactive contents such as nucleic acid and protein. They not only mediate signal transduction and information exchange between cells but are also involved in the pathophysiological processes of various diseases in the human body [28,39]. Exosomes contain four transmembrane proteins (CD9, CD63, CD81, CD82), membrane transport and fusion proteins (GTPases, annexins, flotillin), major histocompatibility complex (MHC Class I/Class II), and heat shock proteins (HSP70, HSP90) [40]. Surface proteins and internal biological substances (such as protein, mRNA, miRNA, and DNA) determine the specificity of exosomes [28,41].

Exosome delivery

Exosomes are small extracellular vesicles that are secreted by cells and are involved in intercellular communication. Exosomes can deliver various types of biomolecules, including proteins, lipids, and nucleic acids, to target cells [42]. The process of exosome delivery can be divided into three main steps [43,44].

(1) Biogenesis and packaging: Exosomes are formed by the release of intraluminal vesicles after the fusion of multivesicular endosomes (MVEs) with the plasma membrane. In the early stage, extracellular matrix or membrane proteins are encapsulated by the plasma membrane to form early endosomes, which form intraluminal vesicles and are encapsulated by MVEs over time [29,39,45,46]. During this process, specific proteins, lipids, and nucleic acids are selectively packaged into the exosomes; (2) Release and uptake: Once exosomes are formed, they are released into the extracellular space and can be taken up by neighboring or distant cells. Exosomes can be internalized by target cells through various mechanisms, including endocytosis, direct fusion, and ligand-receptor interaction with the plasma membrane; and (3) Targeting and function: Once exosomes are internalized, the biomolecules they contain can exert various effects on target cells. For example, exosomes can deliver miRNAs that regulate gene expression, proteins that modulate cell signaling pathways, or lipids that affect membrane properties [47].

Therefore, exosomes can serve as a delivery system for therapeutic agents such as drugs or nucleic acids [48], which can target specific cells and promote healing. Studies have suggested that exosomes derived from various cell types such as adipose-derived stem cells, mesenchymal stem cells can promote skin regeneration and repair [49]. These studies demonstrate that exosome therapy can promote wound healing, reduce scar formation, and reduce inflammation in acute skin injury. In sum, exosomes are well tolerated and stable, and have been considered as ideal nanocarriers that can penetrate deep tissues and evade immune cell attacks to deliver drugs to the injured site [44].

Mechanisms of exosome-mediated intercellular communication

Exosomes have the biological function of mediating intercellular communication. They can directly activate cell surface receptors through proteins and bioactive lipid ligands, and fuse with target cell membranes and target cells or act as signal bodies, transfer effector molecules and multifunctional signaling complexes to maintain normal cell physiological functions, i.e., immune surveillance, blood coagulation, stem cell maintenance, tissue repair, and communication functions [40]. When the normal biological function of exosomes suffers unbalanced, it will induce pathological changes, including tumor occurrence and metastasis, autoimmune diseases, neurodegenerative diseases, and HIV infection [29,40].

The involvement of exosomes in skin acute wound healing

Acute skin injury, such as a cut or burn, triggers a series of pathophysiological changes that are part of the body’s natural wound healing response. These changes can be divided into four overlapping phases: hemostasis, inflammation, proliferation, and remodeling [50-52]. Here is a brief overview of the pathophysiological changes that occur during each phase (Fig. 1).

Fig. 1.

Pathophysiological changes at different stages of skin wound healing. (A) At the hemostasis stage, platelets are activated and release various cytokines and growth factors, recruit immune cells and induce vasoconstriction. (B) In the stage of inflammatory response, macrophages and neutrophils gather at the site of injury, macrophages release inflammatory factors, and the M1 phenotype transitions to M2 phenotype to promote wound healing. (C) Endothelial cell vascularization and fibronectin migration promote collagen formation during the proliferation stage. (D) The main process during the remodeling stage involves the formation of type I and II collagen, as well as the reorganization of collagen and ECM. ECM, extracellular matrix.

(1) Hemostasis phase: This is the initial phase of wound healing that occurs immediately after the injury. The body’s first response is to stop bleeding and promote clotting. Platelets aggregate at the site of injury and release various cytokines, growth factors, and chemokines that promote vasoconstriction and recruit immune cells to the site of injury [51]. (2) Inflammatory phase: This phase begins within hours of injury and lasts for up to 5–7 days. During this phase, neutrophils, monocytes, and macrophages migrate to the wound site and remove debris, foreign particles, and pathogens [53,54]. These immune cells release various pro-inflammatory cytokines and chemokines that activate the immune response and recruit more immune cells to the site of injury [51]. (3) Proliferation phase: This phase begins around 3–5 days and lasts up to 3 weeks [54]. During this phase, fibroblasts migrate to the wound site and begin to synthesize collagen and other extracellular matrix components that provide structural support for the healing tissue [54]. New blood vessels form to supply nutrients and oxygen to the healing tissue [55]. Epithelial cells at the wound edges proliferate and migrate across the wound bed to cover the wound. (4) Remodeling phase: This phase can last for up to several months and involves the reorganization of the extracellular matrix and the remodeling of collagen fibers [56]. Collagen synthesis and degradation are balanced to strengthen and align the collagen fibers in the healing tissue. The wound site becomes less vascularized and less cellular as the healing progresses. Those pathophysiological changes are essential for the normal wound healing response.

Studies have shown that exosome signal transduction plays an important role in different stages of wound healing. Exosomes by themselves contain various bioactive molecules such as growth factors, cytokines, and nucleic acids, cannot only promote wound healing by inducing angiogenesis, cell proliferation and differentiation, and vascular remodeling by mediating signal communication between cells, but also play an effective therapeutic role as vehicles for the delivery of drug payload(s) [27,28]. Therefore, exosome therapy has shown promise for treating acute skin injury by promoting tissue regeneration and reducing inflammation. Here are some of the ways in which exosome therapy can potentially treat acute skin injury (Fig. 2).

Fig. 2.

The mechanism of exosomes therapy for acute skin wound. (A) Exosomes of different cellular origins, such as fibroblast, macrophage, stem cell and platelet etc. (B) The role of exosomes in wound healing of the skin includes alleviating inflammatory reactions, promoting proliferation and migration of fibroblasts, promoting angiogenesis of endothelial cells, and accelerating remodeling of extracellular matrix and collagen. (C) Exosomes accelerate skin wound healing and reduce scar formation.

Wound healing is a complex biological process involving multiple cell types and signaling molecules, including cytokines, to orchestrate tissue repair and regeneration. Exosomes, small extracellular vesicles secreted by many cell types, are increasingly recognized for their role in intercellular communication and tissue repair. These exosomes can carry bioactive molecules, including cytokines, growth factors, and microRNAs, modulating the wound healing process. The cascade of cytokine expression in wound healing is a dynamic and coordinated process involving multiple steps. Here is a simplified overview of how cytokines in exosomes are thought to participate in normal wound healing.

Exosomes in hemostasis phase

Platelets can release a variety of cell growth factors, cytokines, and extracellular matrix to promote vasoconstriction and play an important role in hemostasis. Blood contains a large number of exosomes, and platelet-derived exosomes are the most abundant in circulation. When platelet-derived exosomes in circulation are stimulated by inflammation and stress, their secretion increases [57]. Studies have shown that the surface coagulants of exosomes derived from platelets in circulation are 50–100 times more than activated platelets, and there are binding sites of coagulant promoters [58]. Therefore, platelet-derived exosomes can accelerate wound hemostasis.

In addition, platelet-derived exosomes are rich in platelet-derived growth factors (PDGF), fibroblast growth factor (FGF), transforming growth factor-β (TGF-β), and vascular endothelial growth factor (VEGF) [59]. Therefore, platelet-derived exosomes can enhance tissue regeneration through paracrine mode [60], and platelet-rich plasma exosomes can promote the healing of diabetic chronic skin wounds by upregulating collagen synthesis, inducing the proliferation and migration of endothelial cells and fibroblasts, and angiogenesis [61,62]. These studies highlight the role of platelet-derived exosomes in skin and vascular regeneration.

In conclusion, platelet-derived exosomes have been used in the field of skin wound healing. In addition, platelet-derived exosomes do not have nuclei, which can avoid the occurrence of teratogenesis. Currently, the downstream industrial manufacturing process, pathogen safety measures, quality control and release standards of platelet-derived exosomes are under development [57], which are worthy of further research.

Exosomes in inflammation phase

The wound healing process begins with an inflammatory response to remove debris and bacteria from the wound site. During this phase, immune cells and other cell types secrete pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6). Exosomes released by various cells can also contain these cytokines and deliver them to target cells, amplifying the inflammatory response.

In addition, exosomes can regulate immune responses by influencing gene expression and signaling pathways in recipient cells [29,48], and mediate the transformation of M1-proinflammatory phenotype to M2-anti-inflammatory phenotype to accelerate wound healing [63-65]. Cheng et al. has found that exosome PD-L1 can inhibit cytokine production of CD8⁺ T cells and suppressed CD8⁺ T cell numbers in spleen and peripheral lymph nodes to promote tissue repair [66]. Macrophage-derived exosomes can induce the reprogramming of macrophages from Ml to M2, which not only enhances the anti-inflammatory ability, but also accelerates the migration of fibroblasts and the tubular forming ability of vascular endothelial cells, thus promoting wound healing [63,67].

Exosomes in proliferation phase

During this phase, cell proliferation and tissue remodeling occur. Exosomes released from various cell types can carry cytokines such as transforming growth factor-beta (TGF-β), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF). These cytokines play an essential role in promoting cell proliferation, angiogenesis (formation of new blood vessels), and collagen synthesis. Mesenchymal stem cells derived exosomes (MSC-Exos) cultured from neonatal serum demonstrated superior therapeutic efficacy in skin wound healing by targeting endothelial cells through the regulation of the AKT/eNOS signaling pathway to enhance angiogenesis [68]. Additional studies have shown that human MSC-Exos promote wound healing by stimulating angiogenesis [69,70]. Therefore, exosomes from different cell origins have a positive effect on skin healing.

Exosomes also can promote the proliferation and migration of various cell types involved in wound healing, including keratinocytes, fibroblasts, and endothelial cells [65,71]. This can accelerate the formation of new tissue and promote re-epithelialization of the wound. Studies have shown that exosomes derived from human adipose stem cells promote the proliferation and migration of human dermal fibroblasts in a dose-dependent manner and by inducing the expression of genes involved in dermal cell proliferation [49]. MSC-Exo promotes the growth and migration of diabetic wound fibroblasts and the formation of blood vessels to promote wound healing through AKT, ERK1/2, STAT3 and other signaling pathways [72], and it can enhance the proliferation and migration of keratinocytes and fibroblasts by regulating their biological characteristics, thus promoting the healing of skin wounds [69].

Exosomes in remodeling phase

The final phase of wound healing involves tissue remodeling. Exosomes can deliver cytokines that modulate matrix metalloproteinases (MMPs) to regulate the degradation and remodeling of the extracellular matrix.

Collagen synthesis plays an important role in wound remodeling. Exosomes can stimulate the synthesis of collagen, a major component of the extracellular matrix [71,73,74], which provides structural support for the healing tissue. Human adipose MSC-Exo enhance collagen synthesis by downregulating MMP3 while promoting skin barrier function recovery [73]. Exosomes released from human pluripotent stem cell derived MSCs promote skin wound healing through type I and III collagen synthesis and angiogenesis [74]. In addition, fibroblast-derived exosomes can increase collagen synthesis, migration, and vascular endothelial formation in injured skin to promote wound healing [75].

Exosomes act as mediators of intercellular communication by transferring their cargo of cytokines and other bioactive molecules to target cells. This process allows cells involved in wound healing to communicate with each other, promoting a coordinated and regulated healing response. This evidence highlights the role of exosomes in various stages of wound healing. It is important to note that the exact composition and functions of exosomes in wound healing are still an active area of research, and our understanding of their role in different stages of tissue repair is still evolving. In addition, different wound types and individual patient factors may influence the specific cytokines carried by exosomes and their overall impact on wound healing.

Comparison of stem cell therapy and exosome therapy

In general, both exosome therapy and stem cell therapy have the potential for treating skin injury, and the choice of therapy may depend on the severity of the injury, the therapeutic goal, and the patient’s individual circumstances. Exosome therapy for acute skin injury has several potential advantages and disadvantages compared to stem cell therapy (Table 1), as follows.

Table 1.

Comparison of stem cell therapy and exosome therapy

	Stem cell therapy	Exosome therapy
Advantages	Extensively studied	No ethical concerns for ESCs/iPSCs
	Cell differentiation capacity	Less invasive
	Self-renewal	Fewer safety concerns
	Longer-lasting effect	No need for genetic manipulation or expansion
		Storage and transportation of exosome more easily
Disadvantages	Time-consuming, expensive	Shorter half-life
	Safety concerns	Require multiple doses or sustained release formulations
	Ethical concerns for iPSCs/ESCs	Failure to replicate the regenerative potential of stem cells in severe tissue damage
	Risk of immune rejection

Abbreviations: ESCs, embryonic stem cells; iPSCs, induced pluripotent stem cells.

Advantages of exosome therapy

Exosomes have regenerative potential and can promote tissue regeneration and healing in the wound bed, but less invasive compared to stem cell therapy that requests wound cleaning procedures. Injection of exosomes into the wound bed makes it an attractive option for patients who may not be suitable for more invasive treatments.

Importantly, exosome therapy avoids the ethical concerns associated with the use of embryonic stem cells or induced pluripotent stem cells. In addition, there is a reduced risk of immune rejection compared to other treatments as exosomes are derived from the patient’s own cells or from a compatible donor [76]. Furthermore, exosome therapy is generally considered to be safe, with minimal side effects reported in clinical trials to date.

Exosome can be isolated and purified from various sources, including mesenchymal stem cells, without the need for genetic manipulation or expansion. Exosome can be stored and transported more easily than living cells, allowing for more flexible treatment options [76].

Limitations of exosome therapy

Exosome therapy is a relatively new field, and further research is needed to determine the optimal dose, delivery method, and therapeutic potential of exosomes for different types of chronic skin injury. Exosomes may have a shorter half-life than stem cells and may require multiple doses or sustained release formulations to achieve optimal therapeutic effects [77]. Exosomes may not be able to replicate the regenerative potential of stem cells, especially in cases of severe or extensive tissue damage. In addition, currently, the availability of exosomes for clinical use is limited, which may restrict the widespread use of exosome therapy [78]. Furthermore, there is currently no standardized protocol for producing, isolating, and characterizing exosomes, which can lead to variability in the quality and effectiveness of exosome therapy [42,78]. Finally, exosome therapy can be expensive, which may limit access for some patients.

Advantages of stem cell therapy

Stem cell therapy has been extensively studied and has demonstrated a high potential for tissue regeneration and repair in preclinical and clinical studies. Stem cell can differentiate into multiple cell types and have the ability to self-renew [79], making them a versatile tool for tissue engineering and regenerative medicine [80]. Stem cell therapy may have a longer-lasting effect compared to exosome therapy.

Disadvantages of stem cell therapy

Stem cell therapy requires the isolation, culture, and expansion of living cells, which can be time-consuming, expensive, and may have safety concerns [81,82]. Stem cell therapy may have ethical concerns associated with the use of embryonic stem cells or induced pluripotent stem cells. In addition, stem cell therapy may have a risk of immune rejection, requiring immunosuppressive therapy in some cases [81,82].

Optimization of exosome therapy for skin acute wound healing

Exosome therapy has shown potential for promoting skin regeneration and reducing inflammation in various models of skin injury. We proposed the strategies to optimize adult stem cell therapy to enhance greatly for tissue regeneration [80]. Similarly, exosomes are a promising therapeutic tool, exosome optimization can better promote tissue regeneration and accelerate wound healing, but optimizing their yield and quality is crucial to the success in the improvement of their therapeutic efficacy and makes exosomes a more viable option for clinical use (Table 2).

Table 2.

Optimization of the yield and quality of exosomes as well as exosome therapy

Optimizing their yield	Optimizing their quality	Optimizing their therapy
Cell source	Purity	Timing
Culture conditions	Cargo loading	Dose
Isolation methods	Targeting	Frequency
Identification	Characterization	Interval
Storage conditions	Stability	Route of administration

First, optimizing the yield of exosomes for therapy requires careful consideration of the cell source, culture conditions, isolation methods, characterization, and storage conditions (Fig. 3). By optimizing these factors, researchers can improve the yield and quality of exosomes and enhance their therapeutic potential.

Fig. 3.

Optimization of exosomes yield and therapy for skin acute wound healing, including optimization of exosome sources, culture conditions, storage conditions, isolation, genetic engineering and optimal does, timing, frequency, administration routine of exosomes.

Optimizing exosomes yield

Selection of exosome sources

The yield and quality of exosomes can vary depending on the cell type used for their isolation. Exosomes can be derived from blood, urine and a variety of cell types [28], including stem cells [83], fibroblasts [84], immune cells [85], etc. Different cell types produce exosomes with different cargoes, so it is essential to select a cell type that can secrete exosomes with the desired therapeutic payload. Stem cells derived exosomes (SC-Exos) include adipose-derived exosomes (AD-Exos), human umbilical mesenchymal stem cells derived exosomes (hUMSC-Exos), bone mesenchymal stem cells derived exosomes (BMSC-Exos), etc. Hence, in addition to containing abundant biomolecules of donor cells, such as RNA, DNA, nucleic acid, lipids, metabolites, and cytosolic, SCExos also contain plentiful growth factors, such as TGF-β, fibroblast growth factor, vascular endothelial growth factor, and neurotrophic factor, which can deliver a large number of functional biological molecules to the recipient cells to do their job. For example, human AD-Exos accelerate the healing of full-layer skin wounds in mice by reducing macrophage infiltration and pro-inflammatory cytokine secretion and increasing the formation and proliferation of new blood vessels [86,87]. Human amniotic fluid derived exosomes can accelerate the wound healing in the full skin wound rats, improve the regeneration of hair follicles, nerves, and blood vessels, and the natural distribution of collagen, and increase the proliferation of skin cells, as well as inhibit the TGF-β signaling pathway by targeting TGF-β receptors to reduce the formation of fibrosis scar [88]. Similarly, epidermal stem cells derived exosome specific microRNA can inhibit the activity of TGF-β1 and its downstream genes to promote wound healing and reduce scar formation [89]. Xu et al. summarized the therapeutic effects of SC-Exos in wound healing, indicating that SC-Exos are small size, high efficiency, low immune repulsion, and have more biological functions, which have significant advantages in promoting wound healing [90]. Recent research suggested that skin cells can release extracellular vehicles (EVs) to regulate intercellular skin communication [91]. Wang et al. described the therapeutic role of exosomes derived from specific skin cells in skin wound healing, and found that exosomes derived from epidermal keratinocytes, epidermal stem cells, and dermal fibroblasts play a unique role in promoting skin wound repair and can reflect cross-talk between cells in the wound healing environment, but the potential risks and more still need to be clarified [91]. Inflammation plays an important role in the process of wound healing. Macrophages are the main immune cells in the process of wound healing and play a key regulatory role in the inflammatory and proliferative stages of skin wound repair. Studies have shown that M2-macrophage-derived exosomes promote angiogenesis in vitro by targeting PTEN via miR-21 to activate the AKT/mTOS signaling pathway, and significantly promote angiogenesis, reduce scar formation, and accelerate wound healing in a mouse full-layer skin injury model [92].

In sum, exosomes from different cell sources have been proven to promote and accelerate wound healing during acute skin wound healing, but most of the current research is still focused on exosomes from stem cells, and some of the studies have entered clinical trials. Although skin- or immune-cells-derived exosomes may promote acute skin wound healing, the underlying mechanisms and clinical applications require further investigation. Therefore, SC-Exos show greater advantages and clinical applications in the treatment of acute skin wound healing.

Optimizing cell culture conditions

The culture conditions of the cells used for exosome production can also influence the yield and quality of exosomes. Optimizing cell culture conditions, such as pH, oxygen concentration, and nutrient availability, can enhance exosome production.

Cell starvation

It has been shown that depletion of the culture conditions required for cell growth can promote exosome biogenesis. Oxygen, glucose, and serum deprivation are common starvation methods that can increase exosome biogenesis. Serum deprivation of MSC-Exos increased 22 times in the delivery efficiency of exosomes compared with control [93]. In addition, Jeremy et al. suggested that serum has an inhibitory effect on exosomes production, and serum-free Opti-MEM is the optimal cell culture media for exosomes production [94]. Furthermore, hypoxia also effect exosome biogenesis. For instance, hypoxic can stimulate the biogenesis of exosomes derived from adipose and stem cells, etc. Gonzalez-King et al. overexpressed hypoxia-inducing factor-1a (HIF-1a) in MSC by transduction, and overexpressed HIF-1a stimulated stem cells to produce more exosomes [95]. Stem cells are more bioactive and can secrete more exosomes when cultured in hypoxia environment [96]. Overall, serum-free Opti-MEM promotes exosome biogenesis due to the combination of physical, molecular, and transcriptional effects. In the future, optimizing its production can be developed to exert more therapeutic effects of exosomes.

pH and others

Exosomes are also affected by pH. Researches have suggested that the loss of exosome concentration at pH 4 and pH 10 was more than at pH 7; moreover, exosomes stored at pH 4 degrade faster than at pH 10 [97]. Moreover, studies have observed that MSC-EV production depends on cell seeding density, per cell between MSCs seeded at 1E2 or 1E4 cells/cm² for P2, P3, P4, and P5 MSCs measuring ~126-fold, ~152-fold, ~201-fold, and ~126-fold, respectively; and increased EV collection frequency can augment EV production. When the inoculation density was fixed, increasing the collection frequency could increase the total exosomes by 2.0–2.4 fold [98].

Optimizing the isolation of exosomes

At present, the traditional separation and purification methods of exosomes have some shortcomings. There are several methods available for exosome isolation, including ultracentrifugation, size-exclusion chromatography, and polymer-based precipitation, etc., and we showed the pros and cons of different separation methods (Table 3).

Table 3.

Exosome isolation techniques and their comparison

Isolation methods	Advantages	Limitations	Sample matrix
UC	Golden standard	Time consuming	Cell culture medium Serum Urine
	Easily required	High requirements for equipment	Serum
	Simplicity of operator	Decreased in biological activity	Urine
		Limited mass production
Differential centrifugation	Rapid	Heterogeneity	Cell culture medium
	Mass production	Easy to drain	Serum
			Urine
UF	Rapid	Protein contamination	Cell culture medium
	Simplicity of operation	Exosomes are damaged	Serum
			Urine
			Cerebrospinal fluid
Immuno-isolation	Rapid	Additional separation and purification are required	Cell culture medium
	High purity and specificity	Not suitable for mass generation	Serum
Polymer precipitation	Mass production	Decreased purity	Cell culture medium
	Simplicity of operator	Protein contamination
		Expensive kit
SEC	High purity	Co-separation of proteins with similar diameters	Cell culture medium
	Commercial kits available	Not satisfy the downstream application	Serum
	High productivity		Urine
			Cerebrospinal fluid
			Saliva, etc.
AF4	High purity	High requirements for equipment and personnel	Cell culture medium
	High efficiency	Limited mass production	Serum
	Identify subset		Urine
Ion exchange	Simplicity of operator	Unknown	Cell culture medium
	High purityy
Microfluidic technology	Rapid	Not suitable for mass generation	Cell culture medium
	Save samples and reagents	Methods need to be further standardized	Serum
	High purity and efficiency

Abbreviations: UC, ultracentrifugation; UF, ultrafiltration; SEC, size-exclusion chromatography; AF4, asymmetric flow field-flow fractionation.

Traditional methods are density-based capture using ultra or differential centrifugation [99], immunoaffinity based capture using specific membrane proteins (e.g., CD9, CD63, and CD31) [100], and size based capture using volume exclusion chromatography, such as ultracentrifugation (UC), size-exclusion chromatography (SEC), ultrafiltration (UF), precipitation, etc. Although exosomes separation technology is constantly updated, there are some shortcomings in the separation and purification process of exosomes, such as low purity, contamination, coexistence of immunoglobulins, vesicle destruction and heterogeneity, which affect the biological function of exosomes.

Therefore, some studies have optimized SEC, using longer columns (56 mm to 222 mm), reduced protein and immunoglobulin contamination by 90% without affecting EV particle size distribution and number, and improved EV purity [101]. In addition, 2D SEC (including 2 columns) can be used for further isolation of exosome subpopulations, which has been achieved in the isolation of urine exosomes [102]. However, the separation of EV subpopulations by SEC separation techniques above is limited, which hinders characterization of their molecular composition and biogenesis. Recent studies describing new isolation strategies for exosomes show great progress in harvesting high quality EVs with increasingly convenient operating steps and high-level devices. For example, the methods of microfluidics, asymmetric flow field-flow fractionation (AF4), ion charge and combined multi-step methods are becoming increasingly popular [103-105].

Flow field-flow fractionation

Flow field-flow fractionation (FFF) is a size-based exosome separation technique, of which asymmetric flow field-flow fractionation (AF4) is most common in FFFS. David et al. [106] successfully isolated and identified EV subsets with asymmetric flow field-flow fractionation (AF4), thus identifying their heterogeneity, and compared other isolate methods, AF4 can obtain high purity, efficiency and integrity, but the technology has high requirements for equipment and personnel and fail to dispose large volume sample [103].

Microfluidics

Microfluidics, a separation technology that can integrate separation and detection functions into a single chip, is widely used for the separation and detection of micro-nanoparticles, biomarkers, cells, and proteins. The separation of exosomes by microfluidic chip technology is mainly related to the size, density, and surface antigen of exosomes. Current microfluid-based exosome separation techniques include capture, filtration, magnetic separation, and acoustic fluid separation [104]. (1) Capture: The capture is mainly to modify the antibodies on the inner wall of the microchannel, and anti-CD63 and CD81 antibodies are often used for exosome capture [107]. Other studies have shown that it can be optimized, such as filling microchannels with proteins, novel GO/polydopamine, etc., which can improve the separation of exosomes [108]. (2) Filtration: Microfluid-based filtration is similar to traditional ultrafiltration in that the microfluid-based filtration chip is driven by direct current to separate exosomes from plasma. Microfluid-based filtration can achieve high throughput, but its purity is limited. (3) Magnetic: Magnetic separation begins with the capture of exosomes by magnetic nanoparticles modified with antibodies, and then magnetically separates the resulting exosomes [109], mainly used for cell separation, can achieve high specificity, high throughput, and so on [104]. In addition, compared with UC, Magnetic bead-mediated selective adsorption strategy can obtain more than 2 times the yield and relatively stable purity, could meet the requirements of various EV-associated downstream applications [110]. (4) Acoustic fluid: Acoustic fluid separation is the separation of exosomes of different sizes by acoustic force in the environment of sound field, which is often used in cells culture [104].

Ion exchange

Ion exchange is a charge-based separation technique in exosome separation mainly through the interaction between a negatively charged EV membrane component and a positively charged functional group or cation anion exchanger. The team of Hiroshi Shiku used an anion-exchange method to obtain high-performance exosomes, and showed that it is suitable for the large-scale separation of bioactive exosomes and micro vesicle (MV)-like EVs as a cargo for dangerous nucleic acids at high-purity [111]. The all-in-one nanowire-integrated well plate assay system can capture and analyze EVs based on the surface charge of vesicle membranes, especially in EV-mediated tumor-microenvironment crosstalk [112,113]. But ion exchange is challenging for exosome separation in blood or plasma, which contains other charged materials.

In addition, the combined exosome isolation technology is recommended because it clearly outperformed single exosome isolation method to obtain higher purity and quantity, such as UC combination with microfiltration, SEC combined low-speed centrifugal method, combination of iodixanol density gradient ultracentrifugation and bind-elute chromatography [114-116].

Overall, the method of isolation and purification can affect the purity and quality of the exosomes, and it is important to use standardized and validated methods to ensure consistent and high-quality exosomes. Each method has its own advantages and limitations, and choosing the appropriate isolation method can optimize the yield and purity of exosomes.

Selection of exosome identification methods

Proper identification of the exosomes is crucial to ensure their quality and efficacy. Techniques such as nanoparticle tracking analysis (NTA), electron microscopy (EM), flow cytometry (FCM), and western blotting (WB) can be used to characterize the size, morphology, and concentration of exosomes. EM can identify the morphology of exosomes; ANT can track the distribution of exosomes; FCM and WB can identify molecular phenotyping with high sensitivity and specificity [117]. However, each identification method has certain disadvantages, such as: transmission electron microscopy destroys the morphology of exosomes in the identification process; NTA shows poor sensitivity, low efficiency; and WB takes a long time [118]. Therefore, combination of the four methods can better characterize the characteristics of exosomes and improve the quality.

Engineered exosomes for wound healing

Exosomes are promising therapeutics for skin injury due to their ability to modulate cellular processes such as inflammation, proliferation, and differentiation. However, their efficacy may be limited by factors such as low bioavailability, poor targeting, and short half-life in vivo. To address these limitations, researchers are exploring the use of engineered exosomes to improve therapeutic outcomes for skin injury, and we summarize some methods for optimizing exosome quality, as follows (Fig. 4, Table 4).

Fig. 4.

Engineered exosomes for skin wound healing, including surface and genetic engineering, precondition and loaded.

Table 4.

Engineered exosomes better improve skin wound healing

Methods	Mechanism	Outcomes	References
Surface engineering	Modified with the surface antibodies	Modulate inflammatory response and promote skin tissue repair in immune cells, keratinocytes or fibroblasts	[44,119,120]
Genetic engineering	Modify donor cells cargo	Overexpressed or loaded with growth factors or miRNA can promote cell proliferation and differentiation, angiogenesis, and extracellular matrix formation	[121-128]
Hydrogels combined with exosomes	Exosomes are transported to the site of injury by hydrogels	Loaded Exos on hydrogel facilitate cell proliferation and accelerate collagen synthesis to promote wound healing	[129,130]
Preconditioning	Preconditioning donor cells	ATV-pretreated MSC-Exos upregulating miR-221-3p and AKT/eNOS pathway to accelerate diabetic wound repair	[131-137]
		Fe3O4 nanoparticles preconditioning BMSc-Exos upregulating miR-21-5 to promote wound healing
		HGF-ADSC-Exos promote wound healing by increasing neovascularization

Abbreviations: Exos, Exosomes; ATV, atorvastatin; MSC-Exos, mesenchymal stem cells derived exosomes; BMSC-Exos, bone mesenchymal stem cells derived exosomes; HGF-ASC-Exos, exosomes from hepatocyte growth factor-modified human adipose mesenchymal stem cells.

Surface engineering

One approach to engineering exosomes for skin injury is to modify their surface with targeting ligands or antibodies that can enhance their uptake by specific cell types in the skin. For example, exosomes can be modified with antibodies that target specific markers on immune cells, such as macrophages, to modulate the inflammatory response and promote tissue repair [119]. Similarly, exosomes can be modified with peptides or proteins that target specific cell types in the skin, such as keratinocytes or fibroblasts, to promote wound healing and tissue regeneration [44,120].

Genetic engineering

Engineered exosomes for skin wound are transfected into donor cells with specific proteins, growth factors, or small molecules to modify their cargo to enhance their therapeutic efficacy. Studies have shown that exosome-based genetic modification has higher sensitivity and specificity for the treatment of certain diseases and can improve the efficiency of drug delivery. For example, exosomes can be overexpressed or loaded with growth factors such as TGF-β or PDGF, which can promote cell proliferation and differentiation, angiogenesis, and extracellular matrix formation [121,122]. Exosomes can also be loaded with small molecules such as microRNAs or other non-coding RNAs that can modulate gene expression and promote tissue regeneration [123-126]. For instance, Chen et al. have found that AD-Exos can highly express miR-21 in process of mouse full layer wound healing, miR-21 plasmid was used to overexpress miR-21 by transfection of HaCaT cells (representative of keratinocytes in vitro), and miR-21 can promote the migration and proliferation of the keratinocytes via PI3K/AKT signal pathway to improve wound healing [127]. However, the modification of exosome genes is mainly used in cancer research, and more investment is needed to research skin wound healing in the future.

Hydrogels combined with exosomes

Researchers are exploring the use of exosome-based hydrogels or scaffolds for skin injury [138]. These hydrogels or scaffolds can be engineered to release exosomes in a controlled manner over time, providing a sustained therapeutic effect [67]. Furthermore, these hydrogels or scaffolds can be designed to mimic the structure and function of the extracellular matrix in the skin, providing a favorable environment for tissue regeneration. In addition, recent studies have shown that a YARA peptide based equipping technique can enhance the cargo carrying potential of exosomes. YARA peptide covalently conjugated with mammalian miR-21-5p to form the conjugate YARA-miR-21-5p and incubated with mesenchymal stem cells-derived exosomes, which significantly enhanced the proliferation, migration of fibroblasts in human and mouse wound healing [129]. Zhu et al. has suggested that loaded human umbilical mesenchymal stem cell (UMSC) derived exosomes on hydrogel can facilitate cells proliferation and accelerate collagen synthesis to promote wound healing through upregulating oxidative phosphorylation-related proteins in mitochondria [130]. Therefore, loading exosomes on hydrogels may be a potential treatment strategy for wound healing.

Preconditioning

Donor cells preconditioned using drugs, factor, and hypoxia, etc. can accelerate skin wound healing. For example, exosomes derived from atorvastatin-pretreated mesenchymal stem cell accelerate diabetic wound repair by enhancing biological function of endothelial cells and angiogenesis via upregulating miR-221-3p and AKT/eNOS pathway [131]. BMSC-Exos preconditioned with Fe₃O₄ nanoparticles and a static magnetic field can improve angiogenesis and fibroblast function by upregulating miR-21-5 to promote wound healing [132]. In addition, growth factor-stimulated exosomes also can facilitate skin wound healing. For example, exosomes from hepatocyte growth factor-modified human adipose mesenchymal stem cells can reduce exudate in early stage, and significantly promote wound healing by increasing neovascularization in wound tissue, and the mechanism may be related to activate PI3K/Akt signaling pathway [137].

Electrical stimulation

Electrical stimulation (ES) is a non-invasive operation that can stimulate cell differentiation, migration, proliferation, and so on. ES as a physical stimulator can regulate cellular behavior for applying to regenerative medicine and tissue engineering [139]. Studies have found that ES can increase EV secretion and cargo loading [140,141]. However, different cells receive different electric fields, which can change the size, concentration, and cargo of exosomes. In addition, the application of electric field can also impair the normal physiological function of cells. Therefore, the intensity of the electric field needs to be controlled according to different cell types. In the future, the role of electrical stimulation in promoting skin wound healing needs to be further explored.

Ultraviolet B

Ultraviolet B (UVB) can stimulate the generation of exosomes by increasing HaCaT exosome secretion in dose- and time-dependent manners, and promote the expression of miRNA, especially miR-126, in skin cells-derived exosomes in diabetic mice [142]. However, UVB does not always promote the treatment of skin diseases. It was found that UVB could stimulate the secretion of exosome miR-769-5p in human skin fibroblasts, but miR-769-5p could aggravate the oxidative damage of human skin fibroblasts and accelerate cell apoptosis, while inhibition of miR-769-5p can reverse those effects [143]. Therefore, more experiments are needed to further confirm the effect of UVB stimulation on exosomes.

In addition, exosomes secreted from hypoxic-preconditioned mesenchymal stem cells show great therapeutic potential in the treatment of diseases [144]. Hypoxic microenvironment plays a negative role in wound healing, and prolonged hypoxia can also lead to delayed wound healing. Hypoxic adipose-derived exosomes can regulate the function of cell metabolism, growth and transformation factors by upregulating and downregulating the expression of miRNA related to skin healing, and promote wound healing and inhibit inflammation through PI3K/AKT signaling pathway [133]; and compared with MSC-Exo, hypoxic MSc-Exo has greater potential to promote angiogenesis, cell proliferation and differentiation [134]. Besides, BMSC-Exos facilitates human umbilical vein endothelial cells proliferation, migration and angiogenesis by increasing the expression of high mobility box 1 protein to stimulate JNK signaling pathway [135]. Exosomes derived from hypoxic umbilical cord mesenchymal stem cells could induce the expression of miR-125b, which targeted inhibition of the expression of tumor protein p53 inducible nuclear protein 1 for increasing the proliferation and migration of endothelial cells, and inhibiting cell apoptosis to promote wound healing [136]. Therefore, preconditioning stem cells-derived exosomes may open a new strategy for skin wound treatment.

Overall, engineering exosomes for skin injury has the potential to improve therapeutic outcomes by enhancing their bioavailability, targeting, and therapeutic effects. However, further research is needed to optimize these strategies and evaluate their safety and efficacy in preclinical and clinical settings.

Storage of exosomes

Exosomes can be sensitive to changes in their environment, such as temperature and pH levels. Therefore, it is important to optimize the storage conditions to ensure their stability and efficacy over time. Currently, the storage of exosomes mainly contains freezing, freeze-drying, and spray-drying [145,146]. Studies have shown that more quantity of exosomes were stored at 4 °C for 24 h than at −80 °C, but the best long-storage method for exosomes is frozen storage at −80 °C [97]; and minimizing the storage time can avoid structural changes and bioactivity degradation of exosomes [145]. In addition, a certain concentration of antifreeze can be added in the freezing process to extend the shelf life of exosomes. The study found that storing exosomes in disaccharide antifreeze solution trehalose is the best antifreezing method [147,148].

In short, by optimizing the factors affecting the quantity and quality, therapeutic efficacy of exosomes can be improved as a more viable option for clinical use.

Optimizing exosome therapy for skin wound healing

Although exosomes show great advantages in the treatment of skin wounds, the dosage, mode of administration, safety and efficacy of exosomes should be considered (Fig. 3). The optimal dose and route of delivery of exosomes vary depending on the severity and location of the skin injury. Exosomes can be delivered topically, injected directly into the injured area, or administered systemically. The timing of exosome therapy may also be important, as early treatment can be more effective in promoting skin regeneration.

Doses of exosome therapy

The optimal dose of exosome therapy for wound healing is still an area of ongoing research, and there is no consensus on the ideal dose. The dose of exosome therapy may depend on various factors such as the type and severity of the wound, the characteristics of the exosomes used, and the mode of administration.

A systematic review has shown that various doses of exosomes have been used to treat skin injury, ranging from 1 μg to 200 μg per treatment, depending on the study design and animal model used [27]. The higher doses of exosomes may result in improved therapeutic outcomes, while lower doses may be sufficient for achieving therapeutic effects [149]. For example, MSC-Exos eye drops were formulated at 0.5 × 10¹⁰, 2.5 × 10¹⁰, or 12.5 × 10¹⁰ particles/mL in 5 μL per drop. When administrated twice a day for 7 days, the 2.5 × 10¹⁰ particles/mL formulation was the most optimal and effective dose for the mouses with dry eyes [150]. On the contrary, human corneal mesenchymal stem cell-derived exosomes with 1.0 × 10⁸ exosome/mL treated a monolayer of confluent human corneal endothelial cells (HCECs) were scratched for 24 h can accelerate the re-epithelialization of HCECs, while 10⁵ and 10⁷ exosomes/mL has a slight dose dependent effect, but this was not statistically significant [151]. In clinical studies, the optimal dosage of exosomes for skin injury has not yet been established, as these studies are still in the early stages of development. However, some clinical trials have used doses ranging from 10¹¹ to 10¹³ exosomes in antitumor [152]. In a clinical trial of human umbilical cord mesenchymal stem cell-derived exosomes (UCSC-Exos) for 28 patients with graft-versus-host disease (GVHD)-associated dry eye, UCSC-Exos at a concentration of 10 μg/50 μL and administrated 4 times a day per eye for 2 weeks can significantly remission dry eye symptoms and improve quality of life [150].

In the end, the optimal dose of exosome therapy for wound healing may depend on the specific wound and the treatment protocol. Further research is needed to determine the best dose and dosing schedule of exosome therapy for different types of wounds.

Timing of exosome therapy

Exosome therapy for wound healing is an emerging field of research, and there is still much to learn about the optimal timing of exosome therapy. However, based on current research, the timing of exosome therapy may depend on the type and severity of the wound. Similarly, to stem cell therapy, exosome therapy may be most effective when administered during the early stages of wound healing, when inflammation and angiogenesis are occurring. At this stage, exosomes may help to promote the recruitment of immune cells and the formation of new blood vessels, which are important steps in the wound healing process.

However, it is also possible that exosome therapy could be beneficial during the later stages of wound healing, when tissue remodeling and maturation are occurring. Exosomes may help to promote the production of extracellular matrix components and the organization of new tissue, which are critical for the proper healing of wounds. Thus, the optimal timing of exosome therapy for wound healing depends on the specific characteristics of the wound and the underlying tissue damage. Further research is needed to determine the best timing and dosing of exosome therapy for different types of wounds.

The timing of exosome therapy may depend on various factors such as the type and severity of the wound, the characteristics of the exosomes used, and the mode of administration. In general, the timing of exosome therapy may be influenced by the phases of wound healing, which include inflammation, proliferation, and remodeling. During the inflammation phase (early phase), which typically lasts up to 3–7 days after injury, exosomes may help to modulate the immune response and promote angiogenesis, which is the formation of new blood vessels. In the proliferation phase, which occurs after the inflammation phase and lasts for several weeks, exosomes may promote cell growth and tissue regeneration. In the remodeling phase, which can last for several months, exosomes may help to promote tissue remodeling and the organization of new tissue.

However, it is important to note that the optimal timing of exosome therapy for wound healing may vary depending on the specific wound and the treatment protocol. It is likely that further research will be needed to determine the best timing and dosing of exosome therapy for different types of wounds.

Administration of exosome therapy

The optimal administration of exosome therapy for wound healing may depend on various factors such as the type and location of the wound, the characteristics of the exosomes used, and the desired therapeutic effect. However, several administration routes have been studied for exosome therapy for wound healing (Table 5): (1) Local injection: Exosomes can be injected directly into the wound site or the surrounding tissue. This mode of administration can deliver exosomes directly to the site of injury, promoting local tissue repair and regeneration [153]. (2) Topical application: Exosomes can be applied topically to the surface of the wound or incorporated into a wound dressing. This mode of administration can promote healing of the wound surface and prevent infection [154]. (3) Systemic injection: Exosomes can be injected systemically, such as intravenously or intramuscularly. This mode of administration can deliver exosomes to the entire body, promoting general tissue repair and regeneration [87,155]. (4) Inhalation: Exosomes can be delivered to the lungs through inhalation. This mode of administration has been studied for pulmonary diseases but may also have potential for wound healing of respiratory tissues [156]. (5) Oral administration: Exosomes can be administered orally, such as in the form of a capsule or tablet [157]. Due to the poor oral bioavailability, further studies are needed to characterize the efficacy of this delivery method. However, recent studies have shown that milk exosomes can survive the highly acidic conditions in the stomach and degradation conditions in the gut. In addition, they can cross biological barriers to reach the target tissue. The ability of milk exosomes to cross the gastrointestinal barrier makes them a promising drug delivery vehicle for oral delivery [158].

Table 5.

Comparison of exosome administration

Administration	Diseases	Outcomes	References
Local injection (dose: 1 g/L)	Diabetic wound	Enhanced the biological functions of HUVECs in vitro; enhanced angiogenesis to accelerated diabetic wound healing in vivo.	[153]
Topical application	Diabetic wound	Less inflammation, collagen deposition, and neovascularization to drive wound healing	[154]
Systemic injection (dose: 200 μg)	Acute skin wound	Stimulate cell migration, proliferation and collagen synthesis to accelerate wound healing	[87,155]
Inhalation (dose: 10 × 109 particles/kg, 30 min/day, successive 10 days)	Idiopathic pulmonary fibrosis	Reestablishing normal alveolar structure and decreasing both collagen accumulation and myofibroblast proliferation	[156]
Oral administration (dose: 3 mg/day, successive 7 days)	Ulcerative colitis	Ameliorate mice colitis and accelerate colitis resolution via regulating the expression of the pro-inflammatory cytokines	[157]

In summary, the above exosome delivery methods can better promote disease healing. However, the current study shows that inhalation is mainly used for lung diseases, and due to the low bioavailability of oral methods, more studies are needed to further confirm the effectiveness of promoting skin wound healing. Local application, local administration and systemic intravenous administration of exosomes all play a positive role in promoting skin wound healing, but studies have shown that the therapeutic effect of intravenous injection and local application is superior to that of local injection [87,155]. This may be related to the loss of exosomes during local injection, and in addition, local injection will inevitably interfere with the wound, thus interfering with wound healing. Intravenous exosomes may recruit injured areas via the membrane surface of receptors or adhesion molecules, targeting fibroblasts and promoting wound healing.

The optimal administration of exosome therapy for wound healing may depend on the specific characteristics of the wound and the exosomes used. However, intravenous injection and topical application may be particularly effective for promoting wound healing, as they allow direct delivery of exosomes to the site of injury. Further research is needed to determine the most effective administration route for exosome therapy for wound healing.

Frequencies of exosome therapy

The optimal frequency of exosome therapy for wound healing may depend on various factors such as the type and severity of the wound, the characteristics of the exosomes used, and the mode of administration. Frequencies of exosome therapy for wound healing, including: (1) Single administration: Exosome therapy can be given as a single dose, typically at the time of injury or after the initial wound healing phase. This mode of administration may be effective in promoting wound healing and tissue regeneration. (2) Repeated administration: Exosome therapy can be given multiple times, either at regular intervals or as needed depending on the wound healing progress. This mode of administration may be more effective in promoting sustained tissue repair and regeneration. (3) Continuous administration: Exosome therapy can be given continuously over an extended period of time, such as through a slow-release implant or infusion. This mode of administration may be effective in promoting long-term tissue repair and regeneration. Zhang et al. compared the administration frequency of ASC-Exos in skin wounds and found that increasing the administration frequency (3 times a day) could promote skin wound healing more than once a day and twice a day [87].

The optimal frequency of exosome therapy for wound healing may depend on the specific characteristics of the wound and the exosomes used. However, repeated administration may be particularly effective for promoting sustained tissue repair and regeneration, as it allows for ongoing delivery of exosomes to the site of injury. Further research is needed to determine the most effective frequency and dosing schedule of exosome therapy for wound healing.

Interval between repeated exosome therapies

The optimal interval between two exosome therapies for wound healing may depend on various factors such as the type and severity of the wound, the characteristics of the exosomes used, and the mode of administration. However, several general guidelines have been suggested based on existing studies.

For repeated administration of exosome therapy, the optimal interval can be vary depending on the desired therapeutic effect and the duration of exosome activity. If exosomes have a short half-life, more frequent administration is necessary to maintain their therapeutic effect. Exosome therapy has been administered at intervals ranging from daily to weekly [149]. Injections of ASC-Exos every three times one days for two weeks were effective in promoting full-skin wound healing [87]. Another study on diabetic mice with impaired wound healing found that topical application of ASC-exosomes every two days for two weeks was effective in promoting wound healing. However, the optimal interval between two exosome therapies for wound healing may vary depending on the specific characteristics of the wound and the exosomes used. Further research is needed to determine the most effective dosing schedule for exosome therapy for different types of wounds.

In summary, optimizing exosomes for therapy involves a comprehensive approach that includes isolation and purification, characterization, loading of therapeutic cargo, targeting, quality control, and scale-up. By optimizing these parameters, exosomes can be developed as effective and safe therapeutic agents for a wide range of diseases.

Safety and efficacy

The safety and efficacy of exosome therapy should be carefully evaluated in preclinical and clinical studies. Although local administration is safer and more bioavailable than systemic administration, which can reduce the influence on other cells of the body and avoid the influence of phagocytes and cyclic clearance in vivo, drug concentration is easily interfered by external factors. Moreover, whether local or systemic administration, different concentrations of administration will produce different biological effects [159,160]. Adverse effects such as immune reactions or unintended effects on other tissues or organs should be monitored, and the efficacy of exosome therapy should be assessed using objective measures such as wound healing time and scar formation. To optimize exosome therapy for skin injury, it is important to carefully consider the source of exosomes, isolation and purification methods, dosing and delivery, and safety and efficacy in preclinical and clinical studies. Further research is needed to fully evaluate the potential of exosome therapy for promoting skin regeneration and reducing inflammation in various types of skin injuries.

Exosome therapy used in other diseases

Exosome therapy has been widely studied in the management of other diseases, such as cancer, ischemia-induced diseases, and inflammatory-related diseases (Table 6).

Table 6.

Exosome therapy used in other diseases

Indication	Diseases	Exosome source	Effective molecules	Outcomes	References
Cancer	Glioma	Neutrophils	–	Loaded with doxorubicin, inhibit glioma progression via BBB transfer to the TME	[162]
	Colorectal cancer	293T cells	miR-21↓, PTEN and hMSH2↑	Decrease tumor proliferation and increase apoptosis to inhibit tumor growth	[164]
	Gastric cancer	HEK293T cells culture media and plasma	miR-214↓	Induce cell apoptosis, reduce proliferation and migration, and increase drug sensitivity	[165]
	BC	Breast tumor cells	miRNA-16-5p↓, CD73↑	BC-derived exosome SNHG16/miR-16-5p/SMAD5-regulatory axis potentiates TGF-β1/SMAD5 pathway activation, to induce CD73 expression in Vδ1 T cells	[168]
	BC	CAF	miR-92, PD-L1↑	Promotes apoptosis and impaired proliferation of T cells	[169]
Ischemia-induced injury or diseases	MI	hUSC	Cx43, Ki67, CD31, α-SMA, Vwf, TGF-β1, MMP-9, etc.↑	Promote cell proliferation and angiogenesis, enhancing ejection fraction	[171]
	IS	IPAS	circSHOC2, miR-7670-3p, SIRT1↑	Regulate autophagy and reduce neuronal apoptosis	[172]
	IS	Lactobacillus plantarum	miR-101a-3p↑	Inhibit neuron apoptosis to protect against ischemic brain injury through the micro RNA-101a-3p/c-Fos/TGF-β axis	[173]
	Kidney I/R	HUVEC	NF-κb activity↓	Decreased NF-κB activity, expression of pro-inflammatory cytokines and adhesion molecules	[174]
Inflammatory-related diseases	Atherosclerosis	HEK293T cells	miR-155 ↑	Exosome-based delivery of the engineered IL-10 could alleviate the atherosclerosis in ApoE mice	[180]
	AA	M2 macrophage	Inflammatory cytokines ↓	Ameliorated AA with a marked reduction of lung inflammation	[181]
	TBI	Astrocyte	miR-873a-5p↑	Inhibited LPS-induced microglial M1 phenotype transformation and inflammation through decreased phosphorylation of ERK and NF-κB p65	[182]
	Murine colitis	M2 macrophage	miR-590-3p, LATS1↑	Reduces inflammatory signals and promotes epithelial regeneration	[183]
	NASH	HLSCs	α-SMA, Col1a, Tgf-β1, TNF, IL-1β↓	Display anti-fibrotic and anti-inflammatory effects to improve liver function	[186]
	EAE	HUCB	IL-2, MMP-9, HSP-72↑	Inhibited T cell proliferation and EAE development by regulating IL-2 signaling	[189]

Abbreviations: BBB, blood-brain barrier; TME, tumour microenvironment; PTEN, phosphatase and tensin homolog; hMSH2, human DNA MutS homolog 2; BC, breast cancer; CAF, cancer-associated fibroblasts; hUSC, human umbilical cord mesenchymal stem cells; MI, myocardial infarction; IS, ischemic stroke; IPAS, ischemic-preconditioned astrocyte; I/R, ischemia-reperfusion; HUVEC, human umbilical vein endothelial cells; CIRI, cerebral ischemia-reperfusion injury; AA, allergic asthma; TBI, traumatic brain injury; NASH, non-alcoholic steatohepatitis; HLSCs, human liver stem cells; EAE, experimental autoimmune encephalomyelitis; HUCB, human umbilical cord blood.

Exosome therapy used in the treatment of cancer

Exosome therapy is an emerging approach in cancer treatment in skin or other tissues, which involves the use of exosomes or small extracellular vesicles released by cells [161]. These exosomes can carry various molecules, such as proteins, nucleic acids, and lipids, which can influence cellular behavior and signaling [43].

In cancer treatment, exosome therapy holds promise for several reasons. First, exosomes can be isolated from various cell sources, including immune cells, stem cells, and tumor cells, allowing for the potential delivery of therapeutic cargo to cancer cells [43]. Second, exosomes naturally have the ability to cross biological barriers, including the blood–brain barrier, which can enhance their accessibility to tumors located in challenging anatomical sites [162]. Third, exosomes can serve as mediators of intercellular communication, facilitating the transfer of information between cells and modulating tumor growth, immune response, and drug resistance [163].

Exosome-based therapies in cancer treatment can be categorized into two main strategies: therapeutic cargo delivery and immunomodulation. In therapeutic cargo delivery, exosomes can be loaded with anticancer agents, such as chemotherapeutic drugs or small interfering RNAs (siRNAs), and delivered to cancer cells to exert their effects. Studies have shown that loading 5-fluorouracil and miRNA-21 inhibitors into exosomes through electroporation can effectively promote cellular uptake, reduce miRNA-21 expression, decrease tumor proliferation, increase apoptosis, and reduce expression of PTEN and hMSH2 to inhibit tumor growth [164]. In addition, exosome delivering anti-miRNA-214 to gastric cancer cells can induce cell apoptosis, reduce proliferation and migration, and increase drug sensitivity [165]. Exosome can handle the payload of anti-tumor drugs, and can also be used as a carrier of genetic tools to increase the effectiveness of anti-tumor treatment. Exosome loaded siRNAs may solve the drawbacks of RNA enzyme degradation, tumor barrier, and off-target effects [166]. This approach can potentially enhance drug delivery specificity and reduce off-target toxicity to improve the effectiveness of treatment.

On the other hand, exosomes can also be utilized to modulate the immune response against cancer. They can be engineered to carry immune-stimulating molecules or tumor antigens to activate the immune system and promote antitumor immune responses [167]. Additionally, exosomes derived from immune cells can be modified to enhance their immunosuppressive properties, which may help counteract the immunosuppressive tumor microenvironment and promote better immune surveillance against cancer cells. Exosome transmits SNHG16 to induce the TGF-β/SMAD16 axis, reducing miRNA-16-5p and mediating CD73 overexpression on Treg cells, leading to immune suppression in breast cancer [168]. Study has shown that miRNA-92 mediated interaction between LATS2 and YAP1 in breast cancer cells is facilitated by the exosome contained in cancer-associated fibroblasts. YAP1 is translocated to the cell nucleus and binds to the PD-L1 promoter to enhance its expression, leading to T cell apoptosis and decreased proliferation [169].

Exosome therapy used in ischemia-induced injury or diseases

Ischemia refers to a condition where there is inadequate blood supply to the skin, other tissues or organs, resulting in reduced oxygen and nutrient availability. This can lead to skin ischemia or tissue damage and various diseases, such as ischemic stroke, myocardial infarction, renal failure, liver dysfunction, severe acute respiratory syndrome and peripheral artery disease. Exosome therapy has shown promise in the context of ischemia-induced injury or diseases [170].

In preclinical studies, administration of MSC-derived exosomes has demonstrated beneficial effects in ischemia-induced injury models. For example, Zou et al. has found that hydrogel USC-Exos can improve cardiac function and promote blood vessel formation in myocardial infarction models by promoting cell proliferation and angiogenesis [171]. Chen et al. discovered that the expression of circSHOC2, an exosome derived from astrocytes, was significantly upregulated, which can promote neuroprotection and enhance functional recovery via the miR-7670-3p/SIRT1 axis to regulate autophagy and reduce neuronal apoptosis [172]. In addition to mammalian-derived exosomes showing a positive therapeutic effect on ischemic diseases, Lactobacillus plantarum-derived exosome can inhibit neuron apoptosis to protect against ischemic brain injury through the microRNA-101a-3p/c-Fos/TGF-β axis [173]. Furthermore, exosomes have emerged as a promising delivery tool for treating ischemic-related diseases. For example, exosome can delivery super-repressor IκBα via decreasing NF-κB activity and reducing apoptosis to ameliorates kidney ischemia-reperfusion injury [174].

Exosome therapy used in chronic inflammatory-related diseases

Chronic inflammatory conditions are characterized by persistent inflammation in the affected tissues or organs, leading to tissue damage and dysfunction. Examples of such skin disease include eczema, seborrheic dermatitis, and psoriasis. Other diseases include rheumatoid arthritis, inflammatory bowel disease, and asthma. Some studies have reported that exosome therapy holds promise for the treatment of chronic inflammatory-related diseases in skin or other tissues, including inflammatory bowel disease, neuroinflammation, osteoarthritis, atherosclerosis, allergic asthma, etc. [36,175-181].

Exosomes derived from immune cells, such as dendritic cells and macrophages, can influence the immune response by delivering specific molecules that either promote or suppress inflammation [182,183], while exosome also can act as transport vehicles to deliver cargo to the inflammation site for better anti-inflammatory effects [184,185]. Bruno et al. found that EVs derived from human liver stem cells can reduce pro-inflammatory genes such as TNF, IL-1β, and IFN-γ in the mouse model of non-alcoholic steatohepatitis (NASH), thereby alleviating the inflammatory response of NASH [186]. Other research has shown that EVs released by inflammasome activated macrophages contain interferon beta and induce interferon signatures to inhibit NLRP3 inflammasome activation to prevent systemic inflammatory responses [187].

Furthermore, the unique properties of exosomes are involved in immune regulation of inflammatory or autoimmune diseases and are considered as potential alternatives to cell therapy [188]. For example, EVs derived from human umbilical cord blood cells can suppress T cell proliferation by reducing the production of IL-2 and express MMP-9 and HSP-72 to achieve significant T cell immune suppression, improving experimental autoimmune encephalomyelitis (EAE) [189]. EVs derived from oligodendrocytes protect EAE mice from demyelination and axonal damage in a myelin self-antigen dependent manner, and reduce infiltration of CD45 and CD4 in the central nervous system [190].

The therapeutic cargo within exosomes, such as microRNAs, anti-inflammatory proteins, and growth factors, can modulate immune cell activity, reduce pro-inflammatory cytokines, and promote tissue repair and regeneration [191]. These exosomes can also influence the communication between immune cells and damaged tissues, helping to restore tissue homeostasis and alleviate chronic inflammation.

Taken together, the use of extracellular vesicles as a delivery tool and their direct action on cancer, ischemic-related diseases, and chronic inflammatory-related diseases, etc., make them a promising therapeutic approach for treating a range of conditions. To fully understand the potential of these tiny vesicles, and early results are highly encouraging. While the field of exosome therapy is still in its early stages, these findings highlight the potential of utilizing exosomes as a therapeutic approach. Further research and clinical studies are necessary to optimize exosome isolation, characterization, and delivery methods to harness their full therapeutic potential in treating diseases.

Future work and conclusions

Exosome therapy for acute skin injury is an area of active research, and there are several promising directions that this research is taking. Here are some future directions of exosome therapy for acute skin injury.

Potential for clinical translation of exosome therapy

Exosomes have emerged as promising candidates for clinical translation due to their unique properties and potential therapeutic applications. The potential for clinical translation of exosomes is listed below.

(1). Natural intercellular communication:

Playing a critical role in natural intercellular communication, exosomes act as shuttles to transfer bioactive molecules from donor cells to recipient cells. This unique feature allows exosomes to influence cellular behavior, promote tissue repair, and regulate immune responses. Harnessing this natural communication system offers great potential for targeted therapies.

(2). Minimal immunogenicity:

Exosomes have been shown to have minimal immunogenicity, making them attractive for therapeutic use. They have a reduced likelihood of eliciting immune responses when administered to patients, even across allogeneic or xenogeneic systems. This characteristic increases their compatibility for clinical translation and reduces the risk of adverse reactions.

(3). Specific targeting and payload delivery:

Exosomes can be engineered to carry specific cargo, such as therapeutic proteins, small RNAs, or drugs. By modifying the parent cells, researchers can load exosomes with the desired cargo and direct them to specific target tissues or cells. This targeted delivery system minimizes off-target effects and enhances the therapeutic potential of various treatments.

(4). Combined with biomaterials:

Combined exosomes with hydrogels or 3D scaffolds, exosomes can achieve the effect of continuous and even distribution on the wound, or exosomes can be used as drug carriers to deliver drugs or genes to the wound site, regulate the activity of related growth factors and the formation of blood vessels, thus shortening the time of wound healing and improving wound healing.

(5). Potential for regenerative medicine:

Exosomes derived from stem cells, mesenchymal stromal cells, and other cell types have demonstrated promising regenerative properties. They can promote tissue repair, reduce inflammation, and stimulate cell proliferation and differentiation. These characteristics make exosomes potential candidates for treating various degenerative diseases, such as cardiovascular diseases, neurodegenerative disorders, and tissue injuries.

(6). Drug delivery applications:

Exosomes’ ability to cross biological barriers, such as the blood–brain barrier, and their favorable biodistribution properties make them excellent candidates for drug delivery applications. They can protect their cargo from degradation, extend circulation time, and enhance drug bioavailability, leading to improved therapeutic outcomes.

(7). Non-invasive biomarkers:

Exosomes carry unique biomolecules that reflect physiological status of their parent cells. As such, exosomes provide non-invasive and readily accessible source of biomarkers for diagnosing diseases and monitoring treatment response. This potential for liquid biopsy and disease monitoring holds great promise for personalized medicine.

(8). Clinical trials:

As the safety and efficacy of exosome therapy for acute skin injury is further established, more clinical trials will be conducted to determine the optimal dosage, frequency, and delivery methods for exosome therapy.

While the potential for clinical translation of exosomes is significant, several challenges must be addressed. These include standardizing isolation and purification methods, optimizing loading and targeting strategies, ensuring scalability and reproducibility, and understanding potential long-term effects.

In summary, exosomes represent a promising avenue for clinical translation in several therapeutic areas, including regenerative medicine, targeted drug delivery, and disease diagnostics. Their ability to mediate intercellular communication, minimal immunogenicity, and potential for specific targeting make them attractive candidates for future therapeutic interventions. However, further research, preclinical studies, and clinical trials are needed to fully understand their safety, efficacy, and long-term implications before they can be widely adopted in the clinical setting. With continuing advancements and innovations in the field, exosomes hold great potential to revolutionize modern medicine and improve patient outcomes.

Challenges and opportunities

Although a large number of techniques have been applied to the separation and purification of exosomes, different techniques have some shortcomings, and some of them are not fully mature. Therefore, how to obtain a large number of exosomes with high purity, specificity, and low cost is the current difficulty. At present, with the continuous update of exosome separation technology, great progress has been made in the separation and purification of exosomes, especially researchers are focusing on how to obtain high quality and low cost exosomes through simple procedures. Secondly, exosomes were obtained by combining different separation methods to meet the accuracy of downstream experimental research results. Finally, we need to evaluate the composition of exosomes, although Dennis et al. have identified that annex A1 as a specific marker for microvesicles shed directly from the plasma membrane and found that small extracellular vesicles are not vectors for active DNA release. Therefore, future studies are needed to evaluate exosome composition and better understand exosome heterogeneity [192]. In addition, the activity of exosomes decreases over time. Therefore, it is necessary to develop methods to maintain exosome activity in the future to increase the therapeutic effect of exosomes. We expect that the separation and purification techniques of exosomes will be developed more rapidly and achieve great success in the future, so as to solve the key problems that have long hindered the study of exosomes.

In addition, increasing studies have shown that exosomes can promote skin wound regeneration. But in animal studies, exosomes show fatal shortcomings such as low bioavailability, poor targeting, and short half-life in vivo. Therefore, how to optimize exosomes is a hot and difficult topic in current research, and more studies are needed to confirm the therapeutic effect of different methods to optimize exosomes on skin wounds. What’s more, the effects of exosome administration modes, dose, and time on different types of skin wounds also need to be further studied.

The process of exosome delivery is a complex and dynamic process, involving multiple steps and mechanisms. The specific factors that govern exosome biogenesis, release, and targeting still need research, and a better understanding of these processes could have important implications for the development of new therapies for wound healing. Therefore, optimization of exosomes in isolation and treatment is crucial, and more studies are needed in the future to confirm the therapeutic effect of exosomes in skin wound healing.

The delayed or non-healed skin wound is a common clinical complication. Therefore, effective and rapid promotion of skin wound healing is an urgent clinical problem. Basic medical research provides a powerful theoretical basis for clinical treatment, and clinical research provides human tissue samples and data sources for basic research. Only by combining clinical and basic practice can it be better applied in clinical practice. For example, storing the samples obtained in clinical practice into the biological sample database for basic research; and researchers work with clinicians to better integrate the clinical and basic.

Conclusions

Exosomes are considered as a promising regenerative therapy for skin tissue repair and beyond as they carry a variety of bioactive substances from the parental cell, which can mediate intercellular communication. Exosomes accelerate skin wound healing by promoting vascular regeneration, collagen synthesis, cell proliferation and migration. In this review, we mainly describe how to optimize the regeneration potential of exosomes in skin wounds. Continued research in this area is expected to lead to new breakthroughs in the treatment of acute skin injury and prevent complications.