A bacterial vesicle-coated core-shell structure ZnO2-Fe3O4 nanocomposite material, preparation method and application thereof

Abstract

The present invention discloses a kind of bacterial vesicle-coated core-shell structure ZnO2-Fe3O4 nanocomposite material, preparation method and application thereof. The core-shell structure ZnO2-Fe3O4 nanocomposite material disclosed by the present invention is ZnO2-Fe3O4@MVs, which is composed of Streptococcus gordonii (S.gordonii) vesicle-coated ZnO2-Fe3O4 nanoparticles; the ZnO2-Fe3O4 nanoparticles are assembled by Fe3O4 nanoparticles with surface modification of 3,4-dihydroxyphenylpropionic acid and ZnO2 nanoparticles with surface modification of polyvinylpyrrolidone. It has homologous targeting to enhance the effect of selective uptake for Streptococcus gordonii (S.gordonii), and preferentially removes Streptococcus gordonii (S.gordonii), a basal bacteria of the symbiotic biofilm in the oral cavity, thereby causing the structural collapse of the symbiotic biofilm, thereby removing the main pathogenic bacteria of periodontitis (Porphyromonas gingivalis), providing a new strategy for treating periodontitis.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and relates to a bacterial vesicle-coated core-shell structure ZnO2-Fe3O4 nanocomposite material, a preparation method and an application thereof. Background Art

[0002] The unique structure of the oral cavity allows microorganisms to colonize and form symbiotic biofilms (dental plaque) within the dental crevices and gingival sulci. The formation of symbiotic biofilms involves the participation of multiple bacteria within the oral cavity and typically follows a specific sequence: initial colonizing bacteria accumulate in situ with the assistance of saliva and gingival crevicular fluid, and then interact with subsequent bacteria through adhesins or nutrient transfer, mediating secondary colonization.

[0003] Conventional treatment for periodontitis involves physical removal, surgical scaling, and systemic antibiotic therapy, but this is associated with low efficacy, antibiotic resistance, and incomplete eradication. Furthermore, periodontitis-causing bacteria are present at low abundance in early-stage periodontal biofilms, making them difficult to target.

[0004] Streptococcus gordonii is a typical initial colonizing bacterium. It first forms a biofilm on the tooth surface and then uses its own multiple adhesion proteins to provide nutrient supply and adhesion basis for the secondary colonization of Porphyromonas gingivalis (the main pathogen of periodontitis).

[0005] Nanomaterials using exosomes or membrane vesicles as nano-delivery vehicles currently hold great potential for application. These materials share similar structures and DNA, RNA, and protein components with their parent cells, enabling homologous targeting. Naturally secreted membrane vesicles from cells or bacteria offer advantages such as high yields, no modification requirements, and excellent biocompatibility. Furthermore, outer membrane vesicles protect their contents from external interference. Currently, the design of bacterial membrane vesicles as delivery vehicles focuses on engineering bacteria to enhance their functional properties. However, limited research has exploited the natural advantages of bacterial membrane vesicles for targeted drug delivery. Summary of the Invention

[0006] Objectives of the Invention: The first objective of the present invention is to provide a composite material based on the unique symbiotic relationship between Porphyromonas gingivalis (P. gingivalis) and Streptococcus gordonii (S. gordonii) in the oral cavity. This composite material comprises ZnO2-Fe3O4 nanoparticles loaded with homotypic S. gordonii vesicles, targeting S. gordonii to kill the symbiotic biofilm system, thereby eliminating P. gingivalis. The second objective of the present invention is to provide a method for preparing this nanocomposite material. The third objective of the present invention is to provide applications of this nanocomposite material.

[0007] Technical solution: The bacterial vesicle-coated core-shell structure ZnO2-Fe3O4 nanocomposite material described in the present invention is ZnO2-Fe3O4@MVs, which is composed of Streptococcus gordonii (S.gordonii) vesicles coated with ZnO2-Fe3O4; the ZnO2-Fe3O4 nanoparticles are assembled from Fe3O4 nanoparticles with surface modification of 3,4-dihydroxyphenylpropionic acid (DHCA) and ZnO2 nanoparticles with surface modification of polyvinylpyrrolidone (PVP).

[0008] In the symbiotic system of oral biofilm, the special interaction between Streptococcus gordonii and Porphyromonas gingivalis provides a basic possibility for the vesicle transport system. Targeted materials targeting Streptococcus gordonii can effectively kill the colonization basis of Porphyromonas gingivalis, causing it to lose its main nutrient supply and adhesion basis, and use high-abundance intake materials to kill low-abundance pathogens.

[0009] The preparation method of the bacterial vesicle-coated core-shell structure ZnO2-Fe3O4 nanocomposite material of the present invention comprises the following steps: assembling Fe3O4 nanoparticles with DHCA surface modification and ZnO2 nanoparticles with PVP surface modification onto ZnO2-Fe3O4 nanoparticles through electrostatic interaction; and coating Streptococcus gordonii (S.gordonii) vesicles onto the ZnO2-Fe3O4 nanoparticles by a squeezing and pushing method.

[0010] Furthermore, the method further includes a step of collecting vesicles of Streptococcus gordonii (S. gordonii).

[0011] Furthermore, the preparation steps of the surface-modified PVP ZnO2 nanoparticles include: dissolving zinc acetate dihydrate (Zn(CH3COO)2·2H2O) and PVP in water, adding hydrogen peroxide (H2O2), and obtaining the surface-modified PVP ZnO2 nanoparticles after the reaction is completed.

[0012] Furthermore, the reaction condition is 1000 rpm, and the reaction time is 12 hours.

[0013] Furthermore, the preparation steps of the Fe3O4 nanoparticles include: mixing triacetylacetonate iron (Fe(acac)3), 1,2-hexadecanediol, oleic acid (OA), oleylamine (OM) and benzyl ether, stirring and heating in a nitrogen atmosphere to obtain a black mixture, cooling the black mixture to room temperature, adding ethanol, and centrifuging. The resulting precipitate is Fe3O4 nanoparticles.

[0014] Furthermore, the stirring condition is 600 rpm, and the heating condition is first heating at 200° C. for 2 hours and then heating at 300° C. for 1 hour.

[0015] Furthermore, the preparation steps of the surface-modified DHCA Fe3O4 nanoparticles include: dissolving DHCA in tetrahydrofuran (THF), adding the THF solution containing the surface-modified DHCA Fe3O4 nanoparticles, stirring under a nitrogen atmosphere, heating and maintaining for 3 hours, continuing stirring until cooling to room temperature, and then adding NaOH. The resulting precipitate is the surface-modified DHCA Fe3O4 nanoparticles.

[0016] Furthermore, the stirring condition is 600 rpm, and the heating condition is 50°C.

[0017] Furthermore, the specific steps of coating Streptococcus gordonii vesicles on ZnO2-Fe3O4 nanoparticles by the squeezing method are: using a handheld micro-extruder with a polycarbonate porous membrane with a pore size of 400 nm to extrude the Streptococcus gordonii vesicles and ZnO2-Fe3O4 nanoparticles 20 times.

[0018] The invention discloses an application of the bacterial vesicle-coated core-shell structured ZnO2-Fe3O4 nanocomposite material in the preparation of medicines for treating diseases associated with Streptococcus gordonii (S. gordonii).

[0019] Furthermore, the application targets specific bacteria in the biofilm by coating ZnO2-Fe3O4 nanoparticles with Streptococcus gordonii (S. gordonii) vesicles, thereby increasing the uptake / killing of specific bacteria and causing the overall collapse of the symbiotic biofilm, thereby eliminating pathogenic bacteria.

[0020] Furthermore, the pathogenic bacteria is Porphyromonas gingivalis (P. gingivalis) or other secondary pathogenic bacteria that depend on Streptococcus gordonii (S. gordonii).

[0021] Furthermore, the disease is periodontitis.

[0022] This application is based on vesicle-coated ZnO2-Fe3O4 nanomaterials and uses its homologous targeting effect to construct a killing strategy targeting the basal bacteria Streptococcus gordonii in the symbiotic biofilm, aiming to destroy the living conditions and colonization basis of Porphyromonas gingivalis, the main pathogen of periodontitis, and thereby cause collapse-like damage to the symbiotic biofilm system.

[0023] Beneficial effects: Compared with the existing technology, the present invention has the following outstanding significant advantages: the core-shell structure ZnO2-Fe3O4 nanocomposite material disclosed in the present invention has a homologous targeting effect on Streptococcus gordonii to enhance selective uptake, and preferentially eliminates Streptococcus gordonii (S. gordonii), a bacterium in the basal layer of the oral symbiotic biofilm, thereby causing the structural collapse of the symbiotic biofilm, thereby eliminating the main pathogenic bacteria of periodontitis (Porphyromonas gingivalis), providing a new strategy for the treatment of periodontitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the synthesis process of the material;

[0025] Figure 2 Transmission electron microscopy morphology characterization of ZnO2-Fe3O4 NPs (a) and ZnO2-Fe3O4@MV NPs (b);

[0026] Figure 3 High-resolution transmission electron microscopy elemental analysis (a) and EDS energy spectrum analysis (b) of single ZnO2-Fe3O4@MV nanomaterials;

[0027] Figure 4 Verification of the Fenton effect of ZnO2-Fe3O4@MV NPs (where (a) is the result at different pH values, and (b) is the result at different ZnO2-Fe3O4@MV NPs concentrations);

[0028] Figure 5 The results of in vitro antibacterial experiments of ZnO2-Fe3O4@MV NPs ((a) is the antibacterial results of ZnO2-Fe3O4@MVNPs at different concentrations, and (b) is the antibacterial results of tetracycline, ZnO2-Fe3O4@MV NPs and ZnO2-Fe3O4NPs);

[0029] Figure 6 Representative confocal fluorescence images of colocalization of S. gordonii after co-incubation with S. gordonii MVs (a), bacterial uptake efficiency of S. gordonii MVs (b), and bacterial uptake efficiency of S. gordonii MVs when normalized by DiI fluorescence (c) are shown;

[0030] Figure 7Figure 3 shows the dispersion of the symbiotic biofilm of S. gordonii and P. gingivalis after 48 hours of growth and exposure to different concentrations of materials for 2 hours (a), the normalized statistical graph of the fluorescence values ​​of the biofilm formed by 5(6)-FAM-labeled Streptococcus gordonii after treatment (b), and the normalized statistical graph of the fluorescence values ​​of the biofilm formed by HI-labeled Porphyromonas gingivalis after treatment (c). DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0032] Example 1 Preparation of ZnO2-Fe3O4@MV NPs

[0033] Step 1: Collect Streptococcus gordonii (S. gordonii) vesicles (purchased from Ningbo Mingzhou Biotechnology Co., Ltd.): After the bacteria are cultured to the third generation, they are inoculated into 500 mL of tryptone soy broth (TSB) medium at a ratio of 1:100 and incubated for about 48 hours until the peak bacterial concentration is about 1×10 9 CFU / mL. The bacterial culture was then centrifuged at 1,800 × g for 10 minutes to remove the bacterial bodies. The resulting supernatant was concentrated three times using a 100 kDa molecular weight ultrafiltration tube. Finally, the membrane vesicle precipitate in the concentrated supernatant was collected by ultracentrifugation (150,000 × g, 2 hours) at 4°C. The resulting precipitate was resuspended in sterile 1× PBS and the protein concentration was determined by the BCA assay (approximately 0.3 mg / mL). The precipitate was aliquoted and stored at -80°C to obtain S. gordonii MVs for subsequent experiments.

[0034] Step 2: Preparation of ZnO2 nanoparticles: Dissolve 0.1 g of Zn(CH3COO)2·2H2O and 0.15 g of PVP (Mw=10,000) in 5.0 mL of water. Then, add 0.5 mL of H2O2 (30 wt.%) and stir vigorously. After 24 h of reaction, PVP-modified ZnO2 nanoparticles are obtained. These are then washed three times and redispersed in ultrapure water to obtain a ZnO2@PVP nanoparticle solution.

[0035] Preparation of Fe3O4 nanoparticles: Fe(acac)3 (2 mmol), 1,2-hexadecanediol (10 mmol), OA (6 mmol), OM (6 mmol), and benzyl ether (20 mL) were mixed and stirred under a nitrogen stream. The mixture was heated at 200°C for 2 h and then heated to 300°C under a nitrogen atmosphere for 1 h. After the resulting black mixture was cooled to room temperature, ethanol (40 mL) was added to the mixture, and the precipitate was isolated by centrifugation (15,000 × g, 10 min). The black product was dissolved in a mixture of n-hexane and ethanol (1:1, volume ratio) in the presence of OA (0.05 mL) and OM (0.05 mL). The solvent was removed by centrifugation (15,000 × g, 10 min), washed three times, and then dispersed in THF for storage to obtain a THF solution containing Fe3O4 NPs.

[0036] Step 3: Preparation of ZnO2-Fe3O4 NPs: DHCA (50 mg) was dissolved in 6 mL of THF and slowly added to the THF solution containing 20 mg of Fe3O4 NPs (20 mg / mL) prepared in Step 2, followed by stirring under a nitrogen atmosphere. The mixture was slowly heated to 50°C for 3 h and maintained with stirring until cooled to room temperature. 0.5 mL of NaOH (0.5 mmol) was added to precipitate a black product, which was washed three times and then dispersed in ultrapure water to obtain an aqueous Fe3O4-DHCA solution.

[0037] 5 mL of the ZnO2 nanoparticle solution (1 mg / mL) prepared in step 2 was added to 1 mL of Fe3O4-DHCA (10 mg / mL) aqueous solution under ultrasound for 5 min. The mixture was then transferred to a magnetic stirrer and stirred at 1000 rpm for 12 h. The mixture was washed three times with ultrapure water to remove excess Fe3O4-DHCA and stored in ultrapure water to obtain ZnO2-Fe3O4 NPs.

[0038] Step 4: Preparation of ZnO2-Fe3O4@MVs: The ZnO2-Fe3O4 NPs prepared in Step 3 were first centrifuged and resuspended in 1× PBS. S. gordonii MVs collected in Step 1 were then mixed with the ZnO2-Fe3O4 NPs prepared in Step 3 at a volume ratio of 1:3 and pretreated with ultrasound for 3 minutes. An extrusion apparatus was then assembled, and the mixture was extruded 20 times using a handheld microextruder with a polycarbonate porous membrane with a 400 nm pore size. The mixture was then centrifuged at 3,500 × g and 4°C for 10 minutes to precipitate the nanoparticles. The supernatant was removed to obtain a concentrated suspension of ZnO2-Fe3O4@MVs, which was then suspended in 1× PBS. The resulting concentrated suspension (1 mL) was placed in a vacuum oven and dried overnight at room temperature.

[0039] Example 2 Characterization and performance analysis of ZnO2-Fe3O4@MV NPs

[0040] (1) The morphologies of ZnO2-Fe3O4 NPs and ZnO2-Fe3O4@MV NPs were characterized by transmission electron microscopy ( Figure 2 The morphology of ZnO2-Fe3O4 NPs exhibits a typical core-shell structure with a regular and uniform spherical core of ZnO2 nanoparticles surrounded by a large number of individual Fe3O4 nanoparticles. No excess Fe3O4 aggregates are evident in the background, demonstrating the effectiveness of the electrostatic adsorption strategy. The size of the ZnO2-Fe3O4 NPs (a) is approximately 80 nm. ZnO2-Fe3O4@MV NPs prepared using a standard handheld micro-extruder exhibit complete encapsulation, forming small aggregates of single or multiple particles, with individual particles measuring approximately 110 nm under transmission electron microscopy (b).

[0041] (2) In order to determine whether ZnO2-Fe3O4 NPs can be successfully loaded into S.gordonii MVs without destroying the electrostatic binding ability of ZnO2 and Fe3O4, this experiment was combined with elemental analysis to determine the content of each element in ZnO2-Fe3O4@MV NPs, such as Figure 3 As shown in (a), in addition to the outer edge outline of the outer membrane vesicles in high-angle annular dark field (HAADF) mode, nitrogen (N) was selected in this example to characterize the main components of the membrane vesicles. The energy spectrum analysis (b) also confirms the coexistence and distribution of Fe, Zn, and N in the ZnO2-Fe3O4@MV nanocomposite, thus demonstrating the successful preparation of the ZnO2-Fe3O4@MV NPs.

[0042] (3) ZnO2-Fe3O4@MV NPs can undergo Fenton reaction under acidic conditions, producing a large amount of highly reactive oxygen species (ROS) and achieving a bactericidal effect. In order to verify this conclusion, the following two sets of experiments were designed. Acetic acid-sodium acetate buffer solutions with pH values ​​of 4.5, 5.0, 5.5 and 7.4 were prepared to verify the color change of 3,3',5,5'-tetramethylbenzidine (TMB) in ZnO2-Fe3O4@MV NPs at a concentration of 20 μg / mL under different pH conditions. The UV-visible spectrum at 652 nm was measured and compared with the actual image. The results are shown in the figure below. Figure 4(a). It can be seen that the lower the pH value, the more obvious the color development is at the same concentration and time, while there is almost no change under a neutral environment, which is consistent with the acid response characteristics of the nanomaterial and has a significant Fenton effect under pH 5.0. Then, the Fenton reaction of different concentrations of ZnO2-Fe3O4@MV NPs (5, 10, 20μg / mL) under pH 5.0 was verified, and the measurement method and principle are the same as above. From the UV-visible spectrum and the actual picture ( Figure 4 (b) shows that the higher the concentration of ZnO2-Fe3O4@MV NPs, the more obvious the Fenton effect is at the same time, and a significant Fenton effect is already evident at 20 μg / mL. In the above experiments, the control group was replaced by an equal amount of sterile 1× PBS.

[0043] (4) In order to verify the in vitro killing effect of ZnO2-Fe3O4@MV NPs on planktonic Streptococcus gordonii, the following experiments were designed:

[0044] 4.1 First, TSB medium with pH 5.0 was prepared to meet the previously reported acid response conditions and the environmental basis for the spontaneous decrease in pH of oral biofilms. The CFU plate count method was used to evaluate the interaction of ZnO2-Fe3O4@MV NPs (at concentrations of 0, 10, 20, 50, 100, 200, 500, and 1000 μg / mL) with Streptococcus gordonii (approximately 1×10 7 CFU / mL) after 2h of incubation, the results are as follows Figure 5 As shown in (a), when the concentration of ZnO2-Fe3O4@MV NPs reached 100 μg / mL, the killing of planktonic bacteria exceeded 90%, and the subsequent increase in concentration did not significantly improve the killing effect, so it was selected as the final inhibitory concentration.

[0045] 4.2 The killing effect of ZnO2-Fe3O4@MV NPs and ZnO2-Fe3O4 NPs at 100 μg / mL and tetracycline at 3 μg / mL on Streptococcus gordonii was also verified. The third generation of logarithmic phase Streptococcus gordonii was collected by centrifugation at 1,500 × g for 5 min and washed twice with sterile 1× PBS. The bacteria were diluted proportionally and resuspended in 10 mL of TSB at a concentration of 1×10 7CFU / mL. Then, 100 μL of TSB medium was added to each well. High concentrations of tetracycline (200 μg / mL), ZnO2-Fe3O4 NPs (1,000 μg / mL), and ZnO2-Fe3O4@MV NPs (1,000 μg / mL) were added to the first well of each row, and continuous gradient dilution was performed. Finally, 100 μL of evenly mixed Streptococcus gordonii liquid was added to each well. The cells were placed in an incubator at 37°C with 5% CO2 and incubated for 24 hours. The turbidity of each well was observed. The results are shown in Figure 2. Figure 5 As shown in (b), compared to tetracycline antibiotics commonly used in periodontitis treatment, the addition of ZnO2-Fe3O4 NPs significantly reduced the survival rate of S. gordonii. This is primarily due to the highly reactive ·OH produced by the Fenton reaction, which has a killing efficiency higher than that of tetracycline at the MIC concentration. Encapsulation in membrane vesicles further enhanced the antibacterial efficacy of the nanocomposite, which can be explained by more efficient bacterial uptake of the membrane vesicles and their contents, accelerating the release of ZnO2-Fe3O4 NPs and the Fenton effect. Furthermore, the nanomaterial encapsulated by membrane vesicles avoids premature release in the surrounding environment, improving its release efficiency and demonstrating more effective killing of S. gordonii compared to unencapsulated materials and antibiotics.

[0046] (5) To verify the targeting effect of membrane vesicles, DiI-labeled S. gordonii MVs or liposomes were co-incubated with Syto9-labeled Streptococcus gordonii for 2 hours to observe the interaction between Streptococcus gordonii and MVs. Figure 6 As shown in (a), the excitation wavelength of the DiI fluorescent probe is 561 nm, and the detection wavelength of the Syto9 fluorescent probe used to label bacteria is 488 nm. After incubation for 2 h, it can be observed that there are many co-localization areas (shown in yellow) between Syto9-labeled bacteria and DiI-labeled S.gordonii MVs. As a control, very little co-localization was detected on the surface of bacteria co-incubated with DiI-labeled liposomes (Lipo@DiI), while the bacterial group treated with only DiI dye did not show co-localization fluorescence signals. For further comparison, the fluorescence area of ​​membrane vesicle uptake in Zeiss confocal images was calculated by ImageJ software. The fluorescence images were compared by semi-quantitative analysis to calculate the percentage pixels of co-localized fluorescence and Syto9 staining or DiI staining. The results showed that the bacterial uptake efficiency of S.gordonii MVs was 5 times higher than that of liposomes ( Figure 6 (b) When normalized by DiI fluorescence, the utilization of S. gordonii MVs was 3-fold higher than that of liposomes ( Figure 6 (c)). The above results indicate that the uptake efficiency of Streptococcus gordonii into its own biomembrane is higher than that of traditional liposomes.

[0047] (6) In order to evaluate the antibacterial effect of ZnO2-Fe3O4@MV NPs on bacteria in composite symbiotic biofilms and the strategy of destroying the colonization conditions of pathogenic bacteria Porphyromonas gingivalis by killing the substrate biofilm of S. gordonii, the present invention successfully established an in vitro biofilm model and compared it with ZnO2-Fe3O4 NPs and tetracycline. According to the bacterial colonization rules in the oral cavity, this example prepared a composite biofilm with Streptococcus gordonii as the biofilm substrate and combined with Porphyromonas gingivalis in vitro to verify the killing strategy of ZnO2-Fe3O4@MV NPs. The composite biofilm was cultured on a circular cell slide with a diameter of 18 mm placed in a 12-well plate for subsequent experiments. In short, logarithmic Streptococcus gordonii (about 1×10 8 CFU / mL) was washed twice with sterile 1×PBS and incubated with ethidium iodide (HI, 15μM) at 37°C in the dark for 40 minutes, followed by washing 3 times with PBS (1,500×g, 5min) to remove unbound dye. The bacteria were resuspended in TSB medium specially prepared for Streptococcus Gordonii, 2mL of bacterial solution was added to the 12-well plate and allowed to stand at 37°C in the dark for 2 hours, allowing the bacteria to settle on the cell slide and begin initial adhesion. After 2 hours, the supernatant was aspirated and 2mL of new TSB medium with additional glucose (1g / mL) and yeast extract (1g / mL) was added to each well. The culture was incubated in the dark for 12 hours in a bacterial incubator, and then the supernatant was removed and replaced with TSB medium, and the culture was continued in the dark for 10 hours. The same concentration of Porphyromonas gingivalis (about 1×10 8 CFU / mL) and washed once, then labeled with 5(6)-carboxyfluorescein succinimidyl ester fluorescent dye (5(6)-FAM, 5μM) for 40 minutes. After incubation, unbound fluorescent dye was removed by centrifugation (1,500×g, 5min) and resuspended in fresh TSB medium. All operations on Porphyromonas gingivalis were carried out in a nitrogen atmosphere in the dark, and sealed with sealing film during centrifugation to prevent oxygen from entering. All operations should be carried out as quickly as possible. In the nitrogen operation box, when the S. gordonii biofilm grew for 24 hours, the supernatant was removed and replaced with 2mL of the stained P. gingivalis bacterial solution, and continued to grow for 24 hours to form a composite biofilm. After 24 hours of co-culture, the supernatant was removed and replaced with pH 5.0 Porphyromonas gingivalis TSB medium containing different concentrations of materials. The concentrations of tetracycline, ZnO2-Fe3O4 NPs, and ZnO2-Fe3O4@MV NPs were 3, 100, and 100 μg / mL, respectively, to evaluate the killing effect of the materials on the composite biofilm. After co-incubation for 2 hours in an anaerobic culture bag in the dark, the medium was discarded and images were taken at 40x using a Zeiss LSM 800 confocal microscope to evaluate biofilm damage. Figure 7As shown in (a), the 3D CLSM image shows that the symbiotic biofilm in the blank control group exhibited a dense structure after 48 hours of culture, with large areas of symbiotic growth (shown in yellow). Tetracycline, an antibiotic commonly used to treat periodontal infections, partially killed Streptococcus gordonii and reduced the colonization survival rate of Porphyromonas gingivalis. ZnO2-Fe3O4 NPs exhibited slightly greater killing efficacy than tetracycline, likely due to the indiscriminate killing of bacteria by the Fenton reaction. Under this effect, the fluorescence area of ​​P. gingivalis and S. gordonii was significantly reduced. Compared with the above results, ZnO2-Fe3O4@MV NPs coated with S. gordonii membrane vesicles exhibited significant killing and dispersion effects on the composite biofilm, with the most significant disruption of the S. gordonii substrate biofilm. The mechanism of bacterial uptake of membrane vesicles enhanced the bactericidal effect of the nanocomposite, significantly reducing the survival rate of P. gingivalis. In vitro experiments demonstrated that the strategy of killing the composite biofilm substrate to limit the colonization and survival of P. gingivalis is effective. The normalized fluorescence statistics of 3D CLSM images were analyzed using ImageJ software ( Figure 7 (b) is a biofilm substrate formed by Streptococcus gordonii labeled with 5(6)-FAM fluorescent dye. Figure 7 (c) Biofilm formed by Porphyromonas gingivalis labeled with HI fluorescent dye). ZnO2-Fe3O4@MV NPs have a significant effect on reducing the thickness of the biofilm and inhibiting the colonization of the pathogen Porphyromonas gingivalis. Both the Streptococcus gordonii and Porphyromonas gingivalis layers in the composite biofilm showed high fluorescence values ​​and biofilm thickness. After treatment with ZnO2-Fe3O4 NPs and tetracycline, the overall thickness and fluorescence intensity of the Streptococcus gordonii biofilm decreased, and Porphyromonas gingivalis showed a certain killing effect; treatment with ZnO2-Fe3O4@MV NPs significantly enhanced the destruction of the Streptococcus gordonii biofilm and effectively reduced the number of Porphyromonas gingivalis colonization.

Claims

1. A bacterial vesicle-coated core-shell structure ZnO2-Fe3O4 nanocomposite material, characterized in that: The nanocomposite material is ZnO2-Fe3O4@MVs, which is composed of Streptococcus gordonii ( S. gordonii ) vesicles are coated with ZnO2-Fe3O4 nanoparticles; the ZnO2-Fe3O4 nanoparticles are assembled from Fe3O4 nanoparticles with 3,4-dihydroxyphenylpropionic acid modified on the surface and ZnO2 nanoparticles with polyvinylpyrrolidone modified on the surface.

2. A method for preparing the bacterial vesicle-coated core-shell structure ZnO2-Fe3O4 nanocomposite material according to claim 1, characterized in that: The following steps are involved: The ZnO2-Fe3O4 nanoparticles were assembled by electrostatic interaction between Fe3O4 nanoparticles modified with 3,4-dihydroxyphenylpropionic acid and ZnO2 nanoparticles modified with polyvinylpyrrolidone. Streptococcus gordonii ( S. gordonii ) vesicles coated on ZnO2-Fe3O4 nanoparticles.

3. The preparation method according to claim 2, characterized in that The preparation steps of the ZnO2 nanoparticles with surface modified polyvinyl pyrrolidone include: dissolving zinc acetate dihydrate and polyvinyl pyrrolidone in water, adding hydrogen peroxide, and obtaining the ZnO2 nanoparticles with surface modified polyvinyl pyrrolidone after the reaction is completed.

4. The preparation method according to claim 3, characterized in that The reaction condition is 1000 rpm, and the reaction time is 12 to 24 hours.

5. The preparation method according to claim 2, characterized in that The preparation steps of the Fe3O4 nanoparticles include: mixing iron triacetylacetonate, 1,2-hexadecanediol, oleic acid, oleylamine and benzyl ether, stirring and heating in a nitrogen atmosphere to obtain a black mixture, cooling the black mixture to room temperature, adding ethanol, and centrifuging to obtain a precipitate that is the Fe3O4 nanoparticles.

6. The preparation method according to claim 2, characterized in that The preparation steps of the Fe3O4 nanoparticles surface-modified with 3,4-dihydroxyphenylpropionic acid include: dissolving 3,4-dihydroxyphenylpropionic acid in tetrahydrofuran, adding the solution to the tetrahydrofuran solution containing the Fe3O4 nanoparticles, stirring under a nitrogen atmosphere, heating and maintaining for 3 hours, continuing stirring until cooling to room temperature, and then adding sodium hydroxide. The resulting precipitate is the Fe3O4 nanoparticles surface-modified with 3,4-dihydroxyphenylpropionic acid.

7. A ZnO2-Fe3O4 nanocomposite material with a core-shell structure coated with the bacterial vesicles according to claim 1 for use in the preparation of a treatment for Streptococcus gordonii ( S. gordonii ) in a drug for a related disease, characterized in that, The disease is periodontitis.

8. The use according to claim 7, characterized in that The application is carried out by Streptococcus gordonii ( S. gordonii ) The vesicles are coated with ZnO2-Fe3O4 nanoparticles, which target specific bacteria in the biofilm, increase the uptake / killing of specific bacteria, and cause the overall collapse of the symbiotic biofilm, thereby eliminating pathogenic bacteria.

9. The use according to claim 8, characterized in that The pathogenic bacteria is Porphyromonas gingivalis ( P. gingivalis ).

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