Application of probiotic exosome in preparation of antibacterial product

Abstract

The invention discloses application of probiotic exosomes in preparation of antibacterial products, and belongs to the technical field of biological medicines. According to the invention, the exosome secreted by lactobacillus probiotics is used for resisting bacterial and fungal mixed infection; the probiotic exosome effectively inhibits mixed infection of bacteria and fungi through various mechanisms of inhibiting adhesion of bacteria and fungi, resisting formation of a biological membrane, promoting dispersion of the biological membrane and the like. According to the invention, probiotic exosome resources with antibacterial, anti-inflammatory and mixed infection treatment functions are expanded, and a new scheme is provided for preparing products for treating related infections.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of probiotic exosomes in the preparation of antibacterial products. Background Technology

[0002] Mixed bacterial and fungal infections have been reported in various infections, including vaginitis, cervicitis, endometritis, periodontitis, wound infections, and medical device infections. Interactions between pathogens can further enhance their pathogenicity, drug resistance, and immune evasion capabilities. Therefore, mixed bacterial and fungal infections often present with more severe symptoms than single-pathogen infections, easily leading to high morbidity and mortality. Staphylococcus aureus and Candida albicans are known as a very important and dangerous combination, having been co-isolated from infections such as periodontitis, burn wound infections, and medical device infections, and exhibiting enhanced resistance to antibiotics such as vancomycin, fluconazole, and miconazole. Biofilms consist of microbial cells and extracellular matrix, and their formation process includes early microbial adhesion, release of extracellular matrix to form a biofilm, biofilm maturation, and biofilm dispersion. Staphylococcus aureus alone is difficult to form a biofilm, while the mycelium of Candida albicans provides a scaffold structure. The two readily form mixed biofilms, significantly limiting antibiotic penetration, which is a major reason for the increased drug resistance in mixed Staphylococcus aureus and Candida albicans infections.

[0003] The increasing drug resistance of pathogens has prompted researchers to seek new antimicrobial strategies. In contrast to pathogenic microorganisms, probiotics are defined as live microorganisms that, at appropriate doses, provide health benefits to the host. They mainly include two types: lactobacilli (such as *Lactobacillus acidophilus*, *Lactobacillus casei*, *Lactobacillus curvatureii*, and *Lactobacillus gasseri*) and bifidobacteria (such as *Bifidobacterium longum*, *Bifidobacterium breve*, *Bifidobacterium ovale*, and *Bifidobacterium thermophilum*). Existing research has shown that probiotics exert their antimicrobial activity through mechanisms such as enhancing the integrity of the epithelial barrier, competitively reducing pathogen adhesion, secreting antimicrobial substances, regulating the immune system, and interfering with the expression of pathogen virulence genes. They can also help restore normal flora ecology, making them a major research hotspot in the field of antimicrobial research. However, the antimicrobial activity of probiotics themselves is relatively weak, and they still need to be used in combination with antifungal drugs. Antifungal drugs are generally poorly water-soluble and difficult to effectively overcome biofilm barriers; therefore, there is an urgent need to develop antifungal drug delivery strategies that enhance biofilm permeability.

[0004] Exosomes (EVs) are tiny vesicle-like bodies released by cells, including microorganisms, and enclosed in a phospholipid bilayer membrane, containing substances such as proteins, lipids, and nucleic acids. Various methods for the isolation and purification of EVs have been developed, including differential ultracentrifugation, density gradient centrifugation, and size exclusion chromatography. Increasing evidence suggests that EVs play a crucial role in communication between microorganisms and between microorganisms and the host. Probiotic-derived EVs have been shown to enhance the integrity of the intestinal barrier, regulate immune responses, inhibit pathogens, and influence the host microbiome composition, making them a promising therapeutic platform for treating infections, allergies, cancer, autoimmune diseases, and neurodegenerative diseases. Furthermore, the nanoscale size and phospholipid bilayer structure of exosomes themselves have the potential to overcome biomembrane barriers and can be used for antifungal drug delivery. However, few studies have applied probiotic EVs to mixed bacterial and fungal infections or to the delivery of antifungal drugs. Therefore, exploring the activity, mechanism of action, and carrier capacity of probiotic EVs against mixed bacterial and fungal infections can further verify the application value of EVs in antibacterial applications and provide new treatment options for mixed bacterial and fungal infections. Summary of the Invention

[0005] The purpose of this invention is to provide the application of probiotic exosomes in the preparation of antibacterial products. These antibacterial products can be used to treat one or more of the following: vaginitis, cervicitis, endometritis, periodontitis, wound infections, and medical device infections.

[0006] Furthermore, the probiotic exosomes are prepared from lactobacillus or bifidobacteria probiotics. The lactobacillus probiotics are *Lactobacillus acidophilus*, *Lactobacillus curvatureii*, *Lactobacillus gasseri*, *Lactobacillus bulgaricus*, *Lactobacillus casei*, or *Lactobacillus plantarum*. Preferably, they are *Lactobacillus acidophilus* CICC 6005, *Lactobacillus curvatureii* CICC 24879, or *Lactobacillus gasseri* ATCC 33323.

[0007] The preparation method of the above-mentioned probiotic exosomes is as follows: The activated bacterial suspension is inoculated into a culture medium for expansion culture. The cultured bacterial solution is centrifuged at low speed, and the supernatant is collected. Bacteria are removed by a membrane filter, and the filtrate is concentrated by ultrafiltration. After centrifugation of the concentrate, the precipitate is resuspended to obtain the probiotic exosomes. Further, the expansion culture uses MRS medium, with culture conditions of 37℃ and 220 rpm for 24-36 hours; the low-speed centrifugation conditions are 4℃, 8000-11000g, 10-20 min; the membrane filter is a 0.22 μm membrane filter; the ultrafiltration concentration conditions are: ultrafiltration membrane with a molecular weight cutoff of 100 kDa, 3000-6000g, centrifugation at 4℃ for 10-20 min; the concentrated solution centrifugation conditions are 4℃, 150000g, 150-200 min.

[0008] Furthermore, the product is a pharmaceutical composition, which further includes a pharmaceutically acceptable carrier selected from one or more of poloxamer, polyacrylic acid derivatives, cellulose derivatives, polyvinyl alcohol, gelatin, and sodium alginate. The weight ratio of the probiotic exosomes to the pharmaceutical carrier is 1:1000-1:5000.

[0009] Furthermore, the probiotic exosomes also contain an active drug, with a weight ratio of probiotic exosomes to active drug of 5:1 to 1:1; the active drug is selected from one or more of polyene, triazole, allylamine, echinocandin, and pyrimidine antifungal drugs.

[0010] In one specific embodiment of the present invention, amphotericin B is first encapsulated in Lactobacillus gasseri exosomes to obtain drug-loaded exosomes. These drug-loaded exosomes are then dispersed in a thermosensitive gel matrix to prepare a vaginal gel. The encapsulation of exosomes with a polymer carrier prolongs the retention time of amphotericin B in vivo. Simultaneously, the antibacterial effect is enhanced by utilizing the antibacterial and fungal mixed infection activity of Lactobacillus gasseri exosomes and their ability to enhance biofilm and vaginal mucus penetration. This method has significant application value in the treatment of mixed vaginitis.

[0011] This invention utilizes exosomes secreted by probiotic lactobacilli and verifies the ability and mechanism of action of probiotic lactobacilli exosomes against mixed bacterial and fungal infections by analyzing the adhesion of bacteria and fungi, the formation and dispersion of mixed biofilms, and the macrophages induced by bacteria and fungi. This expands the resources of probiotic exosomes with antibacterial, anti-inflammatory and therapeutic effects on mixed infections, and provides new treatment options for related infections.

[0012] The probiotic exosome preparation method provided by this invention produces exosomes with uniform particle size and good stability. The probiotic exosomes prepared by this invention can inhibit the expression of bacterial and fungal adhesion-related genes, reducing bacterial and fungal adhesion to vaginal epithelial cells. Furthermore, the probiotic exosomes prepared by this invention can inhibit the expression of bacterial and fungal biofilm formation-related genes and increase the expression of biofilm dispersion-related genes, thus exerting an anti-mixed biofilm formation and dispersing effect on mature biofilms. Finally, the probiotic exosomes prepared by this invention can reduce the level of pro-inflammatory cytokines secreted by bacterial and fungal-induced macrophages and increase the level of anti-inflammatory cytokines, thus exhibiting anti-inflammatory activity.

[0013] This invention, through the encapsulation of polymer carriers and exosomes, can prolong the retention time of active drugs in the body. At the same time, by utilizing the antibacterial and fungal mixed infection activity of Lactobacillus gasseri exosomes and their ability to enhance the penetration of biofilms and vaginal mucus, the antibacterial effect is enhanced, which has great application value in the treatment of mixed vaginitis. Attached Figure Description

[0014] Figure 1Transmission electron micrographs of exosomes from different lactobacillus probiotics.

[0015] Figure 2 This is a particle size diagram of exosomes from different lactobacillus probiotics.

[0016] Figure 3 This figure shows the particle size changes of exosomes from different lactobacillus probiotics over 14 days at -80℃.

[0017] Figure 4 This is the result of Example 5 showing the effect of Lactobacillus gasseri on the adhesion of Staphylococcus aureus and Candida albicans to vaginal epithelial cells.

[0018] Figure 5 The crystal violet staining results show the effect of Lactobacillus gasseri exosomes on the formation of a mixed biofilm of Staphylococcus aureus and Candida albicans in Example 6.

[0019] Figure 6 This is a crystal violet staining result of the effect of Lactobacillus gasseri exosomes on the mixed mature biofilm of Staphylococcus aureus and Candida albicans in Example 7.

[0020] Figure 7 This is a graph showing the results of the effect of Lactobacillus gasseri exosomes on the cytokine levels secreted by macrophages induced by Staphylococcus aureus and Candida albicans in Example 8.

[0021] Figure 8 This is a scanning electron microscope image of a vaginal drug delivery system.

[0022] Figure 9 This is the drug release curve for the vaginal delivery system.

[0023] Figure 10 This image shows the staining of live and dead cells formed by a mixed biofilm of drug-loaded exosomes against Staphylococcus aureus and Candida albicans.

[0024] Figure 11 This image shows the staining of live and dead cells in a mixed mature biofilm of drug-loaded exosomes against Staphylococcus aureus and Candida albicans.

[0025] Figure 12 This is a graph showing the retention time of the vaginal delivery system in the mouse vagina.

[0026] Figure 13 This is a diagram showing the penetration depth of the vaginal drug delivery system in the vaginal mucosa of mice.

[0027] Figure 14 Image showing the vaginal appearance of a mouse model of mixed vaginitis after treatment with a vaginal delivery system.

[0028] Figure 15A quantitative graph showing the levels of inflammatory cytokines in the vaginal fluid of mice with a mixed vaginitis model after treatment with a vaginal delivery system. Detailed Implementation

[0029] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] The Lactobacillus acidophilus, Lactobacillus curvature, Lactobacillus tarda, and Lactobacillus gasseri used in the following examples were all purchased from Taisto Biotechnology. Example 1

[0033] Culture of Lactobacillus acidophilus and preparation of exosomes (LA-EVs) 1. Activation culture of Lactobacillus acidophilus: Spread 50 μL of Lactobacillus acidophilus solution onto MRS solid medium for activation and incubate statically at 37°C for 48 h. Inoculate two loops of the activated strain into 5 mL of MRS liquid medium and incubate at 37°C and 220 rpm for 30 h. 2. Expanded culture of Lactobacillus acidophilus: Take 5 mL of activated Lactobacillus acidophilus bacterial solution and inoculate it into 100 mL of MRS liquid medium. Expand the culture at 37℃ and 220 rpm for 30 h. 3. Low-speed centrifugation: Centrifuge the expanded culture medium at 4℃ and 10000g for 20 minutes, take the supernatant and remove larger impurities; 4. Membrane sterilization: Pass the supernatant through a 0.22μm filter membrane to remove residual bacteria, cell debris, polymers, and other larger particles and impurities; 5. Ultrafiltration concentration: The filtrate was centrifuged in a 100kDa ultrafiltration centrifuge tube at 4℃ and 5000g for 15 minutes, and the concentrate was collected. 6. Ultra-high speed centrifugation: The obtained concentrate was centrifuged in an ultra-high speed centrifuge tube at 4℃ and 150,000g for 150 hours, and the precipitate after ultra-high speed centrifugation was collected.

[0034] 7. Washing and resuspending: Wash the precipitate three times with sterile PBS buffer, and then resuspend it with 1 mL of sterile PBS buffer to obtain Lactobacillus acidophilus exosome solution. Example 2

[0035] Culture of Lactobacillus curvature and preparation of exosomes (LC-EVs) The difference from Example 1 is that only the Lactobacillus probiotics used are replaced with Lactobacillus curvifolius. Example 3

[0036] Culture of Lactobacillus gasseri and preparation of exosomes (LG-EVs) The difference from Example 1 is that only the lactobacillus probiotics used are replaced with Lactobacillus gasseri. Example 4

[0037] Characterization analysis of exosomes of lactobacillus probiotics 1. Electron microscopy identification The three types of exosomes obtained in Examples 1-3 were placed on a 300-mesh copper grid, stained with 1% phosphotungstic acid, and their morphological characteristics were observed under a transmission electron microscope. The results are shown in the figure. Figure 1 All three types of exosomes exhibit a goblet-shaped vesicle-like structure.

[0038] 2. Particle size analysis The particle size distribution of the three types of exosomes obtained in Examples 1-3 was detected using nanoparticle tracking analysis (NTA). The results are shown in [Figure 1]. Figure 2 The particle size of the three exosomes was found to be concentrated in the range of 100-150 nm, and the particle size was uniform, which was basically consistent with the results observed under transmission electron microscopy.

[0039] 3. Stability The three types of exosomes prepared in Examples 1-3 were placed in a -80°C freezer for 14 days. The particle size of the LG-EVs was then measured using nanoparticle tracking analysis (NTA). The results are shown in [Figure number missing]. Figure 3 The particle size of the three exosomes did not change significantly within 14 hours, indicating that the three exosomes have good stability and are suitable for application. Example 5

[0040] Inhibitory effect of Lactobacillus gasseri exosomes (LG-EVs) on the adhesion of Staphylococcus aureus and Candida albicans to vaginal epithelial cells 1. Cell Culture Human immortalized vaginal epithelial cells (VK2 / E6E7) were purchased from Shanghai Guandao Biotechnology Co., Ltd. Cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and incubated at 37°C in a 5% CO2 incubator.

[0041] 2. Determination of Adhesion Amount VK2 / E6E7 cells in logarithmic growth phase were fed at a rate of 4 × 10⁻⁶.5 Cells were seeded per well in 12-well plates, divided into control and experimental groups. Incubation was performed at 37°C and 5% CO2 for 12 hours to allow cell adhesion. After 12 hours, 1 mL of PBS was added to the control group, and 1 mL of LG-EVs solution with a protein concentration of 128 μg / mL was added to the experimental group. Three replicates were performed in each group, and incubation continued for another 12 hours. Then, a 10:1 ratio of Candida albicans and Staphylococcus aureus bacterial suspension (bacterial suspension concentration 10) was added. 6 (CFU / mL) and incubate for 3 hours. Discard the supernatant and collect adherent VK2 / E6E7 cells from the bottom of the wells by pipetting PBS. Spread 50 μL of the cell suspension onto YM solid medium and BHI solid medium, respectively, and incubate at 37°C for 48 hours. Count the colonies of Candida albicans and Staphylococcus aureus.

[0042] like Figure 4 As shown, compared with the control group, the colony counts of Staphylococcus aureus and Candida albicans in the experimental group were significantly reduced. The experiment indicates that LG-EVs can inhibit the adhesion of Staphylococcus aureus and Candida albicans to VK2 / E6E7 cells, demonstrating potential against mixed bacterial and fungal infections. Example 6

[0043] Inhibitory effect of Lactobacillus gasseri exosomes (LG-EVs) on the formation of mixed biofilms of Staphylococcus aureus and Candida albicans 1. Formation and administration regimen of mixed biofilm of Staphylococcus aureus and Candida albicans Take a 96-well plate and add 50 μL of 4 × 10⁻⁶ solution to each well. 6 CFU / mL Candida albicans suspension and 50 μL 4×10 6 Staphylococcus aureus suspension at CFU / mL was divided into a control group and an experimental group. The control group was added with 100 μL of PBS solution, and the experimental group was added with 100 μL of LG-EVs solution. The mixtures were incubated at 37°C for 24 h.

[0044] 2. Crystal violet staining Take the above 96-well plate, aspirate and discard the upper bacterial suspension, wash twice with PBS, add 100 μL of 10% methanol aqueous solution for fixation for 15 min, aspirate the methanol, and air dry. Then add 200 μL of 0.1% (w / v) crystal violet solution for staining for 15 min, aspirate the crystal violet, wash three times with PBS, air dry, and observe the biofilm morphology under a fluorescence upright microscope.

[0045] like Figure 5 As shown, in the control group, the biofilm was entangled with hyphae and covered by the extracellular matrix, making the internal structure unclear. Compared with the control group, the experimental group showed a significant reduction in biofilm formation, with clearly visible hyphal structures and a decrease in the number of microcolonies covered by the extracellular matrix.

[0046] 3. Analysis of genes related to biofilm formation in Staphylococcus aureus and Candida albicans Take a 6-well plate and add 0.5 mL of 4 × 10⁻⁶ ppm to each well. 6 CFU / mL Candida albicans suspension and 0.5 mL 4×10 6 CFU / mL Staphylococcus aureus bacterial suspension was divided into control and experimental groups. The control group received 1 mL of PBS solution, while the experimental group received 1 mL of LG-EVs solution. Incubation was continued at 37°C for 24 h. Biofilm from each well was collected, and RNA was extracted according to the kit (purchased from Novizan Biotechnology), reverse transcribed into DNA, and then subjected to qRT-PCR to detect the expression levels of target genes, including the Candida albicans biofilm formation promoting genes ALS3 and HWP1 and hyphal formation inhibiting genes NRG1 and TUP1, and the Staphylococcus aureus biofilm formation promoting genes icaA and FnbA and inhibiting genes icaR and LuxS. Gene normalization was performed using 18S rRNA as an internal control. -ΔΔCt The method was analyzed.

[0047] The primer sequences for the relevant genes are shown in Table 1 below: Table 1 Primers for qRT-PCR analysis

[0048] The experimental results are shown in Table 2. Compared with the control group, the experimental group significantly reduced the expression of Candida albicans biofilm formation promoting genes ALS3 and HWP1, and significantly increased the expression of Candida albicans hyphal formation inhibiting genes NRG1 and TUP1. Compared with the control group, the experimental group significantly reduced the expression of Staphylococcus aureus biofilm formation promoting genes icaA and FnbA, and significantly increased the expression of Staphylococcus aureus biofilm formation inhibiting genes icaR and LuxS.

[0049] Table 2. Effects of each group on the expression levels of genes related to biofilm formation in Staphylococcus aureus and Candida albicans.

[0050] The results showed that Lactobacillus gasseri exosomes could inhibit the formation of mixed biofilms of Staphylococcus aureus and Candida albicans, demonstrating the ability to resist mixed bacterial and fungal infections.

[0051] Example 7 Dispersion of Lactobacillus gasseri exosomes (LG-EVs) on mixed mature biofilms of Staphylococcus aureus and Candida albicans 1. Culture and drug administration regimen for a mixed mature biofilm of Staphylococcus aureus and Candida albicans Take a 96-well plate and add 50 μL of 4 × 10⁻⁶ solution to each well. 6 CFU / mL Candida albicans suspension and 50 μL 4×10 6 A CFU / mL suspension of Staphylococcus aureus was cultured for 24 h to obtain a mature biofilm. The bacterial suspension was discarded, and the cells were washed twice with PBS. 100 μL of 1640 complete culture medium was added, and the cells were divided into a control group and an experimental group. 100 μL of PBS was added to the control group, and 100 μL of LG-EVs solution was added to the experimental group. The cells were incubated at 37°C for another 24 h.

[0052] 2. Crystal violet staining Take the above 96-well plate, perform crystal violet staining and observe the biofilm morphology in the same manner as in Example 6-2.

[0053] like Figure 6 As shown, in the control group, the biofilm was entangled with hyphae and covered by the extracellular matrix, making the internal structure unclear. Compared with the control group, the biofilm in the drug-treated group showed some dispersion, with hyphae exposed.

[0054] 3. Analysis of genes related to mature biofilms of Staphylococcus aureus and Candida albicans Take a 6-well plate and add 0.5 mL of 4 × 10⁻⁶ ppm to each well. 6 CFU / mL Candida albicans suspension and 0.5 mL 4×10 6 A CFU / mL Staphylococcus aureus bacterial suspension was incubated at 37°C for 24 hours to form a mature biofilm, which was then divided into a control group and an experimental group. The control group received 1 mL of PBS solution, while the experimental group received 1 mL of LG-EVs solution, and incubation continued at 37°C for another 24 hours. Biofilms from each well were collected, and RNA was extracted according to the kit (purchased from Novizan Biotechnology), and reverse transcribed into DNA. qRT-PCR was then performed to detect the expression levels of target genes, including the Candida albicans biofilm maintenance-related gene UME6 and the dispersion-related gene NRG1, and the Staphylococcus aureus biofilm dispersion-related genes PSMα and Nuc. Gene normalization was performed using 18S rRNA as an internal control, and 2... -ΔΔCt The method was analyzed.

[0055] The relevant gene primer sequences are shown in Table 3 below: Table 3 Primers for qRT-PCR analysis

[0056] The experimental results are shown in Table 4. Compared with the control group, the experimental group significantly reduced the expression of UME6, a gene related to Candida albicans biofilm maintenance, and significantly increased the expression of NRG1, a gene related to Candida albicans biofilm dispersion. Compared with the control group, the experimental group significantly increased the expression of PSMα and Nuc, genes related to Staphylococcus aureus biofilm dispersion.

[0057] Table 4. Effects of each group on the expression levels of genes related to biofilm dispersion in Staphylococcus aureus and Candida albicans.

[0058] The results showed that Lactobacillus gasseri exosomes can help disperse mixed mature biofilms of Staphylococcus aureus and Candida albicans, and have the ability to resist mixed bacterial and fungal infections.

[0059] Example 8 Effects of Lactobacillus gasseri exosomes (LG-EVs) on inflammatory cytokines secreted by macrophages induced by Staphylococcus aureus and Candida albicans 1. Cell Culture Macrophages (RAW264.7) were purchased from the Shanghai Institute of Cell Biology. Cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and incubated at 37°C in a 5% CO2 incubator.

[0060] 2. Measurement of macrophage secretion of inflammatory cytokines The host employs various protective mechanisms to defend against bacterial and fungal invasion, including activating inflammasomes, releasing inflammatory cytokines, and recruiting neutrophils. During infection, the release of pro-inflammatory cytokines such as IL-1β and TNF-α increases; therefore, the level of inflammatory markers in the body can effectively reflect the antibacterial effect of the agent.

[0061] RAW264.7, which is in the logarithmic growth phase, was used with 4 × 10 5 Cells were seeded per well in 12-well plates and incubated at 37°C with 5% CO2 for 12 hours. The plates were divided into control and experimental groups. After 12 hours, 1 mL of PBS solution was added to the control group, and 1 mL of LG-EVs solution was added to the experimental group. Incubation continued for another 12 hours. Then, a mixed bacterial suspension of Candida albicans and Staphylococcus aureus at a ratio of 10:1 (bacterial suspension concentration 4 × 10⁻⁶ cells / well) was added. 6 After stimulating the cells with CFU / mL for 3 hours, the cell culture supernatant was collected, and the levels of pro-inflammatory cytokine IFN-γ and anti-inflammatory cytokine IL-10 in each group were detected according to the instructions of the enzyme-linked immunosorbent assay kit (purchased from Jiangsu Enzyme Immunosorbent Assay Kit).

[0062] like Figure 7 As shown, compared with the control group, the experimental group significantly reduced the amount of pro-inflammatory cytokine IFN-γ secreted by macrophages under mixed induction by Staphylococcus aureus and Candida albicans, and significantly increased the secretion of anti-inflammatory cytokine IL-10.

[0063] Experimental results showed that *Lactobacillus gasseri* exosomes could significantly reduce the adhesion of *Staphylococcus aureus* and *Candida albicans* to vaginal epithelial cells; they could inhibit the expression of genes related to biofilm formation of *Staphylococcus aureus* and *Candida albicans*, increase the expression of genes related to biofilm dispersion, inhibit the formation of mixed bacterial-fungal biofilms, and help disperse mixed mature biofilms; they could inhibit the secretion of the pro-inflammatory cytokine IFN-γ, increase the level of the anti-inflammatory cytokine IL-10, improve the inflammatory environment, and combat mixed bacterial-fungal infections from multiple aspects.

[0064] Example 9 A vaginal delivery system loaded with Lactobacillus gasseri exosomes, wherein the delivery system is loaded with Lactobacillus gasseri exosomes; the Lactobacillus gasseri exosomes contain amphotericin B.

[0065] Its preparation method includes the following steps: Step 1: Mix the Lactobacillus gasseri exosome solution obtained in Example 3 with amphotericin B solution, incubate in the dark, pass through a 0.45 μm membrane filter, collect the filtrate, and obtain the drug-loaded exosome solution; Step 2: Mix the drug-loaded exosome solution and the polymer matrix to obtain a vaginal delivery system loaded with Lactobacillus gasseri exosomes.

[0066] (I) Effect of incubation temperature on the preparation of drug-loaded exosome solutions 1. Mix Lactobacillus gasseri exosome solution with a protein concentration of 128 μg / mL and amphotericin B solution with a concentration of 64 μg / mL at a volume ratio of 1:1. Place the mixture at the different temperatures shown in Table 5 and incubate in the dark for 30 min. Filter the mixture through a 0.45 μm membrane filter and collect the filtrate to obtain the drug-loaded exosome solution. 2. The particle size and PDI of the samples were determined using a nanoparticle size analyzer, with three parallel measurements performed. The encapsulation efficiency of exosomes was determined using ultrafiltration: the drug-loaded exosome solution was taken, and its drug content was determined and denoted as C. 投药量 Centrifuge the drug-loaded exosome solution at 4000 rpm for 15 min, discard the precipitate, and determine the drug content in the supernatant. Record the concentration as C. 总 Take the supernatant obtained from the previous centrifugation step and place it in an ultrafiltration centrifuge tube. Centrifuge at 14,000 rpm until the volume of the lower liquid no longer increases. Take the lower solution and determine the drug content; record the concentration as C. 游离 The encapsulation rate is calculated using the following formula.

[0067]

[0068] Table 5. Effects of different incubation temperatures on particle size, PDI, and encapsulation efficiency of drug-loaded exosomes ( ±SD, n=3)

[0069] The results showed that the encapsulation efficiency of drug-loaded exosomes was low when the incubation temperature was 4℃; when the incubation time was 25℃, the encapsulation efficiency was >95%. Further increasing the incubation temperature did not significantly change the encapsulation efficiency. Therefore, the preferred incubation temperature in this invention is 25℃.

[0070] (II) Effect of incubation time on the preparation of drug-loaded exosome solutions 1. Mix Lactobacillus gasseri exosome solution with a protein concentration of 128 μg / mL and amphotericin B solution with a concentration of 64 μg / mL at a volume ratio of 1:1, incubate at 25°C in the dark for different times as shown in Table 6, pass through a 0.45 μm membrane filter, collect the filtrate, and obtain drug-loaded exosome solution; 2. The particle size and PDI of the samples were determined using a nanoparticle size analyzer, with three parallel measurements performed. The encapsulation efficiency of exosomes was determined using ultrafiltration: the drug-loaded exosome solution was taken, and its drug content was determined and denoted as C. 投药量 Centrifuge the drug-loaded exosome solution at 4000 rpm for 15 min, discard the precipitate, and determine the drug content in the supernatant. Record the concentration as C. 总 Take the supernatant obtained from the previous centrifugation step and place it in an ultrafiltration centrifuge tube. Centrifuge at 14,000 rpm until the volume of the lower liquid no longer increases. Take the lower solution and determine the drug content; record the concentration as C. 游离 The encapsulation rate is calculated using the following formula.

[0071]

[0072] Table 6. Effects of different incubation times on particle size, PDI, and encapsulation efficiency of drug-loaded exosomes ( ±SD, n=3)

[0073] The results showed that the encapsulation efficiency of drug-loaded exosomes increased with increasing incubation time. When the incubation time was 30 min, the encapsulation efficiency was >95%. Further increasing the incubation time did not significantly change the encapsulation efficiency. Therefore, the preferred incubation time in this invention is 30 min.

[0074] Example 10 The vaginal delivery system loaded with Lactobacillus gasseri exosomes was prepared using the optimal process described in Example 9. The preparation method includes the following steps: Step 1: Mix Lactobacillus gasseri exosome solution with a protein concentration of 128 μg / mL and amphotericin B solution with a concentration of 64 μg / mL at a volume ratio of 1:1, incubate at 25°C in the dark for 30 min, pass through a 0.45 μm membrane filter, collect the filtrate, and obtain the drug-loaded exosome solution; Step 2: Mix the thermosensitive gel matrix poloxamer 407 and poloxamer 188 with the drug-loaded exosome solution obtained in Step 1, so that the concentration of poloxamer 407 is 22% (w / v) and the concentration of poloxamer 188 is 8% (w / v). Stir and mix evenly in an ice-water bath, and let stand overnight at 4°C to obtain a gel drug delivery system loaded with Lactobacillus gasseri exosomes.

[0075] Example 11 Morphological characterization of vaginal drug delivery system loaded with Lactobacillus gasseri exosomes Take 3 mL of the gel drug delivery system described in Example 10 into a 5 mL EP tube, place it in a 37°C oven for 5 min to allow the gel to gelleave, then place the EP tube in liquid nitrogen for 5 min to freeze-dry for 48 h. Break the freeze-dried gel with tweezers, sputter-coat it with gold at 3 kV, and observe the surface morphology of the gel cross-section using a scanning electron microscope. The results are as follows. Figure 8 As shown in the figure. The results indicate that the gel has a porous network structure with drug-loaded exosomes dispersed within it.

[0076] Example 12 In vitro drug release curve of the vaginal delivery system loaded with Lactobacillus gasseri exosomes The in vitro release of a vaginal drug delivery system loaded with *Lactobacillus gasseri* exosomes was studied using dynamic dialysis. The drug-loaded exosome solution obtained in step 1 of Example 10 and the gel drug delivery system obtained in step 2 were placed in dialysis bags, sealed at both ends, and immersed in 250 mL of release medium. In vitro release was performed at 37°C and 200 rpm. 2 mL samples were taken at different time points, and the same volume and temperature of release medium were immediately replenished. The drug concentration in the samples was measured, and the cumulative drug release rate was calculated. The results are as follows: Figure 9 As shown in the figure. The results indicate that both drug-loaded exosomes and gel delivery systems have sustained-release effects, with the gel delivery system exhibiting a stronger sustained-release effect.

[0077] Example 13 Inhibitory effect of drug-loaded exosomes on the formation of mixed biofilms of Staphylococcus aureus and Candida albicans 1. Formation and administration regimen of mixed biofilm of Staphylococcus aureus and Candida albicans Take a 12-well plate, add cell spreaders and a concentration of 4×10⁻⁶ to each well. 6 0.5 mL each of CFU / mL Candida albicans and Staphylococcus aureus bacterial suspensions were divided into a control group and an experimental group. 1 mL of PBS solution was added to the control group, and 1 mL of the drug-loaded exosome solution obtained in step 1 of Example 10 was added to the experimental group. The cultures were incubated at 37°C for 24 hours.

[0078] 2. Staining of live and dead cells Prepare the staining solution according to the instructions of the live / dead cell staining kit (purchased from Beyotime). Remove the 12-well plate, discard the supernatant, and stain according to the kit instructions. Aspirate the staining solution, remove the coverslip with a needle, place it on the slide, seal the slide, and observe under a confocal microscope. See attached image. Figure 10 .

[0079] The results showed that the bacteria and fungi in the control group grew vigorously, with entangled hyphae and no obvious dead cells. Compared with the control group, the number of dead cells in the experimental group increased significantly, and the biofilm showed some degree of dispersion. These results indicate that drug-loaded exosomes can inhibit the formation of mixed biofilms of Candida albicans and Staphylococcus aureus, helping amphotericin B penetrate deep into the mixed biofilm to exert its antibacterial effect.

[0080] Example 14 Dispersion of drug-loaded exosomes on mixed mature biofilms of Staphylococcus aureus and Candida albicans 1. Culture and drug administration regimen for a mixed mature biofilm of Staphylococcus aureus and Candida albicans Take a 12-well plate and add cell spreaders and a concentration of 4×10⁻⁶ cells to each well. 6 0.5 mL each of CFU / mL Candida albicans and Staphylococcus aureus bacterial suspensions were incubated at 37°C for 24 h to obtain mature biofilms. The bacterial suspensions were discarded, and the cells were washed twice with PBS. 1 mL of 1640 complete culture medium was added to each well, dividing the cells into control and experimental groups. The control group received 1 mL of PBS solution, while the experimental group received 1 mL of the drug-loaded exosome solution obtained in step 1 of Example 10. The cells were then incubated at 37°C for another 24 h.

[0081] 2. Staining of live and dead cells Prepare the staining solution according to the instructions of the live / dead cell staining kit (purchased from Beyotime). Remove the 12-well plate, discard the supernatant, and stain according to the kit instructions. Aspirate the staining solution, remove the coverslip with a needle, place it on the slide, seal the slide, and observe under a confocal microscope. See attached image. Figure 11 .

[0082] The results showed that the bacteria and fungi in the control group grew vigorously, with entangled hyphae and no obvious dead cells. Compared with the control group, the number of dead cells in the experimental group increased significantly, and the biofilm showed a certain degree of dispersion. The results indicate that drug-loaded exosomes can disperse the mixed mature biofilm of Candida albicans and Staphylococcus aureus, helping amphotericin B penetrate deep into the mixed biofilm to exert its antibacterial effect.

[0083] Example 15 A vaginal delivery system loaded with Lactobacillus gasseri exosomes prolongs the retention time of the formulation in the mouse vagina. 1. Dosing regimen for mice 100 μL of the drug-loaded exosome solution obtained in step 1 of Example 10 with DiI labeling was vaginally instilled into mice as the exosome group; 100 μL of the gel delivery system obtained in step 2 of Example 10 with DiI labeling was vaginally instilled into mice as the gel group.

[0084] 2. In vivo imaging of mice In vivo photographs of mice were taken using a small animal in vivo imaging system at 0 h, 24 h, and 48 h post-drug administration. Before photographing, mice were anesthetized with gas, then placed inside the in vivo imaging system, inhaled the anesthetic, and the software was used to take the images. Results are shown below. Figure 12 .

[0085] The results showed that the fluorescence signal in the exosome group rapidly decayed 24 hours after administration, and essentially disappeared after 48 hours. Compared with the exosome group, the gel group showed significantly longer retention in the vagina, with fluorescence signals still present after 48 hours. These results indicate that the vaginal delivery system of the present invention effectively improves the retention time of the formulation in the vagina.

[0086] Example 16 A vaginal delivery system loaded with Lactobacillus gasseri exosomes increases the penetration depth of the formulation in the mouse vagina. The unique physiological structure of the vagina, such as its self-cleaning behavior and vaginal mucus, significantly hinders the penetration of the formulation. Mice from each group in Example 15 were euthanized by cervical dislocation 24 hours after drug administration. The vaginas were removed, and 10 μm sections were prepared using a cryostat. AF 488 WGA staining was used to label vaginal mucus, and DAPI staining was used to label the vaginal mucosa. Images were taken using a fluorescence microscope. Results are shown below. Figure 13 .

[0087] The results showed that both the exosome group and the gel group had a certain penetration depth. The gel group, however, could uniformly accumulate on the vaginal mucosa surface and penetrate deeper, achieving a greater penetration depth than the exosome group. These results indicate that the vaginal delivery system of this invention can effectively retain mucosa and penetrate mucus, helping the drug to fully exert its antibacterial potential.

[0088] Example 17 A vaginal delivery system loaded with Lactobacillus gasseri exosomes improved inflammatory symptoms in mice with mixed vaginitis. 1. Construction of a mouse model of mixed vaginitis Three days prior to inoculation, mice were subcutaneously injected daily with 0.05 mL of 2 mg / mL estradiol benzoate injection and intraperitoneally injected with 0.15 mL of 1 mg / mL dexamethasone sodium phosphate injection. On the day of inoculation, 10 μL of Candida albicans suspension was collected (the inoculation amount of Candida albicans spores was approximately 4 × 10⁻⁶). 5 CFU) and 10 μL of Staphylococcus aureus bacterial suspension (the inoculum of Staphylococcus aureus is approximately 4 × 10⁻⁶ CFU).5 CFU was injected into the vagina of mice, and the mice were inverted for 5-10 minutes after inoculation to prevent the bacterial suspension from flowing out. Inoculation was repeated for 2 days. Every other day, mice in the model group were subcutaneously injected with 0.05 mL of 2 mg / mL estradiol benzoate injection; every two days, mice in the model group were intraperitoneally injected with 0.15 mL of 1 mg / mL dexamethasone sodium phosphate injection to maintain estrus and an immunosuppressive environment in the mice until the end of treatment.

[0089] 2. Dosing regimen for mice Mice that successfully developed the model were randomly divided into two groups: a control group and an experimental group; mice that did not develop the model were used as a blank control group. Mice in the control group were vaginally instilled with 20 μL of PBS, while mice in the experimental group were vaginally instilled with 20 μL of the gel delivery system obtained in step 2 of Example 10. Administration was repeated for 2 consecutive days. The blank control group received no treatment.

[0090] 3. Appearance of mouse vagina After 1 and 2 days of drug administration, the appearance of the vagina of mice in each group was observed and photographed. Results are shown below. Figure 14 The results showed that, compared with the control group, the vaginal opening closed one day after administration in the experimental group, and two days after administration, there was no obvious redness or swelling or discharge in the vagina, and the appearance of the vagina was similar to that of the blank group.

[0091] 4. Inflammatory cytokine levels in mouse vaginal irrigation fluid During the pathogenesis of vaginitis, the host utilizes multiple protective mechanisms to combat the invasion of pathogenic microorganisms, involving the activation of inflammasomes, the release of inflammatory cytokines, and the recruitment of neutrophils. Two days after drug administration, the vaginas of mice were repeatedly irrigated with 150 μL of sterile PBS 8–10 times to obtain vaginal irrigation fluid. The levels of pro-inflammatory cytokines TNF-α and IFN-γ and anti-inflammatory cytokines IL-10 and TGF-β1 were detected according to the instructions of an enzyme-linked immunosorbent assay (ELISA) kit (purchased from Jiangsu ELISA) to characterize the inflammatory status of the vagina. Results are shown below. Figure 15 The results showed that, compared with the control group, the concentrations of pro-inflammatory cytokines such as TNF-α and IFN-γ were significantly reduced in the experimental group, while the concentrations of anti-inflammatory cytokines IL-10 and TGF-β1 were significantly increased, similar to those in the blank group.

[0092] In summary, the preparation method of this invention is simple to operate, operates under mild conditions, and is easy to scale up for production. *Lactobacillus gasseri* exosomes possess multiple physiological activities, inhibiting the formation of mixed bacterial-fungal biofilms, exhibiting anti-inflammatory activity, and can exert a synergistic antibacterial effect with amphotericin B. The encapsulation of *Lactobacillus gasseri* exosomes enables sustained release of amphotericin B while providing the ability to cross biological barriers, helping amphotericin B overcome vaginal mucus and biofilm barriers, thus enhancing efficacy. The temperature-sensitive gel system prolongs the drug's retention time in the vagina, enhancing the antibacterial effect. The vaginal delivery system loaded with *Lactobacillus gasseri* exosomes prepared in this invention exhibits good anti-inflammatory effects in mice with mixed vaginitis and is expected to become a new strategy for treating vaginal-related infections.

Claims

1. Use of probiotic exosomes in the preparation of an antibacterial product.

2. Use according to claim 1, characterized in that, The probiotic exosomes are prepared from a Lactobacillus or Bifidobacterium probiotic.

3. Use according to claim 2, characterized in that, The Lactobacillus probiotic is Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus bulgaricus, Lactobacillus casei or Lactobacillus plantarum.

4. Use according to claim 3, characterized in that, The Lactobacillus probiotic is Lactobacillus acidophilus, Lactobacillus crispatus or Lactobacillus gasseri.

5. Use according to claim 2, characterized in that, The method for preparing the probiotic exosomes comprises inoculating the activated bacterial suspension into a culture medium for expansion culture, low-speed centrifugation of the cultured bacterial solution, collection of the supernatant, removal of bacteria by a membrane filter, ultrafiltration and concentration of the filtrate, and obtaining the probiotic exosomes from the resuspended precipitate after centrifugation of the concentrated solution.

6. Use according to claim 5, characterized in that, The expansion culture uses MRS medium, and the culture conditions are 37°C and 220 rpm for 24-36 h; the low-speed centrifugation conditions are 4°C, 8000-11000 g and 10-20 min. The membrane filter is a 0.22 μm membrane filter; the ultrafiltration and concentration conditions are an ultrafiltration membrane molecular weight cutoff of 100 kDa, 3000-6000 g and 4°C centrifugation for 10-20 min; and the concentrated solution centrifugation conditions are 4°C, 150000 g and 150-200 min.

7. The use according to claim 1, characterized in that, The product is a pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable carrier selected from one or more of poloxamer, polyacrylic acid, cellulose derivative, polyvinyl alcohol, gelatin and sodium alginate.

8. Use according to claim 7, characterized in that, The weight ratio of the probiotic exosomes to the pharmaceutical carrier is 1:1000-1:5000.

9. Use according to claim 7, characterized in that, The probiotic exosomes further encapsulate an active drug, and the weight ratio of the probiotic exosomes to the active drug is 5:1-1:

1.

10. Use according to claim 9, characterized in that, The active drug is selected from one or more of polyene antifungal drug, triazole antifungal drug, propylene amine antifungal drug, echinocandin antifungal drug and pyrimidine antifungal drug.

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