A bacterial outer vesicle-initiated lbevs-ppy coating, and preparation method and application thereof

Abstract

This invention belongs to the field of biotechnology, specifically relating to an in-situ spray-polymerized coating initiated by bacterial extravesicles (LBEVs), its preparation method, and its application. LBEVs are electrostatically adsorbed by Fe... 3+ Functionalization yields LBEVs-Fe 3+ Solution. Spray LBEVs-Fe onto the wound site. 3+ After reacting with pyrrole monomers, LBEVs-Fe 3+ The process initiates the oxidative polymerization of pyrrole to form polypyrrole, resulting in a polymeric coating LBEVs-PPy. The LBEVs of this invention exhibit effective antibacterial activity against Staphylococcus aureus, Escherichia coli, and Staphylococcus epidermidis. Near-infrared triggered photothermal activation produces mild thermotherapy, significantly upregulating the expression of angiogenesis regulators (such as VEGFA and ANGPT1) in human umbilical vein endothelial cells (HUVECs) and activating the HSP90 / p-eNOS pathway to accelerate angiogenesis. The LBEVs-PPy coating significantly accelerates wound healing through a synergistic mechanism integrating the potent antibacterial activity of LBEVs and the highly efficient photothermal properties of PPy.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a bacterial exovesicle-initiated LBEVs-PPy coating, its preparation method, and its application. Background Technology

[0002] As the host's primary physical barrier, the skin interface plays a crucial role in defending against microbial pathogen invasion and a range of physical and chemical damage. However, when the skin is damaged, harmful bacteria can invade living tissue, leading to wound infection and potentially causing severe tissue damage. To combat bacterial infections, a variety of novel antimicrobial drugs and innovative materials have been developed, such as novel antibiotics, antimicrobial peptides, and antimicrobial nanoparticles. While existing strategies are effective in inhibiting bacterial growth and promoting wound healing, they often indiscriminately kill both pathogenic and beneficial bacteria, disrupting the balance of the skin microbiome. Therefore, developing selective antimicrobial strategies that can inhibit pathogens without harming symbiotic bacteria remains a key challenge.

[0003] In recent years, microbiome-based therapies have gained significant attention for treating various diseases through bacterial interference and immunomodulation. Certain symbiotic and beneficial bacteria can establish unique local microenvironments by secreting large amounts of metabolites and antimicrobial substances, promoting their own proliferation while inhibiting competitors. For example, lactobacilli effectively treat urinary tract infections by restoring the balance of the vaginal microbiota. However, the pathogenicity and immunogenicity of bacteria largely limit the application of biotherapies. Bacterial extravesicular vesicles (BEVs), spherical particles defined by a lipid bilayer, have been shown to carry a variety of bioactive components (such as proteins, nucleic acids, lipids, and virulence factors) directly derived from their parent cells. Vesicles play a role in bacterial-bacterial or bacterial-host interactions and immunomodulation. BEVs secreted from *Synechococcus elongatus* PCC7942 have been shown to accelerate skin wound healing.

[0004] Therefore, it remains very urgent to develop a BEV-based antimicrobial wound dressing that can effectively modulate the wound microenvironment and promote healing. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to provide a method for preparing and applying an in-situ spray-polymerized coating of LBEVs-PPy initiated by bacterial exovesicles, thereby resolving the problems existing in the background technology.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A LBEVs-PPy coating initiated by bacterial exovesicles, wherein the bacterial exovesicles are electrostatically adsorbed by Fe 3+ Functionalization yields LBEVs-Fe 3+Then, it is sprayed onto the wound along with pyrrole monomers, and polymerized in situ at the wound site to form an LBEVs-PPy coating.

[0008] Furthermore, the bacterial extracellular cyst is derived from Lactobacillus reuteri.

[0009] Furthermore, Fe 3+ Ions are adsorbed onto the surface of bacterial exovesicles, and pyrrole monomers are oxidized to form polypyrrole (PPy) to obtain LBEVs-PPy. Spherical bacterial exovesicles are embedded in the coating.

[0010] Furthermore,

[0011] The LBEVs-PPy coating accelerates wound healing through the dual synergistic effect of bacterial exovesicle-mediated antibacterial clearance and polypyrrole photothermal angiogenesis promotion.

[0012] A method for preparing LBEVs-PPy coatings initiated by bacterial exovesicles.

[0013] (1) Extraction of LBEVs

[0014] Lactobacillus reuteri was cultured to produce LBEVs, and the bacterial solution was separated by a series of centrifugations to obtain LBEVs.

[0015] (2) LBEVs-Fe 3+ Preparation

[0016] FeCl3 solution was added to the obtained LBEVs precipitate, and the mixture was incubated at room temperature for 30 minutes to allow for complete reaction. The mixture was then transferred to an ultracentrifuge tube, centrifuged at ultracentrifuge, and the supernatant was discarded to obtain LBEVs-FeCl3. 3+ The precipitate was resuspended in 0.9% physiological saline to obtain LBEVs-Fe. 3+ Solution;

[0017] (3) Preparation of LBEVs-PPy coating

[0018] The pure pyrrole solution was diluted with 0.9% physiological saline, and a low-concentration pyrrole emulsion was obtained by sonication for 5 minutes. The pyrrole emulsion was then mixed with LBEVs-Fe 3+ When the solution is sprayed onto the wound, an LBEVs-PPy coating can be formed in situ on the wound.

[0019] Furthermore,

[0020] The specific steps of (1) include: transferring Lactobacillus reuteri to MRS liquid medium and culturing at 37°C and 150 rpm for 12 hours; taking a small amount of bacterial solution and streaking it on a solid plate overnight to obtain a single colony; taking the single colony and expanding it in 500 ml of MRS liquid medium for 48 hours to produce LBEVs; centrifuging the obtained bacterial solution at 10,000 × g for 30 minutes to remove the precipitated Lactobacillus reuteri and collecting the supernatant; filtering the supernatant through 0.45 µm and 0.22 µm filter membranes to remove residual bacteria, cell debris and large particles; concentrating the filtered supernatant using a 100 kD filter membrane to obtain a concentrate; centrifuging the concentrate at 150,000 × g for 1.5 hours to remove the supernatant and obtain LBEVs precipitate; resuspending the LBEVs precipitate with PBS; determining the protein concentration with diquinoline carboxylic acid; and adjusting the LBEVs solution concentration to 2 mg / mL.

[0021] Furthermore,

[0022] The specific steps of (2) include: rapidly transferring anhydrous ferric chloride powder to 1 mol / L hydrochloric acid to fully dissolve the powder; slowly adding the dissolved FeCl3 solution to 0.9% physiological saline to prepare a concentration of 0.1 mol / L FeCl3; taking 1 mL of LBEVs solution; centrifuging at ultraspeed to obtain LBEVs precipitate; adding 1 mL of 0.1 mol / L FeCl3 to the precipitate; gently blowing to mix thoroughly; and incubating at room temperature for 30 minutes to allow for full reaction; transferring the mixture to an ultracentrifuge tube; centrifuging at 150,000 × g at 4°C for 1.5 hours; and discarding the supernatant to obtain LBEVs-FeCl3. 3+ The precipitate was resuspended in 1 mL of 0.9% physiological saline to obtain LBEVs-Fe. 3+ Solution.

[0023] Furthermore,

[0024] The specific steps of (3) include: mixing equal volumes of 0.9% physiological saline and pure pyrrole solution, sonicating for 5 minutes to obtain a low-concentration pyrrole emulsion, and immediately mixing it with the obtained LBEVs-Fe 3+ When the solution is sprayed onto the wound at a volume ratio of 1:10, an LBEVs-PPy coating is formed in situ at the wound site.

[0025] Application of a bacterial exovesicle-initiated LBEVs-PPy coating in drugs for controlling infection and accelerating wound healing.

[0026] The beneficial technical effects of this invention are as follows:

[0027] The LBEVs of this invention exhibit effective antibacterial activity against Staphylococcus aureus, Escherichia coli, and Staphylococcus epidermidis. Near-infrared triggered photothermal activation produces mild thermotherapy, significantly upregulates the expression of angiogenesis regulators (such as VEGFA and ANGPT1) in human umbilical vein endothelial cells (HUVECs), and activates the HSP90 / p-eNOS pathway to accelerate angiogenesis. The LBEVs-PPy coating significantly accelerates wound healing through a synergistic mechanism that integrates the strong antibacterial activity of LBEVs and the efficient photothermal properties of PPy. Attached Figure Description

[0028] Figure 1 This is a schematic diagram illustrating the preparation and application of LBEVs-PPy according to an embodiment of the present invention;

[0029] Figure 2 The figure shows the characterization results of BEVs and BEVs-PPy in the embodiments of the present invention;

[0030] Figure 3 This is a graph showing the in vitro antibacterial performance results of LBEVs in an embodiment of the present invention;

[0031] Figure 4 This is a diagram showing the results of LBEVs-PPy promoting angiogenesis through photothermal properties in an embodiment of the present invention.

[0032] Figure 5 This is a diagram showing the healing promotion results of LBEVs-PPy in a mouse skin wound model according to an embodiment of the present invention;

[0033] Figure 6 This is a histological image analysis result of wound tissue in an embodiment of the present invention;

[0034] Figure 7 This is a graph showing the in vivo safety assessment results of LBEVs-PPy according to an embodiment of the present invention. Detailed Implementation

[0035] The specific implementation method will be further described below with reference to the accompanying drawings.

[0036] The reagents and instruments used in the following examples are all conventional laboratory reagents and instruments.

[0037] Example:

[0038] A LBEVs-PPy coating initiated by bacterial exovesicles, wherein the bacterial exovesicles are electrostatically adsorbed by Fe 3+ Functionalization yields LBEVs-Fe 3+ LBEVs-Fe 3+When sprayed onto the wound site simultaneously with pyrrole monomers, an LBEVs-PPy coating can be formed in situ. The bacterial exovesicles are derived from *Lactobacillus reuteri*. Spherical bacterial exovesicles are embedded within the polymeric coating. The polymeric coating accelerates wound healing through the synergistic effect of antibacterial clearance mediated by bacterial exovesicles and photothermal angiogenesis-promoting effects of polypyrrole.

[0039] A method for preparing an in-situ spray-polymerized coating initiated by bacterial exovesicles.

[0040] (1) Lactobacillus reuteri was transferred to MRS liquid medium and cultured at 37°C and 150 rpm for 12 hours. A small amount of bacterial solution was streaked on a solid plate and cultured overnight to obtain a single colony. The single colony was cultured in 500 ml of MRS liquid medium for 48 hours to produce LBEVs. The obtained bacterial solution was centrifuged at 10,000×g for 30 minutes to remove the precipitated Lactobacillus reuteri and the supernatant was collected. The supernatant was filtered through 0.45 µm and 0.22 µm filter membranes to remove residual bacteria, cell debris and large particles. The filtered supernatant was concentrated using a 100 kD filter membrane to obtain a concentrate. The concentrate was centrifuged at 150,000×g for 1.5 hours to remove the supernatant and obtain LBEVs precipitate. The LBEVs precipitate was resuspended in PBS and the protein concentration was determined by diquinoline carboxylic acid. The concentration of the LBEVs solution was adjusted to 2 mg / mL.

[0041] (2) Quickly transfer anhydrous ferric chloride powder to 1 mol / L hydrochloric acid to dissolve the powder completely. Slowly add the dissolved FeCl3 solution to 0.9% physiological saline to prepare a FeCl3 concentration of 0.1 mol / L. Take 1 mL of LBEVs solution, centrifuge at ultraspeed to obtain LBEVs precipitate, add 1 mL of 0.1 mol / L FeCl3 to the precipitate, gently blow and mix thoroughly, and incubate at room temperature for 30 minutes to allow it to react completely. Transfer the mixture to an ultracentrifuge tube and centrifuge at 150,000 × g at 4℃ for 1.5 hours. Discard the supernatant to obtain LBEVs-Fe 3+ The precipitate was resuspended in 1 mL of 0.9% physiological saline to obtain LBEVs-Fe. 3+ Solution.

[0042] (3) Mix equal volumes of 0.9% physiological saline and pure pyrrole solution, sonicate for 5 min to obtain a low-concentration pyrrole emulsion, and immediately mix it with the obtained LBEVs-Fe 3+ When the solution is sprayed onto the wound at a volume ratio of 1:10, an LBEVs-PPy coating is formed in situ at the wound site.

[0043] Application of a bacterial exovesicle-initiated LBEVs-PPy coating in drugs for controlling infection and accelerating wound healing.

[0044] Figure 1 Schematic diagram of the preparation and application of LBEVs-PPy. (A) After extracting LBEVs from Lactobacillus reuteri, in-situ polymerization of LBEVs-PPy is initiated by encapsulating ferric ions. (B) LBEVs-PPy, with its excellent antibacterial and angiogenesis-promoting abilities, can promote the healing of infected wounds.

[0045] Experimental verification:

[0046] 1. Characterization of LBEVs and LBEVs-PPy

[0047] The average size and zeta potential of LBEVs were measured by dynamic light scattering (DLS). The total protein concentration of concentrated LBEVs was determined using the BCA method. For transmission electron microscopy (TEM) imaging, samples were loaded onto a copper grid and then photographed. BEVs of *E. coli* strains with mCherry fluorescent labels could be directly observed using a laser confocal microscope.

[0048] LBEVs, polypyrrole, and LBEVs-PPy were freeze-dried into powders and subjected to infrared spectroscopy analysis.

[0049] Figure 2 Characterization results for BEVs and BEVs-PPy. (A) Schematic diagram of LBEVs extraction process. (B) Particle size distribution of LBEVs. (C) Transmission electron microscope image of LBEVs. Scale bar: 200 nm. (D) Transmission electron microscope image of LBEVs-PPy. Scale bar: 200 nm. (E) Confocal microscope image of EBEVs. Scale bar: 10 μm. (F) Confocal microscope image of EBEVs-PPy. Scale bar: 10 μm. (G) LBEVs, LBEVs-Fe 3+ Zeta potentials of LBEVs-PPy. Infrared spectra of (H)LBEVs, PPy, and LBEVs-PPy.

[0050] Figure 2 Figures B and 2C show that the harvested LBEVs were spherical, with a major size distribution of approximately 155.6 ± 6.73 nm. The concentration of LBEVs was determined using the BCA protein assay, with total protein content used as the quantitative standard for subsequent experiments. The obtained LBEVs were mixed with 0.1 mol / L FeCl3; electrostatic attraction promoted the adsorption of iron ions, resulting in LBEVs-Fe... 3+ To prove LBEVs-Fe 3+The oxidation of pyrrole by iron ions on LBEVs-Fe 3+ The mixture formed LBEVs-PPy. TEM images of LBEVs-PPy show spherical vesicles embedded in the PPy coating. Figure 2 D). Observation of EBEVs-PPy coatings prepared from *E. coli*-derived BEVs (EBEVs) expressing mCherry using LSCM. Figure 2 As shown in Figure E, the presence of red fluorescence confirms the successful preparation of EBEVs.

[0051] Following the synthesis method of LBEVs-PPy coating, EBEVs-PPy coating was prepared and exhibited green fluorescence co-localized with red fluorescent BEVs. Figure 2 F). Therefore, the green fluorescence is attributed to the PPy aggregates synthesized on EBEVs, demonstrating the efficient synthesis of BEVs-PPy. To provide more objective evidence for the successful synthesis of the BEVs-PPy coating, the change in zeta potential was measured by dynamic light scattering (DLS). After mixing with FeCl3 solution, the zeta potential of BEVs significantly increased from −7.2 ± 1.0 mV to −4.8 ± 0.1 mV. Figure 2 G). This change is attributed to Fe. 3+ Ions adsorbed onto the vesicle surface, neutralizing the surface negative charge. After surface polymerization, the Zeta potential further decreased to −10.1 ± 0.9 mV, confirming the formation of the polypyrrole coating structure. Furthermore, FTIR spectroscopy was used to further confirm the formation of the LBEVs-PPy coating. Figure 2 As shown in Figure H, LBEVs-PPy exhibits the characteristic peak of PPy. (1540 cm⁻¹) -1 The peak at 1653 cm corresponds to C=C stretching vibration, while the peak at 1653 cm corresponds to C=C stretching vibration. -1 and 1030 cm -1 The peaks at these locations represent C=N and C–H bonds, respectively. Furthermore, the peaks at 2750–3000 cm⁻¹ in LBEVs-PPy... -1 The peaks between them correspond to the complex components of the vesicles.

[0052] 2. Antibacterial properties of LBEVs

[0053] Remove the frozen Staphylococcus aureus from the -80°C freezer. Thaw the vial rapidly in a 37°C water bath until the contents are completely liquefied. Using a sterile pipette, transfer a small amount of the thawed bacterial suspension to sterile LB medium. Incubate the inoculated culture at 37°C with constant shaking at 150 rpm to promote bacterial growth.

[0054] LBEVs at specific concentrations (BCA assay concentrations of 1, 1.5, and 2 mg / ml) were compared with a concentration of 10 mg / ml.5 CFU / mL of Staphylococcus aureus was mixed. The mixture was incubated at 37°C for 6 hours, and the number of surviving bacteria in each group was counted by plate counting to assess antimicrobial activity. Staphylococcus aureus was stained using a Live / Dead Bacterial Activity Kit and imaged using laser scanning confocal microscopy (LSCM), while the morphology of Staphylococcus aureus was examined using scanning electron microscopy (SEM). In addition, Escherichia coli and Staphylococcus epidermidis were also mixed with LBEVs (BCA assay concentration of 2 mg / mL), and the mixture was then serially diluted and plated on agar plates.

[0055] Figure 3 In vitro antibacterial properties of LBEVs. (A) Representative plate image of Staphylococcus aureus and LBEVs after co-culturing at 37°C for 6 hours. (B) Statistical analysis of the number of Staphylococcus aureus colonies after co-culturing with different concentrations of LBEVs at 37°C for 6 hours (*p < 0.05, p < 0.01, ns: no significant difference). (C) Statistical analysis of the inhibition rate of LBEVs (p < 0.01, ***p < 0.001, ****p < 0.0001). (D) Morphological changes of Staphylococcus aureus after co-culturing with LBEVs for 6 hours under a scanning electron microscope. Scale bar: 1 μm. (E) Fluorescence image of Staphylococcus aureus double-stained with calcein and propidium iodide after co-culturing with LBEVs for 6 hours under a confocal microscope (green: live bacteria; red: dead bacteria). Scale bar: 5 μm. (F) Plate colony images of Escherichia coli and Staphylococcus epidermidis after co-culturing with LBEVs. All experiments had a sample size of n=3, and one-way ANOVA was used. Data are expressed as mean ± standard error.

[0056] To evaluate the antibacterial activity of the prepared LBEVs, we used the plate count method. Staphylococcus aureus was co-incubated with different concentrations of LBEVs for 6 hours, and bacterial viability was quantitatively determined by counting the colony-forming units (CFUs) on agar plates after the incubation period. Figure 3 A). For example Figure 3 As shown in Figure B, LBEVs exhibited dose-dependent antibacterial activity; bacterial survival gradually decreased with increasing LBEV concentration in the culture medium. After incubation with 1 mg / ml LBEVs for 6 hours in the culture medium, approximately 60% of Staphylococcus aureus growth was effectively inhibited. When the LBEV concentration was increased to 2 mg / ml, the inhibition rate approached 90%. Figure 3 C).

[0057] Subsequently, the morphology of Staphylococcus aureus was observed using scanning electron microscopy (SEM). For example... Figure 3As shown in Figure D, SEM revealed that untreated Staphylococcus aureus cells exhibited a regular spherical morphology with a smooth surface, indicating their intact native structure. In contrast, most bacteria co-incubated with LBEVs showed ruptured cell membranes. To further explore the antibacterial behavior of LBEVs, Staphylococcus aureus was stained using the Live / Dead Bacterial Activity Kit and imaged by LSCM. Figure 3 As shown in Figure E, untreated Staphylococcus aureus exhibited strong green fluorescence, confirming its high bacterial activity. Conversely, the dominant red fluorescence in the LBEV-treated samples demonstrated membrane damage in Staphylococcus aureus, as propidium iodide only entered cells with damaged membranes. These results confirm effective bactericidal efficacy against this pathogen. Furthermore, the antibacterial activity of LBEVs against Escherichia coli (Gram-negative) and Staphylococcus epidermidis (Gram-positive) was evaluated in vitro. Figure 3 As shown in F, compared with the untreated Escherichia coli and Staphylococcus epidermidis control group, LBEV treatment killed the vast majority of bacteria, demonstrating its excellent broad-spectrum antibacterial ability.

[0058] 3. Photothermal properties of LBEVs-PPy

[0059] Frozen cells were thawed in a 37°C water bath. After removing the cryopreservation solution, DMEM complete medium (89% DMEM, 10% fetal bovine serum, 1% penicillin-streptomycin) was added to the cell suspension. The cells were then cultured in a humidified incubator at 37°C with 5% CO2. Cell passage was performed when the cell density reached 80-90% confluence. HaCaT (human keratinocytes), HUVECs (human umbilical vein endothelial cells), and NIH3T3 (mouse fibroblasts) were all cultured in DMEM medium using the above method.

[0060] LBEVs-PPy were added to cell culture dishes and photothermally treated at 42°C for 1 hour. Cell viability staining was performed 24 hours later. Specifically, the supernatant was discarded, and the cells were washed three times with PBS. 200 μL of staining working solution was added to each well of a six-well plate to cover the cells, and the plates were incubated at 37°C for 15 minutes. Finally, the cells were observed under a fluorescence microscope.

[0061] Figure 4 Study on the role of LBEVs-PPy in promoting angiogenesis through photothermal properties. (A) Photothermal heating curve of LBEVs-PPy under 808 nm near-infrared laser irradiation. (B) Real-time infrared thermal image under 808 nm near-infrared laser irradiation.

[0062] To prepare a near-infrared responsive coating, PPy was used as the photothermal material, synthesized by in-situ spraying polymerization of pyrrole monomers induced by BEVs. To verify the photothermal efficiency of the obtained LBEVs-PPy coating, an irradiation experiment was conducted using an 808 nm near-infrared laser at a power density of 0.8 W / cm². Under the same culture conditions, cells were divided into two groups: a control group (no treatment) and an experimental group (treated with LBEVs-PPy), both maintaining equal culture volumes. Under the same irradiation of 808 nm near-infrared light at 0.8 W / cm² for 1 hour, the temperature of the control group remained almost unchanged, while the LBEVs-PPy treated group exhibited superior heat conversion efficiency. The temperature of the experimental group rapidly increased with the extension of irradiation time, reaching 42℃ and maintaining this temperature for 1 hour. Figure 4 A). Real-time infrared thermal imaging captured the heating curves of the experimental group ( Figure 4 (B) This visually verified the photothermal performance of the coating.

[0063] 4. In vitro biocompatibility and bioregulatory effects of LBEVs-PPy

[0064] Figure 4 Study on the role of LBEVs-PPy in promoting angiogenesis through photothermal properties. (C) Fluorescence microscopy images of human umbilical vein endothelial cells (HUVECs) after co-culturing with LBEVs-PPy and LVEVs-PPy + photothermal (green - live cells, red - dead cells). Scale bar: 100 μm. (D) VEGFA gene expression level of HUVECs and LBEVs-PPy after 1 hour of 808 nm near-infrared laser irradiation (p < 0.01). (E) ANGPT1 gene expression level of HUVECs and LBEVs-PPy after 1 hour of 808 nm near-infrared laser irradiation (*p < 0.001). (F) Expression of HSP90 and p-eNOS proteins in HUVECs after 808 nm near-infrared laser irradiation. (G) Quantitative analysis of HSP90 protein expression (*p < 0.05, ns: no significant difference). (H) Quantitative analysis of p-eNOS protein expression (*p < 0.05, ns: no significant difference). All experimental sample sizes were n=3. Statistical analysis of Figures (D) and (E) was performed using the Student t-test, and Figures (G) and (H) were performed using one-way ANOVA. Data are expressed as mean ± standard error.

[0065] To assess the safety of the materials and thermotherapy, cell viability was measured in three skin-associated cell lines (HaCaT keratinocytes, HUVECs, and NIH / 3T3 fibroblasts). Figure 4As shown in Figures C and S1a and b, the cell death rates of the LBEVs-PPy co-incubation group and the LBEVs-PPy group treated with 42°C photothermal therapy for 1 hour were comparable to those of the blank control group, all within acceptable thresholds. These findings demonstrate the good biocompatibility of the LBEVs-PPy coating and its synergistic photothermal treatment. Mild heat treatment can promote angiogenesis, thereby accelerating wound healing. HUVECs were subjected to mild heat stimulation at 42°C for 1 hour. After treatment, the cells were returned to a 37°C / 5% CO2 incubator for 12 hours, and then the expression of angiogenic factors (including vascular endothelial growth factor VEGFA and angiopoietin-1 ANGPT1) was quantitatively measured. Figure 4 As shown in D and 4E, the expression of VEGFA and ANGPT1 genes significantly increased after photothermal treatment. To elucidate the mechanism by which photothermal treatment promotes angiogenesis, HUVECs were harvested at specified time points after photothermal exposure. Western blot analysis showed that mild heat stimulation significantly upregulated the levels of heat shock protein 90 (HSP90) and phosphorylated endothelial nitric oxide synthase (p-eNOS). Figure 4 F). HSP90 protein not only provides thermal protection but also acts as a key molecular chaperone, regulating the expression of angiogenic molecules such as eNOS and playing a beneficial role in angiogenesis. p-eNOS produces large amounts of nitric oxide, promoting endothelial cell proliferation and migration, and leading to increased vascular permeability. Furthermore, quantitative analysis of these two parameters was performed ( Figure 4 These results suggest that thermal stimulation may stimulate angiogenesis through the VEGF, ANGPT1, and HSP90 / p-eNOS pathways.

[0066] 5. In vivo wound healing experiment

[0067] Six-week-old female BALB / c mice were randomly divided into four groups: no treatment (control group), LBEVs group, PPy-mediated photothermal therapy group (PPy), and LBEVs-induced PPy in-situ spraying polymerization synergistic antibacterial and photothermal therapy group (LBEVs-PPy). Cyclophosphamide (100 mg / kg) was administered intraperitoneally once daily on day 4 and day 1 before the start of the experiment. First, the mice were anesthetized and their back hair was shaved. A full-thickness skin defect model (7 mm in diameter) was created on the back to simulate a wound at a skin graft donor site. To establish an infection model, each wound was inoculated with 10 μL of a 1 × 10⁻⁶ concentration. 9CFU / mL Staphylococcus aureus suspension. Antibiotic treatment was administered to the LBEVs and LBEVs-PPy groups by spraying with LBEVs at 0, 6, 12, and 24 hours post-injury. On days 7, 8, and 9, the PPy and LBEVs-PPy groups received photothermal therapy at 42°C for 1 hour daily. Photographs of the wound area were taken on days 0, 3, 5, 7, 9, 11, and 13. Mice were euthanized by cervical dislocation on day 13. Full-thickness wounds with surrounding tissue were excised and stained with hematoxylin and eosin (H&E). CD31 was detected by immunofluorescence, and VEGF was detected by immunohistochemistry (IHC). Important tissues and blood were then extracted for analysis. Histological evaluation of the liver, spleen, and kidneys was performed by H&E staining. Complete blood counts included white blood cell count, lymphocyte count, neutrophil count, and monocyte count.

[0068] Statistical analysis: All data are presented as mean ± standard deviation (SD). Statistical analysis was performed using Prism 8.0 (GraphPad, USA). Unpaired two-tailed Student's t-test was used for comparisons between two groups. Homogeneity of variance (F-test) was performed prior to the t-test.

[0069] Figure 5 The healing-promoting effect of LBEVs-PPy in a mouse skin wound model. (A) Schematic diagram of wound treatment process. (B) Representative wound images of different treatment groups on days 0, 3, 5, 9, and 13. (C) Illustration of wound healing at corresponding time points. (D) Quantitative analysis of wound closure area in each group during treatment. (E) Statistical analysis of wound healing rate in each group. All experimental sample sizes were n=3. One-way ANOVA was used, and data are expressed as mean ± standard error (*p < 0.05, **p < 0.01, ns: no significant difference).

[0070] The in vivo antibacterial and wound-healing abilities of LBEVs-PPy were evaluated in a mouse model of Staphylococcus aureus infection. Figure 5 As shown in Figure A, circular full-thickness skin wounds with a diameter of approximately 7 mm were created on the dorsal surface of BALB / c mice. These wounds were treated with 10 µL of a 10% concentration of [unspecified substance]. 9 CFU / mL Staphylococcus aureus infection. Mice were randomly assigned to four groups: no treatment (control group), LBEVs group (LBEVs), PPy-mediated photothermal therapy group (PPy), and LBEVs-induced PPy in-situ spraying polymer synergistic antibacterial and photothermal therapy group (LBEVs-PPy). Digital images were captured every 2 days to monitor morphological changes during wound healing. Figure 5As shown in B and 5C, although the wound area gradually decreased during the study, the closure speed differed significantly among the treatment groups. Specifically, on postoperative day 3, significant suppuration was observed at and around the wound site in both the control and PPy groups, indicating bacterial infection. In contrast, at the same time point, the LBEVs and LBEVs-PPy groups showed only slight whitishness, confirming the excellent antibacterial ability of LBEVs. Furthermore, by day 5, mice treated with either LBEVs or LBEVs-PPy coatings showed accelerated wound closure, indicating successful control of Staphylococcus aureus infection. By day 9, the wound area in the photothermal-treated LBEVs-PPy group significantly decreased from 26.5 ± 5.7 mm² to only 6.4 ± 0.9 mm², achieving a healing rate of 75%. In contrast, the wound areas in the control group, LBEVs group, and PPy group remained at 22.3 ± 5.2 mm², 12.0 ± 0.8 mm², and 21.6 ± 4.6 mm², respectively, with corresponding healing rates of only 14%, 56%, and 28%. After 13 days, wounds treated with the LBEVs-PPy coating achieved near-complete closure and were covered by new epidermis. Conversely, 33%, 16%, and 27% of wounds in the control group, LBEVs group, and PPy group, respectively, failed to heal. Figure 5 (D and 5E). These findings indicate that LBEVs-PPy coatings significantly promote wound healing through the synergistic effect of combining LBEVs-mediated antibacterial activity with PPy-driven photothermal effects.

[0071] Figure 6 Histological image analysis of wound tissue. (A) Representative hematoxylin-eosin (H&E) staining image of wound tissue on day 13. Blue lines indicate wound width, black arrows indicate inflammatory cells, and red arrows indicate hair follicle structures. Scale bar: 50 μm. (B) Representative immunohistochemical staining image of vascular endothelial growth factor (VEGF) in wound tissue on day 13. (C) Representative immunofluorescence staining image of CD31 in wound tissue on day 13. Scale bar: 500 μm (panoramic view), 50 μm (local magnification).

[0072] To further assess wound healing progress, histological analysis was performed using hematoxylin and eosin (H&E) staining to observe tissue structure. On postoperative day 13, wound tissues from the control group, LBEVs group, PPy group, and LBEVs-PPy coating treatment group were stained with H&E. Figure 6A). The control group, PPy group, and LBEVs group (especially the control and PPy groups) showed significant inflammation, manifested as immune cell infiltration, possibly due to insufficient antibacterial efficacy and wound non-healing. Conversely, the LBEVs-PPy group showed minimal neutrophils, more granulation tissue regeneration, and a certain density of hair follicles, attributed to the potent antibacterial activity of LBEVs and the enhancing effect of PPy on photothermal angiogenesis, thereby accelerating wound healing. Notably, the significant neovascularization and epidermal regeneration in the LBEVs-PPy group revealed accelerated wound healing through photothermal therapy. Post-inflammatory remission angiogenesis promotes the wound healing process by providing growth factors and nutrients that drive fibroblast activity, collagen synthesis, and re-epithelialization. Neovascularization was assessed by immunohistochemical staining for the angiogenic growth factor VEGF. Figure 6 As shown in Figure B, low VEGF expression was observed in the control group, LBEVs group, and PPy group, while the LBEVs-PPy group exhibited the highest VEGF level among all groups. Correspondingly, immunofluorescence staining results showed that on postoperative day 13, the expression level of CD31 (a typical angiogenesis biomarker) in the LBEVs-PPy group was significantly higher than that in the control group, LBEVs group, and PPy group, confirming enhanced angiogenesis activity. Figure 6 C). In summary, these findings confirm that the LBEVs-PPy coating effectively inhibits bacterial infection and significantly accelerates wound healing.

[0073] Figure 7 In vivo safety assessment of LBEVs-PPy. (A) Morphological changes in different tissues were assessed using hematoxylin and eosin (H&E) staining. Scale bar: 100 μm. (BE) Blood routine test parameters (white blood cells, neutrophils, lymphocytes, and monocytes) in mice after different treatments. All experimental sample sizes n=3, and one-way ANOVA was used. Data are expressed as mean ± standard error. Ns: No significant difference.

[0074] To further evaluate the in vivo safety of LBEVs-PPy treatment, serological and pathological analyses related to biosafety were performed. Histological evaluation of vital organs was conducted using H&E staining. Figure 7 As shown in Figure A, compared with control mice, LBEVs-PPy treated mice showed no significant histological changes or cell damage in the liver, spleen, and kidneys. Furthermore, no significant differences were observed in blood routine parameters, including white blood cell count, neutrophil count, lymphocyte count, and monocyte count, in the LBEVs-PPy treatment group. Figure 7 BE).

[0075] This application successfully developed a near-infrared photothermal responsive in-situ sprayed polymer coating induced by *Lactobacillus reuteri* extravesicles (LBEVs) for synergistic antibacterial therapy and accelerated wound healing. LBEVs are derived from the pathogen-inhibiting probiotic *Lactobacillus reuteri*, and after extraction and purification, Fe… 3+ Surface functionalization. By using Fe 3+ Functionalized LBEVs and pyrrole monomers are co-sprayed onto the wound, triggering in-situ oxidative polymerization to form an LBEVs-PPy coating. Leveraging the probiotic properties of the parent bacteria, the LBEVs-PPy coating exhibits excellent biocompatibility and potent inhibitory activity against various pathogens. Simultaneously, the introduction of polypyrrole (PPy) imparts a photothermal effect to the material, upregulating the expression of VEGFA and ANGPT1 in human umbilical vein endothelial cells (HUVECs), thereby promoting angiogenesis. Validation using a mouse skin wound infection model shows that the LBEVs-PPy coating significantly accelerates wound healing through a dual synergistic mechanism—the pathogen clearance effect of LBEVs and the tissue regeneration-promoting ability of PPy photothermal conversion. This application provides a promising biotherapy strategy for the treatment of infected wounds and tissue repair.

[0076] The above embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made based on the essence of the content of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A LBEVs-PPy coating initiated by bacterial exovesicles, characterized in that: Bacterial extravesicular vesicles (LBEVs) adsorb Fe via electrostatic adsorption 3+ Obtain LBEVs-Fe 3+ Then, it is sprayed onto the wound simultaneously with pyrrole monomer, and polymerized in situ at the wound site to form an LBEVs-PPy coating; The bacterial vesicles are derived from Lactobacillus reuteri.

2. The LBEVs-PPy coating initiated by bacterial exovesicles according to claim 1, characterized in that: Fe 3+ Ions are adsorbed onto the surface of bacterial exovesicles, and the monomer pyrrole is oxidized to form polypyrrole (PPy) to obtain an LBEVs-PPy coating, in which spherical bacterial exovesicles are embedded.

3. The LBEVs-PPy coating for bacterial exovesicle initiation according to claim 1, characterized in that: The LBEVs-PPy coating accelerates wound healing through the dual synergistic effect of bacterial exovesicle-mediated antibacterial clearance and polypyrrole photothermal angiogenesis promotion.

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