Bacterial extracellular vesicles as bioactive nanocarriers for wound treatment

Abstract

Bacterial extracellular vesicles (BEVs) secreted by bacteria are considered as messengers for the crosstalk between gut microbiota and wounded skin via the “gut–skin axis”. The BEVs with the lipid bilayer nanostructures can deliver various bioactive molecules from their parent bacteria to the host cells, modulating the signal pathways related to wound repair and regeneration. Besides, the cutting-edge gene editing strategies and mature bacterial culture methods further endowed their more customizable and scalable manufacture compared to the most commonly used extracellular vesicles from mammalian cells. Therefore, more and more BEVs have been exploited directly as bioactive nanocarriers or engineered as delivery vehicles for wound treatment. Herein, the present review began with an overview of the gut–skin axis to better comprehend the bioactivates of BEVs towards wound healing. Their biogenesis, isolation, and uptake were then introduced. A summary of recent advancements in exploring BEVs for accelerated wound healing was followed, with a focus on their roles as nanocarriers for delivery. Diverse engineering approaches to functionalize BEVs were especially discussed for optimal wound management. Constructive insights regarding the new upsurge of BEVs for wound treatment were provided in the end to boost these innovative therapeutic modalities from bench to bedside.

Graphical abstract

Bacterial extracellular vesicles (BEVs) that are secreted by bacteria can be exploited directly as bioactive nanocarriers or engineered as delivery vehicles for accelerating the wound healing process.

1. Introduction

As the primary external barrier of the human body, skin is the most common site of wounds caused by burns, lacerations, or other traumatic injuries^1,2. Based on the clinical presentation, skin wounds can be classified as acute and chronic. The acute wounds can be repaired and regenerated following an ordered healing process, including hemostasis, inflammation, proliferation, and remodeling within weeks³. The chronic wounds caused by incorrect wound responses generally fail to recover after a prolonged period of three months. The imbalance in one or more healing processes that are disrupted by the extensive trauma or certain pathophysiological conditions, such as peripheral arterial disease and diabetes, is considered to be the main cause of chronic wounds⁴. The compromised healing affects millions of people, imposing heavy burdens on patients, families, and societies. Therefore, wound treatment has attracted worldwide attention for the optimal therapeutic modalities.

Currently, the standard wound treatments include local debridement, pressure offloading, and moist dressing. To drive the healing process of chronic wounds, more and more advanced therapies based on the local hydrogel delivery system have been approved for clinical use. For instance, the growth factor therapies such as Regranex® provide proliferation stimulation, meanwhile the skin substitute therapies such as Apligraf® and Dermagraft® offer extracellular matrix (ECM) scaffolding to restore the structural and functional integrity of chronic wounds. Nonetheless, the inconsistent clinical outcomes and expensive treatment costs of these therapies restrict their long-term use⁵. Moreover, emerging treatments with stem cells and stem cell-derived extracellular vesicles (EVs) have also been widely investigated as novel therapies based on the transdermal nano delivery system. However, the translation of these preclinical investigations into clinical practice is severely hindered by the combined clinical regulation and feasibility⁶.

With the advances in sequencing and bioinformatic techniques, sufficient evidence has confirmed the crosstalk between gut microbiota and skin via the “gut–skin axis”⁷. The gut microbiota, which is in symbiosis with humans, plays an important role in maintaining the homeostasis of skin function. Its dysbiosis is associated with the occurrence and progression of diverse skin disorders, including chronic wounds, atopic dermatitis, psoriasis, and acne⁸. With the increasing understanding of the relationship between gut microbiota and skin, the microbiota-based therapies, such as microbiota modulations and engineered gut bacteria, have been developed for wound treatment9, 10, 11, 12. Nonetheless, the long-term safety profiles of these interventions are still lacking or inadequate, limiting their clinical translation.

Bacterial extracellular vesicles (BEVs) that are secreted by gut microbiota have been demonstrated to be the messengers for regulating the long-distance communication between gut microbiota and skin¹³. The BEVs with nanoscale size and lipid bilayer can deliver various bioactive molecules, including protein, nucleic acid, lipopolysaccharide, and metabolites from their parent strains to the host cells, modulating the signal pathways related to wound repair and regeneration^14,15. As such, BEVs have been considered as a potential alternative to the microbiota-based therapies for wound treatment. Compared to the most commonly used EVs from mammalian cells for wound treatment, BEVs can be manufactured in a more scalable and customizable manner due to the mature bacterial culture methods and cutting-edge gene editing strategies¹⁶. Besides, due to their inherited structures and composites from their parental bacteria, the BEVs also possess immunomodulating capacities and bioactive functions. Moreover, their lipid bilayer nanostructures endow them with services as delivery vehicles as well, facilitating the intracellular delivery of the insoluble or unstable therapeutic agents for wound treatment. Therefore, more and more BEVs have been exploited directly as bioactive nanocarriers or engineered as delivery vehicles for wound treatment. Nonetheless, a comprehensive review of the emerging research field is still lacking.

Herein, the emerging concept of “gut–skin axis” was first expounded to facilitate the understanding of the messenger role of BEVs for the crosstalk between the gut and skin. The biogenesis, isolation, and uptake of BEVs were then reviewed to comprehend their bioactivities towards wound healing. Based on these fundamental cognitions, recent advances in exploiting the BEVs as bioactive nanocarriers for accelerated wound healing were elaborated one by one. The diverse functionalization approaches, including the biological, physical, chemical, and even material engineering, were explained and compared to underline the advantages and limitations of each approach in functionalizing the BEVs for optimal wound management. The unique superiorities and future challenges in engineering the BEVs as bioactive nanocarriers for wound management were discussed by the end. The insights of the presented review would guide the future developments and clinical applications of the BEV-based novel therapeutic modalities for the effective and safe treatment of wounds.

2. Gut–skin axis

Gut–skin axis is conceived as the bidirectional signal communication between the gastrointestinal tract and skin, driven by microorganisms. It is an essential process to achieve distal regulation between the gastrointestinal tract and skin via substance exchange¹⁷.

According to the Human Microbiome Project from the National Institute of Health, the gastrointestinal tract and skin are colonized by a larger portion of dwelling microorganisms of 29% and 21%, respectively¹⁸. Therefore, the dysbiosis at either the gut or skin can lead to the disturbance of homeostasis, which is attributed to the decline of bacterial diversity and stability. Increasing studies have demonstrated that both the intestinal and cutaneous inflammations originated from gut dysbiosis^19,20. The microbial imbalance can directly induce the increased intestinal barrier permeability^21,22, facilitating the bacterial and metabolite translocation into systemic circulation, thus triggering the systemic inflammation, and then aggravating the skin pathologies. For instance, clinical investigations have found that patients with inflammatory bowel disease and celiac disease frequently developed comorbid psoriasis and cutaneous ulcers²³. Similarly, patients who exhibit concurrent small intestinal bacterial overgrowth often suffer from rosacea²⁴. As such, the probiotics have been administered to restore gut homeostasis and subsequently improve skin conditions. For instance, experimental mice that were orally administered with Lactobacillus reuteri containing water have been characterized with improved epidermal thickness, stimulated sebaceous epithelial cell proliferation, as well as increased hair follicle development²⁵. The short-chain fatty acids (SCFAs), especially butyrate, from the Lactobacillus probiotics have been further found to reduce the allergen-induced skin barrier disruption in a murine model mimicking human atopic dermatitis (AD)²⁰.

Even though the crosstalk between gut microbiota and skin via the gut–skin axis has been increasingly confirmed, the underlying mechanisms remain to be fully elucidated. The microbial metabolites were initially considered as the mediator of the gut–skin axis since the dietary components processed by the microbiota were observed for the gut–skin crosstalk²⁶. Subsequent studies have further demonstrated that the gut microbiota-derived metabolites could influence skin health through multiple mechanisms: maintaining intestinal barrier integrity, modulating immune homeostasis, as well as regulating inflammatory responses. For instance, bacterial metabolites, such as vitamin K, vitamin C, and 3-hydroxyphenylacetic acid (3-HPA), have been demonstrated to possess the anti-inflammatory and wound-healing effects^27,28.

Notably, recent investigations suggested that BEVs secreted by the gut microbiota were important messengers for the gut–skin axis (Fig. 1)^29,30. Their nanoscale size and lipid bilayer endow their capacity to deliver various bioactive molecules, including proteins, nucleic acids, lipopolysaccharide, and metabolites from their parent strains to the recipient cells31, 32, 33. Increasing evidence has shown that the BEVs derived from gut microbiota could pass through the epithelial barrier to reach mucosal layers, where they could interact with immune cells, including dendritic cells, neutrophils, and macrophages^14,34. Besides, their systemic dissemination through the blood or lymphatic circulation also allows their access to distant organs, including the skin. Depending on their origins, the bioactivities of BEVs vary significantly. The BEVs derived from the beneficial bacteria contain anti-inflammatory substances and can inhibit the inflammation. For instance, Lee et al.³⁵ demonstrated that the BEVs derived from L. paracasei could down-regulate the tumor necrosis factor-α (TNF-α) induced inflammation in human keratinocytes through decreasing the expression level of IL-1β, IL-6, and IL-8. To the contrary, the BEVs derived from the pathogenic bacteria display the pathogen-associated molecular patterns (PAMPs), which can stimulate the inflammatory response and induce the release of inflammatory factors. Therefore, selecting or engineering the appropriate and applicable bacterial strain to construct BEVs for wound treatment is critical. Even though the bioactive nanocarriers’ properties of BEVs have been well exploited for the construction of meningitis vaccines, which have been successfully translated for clinical use^36,37. The therapeutic potential of BEVs for wound treatment remains to be fully unleashed.

Figure 1.

The crosstalk between gut and skin via BEVs and metabolites. The probiotics and pathogens secrete BEVs that can cross through the epidermal barrier and enter systemic circulation. The probiotics-derived BEVs can enhance the proliferation and angiogenesis, and down-regulate the inflammatory responses. While the pathogen-derived BEVs can stimulate the release of pro-inflammatory cytokines and thus activate and up-regulate the inflammatory responses. Figure was created by http://app.biorender.com/. M1, M2: M1/M2 phenotype of macrophages; SCFA, short-chain fatty acids; SPA, staphylococcal protein A; LPS, lipopolysaccharide; PG, peptidoglycan; VCAM1, vascular cell adhesion molecule 1; mPTP, mitochondrial permeability transition pore; ICAM1, intercellular cell adhesion molecule 1; KRT14, keratin 14; LOR, loricrin; IVL, involucrin; AQP3, aquaporin 3; mTOR, mammalian target of rapamycin.

3. Biogenesis of BEVs

Most bacteria release BEVs that contain various cargoes from the parent strain, including nucleic acids, virulence factors, proteins, and metabolites. However, the structure and composition of the secreted BEVs are typically different. These differences are partly attributed to the diverse biogenesis mechanisms.

Gram-negative bacteria can release three kinds of BEVs, including the outer membrane vesicles (OMVs), outer–inner membrane vesicles (OIMVs), and explosive outer membrane vesicles (EOMVs) through the outer membrane blebbing and explosive cell lysis (Fig. 2). OMVs originate from the outer membrane blebbing. Previous studies have shown that the additional insertion of hydrophobic molecules and imbalanced synthesis of peptidoglycan (PG) could result in cell membrane disturbances, and thus the blebbing occurrence^38,39. It is demonstrated that OMVs consisted of the outer leaflet of LPS and the inner leaflet of phospholipid. Due to the intact inner membrane, the content inside the cell cytosol cannot enter OMVs except for the proteins from the outer membrane40, 41, 42. Besides, the other mechanism for Gram-negative bacteria to secrete BEVs is explosive cell lysis, by which OIMVs and EOMVs are generated^42,43. After weakening of the peptidoglycan layer by endolysin, the cell gradually rounds up and ruptures, and then the membrane fragments self-assemble into OIMVs and EOMVs, allowing cytoplasmic contents to enter vesicles⁴⁴.

Figure 2.

The biogenesis of BEVs. The Gram-negative bacteria secrete OMVs through the outer membrane blebbing mechanism, while secret OIMVs and EOMVs through the explosive cell lysis mechanism. The Gram-positive bacteria secrete CMVs through the bubbling cell death mechanism. Figure was created by http://app.biorender.com/. OMV, outer membrane vesicles; EOMV, explosive outer-membrane vesicle; OIMV, outer-inner membrane vesicle; CMV, cytoplasmic membrane vesicle; OM, outer membrane; PG, peptidoglycan; IM, inner membrane.

In contrast, due to the existence of thicker PG and lack of outer membrane, BEVs from Gram-positive bacteria have only been discovered about two decades after the Gram-negative bacteria⁴⁵. Similar to the explosive cell lysis, the endolysin can also trigger bubbling cell death to generate cytoplasmic membrane vesicles (CMVs) (Fig. 2). After degradation of PG, the plasma membrane can protrude and finally transform into CMVs that carry cytoplasmic membrane proteins, nucleic acid, and virulence factors⁴⁵.

The different subtypes of BEVs feature different structures and cargoes (Table 1). Although the traditional opinions indicate the appearance of nucleic acid is a symbol of explosive cell lysis, many studies have reported that miRNA and plasmid have been observed inside OMVs as well⁴⁶. OMVs generated by outer membrane blebbing do not contain inner membrane (IM) proteins and endolysin. Because of explosive cell lysis triggered by phage-derived endolysin, OIMV and EOMV share the same composition, containing IM proteins and endolysin. While the self-assembling of membrane fragments leads to OIMV with a double and EOMV with one bilayer membrane structure. In comparison, CMVs secreted by Gram-positive bacteria do not have the outer membrane (OM) structure, nor the OM proteins.

Table 1.

The mechanism, structure, and composition of the different bacterial extracellular vesicles (BEVs) from varied biogenesis.

Bacteria	BEVs	Mechanism	Structure	Cargo
Gram-negative bacteria	OMV	Outer-membrane blebbing	Phospholipid bilayer (+) OM proteins (+) IM proteins (−)	Plasmid (+) Nucleic acid (−) Endolysins (−)
	EOMV	Explosive cell lysis	Phospholipid bilayer (+) OM protein (+) IM proteins (+)	Plasmid (+) Nucleic acid (+) Endolysins (+)
	OIMV	Explosive cell lysis	Phospholipid bilayer (++) OM proteins (+) IM proteins (+)	Plasmid (+) Nucleic acid (+) Endolysins (+)
Gram-positive bacteria	CMV	Bubbling cell death	Phospholipid bilayer (+) OM proteins (−) IM proteins (+)	Plasmid (+) Nucleic acid (+) Endolysins (+)

+, present; –, absent; ++, double phospholipid bilayers; OMV, outer-membrane vesicle; EOMV, explosive outer-membrane vesicle; OIMV, outer-inner membrane vesicle; CMV, cytoplasmic membrane vesicle; OM, outer membrane; IM, inner membrane.

The different kinds of BEVs and their composition are closely related to biogenesis. The capacity of loading bioactive cargoes is important for drug delivery and further biomedical applications. Further exploration of the precise mechanism of BEVs’ biogenesis is still required, which is imperative for the isolation and purification of a particular subset of BEVs in a standardized process. In the presented review, BEVs are used to refer to all the types of bacteria-derived extracellular vesicles, which is in accordance with the recommendation of MISEV 2023 guidelines⁴⁷. The specific terms, such as OMVs, were only emphasized when the information related to a specific subset of BEVs.

4. Isolation of BEVs

The efficient isolation and purification of BEVs is a crucial step for their subsequent applications. Diverse techniques are being developed for the isolation of BEVs, including ultracentrifugation, ultrafiltration, precipitation, affinity chromatography, size exclusion chromatography, as well as density gradient centrifugation¹⁶. However, specific issues, such as limited purity, consumed time, and high cost, still exist with one specific technique. As such, the combination of multiple techniques is usually desired. To address the issue of size overlap between BEVs and bacterial flagella, pili, as well as protein aggregates, Tulkens et al.³³ have additionally used density gradient centrifugation to remove the excessive impurities imparted by ultracentrifugation. Besides, since most of the BEVs possess negative membrane surface charges, anion exchange chromatography has also been considered as a feasible approach. It mainly involves the binding of positively charged molecules with negatively charged BEVs, and then the elution of bound BEVs with high ionic strength buffers⁴⁸. Combining the anion exchange chromatography with tangential flow filtration (TFF), Pirolli et al.⁴⁹ have developed a procedure for the orthogonal size and charge purification of the probiotic-derived BEVs with improved production.

Currently, the mostly used procedure combines centrifugation, ultracentrifugation, and ultrafiltration for the isolation of BEVs, was proposed by Liu et al.²⁹ (Fig. 3). BEVs from Akkermansia muciniphila (AKK), Lactobacillus rhamnosus GG (LGG), and Escherichia coli Nissle 1917 have been reported to be successfully isolated in high purity and homogeneity using the procedure. As demonstrated in Fig. 3, fermentation is firstly performed to increase the number of bacteria and their secreted BEVs rapidly. Subsequently, the bacteria and their debris are removed by low-speed centrifugation at 10,000×g for 15 min, followed by filtration through the 0.22 μm filter. The non-BEV proteins are then removed by ultrafiltration with a size of 100 kDa and ultracentrifugation at 150,000 × g for 1.5 h. The obtained crude BEVs are suspended in PBS and ultracentrifuged again at 150,000 × g for another 1.5 h for further purification. The size and concentration of the collected BEVs can be characterized by transmission electron microscopy and nanoparticle tracking analysis. The expression of specific proteins and peptides can also be examined by Western blotting to confirm that the collected BEVs are exactly from the cultured bacteria⁵⁰.

Figure 3.

The isolation of BEVs. After the rapid proliferation of bacteria through fermentation, the bacteria and its debris are firstly removed by the low-speed centrifugation and sterile filtration; the filtered solution is subsequently collected, ultrafiltered, and ultracentrifuged to remove the non-BEV proteins; the precipitated BEVs is then suspended in PBS and ultracentrifuged again for purification; and the ultimately collected BEVs was resuspended and stored at −80 °C until further use. Figure was created with https://app.biorender.com/.

5. Uptake of BEVs

The uptake of BEVs is an important process to facilitate the transportation of varied bioactive substances from the parent strains to the recipient cells. Although the precise internalization mechanisms of BEVs by the recipient cells remain to be fully elucidated, there are currently three main pathways being proposed, including endocytosis, membrane fusion, and receptor-mediated internalization (Fig. 4).

Figure 4.

The uptake of BEVs. Recipient cells internalize BEVs through the receptor-mediated internalization, endocytosis, and membrane fusion pathways. Among these pathways, the predominant endocytosis has been further divided into the clathrin-dependent endocytosis, caveolin-dependent endocytosis, and macropinocytosis. Figure was created by http://app.biorender.com/.

Endocytosis is the primary pathway that the host cells uptake BEVs, including the clathrin-mediated endocytosis (CME), non-clathrin-mediated endocytosis, and micropinocytosis (Fig. 4)⁵¹. CME involves five stages: nucleation, cargo selection, coat assembly, scission, and uncoating⁵². After binding of the BEVs to membrane receptors, clathrin aggregates underneath the membrane, gradually forming a pit. Subsequently, the small GTP-binding protein Dynamin assembles into a ring at the neck of the pit, facilitating GTP hydrolysis and promoting scission. Ultimately, vesicles with the clathrin coating detached from the membrane bind with early endosomes to achieve the uptake of BEVs^52,53. In comparison, non-clathrin-mediated endocytosis includes caveolin-mediated endocytosis and lipid raft-mediated endocytosis. The caveolin-mediated endocytosis process is generally similar to CME, with only differences in the scission and internalization speed⁵⁴. While the lipid rafts are structural domains of the plasma membrane that not only enrich caveolin to facilitate internalization but also mediate endocytosis independently, allowing the membrane to invaginate and uptake vesicles into cells⁵⁵. Additionally, macropinocytosis is characterized by non-selective uptake through the formation of large invaginations with a diameter exceeding 200 nm⁵⁶. Therefore, it is also considered to be one of the pathways through which the BEVs enter host cells¹⁴.

In addition to endocytosis, the BEVs have also been shown to undergo membrane fusion with the lipid bilayer structure of eukaryotic cells for uptake (Fig. 4). Bomberger et al.⁵⁷ observed increased fluorescence of labeled vesicles and lipid bilayer structures upon addition of BEVs to the host epithelial cells. Besides, BEVs can also bind to certain Toll-like receptors (TLRs), including TLR1/6, TLR2, and TLR4, further promoting their uptake by host cells (Fig. 4)^58,59. It is well-known that LPS is one of the most common immune-stimulating components of BEVs. The presence of LPS on the surface of BEVs can attach to the TLR4 receptor, activating the downstream inflammation pathways and thus enhancing the endocytosis of BEVs^59,60. O'Donoghue et al.⁶¹ have found that the O-antigen-containing BEVs could enter host cells faster than those without the O-antigen in the absence of the clathrin-dependent pathways. As such, it is believed that BEVs could also be taken up via receptor-mediated internalization. Typically, the BEVs can enter host cells through multiple pathways. While their primary internalization pathway usually varied depending on their varied size, heterogeneity, as well as immunogenicity¹⁴.

6. Application of BEVs for wound management

Inspired by their successful translation as vaccines for the infection treatment^13,14, BEVs with the manipulating capacity of host response have also been emergingly exploited to manipulate the different phases of wound healing. On the one hand, BEVs derived from the pathogenic bacteria are usually enriched with immunogenic molecules that are responsible for drug resistance, host adherence, and immune escape^62,63. As such, these BEVs can be engineered with appropriately intrinsic antigen presentation to increase the production of pro-inflammatory cytokines from both immune and epithelial cells through the TLRs, thus modulating the inflammatory phase of wound healing^57,64. On the other hand, BEVs derived from the commensal bacteria are usually composed of diverse bioactive substances that are responsible for gut symbiosis⁶⁵. Therefore, these BEVs can also be engineered with desired properties to promote the proliferating phase and optimize the remodeling phase of wound healing^16,66,67 (Fig. 5 and Table 2).

Figure 5.

BEVs accelerate wound healing via modulating inflammation, promoting proliferation, and optimizing remodeling. In the inflammation phase, BEVs can modulate the polarization of macrophages from M1 to M2. In the proliferation phase, BEVs can promote the angiogenesis of endothelial cells and the re-epithelialization of epidermal cells. Moreover, in the remodeling phase, BEVs can optimize the deposition of collagen. Figure was created by https://app.biorender.com/.

Table 2.

List of BEVs exploited as bioactive nanocarriers for wound treatment.

Strain	Bioactive cargoes	Animal model	Administration route	Mechanistic pathways	Therapeutic outcomes	Ref.
L. reuteri	3-HPA	Full-thickness wound	Hydrogel assisted transdermal delivery	Reduced mitochondrial permeability	Anti-inflammation Angiogenesis	28,90
L. plantarum	Muramidase	Full-thickness wound	Hydrogel assisted transdermal delivery	–	Anti-inflammation	86
Photosynthetic bacteria	LPC	Full-thickness wound	Subcutaneous injection	EGFR/PI3K/AKT	Angiogenesis Proliferation	91
L. rhamnosus GG	miR-21-5p	Full-thickness wound	Subcutaneous injection	p-AKT/HIF-1α	Angiogenesis Re-epithelization	95
L. druckerii	–	Full-thickness wound Scleroderma model	Subcutaneous injection	–	Anti-fibrosis	98

–, does not mention in the reference.

6.1. Management of the inflammatory phase

With the cascade of coagulation, surrounding tissues and clots can release pro-inflammatory factors and recruit inflammatory cells to initiate the inflammation phase^68,69. Polymorphonuclear leukocytes and neutrophils clear devitalized tissue and engulf infectious agents, releasing a range of active substances such as reactive oxygen species70, 71, 72. Subsequently, monocytes are recruited from the blood, activated upon extravasation, and differentiate into mature macrophages, further clearing apoptotic neutrophils⁷³. Notably, during the early stage of the inflammation phase, macrophages exhibit M1 characteristics, enhancing the bactericidal capacity and secreting high levels of pro-inflammatory cytokines; while in the later stage, macrophages display M2 characteristics that generate anti-inflammatory and extracellular matrix molecules^74,75. Previous studies have found that the BEVs exhibited double-sided influences on the functions of macrophages⁷⁶. On one hand, the BEVs bearing the PAMPs, especially LPS, can bind to the pattern recognition receptors of macrophages, inducing the increased release of pro-inflammatory factors. However, not all BEVs can stimulate the inflammatory response. For instance, the probiotic Escherichia coli Nissle 1917 and its BEVs bearing a truncated LPS chain have shown obvious inflammation inhibition in the mice model of colitis⁷⁷. On the other hand, BEVs, especially those secreted by the probiotics Lactobacillus, do not carry LPS, thus inhibiting the pro-inflammation and promoting the polarization of macrophages towards the M2 subtype^78,79. Although these studies did not elaborate on the detailed mechanism of the polarization of macrophages induced by BEVs, the expression level of interleukin-10 (IL-10) is often considered as one of the reasons, which is regulated by the signal transducer and activator of transcription-3 activation from the IL-10^80,81.

The Lactobacillus reuteri-derived BEVs were first found to possess the inflammation-modulating effect in wound tissues²⁸. These BEVs delivering 3-HPA could alter the permeability of mitochondria by inhibiting the opening of mitochondrial permeability transition pore (mPTP), reducing the number of pro-inflammatory macrophages, and shifting the M1 macrophages to the M2 phenotype⁸². The decreased level of inflammation significantly promoted the wound healing (Fig. 6A)^28,83. Later studies further suggested that the ratio of IL-10 to TNF-α correlated with the wound healing outcomes, with the increased ratio leading to better healing of adult burn wounds⁸⁴. Based on the inflammation regulatory roles of TNF-α and IL-10 in wound healing, Kuhn et al.⁸⁵ found that the L. plantarum-derived BEVs could significantly decrease the TNF-α/IL-10 ratio when the peripheral blood mononuclear cells were treated under anaerobic conditions, effectively suppressing the persistent inflammation and accelerating the wound healing (Fig. 6B). Recently, Kim et al.⁸⁶ demonstrated that the L. plantarum-derived BEVs could also block the secretion of pro-inflammatory cytokines, including IL-6 and IL-4, exhibiting considerable preventive effects against skin inflammation.

Figure 6.

BEVs modulate the inflammation phase of wound healing. (A) The L. reuteri derived BEVs delivering intrinsic 3-HPA to modulate the shift of macrophage from M1 to M2 for inflammatory response inhibition. Adapted with permission from Ref. 27. Copyright 2024, Wiley-VCH. (B) The L. plantarum derived BEVs embedded inside the hydrogel deliver intrinsic muramidase to reduce inflammatory factors released from PMBCs. Adapted with permission from Ref. 84. Copyright 2024, Elsevier Ltd. Figure was created by https://app.biorender.com/. 3-HPA, 3-hydroxypropionaldehyde; PMBCs, peripheral blood mononuclear cells.

6.2. Management of the proliferating phase

The proliferating phase involves the migration and proliferation of various skin repair cells, such as endothelial cells, epidermal cells, and fibroblasts. The growth factors released by inflammatory cells and platelets can effectively promote the proliferation and migration of these cells, which is imperative for angiogenesis, re-epithelization, and regeneration of ECM^4,87. With the generation of the granulation tissue, the peripheral proliferative zone moves toward the central area until the epidermal wound edges meet⁸⁸. However, in certain pathological conditions, such as diabetes, obesity, and aging, the long-term hyperglycemia and hypoxemia usually lead to a continuous pro-inflammatory state, disturbing the angiogenic response, thus impeding the granulation tissue formation. To inhibit the inadequate vascularization in wounds, Zhou et al.⁸⁹ embedded the L. reuteri-derived BEVs into a hydrogel to deliver bioactive miRNAs. These BEVs exhibited distinct stimulating capacities in cell proliferation, migration, and angiogenesis in vitro by modulating the gene expression in hypoxia inducible factor-1 (HIF-1) pathways (Fig. 7A). Besides, Xiao et al.⁹⁰ have found the elevated levels of lysophosphatidylcholine (LPC) in the photosynthetic bacteria-derived bacterial nanovesicles (BNVs) synthesized by extrusion compared to the OMVs obtained by centrifugation. Lipid metabolism plays a significant role in wound healing⁹¹. The LPC could be converted into phosphocholine under the catalysis of acyltransferase, leading to the alteration of membrane configuration and then activation of the epidermal growth factor receptor (EGFR) signal responses^91,92. Subsequently, the downstream phosphatidyqinositol-3 kinase/protein kinase B (PI3K/AKT) pathway was activated to promote angiogenesis and re-epithelialization (Fig. 7B)⁹³. Most recently, Wang et al.⁹⁴ have found that the L. rhamnosus GG derived BEVs could promote re-epithelialization and angiogenesis via delivering miR-21-5p to both the epithelial and endothelial cells (Fig. 7C). The intracellularly delivered miR-21-5p was demonstrated to activate the p-AKT/HIF-1 pathway, thus accelerating the proliferation and migration of both epithelial and endothelial cells during the proliferating phase of wound healing.

Figure 7.

BEVs modulate the proliferation phase of wound healing. (A) The L. reuteri derived BEVs embedding inside a hydrogel, delivering intrinsic miRNA to activate the HIF-1 pathway for promoting angiogenesis. Adapted with permission from Ref. 88. Copyright 2025, American Chemical Society. (B) The photosynthetic bacteria derived BEVs and BNVs deliver LPC to activate the PI3K/AKT/EGFR pathway for promoting angiogenesis and proliferation. Adapted with permission from Ref. 89. Copyright 2024, KeAi Publishing. (C) The L. rhamnosus GG derived BEVs delivering intrinsic miR-21-5p to activate pAKT/HIF1 pathway for promoting proliferation and migration of HUVEC and HaCat. Adapted with permission from Ref. 93. Copyright 2024, Springer. Figure was created by https://app.biorender.com/. LPC, lysophosphatidylcholine; BNVs, bacterial nanovesicles.

6.3. Management of the remodeling phase

The remodeling phase involves the decrease of wound cells, the resolution of inflammation, as well as the transition of the type III collagen in the granulation tissue to the type I collagen, which is the main matrix component of the dermis⁹⁵. The remodeling processes include the synthesis, deposition, as well as degradation of ECM, ultimately forming a scar composed of tissue similar to skin but with altered architecture⁹⁶. To reduce the formation of hypertrophic scars, Han et al.⁹⁷ have observed significantly inhibited hypertrophic scar fibrosis with the treatment of the L. druckerii-derived BEVs. These BEVs were demonstrated to significantly downregulate the expression of collagen I/III and α-smooth muscle actin (α-SMA). While the precise mechanism remains to be elucidated.

7. Functionalization of BEVs for optimal wound management

Although encouraging results for the local administration of BEVs have been achieved in the mouse model of full-thickness wounds, there are still some shortcomings associated with the BEVs for the wound treatment, such as low targeting, poor penetration, short retention time, as well as unsatisfying therapeutic effect^16,29. Therefore, more and more engineering strategies have been proposed to functionalize the BEVs with improved delivery specificity and efficiency for optimal wound management. These engineering strategies can be summarized and categorized into biological engineering, physical engineering, chemical engineering, as well as material engineering (Fig. 8 and Table 3).

Figure 8.

The engineering strategies to functionalize of BEVs for the optimal wound treatment. The biological engineering approach includes overexpression and knockout. The physical engineering approach includes membrane coating, membrane fusion, electroporation, and sonication. The chemical engineering approach includes the covalent reaction based click chemistry, bioconjugation, and aldehyde amine condensation, as well as the non-covalent reaction based electrostatic interactions, receptor–ligand binding, and hydrophobic insertion. The material engineering approach mainly refers to the hydrogel embedding. Figure was created by https://app.biorender.com/.

Table 3.

Engineering approaches to functionalize BEVs for the wound treatment: techniques, mechanisms, advantages, and limitations.

Approach	Technique	Mechanism	Advantages	Limitation	Ref.
Biological engineering	Overexpression	Fusion of the coding sequences of the interested protein into recombinant plasmids	Standardized manufacture	Limited chassis cell type	101,102,154,155
	Knockout	Specific cleavage of bacterial virulence genes by CRISPR-Cas9	High biosafety	Low production	101,109
Physical engineering	Membrane coating	Recombination of the lipid bilayer of BEVs and surface adhesion to nanoparticles	High biocompatibility	Instable membrane	112,156
	Membrane fusion	Transient destruction and reconstruction of the lipid bilayer of BEVs and other particles	High biocompatibility	Limited drug release	113,157
	Electroporation	Formation of a transient pore and enhanced permeability	High efficiency	Damaged membrane	115,158
	Sonication	Accelerated bilateral fluid exchange between the intramembrane and extramembrane	Simple operation	Damaged membrane	112
Chemical engineering	Click chemistry	Copper-catalyzed or strain-promoted azide-alkyne cycloadditions	High stability	Residual organic reagent	124
	Aldehyde amine condensation	Affinity addition between the amino and carbonyl groups	High stability	Residual organic reagent	125
	Bioconjugation	Interaction between CP05 and interested molecules	Broad application	Need for other markable protein	126
	Hydrophobic insertion	Hydrophobic interactions between amphiphilic conjugates with the lipid bilayers of BEVs	Broad application	High purification cost	132
	Electrostatic interactions	Electrostatic interactions between the positively charged molecules and the negatively charged BEVs	Simple operation	Instability	134
	Receptor–ligand binding	Specific interactions between the paired biomolecules	High specificity	Complex operation	137
Material engineering	Hydrogel embedding	Diffusion and adsorption of BEVs in the network structure	Controllable release	Low loading efficiency	86

7.1. Biological engineering

Due to their special biogenesis mechanism, BEVs can carry quite a lot of bioactive cargoes from their parent strains. As such, the biological engineering of BEVs mainly refers to the gene editing of bacteria via the overexpression or knockout approach, which can change the composition of bacteria and thus BEVs (Fig. 9).

Figure 9.

The advanced therapeutic outcomes imparted from the diverse engineering strategies for the functionalization of BEVs. The biological, physical, chemical, and material engineering strategies were marked in green, yellow, blue, and purple, respectively. All these advanced therapeutic outcomes, including specific targeting, drug loading, antigen displaying, toxicity attenuation, prolonged retention, as well as endosome escaping were marked in grey. Figure was created by https://app.biorender.com/.

Overexpression refers to the upregulation of specific gene expression⁹⁸. The desired genes are first synthesized through PCR or chemical synthesis. Then a suitable vectors, commonly featuring a strong promoter, selected marker, and multiple cloning sites, are chosen to load the desired gene for transfection⁹⁹. Based on the overexpression strategy, bacteria can be engineered to overexpress a therapeutic protein so that it can be packaged inside the BEVs. For instance, the Gram-negative Escherichia coli has been genetically engineered to generate BEVs displaying the hyaluronidase on the surface via the membrane protein cytolysin A. The hyaluronidase could break down hyaluronan, remodeling the ECM and inhibiting the hypertrophic scar fibrosis for the optimal wound management¹⁰⁰. Besides, the Gram-positive Lactobacillus lactis NZ9000 has also been genetically engineered to generate BEVs containing the vascular endothelial growth factor (VEGF). The VEGF could promote angiogenesis and thus accelerate the chronic wound healing¹⁰¹. Most recently, Escherichia. coli has also been genetically engineered to generate BEVs delivering shuttle plasmid, which has enhanced the expression of both bone morphogenetic protein and VEGF in the host cells¹⁰². All in all, the overexpression is a customizable platform for engineering bacteria to generate BEVs that deliver therapeutic proteins for wound treatment. Nonetheless, the scope of BEV engineering remains limited to Gram-negative bacteria. Even though the Gram-positive bacteria like Lactobacillus lactis NZ9000 have also been reported to be engineered, their thick peptidoglycan layer and unique gene expression model decreased the transference efficiency and increased the manufacturing cost, thus limiting their wide applications^101,103,104.

Knockout refers to the inactivation of specific gene expression. CRISPR-Cas9-based and λ-Red recombination systems are important tools for altering the genes^29,105. Based on these knockout strategies, bacteria can be engineered to generate BEVs without the expression of a specific component. For instance, the nlpl gene has been excised from the genome of Escherichia coli by the λ-Red recombination system and replaced with an electroporated dsDNA containing a chloramphenicol cassette. As such, a hyper vesicular strain was produced¹⁰⁰. Besides, two homologous msbB genes have been mutated from the genome of Escherichia coli using the λ-Red recombination system¹⁰⁶. Since the msbB genes encoding the lipid A acyltransferase, the expression of lipid A on the BEVs has been attenuated, reducing their endotoxic activity and decreasing their inflammatory effect. These BEVs secreted by the msbB knockout Gram-negative bacteria could be widely used as detoxified nanocarriers for the optimal wound management^107,108.

7.2. Physical engineering

Physical engineering of BEVs generally involves changes in their membrane structure caused by external mechanical forces. It can be classified into four groups, including membrane coating, membrane fusion, electroporation, and sonication (Fig. 9).

Membrane coating is mainly accomplished by several methods, including the traditional physical extrusion and sonication, as well as the co-incubation of living cells with the desired nanoparticles^109,110. For instance, Wu et al.¹¹¹ have used Escherichia coli-derived BEVs as the outer shell and the rifampicin-loaded mesoporous silica nanoparticles as the inner core. The membrane-coated nanoparticles significantly exerted the targeting ability of BEVs, and thus enhanced the uptake of nanoparticles for the targeted delivery of antibiotics. Besides, membrane fusion is a strategy to create hybrid membrane nanovesicles consisting of both BEVs and other interested membrane structures. It generally involves mixing the interested membrane source with BEVs before co-extrusion. For instance, Zou et al.¹¹² have fused the Escherichia coli-derived BEVs with the membranes of tumor cells. The generated hybrid membrane nanovesicles could utilize the immunogenicity and the adaptive immunity of BEVs to better against the homologous tumors. In addition, electroporation is a common method to load exogenous molecules into BEVs. The external electric field can alter the permeability of the BEVs’ membranes, allowing the translocation of the specific bioactive molecules, such as peptide or miRNA¹¹³. For instance, Pan et al.¹¹⁴ have loaded the neuroprotectant pioglitazone (PGZ) into the low-endotoxin BEVs by electroporation. The generated BEV@PGZ could be internalized by neutrophils via the TLR-mediated endocytosis, thus penetrating the blood–brain barrier into the infarcted brain for the targeted treatment of ischemic stroke. Moreover, sonication is also a commonly used physical engineering approach. It generally involves the accelerated bilateral fluid exchange and the diffusion caused by the mechanical shear stress, after mixing the interested molecules with BEVs.

Although the physical engineering provides feasible approaches to load the bioactive molecules or nanoparticles into BEVs, the mechanical forces used tend to compromise their structural integrity. For instance, improper electroporation conditions can cause the increased leakage of cargoes¹¹⁵. The electronic pulse may also result in the aggregation of cargoes, such as siRNAs, compromising their silencing efficiency¹¹⁶. Therefore, optimized protocols should be developed for the best outcomes.

7.3. Chemical engineering

Based on the different interactions, chemical engineering can be categorized into the covalent reaction methods and the non-covalent reaction methods. The covalent reaction methods include click chemistry, aldehyde amine condensation, and bioconjugation. The non-covalent reaction methods include hydrophobic insertion, electrostatic interaction, and receptor–ligand binding (Fig. 9).

Click chemistry is a commonly used covalent reaction method to modify BEVs. The alkyne group is firstly attached to the BEVs through the condensation reaction of (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide)-(N-hydroxy succinimide) (EDC-NHS), and then the alkyne group is covalently coupled with the azide group of the interested molecules via the copper-catalyzed azide-alkyne cycloaddition (CuAAC)^117,118. For instance, Smyth et al.¹¹⁹ have achieved the conjugation of azide-fluor 545 to MEVs using the CuAAC. To eliminate the possible cytotoxicity brought by the Cu catalysis, strain-promoted azide-alkyne cycloaddition (SPAAC), based on the use of dibenzocyclooctyne (DBCO), has also been developed as an alternative to CuAAC120, 121, 122. For instance, Feng et al. have introduced an azide group onto the surface of Escherichia coli-derived BEVs via N-azidoacetylgalactosamine-tetraacylated (Ac4GalNAz)¹²³. Then DBCO-PEG/Se was modified to the BEV's surface by SPAAC. The PEG/Se layer not only mitigated the immunogenicity but also achieved the X-ray-triggered controlled release. Besides, aldehyde amide condensation has been used to couple MEVs and aptamers¹²⁴. Aptamers are small sequences of oligonucleotides obtained in vitro that can bind to the receptor with high affinity and strong specificity. The membrane proteins of BEVs can be treated to achieve the exposure of the amino group, and then the aldehyde group of aptamers can be utilized to couple with BEVs through an aldehyde amide condensation, empowering the BEVs with high affinity and strong specificity to the target cells and tissues. However, extra purification is necessary to remove residual organic reagents after engineering BEV through click chemistry and aldehyde amide condensation. In addition, the bioconjugation strategy also has promising applications in the modification of BEVs as nanocarriers for drug delivery¹²⁴. For instance, an anchoring peptide CP05 that was identified by phage display has been specifically coupled to the second extracellular loop of the MEV membrane protein CD63, thus enabling the functionalization of MEVs with desired properties for drug delivery¹²⁵. Nonetheless, the specific functionalization biomarkers still need to be sought to precisely apply the modification of BEVs via the bioconjugation approach¹²⁶.

Hydrophobic insertion is a simple non-covalent approach to engineer BEVs. Due to the lipid bilayer of BEVs, the desired molecules with hydrophobic tails can be readily inserted into the membrane of BEVs, enabling the BEVs with additional properties, such as targeting, penetrating, and retaining for advanced drug delivery¹²⁷. Candidates that have been reported for the hydrophobic insertion include distearoyl phosphatidylethanolamine-polyethyleneglycol (DSPE-PEG), cholesterol, and palmitic acid128, 129, 130. For instance, Liu et al.¹³¹ have customized a bone-targeting peptide SDSSD conjugated DSPE-PEG molecule, and inserted it into the Lactobacillus rhamnosus-derived BEVs. These BEVs exhibited bone microenvironment-targeted ability and attenuated the ovariectomy-induced osteoporosis. Besides, electrostatic interaction has also been used to modify BEVs with the positively charged molecules due to the negatively charged membranes of BEVs^38,132. For instance, Nakase et al.¹³³ have attached the cationic lipids along with pH-sensitive GALA peptides onto the surface of MEVs via electrostatic interactions, significantly enhancing the cellular uptake of the modified MEVs. In addition, receptor–ligand binding firstly presents a known receptor protein onto the surface of BEVs, then binds a ligand-modified molecule to achieve the surface modification¹³⁴. The biotin-streptavidin system is one of the most used receptor–ligand pairs¹³⁵. For instance, Weyant et al.¹³⁶ have expressed the biotin-binding protein onto the surface of BEVs using a genetically engineered plasmid, thus all the biotinylated antigen-displaying proteins could be modified to the surface of BEVs.

Chemical engineering offers more possibilities to modify the surface of BEVs and thus enables the additional functionalities of BEVs, such as protecting, targeting, penetrating, as well as antigen displaying. However, to fully unlock the therapeutic potentials of BEVs, mild reaction conditions and reduction reaction steps are required to expedite their clinical translation.

7.4. Material engineering

Material engineering refers to combining the BEVs with suitable dressing materials for optimal wound management. Considering the wound conditions, dressing materials such as hydrogels, films, or even microneedles are desired to provide a moist, protective, and adaptive microenvironment that favors the wound healing (Fig. 9)137, 138, 139.

Among all the materials, hydrogels have been mostly exploited for wound healing due to their excellent biocompatibility as well as adjustable physicochemical and mechanical properties140, 141, 142. Therefore, the nanosized BEVs can be suspended in the hydrogels to assist their sustained and controlled release at the wounds. Depending on the different interaction modes, the BEVs can be embedded into the hydrogel via both physical and chemical approaches. The physical approach typically involves the direct diffusion or electrostatic interaction of the BEVs with hydrogels. While the chemical approach mainly involves the covalent interaction between BEVs and hydrogels, such as the Michael addition between sulfhydryl groups and maleimides¹⁴³. For instance, Kuhn et al.⁸⁵ have directly embedded the Lactobacillus casei-derived BEVs into a hydrogel that consisted of hydroxyethylcellulose and glycerol. The BEVs loading hydrogel exhibited significant anti-inflammatory effects in the mouse model with full-thickness wounds, promoted wound healing, and reduced scar formation. By contrast, Zhou et al.⁸⁹ have covalently bound the carboxylated chitosan-modified Lactobacillus. reuteri-derived BEVs with the oxidized hyaluronic acid via the Schiff reaction form hydrogel. Compared to the physically mixed BEVs in hydrogel, the covalently formed hydrogel system enabled the controlled release of BEVs, facilitating better vascularization for wound healing.

In addition to hydrogels, microneedles have also been exploited for transdermal delivery¹⁴⁴. Most recently, Qi et al.¹⁴⁵ have loaded the Lactobacillus druckerii-derived BEVs into the methacrylate gelatin-based microneedles. The sustained release of BEVs from the microneedles was demonstrated to inhibit pathogenic bacteria and promote symbiotic bacteria, thus accelerating the infected wound healing. Considering their customizable structures and functions, materials self-assembled from peptides should be exploited to incorporate with the BEVs in future^146,147. Through rational design, both the structures and functions of the peptide-based materials could be programmed to work with the BEVs, fulfilling the varied requirements of the different phases during wound healing and thus facilitating the optimal therapeutic outcomes148, 149, 150, 151, 152. As the material engineering involves both the loading and releasing of BEVs, the bioactivates of BEVs should be furtherly examined and controlled to guarantee their effective wound healing potency.

8. Perspectives and outlook

Wound healing is a complicated process that involves sequential and overlapped phases of inflammation, proliferation, and remodeling. However, in the pathological conditions, including aging, obesity, as well as diabetes, the macrophage polarization in the inflammation phase is dysregulated, the angiogenesis, as well as re-epithelization of the proliferation phase are impaired, leading to the occurrence of chronic wounds. Despite the multidisciplinary care treatments, their unsatisfying efficacy and safety underscore the high demands of the optimized therapeutic modalities.

EVs, the bioactive nanocarriers secreted by cells, have emerged as a promising alternative for optimal wound management158, 159, 160. These phospholipid bilayer nanostructures are capable of shuttling bioactive substances, including nucleic acids, proteins, as well as metabolites from their parent cells to the recipient cells, and are responsible for mediating the intercellular communications. Compared to the cell-based therapies, the EVs are less prone to cause immunogenic rejection or tumor formation. Besides, they can be more easily customized on demand and be more feasibly lyophilized for long-term storage. These advantages of EVs have spurred extensive investigations into unleashing their therapeutic potential for chronic wound treatment. The EVs derived from the human mesenchymal stem cells, pluripotent stem cells, as well as endothelial progenitor cells have been widely explored^161,162. Most notably, the human mesenchymal stem cell-derived EVs have recently entered the Phase I Trial for the wound healing of cutaneous ulcers in diabetics (NCT05243368, ClinicalTrials.gov). Nonetheless, the complicated engineering procedures, low isolation efficiencies, and underlying ethical concerns of these mammalian-derived EVs (MEVs) may limit their further clinical translations.

As an alternative, the bacteria-derived EVs (BEVs) can overcome these limitations. Compared to the MEVs, the nanosized BEVs possess several supercities (Table 4). First of all, owing to the advanced genetic, physical, and chemical modification approaches, the engineering of the BEVs is customizable^153,154. Secondly, due to the rapid proliferation and mature culture procedures of bacteria, the manufacturing of BEVs is scalable and cost-effective¹⁶³. Thirdly, the bioactive cargoes and surface PAMPs of BEVs inherited from their parental bacteria empowered their immunomodulatory capacities, which could be furtherly tailored to meet the varied requirements during the shifted phases of the wound healing process. These superiorities, together with the emerging interests on the “gut–skin axis”, aroused a new upsurge in exploiting the BEVs for wound management. More and more BEVs, particularly those derived from probiotics, have been directly used or engineered for wound management. With the successful communalization and clinical use of the probiotics, as well as the BEV-based vaccines Bexsero®, the new upsurge of BEVs for wound treatment holds the highest potential to be pushed from the bench to bedside in the future.

Table 4.

Comparisons between the BEVs and MEVs in terms of source, structure, immunogenicity, isolation efficiency, engineering approaches, mass manufacture, as well as ethical issue.

Item	BEVs	MEVs
Source	Bacteria cells	Mammalian cells
Structure	High stability	High mobility
Immunogenicity	High	Low
Isolation efficiency	High	Low
Engineering approach	Biological, chemical, physical, material	Biological, chemical, physical, material
Mass manufacture	Bacteria fermentation/economical	Cell culture/expensive
Ethical issue	None	Exist

Despite these unique merits of BEVs, there are still several challenges waiting to be addressed for moving the wound treatment with BEVs from bench to bedsides (Fig. 10). First of all, even though the bioactive cargoes of BEVs are considered as the key mediators during wound healing, their compositions and functions remain ambiguous and need to be identified. Based on the high-throughput omics technologies, several databases, including the Vesiclepedia, ExoCarta, and EVAtlas, have already been established for exosomes. Nonetheless, a similar database specialized for the BEVs is still in high demand to facilitate the optimal wound management164, 165, 166. Secondly, the functions and mechanisms regarding these bioactive cargoes require further clarification. Until now, only a limited number of signal pathways have been identified to accelerate wound repair and regeneration by BEVs^167,168. Considering the diversity of gut microbiota, there are still a huge number of unknown pathways waiting to be explored. Thirdly, the manufacturing and evaluation standards need to be unified. It is well known that the variation in bacterial species, culture conditions, and isolation techniques can lead to the highly heterogeneous structures, compositions, and functions of BEVs. Thereby, both quantity and quality standards are required to ensure their consistent therapeutic performance of BEVs. According to the Minimum Information for Studies of Extracellular Vesicles (MISEV), differential centrifugation at 10,000 to 20,000×g is recommended for 10 to 90 min to collect the larger and denser EVs, while for 45 to 150 min to pellet the smaller and lighter EVs⁴⁷. Nonetheless, these recommendations are only applicable to collect BEVs with improved reproducibility for research purposes. As for the large-scale GMP manufacture of BEVs for clinical use, the standards of the isolation conditions still need to be adjusted and established. Fourthly, the immunogenicity of BEVs should be optimized, rather than simply knocking out genes involved in the LPS synthesis. So that the moderate immunostimulatory capacity of BEVs can be preserved with attenuated biotoxicity. Last but not least, since the main administration route for wound treatment is still local administration, the potential risks regarding the systemic BEV administration are underestimated155, 156, 157. The diverse engineering approaches should be exploited to functionalize the BEVs. For instance, the biologically engineered BEVs loaded with growth factors can be further chemically engineered to achieve the targeted delivery into specific cells, or even materially engineered to achieve controlled release at the wound sites. Therefore, BEVs can be endowed with specific targeting, enhanced penetration, improved endosome escape, as well as prolonged retention time to achieve the optimal wound treatment performance via the different administration routes.

Figure 10.

The advantages and challenges of exploiting BEVs for wound treatment. The outstanding features of BEVs include nanoscale size, loading capacity, natural immunomodulatory, mass manufacture, and standard engineering. While there are still challenges related to BEVs, such as undefined standards, ambiguous content, poor targeting, uncertain biosafety, and unclear mechanisms, that need to be addressed for the better treatment of wounds. Figure was created by https://app.biorender.com/.

With the rapid development of clinical interventions for wound treatment, the current research focus has been moved from tissue repair to appendage regeneration169, 170, 171, 172. The BEVs mediated regeneration of appendages, including hair follicles, sweat glands, and sebaceous glands, would be a valuable research direction worthy of further exploration. In addition, the hot research field of organoids is where the BEVs could also contribute. Skin organoids that integrated with the engineered BEVs derived from human microbiota may serve as the optimal alternative for the realistic skin¹⁷³. Together with the emerging Artificial Intelligence algorithms, the investigation on the properties and functions of BEVs on wound treatment is poised to accelerate¹⁷⁴. It is envisioned that these interdisciplinary technologies could be integrated to work out innovative wound healing modalities based on the BEV for satisfactory clinical outcomes.

Author contributions

Yejiao Shi and Xi Liu conceived the manuscript. Honggang Hu and Cuiping Zhang supervised the manuscript. Zelin Zheng, Yuting Li, and Yirong Wang performed the literature investigation. Yejiao Shi, Zelin Zheng, and Xi Liu wrote the original manuscript. Helene Azevedo, Cuiping Zhang, and Honggang Hu revised the manuscript.

Declaration of Generative AI and AI-assisted technologies in the writing process

The authors declare no generative AI in scientific writing.

Conflicts of interest

The authors declare no conflicts of interest.