Biorthogonal click-immobilized probiotic membrane vesicles in a dynamic hydrogel for accelerated and scar-minimized wound healing

Abstract

Chronic and complex wounds require materials capable of simultaneously regulating inflammation and promoting vascularized tissue regeneration. Here, we engineer a bioorthogonal vesicle–hydrogel that covalently integrates Lactobacillus casei–derived membrane vesicles (LCMVs) within a carboxymethyl chitosan/aldehyde-hyaluronic acid network to achieve sustained vesicle presentation and enhanced bioactivity. The resulting Gel-LCMVs composite exhibits an ECM-mimetic porous architecture, stable viscoelasticity, and controlled vesicle release, enabling marked stimulation of cell proliferation, migration, and endothelial tube formation while suppressing macrophage-derived pro-inflammatory signals. In a full-thickness excisional wound model, Gel-LCMVs achieved >50% closure by day 3 and nearly complete healing by day 10, with improved granulation tissue formation, thicker neo-dermis and enhanced collagen deposition, outperforming a commercial dressing (Tegaderm). Transcriptomic profiling reveals activation of PI3K–AKT, Wnt, and JAK–STAT pathways and suppression of inflammatory gene programs. This bioactive and cell-fre hydrogel platform demonstrates how precise materials–microbio-derived vesicle integration can synergetically steer wound microenvironment remodeling and enable scar-minimized wound repair.

Graphical abstract

Highlights

• •SPAAC chemistry enables stable covalent anchoring of LCMVs within CMCS/HA hydrogel networks.
• •Covalent bonding minimizes burst release, providing sustained delivery for wound management.
• •Gel-LCMVs significantly boost wound closure and neovascularization vs. commercial dressings.

1. Introduction

Wound healing is a complex biological cascade involving hemostasis, inflammation, proliferation, and remodeling [1]. In chronic wounds, such as diabetic ulcers and deep burns, this orchestrated process is often stalled in a persistent inflammatory state, leading to delayed recovery and excessive scarring [2]. Conventional interventions, including growth factors and stem cell transplantation, are frequently constrained by short half-lives, high costs, and immunogenic risks [[3], [4], [5], [6], [7]]. Consequently, there is a critical clinical need for biocompatible wound dressings that provide sustained bioactivity and provide a favorable microenvironment for tissue regeneration.

Bacterial membrane vesicles (BMVs), nanosized extracellular vesicles secreted by bacteria, have emerged as a promising cell-free therapeutic strategy offering stability, low production cost, and high scalability [[8], [9], [10]]. Among them, Lactobacillus casei-derived membrane vesicles (LCMVs) retain the potent anti-inflammatory and regenerative properties of the parent probiotic strain while exhibiting markedly lower immunogenicity [11]. Compared to mammalian cell-derived exosomes, which face hurdles regarding scalable manufacturing, high costs, and donor variability, LCMVs offer a cost-effective and scalable alternative for regenerative nanomedicine [12,13]. However, the therapeutic efficacy of free LCMVs is severely compromised by their rapid degradation and clearance when administered directly to the wound site [14].

Hydrogels are currently one of the most promising materials for tissue repair, due to their high-water content, the structure that mimics the extracellular matrix (ECM), and the controllable drug release properties [[15], [16], [17]]. To address the limitations of rapid clearance and uncontrolled leakage, hydrogel-based delivery systems have been widely explored as biomimetic carriers for vesicle delivery [18,19]. Nevertheless, simply mixing vesicles into hydrogel matrices typically leads to an undesirable initial burst release, which limits long-term therapeutic efficacy and may cause localized toxicity. To overcome this delivery bottleneck, it is essential to engineer a system capable of stable vesicle immobilization and controlled release.

In this study, we employed a nanobiotechnological approach to construct a “click-conjugated” hydrogel system (Gel-LCMVs) by utilizing strain-promoted azide-alkyne cycloaddition (SPAAC). Specifically, carboxymethyl chitosan (CMCS) was functionalized with strained alkyne groups, while the surface of LCMVs was precisely modified with azide moieties to enable site-specific covalent anchoring within the aldehyde-hyaluronic acid (AHA) network. Unlike traditional crosslinking methods, this bio-orthogonal click chemistry allows for stable vesicle immobilization under mild physiological conditions, preserving the structural integrity and intrinsic bioactivity of the nanovesicles. This engineered interface not only prevents the burst release but also facilitates a sustained and controlled release profile, transforming the hydrogel into a highly bioactive nano-scaffold capable of orchestrating angiogenesis and modulating the immune microenvironment over an extended period (Scheme 1). By integrating biochemical cues with a structural support matrix, this work establishes a robust, cell-free nanobiotechnological platform. It not only provides a high-performance therapeutic for full-thickness skin injuries but also offers a new paradigm for the functionalization of biomaterials with probiotic-derived nanovesicles.

Scheme 1.

Schematic illustration of Gel-LCMVs hydrogel synthesis and its wound healing ability in vivo.

2. Materials and methods

2.1. Materials

Lactobacillus casei was obtained from the Industrial Microbial Strain Engineering Technology Research Center of Henan Province, China. MRS medium was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Carboxymethyl chitosan (CMCS, degree of carboxylation ≥80%), tris(2-carboxyethyl) phosphine hydrochloride (TCEP·HCl), and DBCO-PEG4-NHS ester were purchased from Aladdin Industrial Corporation (Shanghai, China). Hyaluronic acid (HA) and sodium periodate were obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Scientific (Waltham, MA, USA). All mice were obtained from the Model Animal Research Center of Chongqing University (Chongqing, China).

2.2. Extraction and characterization of LCMVs

Lactobacillus casei was cultured in MRS medium at 37 °C with agitation at 220 rpm under anaerobic conditions for 48 h. Following incubation, the culture was centrifuged at 2000 g for 30 min, and the supernatant was collected. The supernatant was then passed through a 0.22 μm pore-size filter to remove residual cells. Subsequently, the filtrate was ultracentrifuged at 160,000 g for 70 min, and the resulting pellet was resuspended in PBS and subjected to a second round of ultracentrifugation. The final pellet was collected and designated as LCMVs.The morphology of LCMVs was observed using transmission electron microscopy (TEM), and particle size distribution was analyzed by nanoparticle tracking analysis (NTA).

For miRNA profiling, total miRNAs were extracted from LCMVs, and the top 100 miRNAs were selected based on abundance. Target gene prediction was performed for the selected miRNAs. Genes associated with wound healing were identified using the Gene Ontology (GO) database, and the predicted target gene sets were mapped accordingly to identify wound-healing–related miRNAs. The number of target genes corresponding to each miRNA was quantified and ranked. By integrating target gene information with expression levels and interaction networks, the top 10 highly expressed miRNAs with extensive target gene associations were identified. Furthermore, target gene localization analysis was conducted for the selected miRNAs.

2.3. Synthesis of hydrogels

Aldehyde-functionalized hyaluronic acid (AHA) was synthesized as a hydrogel precursor. Briefly, 100 mL 2.5% HA were mixed with 2.5 g NaIO₄, and stirred in the dark for 12 h. The resulting product was dialyzed against ultrapure water for 3 d and then lyophilized.

Hydrogels were prepared through a Schiff base reaction by mixing equal volumes of 2% (w/v) CMCS solution and 3% AHA solution under physiological conditions, yielding hydrogels referred to as Gel. For the Gel-LCMVs, 2% CMCS solution was reacted with DBCO-PEG4-NHS ester (10 mM) for 12 h, dialyzed against ultrapure water for 48 h, and lyophilized to obtain DBCO-modified CMCS. The synthesized materials were characterized by ¹H NMR spectroscopy. The grafting degree (degree of substitution, DS) of DBCO groups on the carboxymethyl chitosan (CMCS) backbone was quantitatively determined using ¹H NMR spectroscopy. By comparing the integral area of the aromatic protons of the DBCO groups (7.2–7.8 ppm) with the integral area of the characteristic protons on the CMCS sugar ring (3.0–4.0 ppm).

LCMVs (100 μg/mL) pretreated with 1 mM TCEP were reacted with NHS-PEG4-azide (final concentration 2 mM) at 26 °C for 2 h to obtain N₃-LCMVs. The DBCO-CMCS was then mixed with N₃-LCMVs, the pH was adjusted to 7.4, and the mixture was gently shaken at room temperature in the dark for 2 h. Finally, the product was combined with AHA to form the Gel-LCMVs hydrogel. In addition, a physically compounded hydrogel loaded with LCMVs (Gel@LCMVs) was prepared as a control. Briefly, the desired amount of LCMVs (100 μg/mL) was added to the CMCS solution, which was then crosslinked with AHA to form a hydrogel.

2.4. Characterization of hydrogels

The morphology and internal structure of the hydrogels were characterized using scanning electron microscopy (SEM, HITACHI S4800, Japan). The swelling ratio of the hydrogels was determined in PBS at 37 °C, while the in vitro degradation rate was evaluated under physiological conditions in PBS using an osmotic method.

For release studies, LCMV-loaded hydrogels were immersed in PBS and incubated at 37 °C on a shaker, and the release behavior of LCMVs was monitored at predetermined time points. The release rate of LCMVs was quantified using a BCA protein assay kit. In addition, LCMVs labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil; Beyotime Biotechnology, China) were incorporated into the hydrogel matrix, and their distribution within the hydrogels was visualized using confocal microscopy.

The in vivo degradation rate of hydrogels was assessed by implanting Gel-LCMVs into the mouse dermis and monitoring the hydrogel state at predetermined time points. Rheological properties of the hydrogels, including storage modulus (G′) and loss modulus (G″), were measured using an Anton Paar MCR92 rheometer (Anton Paar, Austria). The linear viscoelastic region was determined by amplitude sweep measurements in the range of 0.1%–100%.

The antibacterial activity of the hydrogels was evaluated. Briefly, each hydrogel sample was incubated with 1 mL of LB medium containing 1 × 10⁴ CFU/mL of bacteria (Escherichia coli or Staphylococcus aureus) in sterile test tubes at 37 °C for 24 h. Bacterial suspensions without hydrogels served as the control group. After incubation, the bacterial culture was serially diluted (10⁴-fold), spread evenly onto LB agar plates, and incubated for an additional one day. The number of bacterial colonies on each plate was recorded as M₁, while the number of colonies in the control group was recorded as M₀. The antibacterial efficiency (%) was calculated using the following equation:

Antibacterial efficiency (%) = (M₀ – M₁)/M₀ × 100%

2.5. Cell culture and proliferation assay

Human umbilical vein endothelial cells (HUVECs) and mouse fibroblast cells (L929) were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin under standard conditions.

For cell proliferation assays, cells were seeded into 96-well plates at a density of 5 × 10³ cells per well. After 12 h, the culture medium was replaced with fresh complete medium containing different concentrations of LCMVs, while the control group received complete medium only. At predetermined time points, 10% Cell Counting Kit-8 (CCK-8, Beyotime Biotechnology, China) solution was added to each well, followed by incubation at 37 °C for 2 h. The optical density (OD) of each well was measured at 450 nm using a microplate reader (BioTek Synergy H1, USA). Each condition was tested in triplicate, and the average values were used for statistical analysis.

2.6. Cell migration assay

HUVECs and L929 cells were seeded in 6-well plates and cultured for 24 h. Using a sterile 200 μL pipette tip to create a linear scratch. Add PBS to remove the detached cells, and then add fresh complete medium containing different concentrations of LCMV. Control wells contained only complete medium. Cells were incubated for 24 h, and images of the scratch area were captured at 0 h and 24 h using an inverted microscope (Olympus, Japan). Migration was quantified by measuring the change in wound closure using ImageJ software.

2.7. Angiogenesis assay

For the tube formation assay, 96-well plates were precoated with 50 μL of growth factor–reduced Matrigel (Corning, USA) per well and incubated at 37 °C for 30 min. HUVECs (1 × 10⁴ cells per well) were suspended in complete medium with different concentrations of LCMVs and seeded onto the solidified Matrigel. Control groups were treated with complete medium only. After incubation for 6–12 h at 37 °C, tube-like structures were observed with an inverted microscope (Olympus, Japan). Tube total tube length was analyzed using ImageJ software with the Angiogenesis Analyzer plugin.

Quantitative real-time PCR (qRT-PCR) was performed to determine the mRNA expression levels of VEGFA and PECAM1 (CD31) in HUVECs after treatment with Gel-LCMVs. Total RNA was extracted using TRIzol reagent, reverse transcribed into cDNA, and amplified using SYBR Green Master Mix. The primers were as shown in Table S1.

2.8. In vitro anti-inflammatory activity assay

RAW264.7 cells were co-cultured with different concentrations of LCMVs for 24 h, and an inflammatory model was induced using 100 ng/mL lipopolysaccharide (LPS). Cell morphology was observed under an optical microscope. The supernatants from each group were collected for enzyme-linked immunosorbent assay (ELISA). The levels of inflammatory cytokines (TNF-α and IL-6) were determined using mouse interleukin ELISA kits (Mouse Interleukin ELISA kit).

2.9. In vivo wound healing evaluation

Male C57BL/6 mice (6–8 weeks old, wild-type) were used to evaluate the in vivo wound-healing efficacy of the hydrogels. All animal procedures complied with institutional and national ethical guidelines and were approved by the Animal Ethics Committee of the Third Military Medical University. The mice were randomly assigned to four experimental groups and anesthetized via intraperitoneal injection of sodium pentobarbital. Under aseptic conditions, dorsal fur was removed over an area of approximately 3 × 3 cm, and after disinfection, a full-thickness circular wound (10 mm in diameter) was created on the dorsal skin using a biopsy punch, extending to the fascia without injuring underlying muscle. The wounds were treated with Gel, Gel-LCMVs, or Tegaderm (commercial dressing). Wound closure was monitored and photographed at predetermined intervals, and the residual wound area was quantified using ImageJ software. The wound closure rate was calculated using the following formula:

Wound closure rate (%) =(A₀−A₁)/A₀ × 100%,

Where A₀ represents the initial wound area and A₁ represents the wound area at the designated time point.

2.10. Histologic analysis

The wound tissue was collected at the predetermined time point and fixed with 4% (volume ratio) paraformaldehyde for 48 h. Subsequently, the samples were dehydrated and embedded in paraffin. Then, the samples were sectioned, and stained with hematoxylin-eosin (H&E) to observe the overall morphology and the infiltration of inflammatory cells. At the same time, Masson’s trichrome staining was used to assess the deposition of collagen.

Immunohistochemical (IHC) staining was performed using specific antibodies against α-smooth muscle actin (α-SMA), CD31, vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). Subsequently, the corresponding secondary antibodies were used for detection. The stained sections were observed under an optical microscope and quantitatively analyzed using ImageJ software. Parameters such as the thickness of granulation tissue, the density of new blood vessels, the content of newly formed collagen, and the expression levels of inflammatory and angiogenic markers were evaluated to comprehensively assess the wound healing process. For quantitative analysis of IHC staining, ImageJ software was employed. Briefly, five random fields of view per section were captured. The positive staining area was quantified using the ‘Threshold’ tool, and the results were expressed as a percentage of the total tissue area.

2.11. Statistical analysis

All experiments were repeated at least three times, and the data were presented as the mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 9.0 software (GraphPad Inc., USA). Comparisons between two groups were conducted using the unpaired two-tailed Student’s t-test, while multiple group comparisons were analyzed using one-way analysis of variance (ANOVA) combined with Tukey’s post hoc test.

3. Results and discussion

3.1. Extraction and characterization of LCMVs

After large-scale cultivation of Lactobacillus casei, the culture supernatant was collected for further processing. LCMVs were isolated from the supernatant by ultracentrifugation at 160,000 g, as shown in Fig. 1A. Transmission electron microscopy (TEM) (Fig. 1B) and nanoparticle tracking analysis (NTA) (Fig. 1C) confirmed the successful isolation of LCMVs, exhibiting a predominant size distribution of 80-100 nm, comparable to that of exosomes derived from mammalian cells [20]. These nanosized vesicles are consistent with previously reported bacterial membrane vesicles, indicating that Lactobacillus casei secretes extracellular nanovesicles with typical spherical morphology and uniform dispersion [21].

Fig. 1.

(A) Extraction process of LCMVs. (B) LCMVs morphology and (C) particle size characterized by TEM and NTA. (D) LCMVs contain highly expressed miRNAs that may promote wound healing. (E) GO analysis and (F) KEGG analysis predicting the signaling pathways of LCMVs. (G) Mapping relationships between miRNAs involved in wound repair and their target genes.

To further investigate the biological potential of LCMVs, miRNA sequencing was performed using next-generation sequencing (NGS). The top 100 highly expressed miRNAs were identified, and their target genes were predicted using the multiMiR database. Gene Ontology (GO) enrichment analysis (Fig. 1E) demonstrated that these miRNAs were significantly involved in biological processes such as wound healing, vascular development, and fibroblast migration. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Fig. 1F) indicated that the predicted targets of these miRNAs were enriched in MAPK, Wnt, and Rap1 signaling pathways.

Collectively, these findings suggest that LCMVs possess intrinsic biological activity that may promote tissue repair by modulating key regulatory pathways. The enrichment of miRNAs associated with angiogenic and regenerative signaling indicates that bacterial vesicles may not merely act as passive carriers but actively participate in intercellular communication, similar to mammalian exosomes [22]. Based on expression levels and gene connectivity, the top 10 most abundant miRNAs and their corresponding target genes were identified (Fig. 1D). A gene interaction network constructed from these relationships (Fig. 1G) revealed several hub genes, including Hypoxia-Inducible Factor 1 Alpha (HIF1A), vascular endothelial growth factor A (VEGFA), and Transforming Growth Factor Beta 2 (TGF-β2), all of which are recognized mediators of angiogenesis and granulation tissue formation [[23], [24], [25]].

From a mechanistic perspective, these results imply that LCMVs could influence wound repair through the delivery of regulatory miRNAs capable of activating pro-angiogenic signaling cascades. The identification of miRNAs targeting genes within the MAPK, Wnt, and Rap1 pathways aligns with prior evidence suggesting that microbial vesicles can modulate host cell responses to enhance tissue regeneration [[26], [27], [28]]. This observation expands the current understanding of probiotic-derived vesicles, positioning LCMVs as promising bioactive agents for regenerative applications beyond the gut microenvironment.

3.2. Evaluation of LCMVs effects on cellular behavior

To evaluate the effects of LCMVs on cellular behavior, a series of in vitro experiments were conducted using different concentrations of LCMVs (Fig. 2A). The concentration of LCMVs was quantified using the bicinchoninic acid (BCA) assay. Focusing on biosafety for potential applications, the maximum concentration that promoted the proliferation of HUVECs and L929 fibroblasts was determined to be 20 μg/mL (Fig. 2B). Notably, the total tube length of HUVECs increased with rising concentrations of LCMVs but was inhibited when the concentration exceeded 20 μg/mL (Fig. 2C and D). Moreover, as shown in Fig. 2E and F, when the concentration reached 40 μg/mL, the expression of tumor necrosis factor-alpha (TNF-α)—a representative pro-inflammatory cytokine—was significantly upregulated. In contrast, at 20 μg/mL, the expression of the anti-inflammatory cytokine interleukin-10 (IL-10) was significantly enhanced, while TNF-α expression was notably suppressed. Morphological observations of macrophages (Fig. S1) further supported these findings.

Fig. 2.

(A) Scheme of the co-culture model of cells and LCMVs. (B) Proliferation of HUVECs and L929 cells over 3, 5, and 7 days under treatment with different concentrations of LCMVs. (C, D) Angiogenesis images and statistical results of HUVECs after 1 day of treatment with different concentrations of LCMVs. Determine the expression of IL-10 (E) and TNF-α (F) in RAW264.7 macrophages treated with different concentrations of LCMVs for 24 h ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; n = 3.

These results indicate that LCMVs, when present at an appropriate concentration, can function as a functional biomolecule with biological safety properties, and can promote cell proliferation and angiogenesis. Previous studies have highlighted the effectiveness of functionalizing extracellular vesicles to endow them with diverse biological activities [[29], [30], [31]]. For example, mesenchymal stem cell (MSC)-derived exosomes have been shown to significantly enhance HUVEC migration and angiogenesis, thereby accelerating wound healing [32]. However, stem cell–derived exosomes face several limitations, including limited sources, high production costs, and uncertain biosafety, all of which pose significant challenges [33,34]. In contrast, probiotics possess multiple biological functions and stable activity, along with advantages such as low cost and wide availability. Therefore, the development of probiotic-based biomaterials has emerged as an attractive and innovative strategy to enhance therapeutic outcomes [35,36]. Given these insights, it is reasonable to expect that LCMVs may hold a competitive advantage in the development of biomaterials for tissue repair, offering a more accessible and potentially safer alternative to stem cell–derived exosomes.

3.3. Synthesis and characterization of Gel-LCMVs hydrogel

In previous studies, a biocompatible and three-dimensional (3D) crosslinked hydrogel network was successfully prepared through a Schiff-base reaction between carboxymethyl chitosan (CMCS) and aldehyde-functionalized hyaluronic acid (AHA), serving as a promising biomedical material for tissue engineering [37,38]. AHA was synthesized following the protocol illustrated in Fig. S2. Analysis of its ¹H NMR spectrum revealed new proton peaks at 4.5–5.5 ppm (attributed to hemiacetal protons formed between aldehyde and adjacent hydroxyl groups) and a distinct peak at 9.50 ppm corresponding to the aldehyde group, confirming successful AHA synthesis (Fig. S3). To further enhance the binding efficiency and bioactivity of the hydrogel with LCMVs, a click chemistry-based strategy was employed to anchor LCMVs onto CMCS, followed by crosslinking with AHA to generate a novel LCMV-loaded hydrogel (Gel-LCMVs). As depicted in Fig. S4, dibenzocyclooctyne (DBCO) groups were first introduced to CMCS through the reaction of DBCO-PEG4-NHS with amino groups, producing CMCS-DBCO, which was confirmed by ¹H NMR analysis (Fig. S5). Quantitative analysis of the ¹H NMR spectra revealed that the grafting degree of DBCO on CMCS was approximately 9.67%, indicating successful functionalization while preserving sufficient reactive amine groups for subsequent crosslinking. Subsequently, surface amines on LCMVs were exposed using tris(2-carboxyethyl) phosphine (TCEP), and reacted with DBCO-PEG4-Azide to yield N₃-LCMVs. These were then conjugated with CMCS-DBCO under neutral pH for 6 h. Finally, AHA was added at a defined ratio to form the Gel-LCMVs hydrogel (Fig. 3A). As a control, a physically mixed hydrogel containing LCMVs (Gel@LCMVs) was prepared.

Fig. 3.

Characterization of the hydrogel. (A) Schematic illustration of the synthesis process. (B) Representative scanning electron microscopy (SEM) images of the hydrogel microstructure. (C) In vitro degradation rate of the hydrogel. (D) Confocal laser scanning microscopy (CLSM) images showing the distribution of LCMVs within the hydrogel matrix. (E) Water-retention properties of the hydrogel. (F) Release profile of LCMVs from the hydrogel in PBS. (G) Antibacterial activity of the hydrogel and (H) corresponding quantitative analysis.

Scanning electron microscopy (SEM) and rheological analysis revealed that LCMV modification did not significantly alter the native hydrogel microstructure (Fig. 3B) or its viscoelastic properties (Fig. S6). Similarly, there were no significant differences in the degradation (Fig. 3C), swelling (Fig. S7) and moisturizing properties (Fig. 3E) among the various groups. (Fig. 3C). Confocal microscopy demonstrated that, compared with Gel@LCMVs, LCMVs were uniformly distributed within the Gel-LCMVs matrix with improved loading efficiency (Fig. 3D). Sustained release analysis confirmed that LCMVs were successfully grafted and gradually released from the Gel-LCMVs system (Fig. 3F). Notably, the release profile of LCMVs in the Gel-LCMVs group displayed a high degree of correlation with the hydrogel degradation curve (Fig. 3C), suggesting that vesicle liberation is primarily driven by the progressive erosion of the CMCS/AHA covalent network. While the physically mixed Gel@LCMVs group suffered from a significant burst release (∼73%) within the first 12 h, the click-conjugated Gel-LCMVs maintained a near-linear release kinetics over 15 days. This synchronized degradation-release mechanism ensures a stable concentration of bioactive factors in the wound microenvironment, which is essential for orchestrating the sequential stages of tissue repair. To further assess in vivo degradation behavior, Gel-LCMVs were subcutaneously implanted, and the results indicated that the hydrogel maintained structural integrity for at least 15 days (Fig. S8), consistent with the requirements for prolonged therapeutic applications. In addition, the hydrogel exhibited excellent antibacterial properties against both E. coli and S. aureus (Fig. 3G and H). The primary antibacterial activity is attributed to the intrinsic properties of the CMCS matrix, where positively charged amino groups interact with negatively charged bacterial membranes, leading to cell lysis. However, the incorporation of LCMVs may offer supplementary benefits by optimizing the local microenvironment, though the carboxymethyl chitosan remains the dominant antibacterial component.

Notably, the strain-promoted azide–alkyne cycloaddition (SPAAC) strategy employed in this study enables covalent immobilization of LCMVs under mild, copper-free conditions, which are widely recognized to preserve membrane integrity and biological functionality of extracellular vesicles [39,40]. Unlike harsh chemical crosslinking approaches, SPAAC selectively targets surface-exposed amine groups without disrupting vesicle lipid bilayers or internal bioactive cargos. The preserved bioactivity of Gel-LCMVs observed in both in vitro and in vivo experiments strongly suggests that click conjugation does not compromise the regenerative potential of LCMVs. Nevertheless, we acknowledge that direct side-by-side comparisons between native LCMVs and click-modified LCMVs were not conducted in the present study. Future investigations incorporating functional assays following chemical modification will further clarify the precise influence of bioorthogonal conjugation on vesicle activity and optimize immobilization strategies for translational applications. Meanwhile, the immobilization process preserved the hydrogel’s physicochemical properties—including porosity, elasticity, and degradation behavior—indicating that the chemical modification was non-destructive. The resulting Gel-LCMVs system achieved both structural stability and controlled bioactive release, providing a versatile platform for prolonged therapeutic delivery in wound repair applications.

3.4. Evaluation of the biological performance of Gel-LCMVs

To evaluate the potential biological performance of Gel-LCMVs, we investigated the regulation of cellular behaviors in vitro, including proliferation, migration, and angiogenic differentiation (Fig. 4A). As shown in Fig. 4B, the Gel alone exhibited excellent biocompatibility and no cytotoxicity but did not significantly promote cell proliferation compared to the control group. When LCMVs were immobilized within the hydrogel matrix, the resulting Gel-LCMVs markedly enhanced cell proliferation, demonstrating a superior effect compared to the hydrogel alone. Notably, treatment with Gel@LCMVs induced observable cytotoxicity, which was likely due to the sudden burst release of LCMVs, leading to a locally elevated concentration in the microenvironment that adversely affected cellular behavior. These findings were corresponded to the release profiles shown in Fig. 2B.

Fig. 4.

(A) Schematic illustration of the co-culture system of hydrogels and cells. (B) Representative fluorescence microscopy images of HUVECs and L929 cells after 7 days of co-culture with hydrogels, along with quantitative analysis of cell proliferation. (C) In the Transwell model, cell migration after 24 h of co-culture with hydrogels and the corresponding statistical analysis are shown. (D) Under different treatment conditions, the tube formation of HUVECs, the expression of VEFGA and PECAM1 (CD31) genes, and the quantitative assessment results of angiogenesis. Scratch assay images and statistical analysis of HUVEC (E) and L929 (F) cell migration under various treatment conditions are also displayed. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; n = 3.

To assess the influence of Gel-LCMVs on cell migration, both Transwell and scratch assays were performed. The Transwell assay results (Fig. 4C) demonstrated that the number of HUVECs and L929 cells that migrated through the membrane was significantly higher in the Gel-LCMVs group than in the Gel@LCMVs group. Similarly, the scratch assay results (Fig. 4E and F) showed that the wound closure rate of HUVECs and L929 cells in the Gel-LCMVs group was markedly accelerated, indicating enhanced migratory capacity. These observations were consistent with the Transwell results, confirming that Gel-LCMVs significantly promote cell migration, suggesting their potential to accelerate tissue repair at the implantation site. Moreover, tube formation assays revealed that the total tube length of HUVECs cultured with Gel-LCMVs was significantly greater than that of the control and Gel groups (Fig. 4D), further verifying the angiogenesis-promoting capacity of Gel-LCMVs. At the cellular level, Gel-LCMVs significantly upregulated VEGFA and PECAM1 (CD31) mRNA expression in HUVECs compared to the other groups, further confirming their pro-angiogenic effect (Fig. 4D₃, D₄). Together, compared with other groups, Gel-LCMVs exhibited significantly enhanced and sustained promotion of cell proliferation, migration, and angiogenesis, whereas Gel@LCMVs showed transient effects due to burst release.

The enhanced cellular proliferation, migration, and angiogenic differentiation observed in the Gel-LCMVs group can be attributed to the sustained release of bioactive vesicles and the preservation of their functional integrity within the hydrogel matrix. LCMVs, derived from Lactobacillus casei, contain diverse signaling molecules, including miRNAs and proteins, which may regulate angiogenesis and inflammation through key pathways such as MAPK, Wnt, and Rap1 signaling [41,42]. These pathways modulate the expression of crucial reparative mediators, including VEGFA, HIF1A, and TGF-β2, thereby promoting endothelial cell migration, fibroblast activation, and neovascularization [26,43]. Moreover, the covalent immobilization of LCMVs via click chemistry prevents the burst release typically observed in physically mixed systems, maintaining a stable and physiologically relevant concentration of bioactive factors within the local microenvironment [44]. This controlled release behavior likely underlies the superior bioactivity of Gel-LCMVs compared to Gel@LCMVs, ensuring continuous stimulation of angiogenic and regenerative processes. Collectively, these findings suggest that Gel-LCMVs create a favorable biomimetic niche that promotes coordinated angiogenesis and tissue remodeling.

3.5. Evaluation of wound healing efficacy of Gel-LCMVs in vivo

Based on the excellent biological performance of Gel-LCMVs observed in vitro, we further investigated their potential wound-healing efficacy in a full-thickness skin defect model in mice (Fig. 5A). The excisional wounds were treated with Gel, Gel-LCMVs, or a commercial dressing (Tegaderm), while untreated wounds served as controls. As shown in Fig. 5B and C, Gel-LCMVs exhibited superior wound closure efficiency, achieving over 50% closure by day 3 and nearly complete healing by day 10, with no evident inflammation or scar formation. Throughout the healing process, the Gel-LCMVs group consistently demonstrated enhanced wound closure compared to other groups. Histological examination of wound tissue sections revealed that, at both day 7 and day 14, the Gel-LCMVs group displayed denser granulation tissue formation (Fig. 5D and E), smaller wound diameter (Fig. 5F), and a thicker neo-dermal layer at the wound site (Fig. 5G). Hematoxylin–eosin (H&E) staining showed no visible damage to major organs, indicating that Gel-LCMVs possessed excellent in vivo biosafety (Fig. S9). Moreover, during the healing process, collagen deposition was significantly higher in the Gel-LCMVs group than in the other groups (Fig. 5H). These results highlight that Gel-LCMVs effectively shorten the healing time and improve the quality of wound repair, exhibiting a distinct advantage over commercial Tegaderm dressings.

Fig. 5.

Gel-LCMVs significantly enhance in vivo tissue regeneration and collagen deposition at the wound site. (A) Schematic illustration of the experimental timeline for the full-thickness skin wound model, in which a single-dose treatment was administered and wound closure was monitored over 14 days. (B) Representative macroscopic images showing the wound healing process under different dressing treatments, and (C) corresponding quantitative analysis of wound closure rates. (D) H&E staining and (E) Masson’s trichrome staining images of wound tissues collected on days 7 and 14, revealing progressive tissue regeneration and collagen matrix remodeling. (F) Quantitative analysis of wound diameter, (G) dermal thickness, and (H) collagen deposition rate, demonstrating the superior regenerative performance of Gel-LCMVs compared with commercial dressing. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; n = 3.

In addition, histological observations on days 7 and 14 revealed the presence of more newly formed blood vessels in the Gel-LCMVs-treated wounds (Fig. 6A), indicating that Gel-LCMVs promoted angiogenesis during wound healing. Two angiogenesis-related proteins, vascular endothelial growth factor (VEGF) and cluster of differentiation 31 (CD31), play critical roles in vascular reconstruction. On day 7, the expression of VEGF and CD31 in the Gel-LCMVs group were markedly higher (Fig. 6B and C), suggesting that Gel-LCMVs effectively modulated angiogenesis-related cellular behavior and enhanced vascular formation. By day 14, however, VEGF and CD31 expression levels had decreased in the Gel-LCMVs group but remained elevated in other groups (Figs. S10 and S11), suggesting that angiogenesis had already been completed in the Gel-LCMVs group, while other groups were still in the vascular remodeling phase. Notably, Gel-LCMVs markedly suppressed TNF-α expression during the healing process (Fig. 6D–Fig. S12), indicating effective inhibition of inflammatory responses. Collectively, these findings demonstrate that Gel-LCMVs uniquely accelerate early-stage angiogenesis and mitigate inflammation, thereby promoting more efficient and higher-quality tissue regeneration.

Fig. 6.

Gel-LCMVs hydrogels promote angiogenesis and suppress inflammation at the wound site in vivo. (A) Representative images and quantitative analysis showing the extent of neovascularization in wound tissues. Immunohistochemical staining and corresponding quantitative analysis of (B) CD31, (C) VEGF, and (D) TNF-α expression in wound sections at day 7 post-treatment. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; n = 3.

The enhanced wound-healing efficacy of Gel-LCMVs can be attributed to the synergistic effects of the bioactive LCMVs and the hydrogel’s sustained-release properties. LCMVs contain a variety of biologically active components, including proteins, lipids, and small RNAs that modulate inflammation, angiogenesis, and tissue remodeling. When immobilized within the hydrogel matrix via click chemistry, these vesicles can be released in a controlled manner, maintaining local bioactivity over an extended period. This sustained release likely facilitates continuous stimulation of endothelial and fibroblast activity, thereby promoting neovascularization and collagen deposition. Moreover, the Gel-LCMVs hydrogel exhibited the ability to downregulate proinflammatory cytokine TNF-α, suggesting that it can create a favorable microenvironment for wound regeneration by balancing inflammation and repair. Collectively, these findings indicate that Gel-LCMVs act through both biochemical signaling and biophysical support to orchestrate efficient tissue regeneration.

3.6. Gel-LCMVs promotes the enhancement of signaling pathways related to wound healing

To elucidate the mechanisms by which Gel-LCMVs regulate angiogenesis and inflammation during wound healing, RNA sequencing (RNA-seq) was performed on wound tissues from Gel-LCMVs–treated and control mice. Principal component analysis (PCA) demonstrated high reproducibility and strong inter-sample correlation among biological replicates (Fig. 7A). Compared with the control group, the Gel-LCMVs group exhibited 1228 upregulated and 1449 downregulated genes, indicating that the sustained release of LCMVs by Gel-LCMVs exerts a profound regulatory effect on the wound healing process (Fig. 7B–D). GO enrichment analysis of Biological Processes revealed significant enrichment in pathways associated with immune responses, cell proliferation and migration, angiogenesis, and extracellular matrix (ECM) organization and metaboliscellm (Fig. 7E). These results suggest that Gel-LCMVs effectively enhance anti-inflammatory activity, cellular proliferation, and ECM remodeling, highlighting their potential to promote wound repair. Furthermore, GO enrichment in the Molecular Function and Cellular Component categories indicated that differentially expressed genes were enriched in cytokine activity, epidermal growth factor receptor binding, extracellular matrix components, and protein adhesion (Fig. 7F and G). KEGG pathway analysis identified PI3K–AKT, Wnt, and JAK–STAT signaling pathways as potential mediators of the pro-healing effects of Gel-LCMVs (Fig. 7H). Consistently, gene set enrichment analysis (GSEA) showed downregulation of inflammatory response and immune response (Fig. 7I and J)., along with upregulation of epidermis development, skin development, keratinization, and angiogenesis (Fig. 7K–N). Collectively, these findings indicate that Gel-LCMVs promote gene expression programs associated with efficient wound healing.

Fig. 7.

Transcriptomic analysis of full-thickness skin wound tissues (n = 3). (A) PCA of transcriptomic data. (B) Venn diagram showing the distribution of genes among different groups. (C) Volcano plot and (D) heatmap of differentially expressed genes. GO enrichment analysis of differentially expressed genes for (E) biological process, (F) cellular component, and (G) molecular function categories. (H) KEGG pathway enrichment analysis of differentially expressed genes. Gene set enrichment analysis (GSEA) analysis of the inflammatory response (I), immune response (J), epidermis development (K), skin development (L), keratinization (M), and angiogenesis (N).

The transcriptomic profiling further supports that Gel-LCMVs create a favorable wound microenvironment by orchestrating a coordinated balance between inflammation resolution, fibroblast activation, and angiogenesis. The activation of PI3K-AKT and Wnt signaling cascadesprovides a molecular basis for the enhanced cell proliferation and migration observed in our in vitro assays. Specifically, the upregulation of key pro-angiogenic hub genes, such as VEGFa and HIF1a (Log2FC > 1.6), directly supports the dense neovascularization and high CD31/VEGF expression revealed by immunohistochemical staining. Furthermore, the modulation of the JAK-STAT pathway likely mediates the observed suppression of TNF-α and promotion of IL-10, facilitating a transition from a pro-inflammatory to a pro-regenerative microenvironment. Additionally, the upregulation of epidermal development and keratinization–related genes implies accelerated re-epithelialization and restoration of the skin barrier. Taken together, these transcriptomic results provide mechanistic evidence that Gel-LCMVs accelerate wound healing through multifaceted regulation of immune responses, fibroblast behavior, and epidermal regeneration. The sustained release of LCMVs within the hydrogel matrix allows for prolonged bioactivity, leading to improved tissue remodeling and functional repair. This integrated strategy demonstrates the potential of Gel-LCMVs as a promising biomaterial for promoting efficient and scar-free wound healing.

4. Conclusion

In summary, we developed a click-conjugated Gel-LCMVs hydrogel that combines the regenerative potency of Lactobacillus casei–derived membrane vesicles with the biomimetic and protective features of a CMCS/HA hydrogel network. The covalent immobilization of LCMVs via strain-promoted azide–alkyne cycloaddition enables stable vesicle retention and sustained release without compromising the physicochemical integrity of the hydrogel. Both in vitro and in vivo evaluations demonstrated that Gel-LCMVs effectively enhance cell proliferation, migration, and angiogenic activity, while attenuating inflammatory responses to facilitate accelerated wound closure and tissue remodeling. Transcriptomic analyses further revealed activation of key pathways involved in angiogenesis, fibroblast activation, and re-epithelialization, confirming the multi-level pro-regenerative effects of this system. By integrating biochemical cues with a structural support matrix, the Gel-LCMVs hydrogel provides a robust, cell-free therapeutic strategy for efficient, scar-free wound repair. This work highlights the translational promise of probiotic-derived vesicle–based biomaterials as next-generation platforms for advanced wound management and regenerative medicine.

CRediT authorship contribution statement

Siqi Zhang: Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Writing – original draft, Writing – review & editing. Menglin Zhou: Data curation, Formal analysis, Investigation, Software, Writing – review & editing. Man-Yuan Li: Data curation, Funding acquisition, Investigation, Resources, Software. Renyu Wang: Data curation, Formal analysis. Chengfei Yang: Formal analysis, Validation. Liuquan Yang: Data curation, Methodology, Resources. Jingsi Wang: Formal analysis, Investigation. Xufeng Deng: Conceptualization, Data curation, Project administration, Writing – review & editing. Jigang Dai: Funding acquisition, Project administration, Supervision, Writing – review & editing. Quanxing Liu: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.