Preparation method and application of multifunctional nanospheres targeting bacteria

Abstract

The present invention is applicable to the field of biomedical technology, and provides a preparation method and application of multifunctional nanospheres targeting bacteria. The prepared targeted multifunctional nanospheres have both PDT / PTT functions and target harmful bacteria for antibacterial treatment of periodontitis. By using bismuth silver sulfide hollow nanospheres, photosensitizers and bacterial outer membrane vesicles as raw materials, the raw materials are cheap and the preparation process is simple. The prepared multifunctional nanospheres have unique advantages. They will not cause damage to probiotics, but can accurately target harmful bacteria and exert antibacterial effects, thereby effectively solving the problem of dysbacteriosis. In addition, the direct contact between the multifunctional nanospheres and bacteria will interfere with the electron transfer behavior of the bacteria, which is conducive to strengthening the bactericidal effect on bacteria. The adopted targeting strategy is extremely suitable for the treatment of dysbacteriosis diseases, showing a broad application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine technology, and in particular relates to a preparation method and application of multifunctional nanospheres targeting bacteria. Background Art

[0002] Periodontitis is a multifactorial inflammatory disease characterized by bone resorption and connective tissue damage. Globally, more than 1.1 billion people suffer from severe periodontitis, which leads to alterations in oral physiological function and psychological health. This condition can significantly affect the patient's quality of life and psychological health for a long time. The main cause of periodontitis is oral dysbiosis. Dysbiosis occurs when bacteria accumulate into polymicrobial biofilms, form different structures and reach a quasi-biotic state. Typically, pathogens use virulence factors, such as adhesion molecules, to colonize and form biofilms, subsequently harming the host. In contrast, these damaged bacteria upregulate the activity of genes for lipid transport and DNA repair to mitigate DNA and outer membrane damage.

[0003] At present, there are many difficult-to-solve problems in traditional antibacterial treatments for periodontitis, such as mechanical debridement assisted by antibiotic treatment. Mechanical debridement mainly removes large pieces of dental calculus and plaque by physical means. However, due to the thickness of the instrument, it is difficult to penetrate into the irregularly shaped periodontal pockets, especially to remove some bacteria hiding deep inside. Therefore, biofilms that are difficult to remove are prone to proliferate and accumulate again, leading to recurrence of local inflammation; improper operation of mechanical treatment may also cause damage to the patient's periodontal soft tissue and lead to gingival atrophy. In addition, mechanical treatment has no specificity for the removal of bacterial biofilms. The use of antibacterial drugs also has certain disadvantages. First, some antibiotics usually have broad-spectrum antibacterial properties and kill bacteria indiscriminately. Secondly, the structure of plaque biofilms is dense, and it is difficult for conventional antibiotics to break through biofilms. In addition, long-term use may lead to antibiotic resistance. Some periodontitis pathogens such as Porphyromonas gingivalis and Actinomycetemcomitans become resistant to antibiotics through the acquisition and expression of resistance genes, resulting in treatment failure.

[0004] Emerging antibacterial therapies for periodontitis also have certain limitations. Due to the particularity of periodontitis, antibacterial treatment using PDT or PTT still faces challenges. For example, it is difficult for photosensitizers or photothermal agents to penetrate dense bacterial biofilms, and the limited lifespan and radius of ROS hinder their effective antibacterial activity. The essence of periodontitis is dysbiosis. However, the broad-spectrum bactericidal effect of PDT or PTT is difficult to specifically target and eliminate periodontal pathogens without damaging probiotics. Gas antibacterial therapy also has the problem of broad-spectrum bactericidal effect. At present, antibacterial research on the treatment of periodontitis mainly focuses on bacterial killing and biofilm eradication, but ignores the necessity of selective antibacterial function. Although some antibacterial agents have achieved selective antibacterial effect, such as quaternary ammonium salt antibacterial agents, they mainly exert selective effects through surface charge. This antibacterial agent has broad-spectrum bactericidal activity against Gram-positive bacteria, but has poor bactericidal effect against Gram-negative bacteria, and cannot play a good role in selectively combating harmful bacteria. Summary of the invention

[0005] The purpose of the embodiments of the present invention is to provide a preparation method and application of multifunctional nanospheres targeting bacteria, aiming to solve the problems raised in the above background technology.

[0006] The embodiment of the present invention is implemented as follows: a method for preparing a multifunctional nanosphere targeting bacteria comprises the following specific steps:

[0007] Step 1: Mix and stir a certain amount of zinc acetate and polyvinyl pyrrolidone aqueous solution to obtain a clear solution; quickly add thioacetamide solution, increase the system temperature, and stir at a constant temperature; cool and centrifuge to obtain a product, wash several times, and dry to obtain a template material zinc sulfide;

[0008] Step 2: adding a certain amount of zinc sulfide and thiourea to ethylene glycol, stirring vigorously to obtain a milky white solution; raising the system temperature, adding a mixed solution of silver nitrate and bismuth nitrate prepared with ethylene glycol to the above solution, and stirring at a constant temperature; cooling and centrifuging to obtain a product, washing several times, and drying to obtain bismuth sulfide silver hollow nanospheres;

[0009] Step 3: dropping the indocyanine green aqueous solution into the bismuth silver sulfide hollow nanosphere dispersion, stirring at room temperature, then standing, centrifuging to obtain the product, washing several times, and drying to obtain the composite nanosphere;

[0010] Step 4: Cultivate Fusobacterium nucleatum to the logarithmic phase, and use ultracentrifugation to separate and extract bacterial outer membrane vesicles;

[0011] Step 5: The dispersion of the product in step 3 and the dispersion of bacterial outer membrane vesicles are mixed and vortexed, and then the bacterial outer membrane vesicles are encapsulated by ultrasound, and the targeted multifunctional nanospheres are collected by low-temperature centrifugation.

[0012] A further technical solution is that in step 1, the molar ratio of zinc acetate, polyvinyl pyrrolidone and thioacetamide is 100:(0.5-2):500; the reaction temperature is 100 o C to 120 o C; the reaction time is 2 hours.

[0013] A further technical solution is that in step 2, the molar ratio of zinc sulfide to thiourea is 1.14:1; the molar ratio of silver nitrate to bismuth nitrate is 1:1; the reaction temperature is 110 o C to 150 o C; the reaction time is 2 hours.

[0014] A further technical solution is that in step 3, the molar ratio of indocyanine green to bismuth silver sulfide hollow nanospheres is 1:20.

[0015] According to a further technical solution, in step 4, the mass ratio of the composite nanospheres to the bacterial outer membrane vesicles is 2:1.

[0016] According to a further technical solution, in steps 1 to 3, the rotation speed of the centrifugal separation is 6000-9000 rpm / min.

[0017] According to a further technical solution, in step 4, the rotation speed of the centrifugal separation is 6000 rpm / min.

[0018] Another object of an embodiment of the present invention is to provide a nanomedicine, which partially or completely contains the above-mentioned targeted multifunctional nanospheres.

[0019] A further technical solution is to use the nanomedicine in the preparation of drugs for treating targeted harmful bacteria.

[0020] A further technical solution is to use the nanomedicine in the preparation of drugs for treating periodontitis.

[0021] The preparation method and application of a multifunctional nanosphere targeting bacteria provided in an embodiment of the present invention, the prepared targeted multifunctional nanosphere has both PDT / PTT function and targets harmful bacteria for antibacterial treatment of periodontitis, and uses bismuth sulfide silver nano hollow spheres, photosensitizers and bacterial outer membrane vesicles as raw materials, and the raw materials are cheap and the preparation process is simple. Compared with the existing synthesis method of bismuth sulfide silver nano hollow spheres, the synthesis process of zinc sulfide template is further simplified; the particle size of bismuth sulfide silver nanospheres is smaller, which is conducive to its performance improvement and biological application. The prepared multifunctional nanospheres have unique advantages. They will not cause damage to probiotics, but can accurately target harmful bacteria and exert antibacterial effects, thereby effectively solving the problem of dysbacteriosis. In addition, the direct contact between the multifunctional nanospheres and bacteria will interfere with the electron transfer behavior of the bacteria, which is conducive to strengthening the bactericidal effect on bacteria. The adopted targeting strategy is extremely suitable for the treatment of dysbacteriosis diseases, showing a broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 TEM and HRTEM images of AgBiS2 samples, and TEM images of OMV-AgBiS2 / ICG samples;

[0023] Figure 2 XRD pattern of AgBiS2 sample;

[0024] Figure 3 This is the mapping analysis spectrum of the AgBiS2 / ICG sample elements;

[0025] Figure 4 Protein SDS-PAGE analysis of OMV samples, AgBiS2 / ICG samples and OMV-AgBiS2 / ICG samples;

[0026] Figure 5 This is the photothermal effect diagram of the AgBiS2 / ICG sample;

[0027] Figure 6 The graph is the relationship between the UV-visible absorption spectra of AgBiS2 sample and AgBiS2 / ICG sample and the time when they react with the probe;

[0028] Figure 7 This is the cell viability result diagram of OMV-AgBiS2 / ICG sample for different cells;

[0029] Figure 8 This is the blood compatibility result diagram of OMV-AgBiS2 / ICG sample;

[0030] Fig. 9 It is the statistical result diagram of CFU value of product samples in each stage against single bacterial species biofilm;

[0031] Fig.10 TEM images of the effects of product samples at each stage on multiple bacteria;

[0032] Fig.11 The FISH staining diagram of the product samples at each stage for selective antibacterial activity against bacteria;

[0033] Fig.12 Linear sweep voltammetry curves and electrochemical impedance spectroscopy of AgBiS2 / ICG sample and OMV-AgBiS2 / ICG sample;

[0034] Fig.13 GO enrichment analysis of upregulated genes in the transcriptome of Porphyromonas gingivalis by OMV-AgBiS2 / ICG samples;

[0035] Fig.14 The microCT acquisition images and bone volume fraction statistics of the effects of product samples at each stage on the alveolar bone;

[0036] Fig.15 The Masson staining results of the product samples at each stage are shown. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0038] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0039] An embodiment of the present invention provides a method for preparing a multifunctional nanosphere targeting bacteria, wherein the multifunctional nanosphere comprises the following main raw materials: bismuth silver sulfide hollow nanospheres, a photosensitizer, and bacterial outer membrane vesicles;

[0040] The preparation method comprises the following specific steps:

[0041] Step 1: Mix and stir a certain amount of zinc acetate and polyvinyl pyrrolidone aqueous solution to obtain a clear solution; quickly add thioacetamide solution, increase the system temperature, and stir at a constant temperature; cool and centrifuge to obtain a product, wash several times, and dry to obtain a template material zinc sulfide;

[0042] Step 2: adding a certain amount of zinc sulfide and thiourea to ethylene glycol, stirring vigorously to obtain a milky white solution; raising the system temperature, adding a mixed solution of silver nitrate and bismuth nitrate prepared with ethylene glycol to the above solution, and stirring at a constant temperature; cooling and centrifuging to obtain a product, washing several times, and drying to obtain bismuth sulfide silver hollow nanospheres;

[0043] Step 3: dropping the indocyanine green aqueous solution into the bismuth silver sulfide hollow nanosphere dispersion, stirring at room temperature, then standing, centrifuging to obtain the product, washing several times, and drying to obtain the composite nanosphere;

[0044] Step 4: Cultivate Fusobacterium nucleatum to the logarithmic phase, and use ultracentrifugation to separate and extract bacterial outer membrane vesicles;

[0045] Step 5: The dispersion of the product in step 3 and the dispersion of bacterial outer membrane vesicles are mixed and vortexed, and then the bacterial outer membrane vesicles are encapsulated by ultrasound, and the targeted multifunctional nanospheres are collected by low-temperature centrifugation.

[0046] As a preferred embodiment of the present invention, in step 1, the molar ratio of zinc acetate, polyvinyl pyrrolidone and thioacetamide is 100:(0.5-2):500; the reaction temperature is 100 o C to 120 o C; the reaction time is 2 hours.

[0047] As a preferred embodiment of the present invention, in step 2, the molar ratio of zinc sulfide to thiourea is 1.14:1; the molar ratio of silver nitrate to bismuth nitrate is 1:1; the reaction temperature is 110 o C to 150 o C; the reaction time is 2 hours.

[0048] As a preferred embodiment of the present invention, in step 3, the molar ratio of indocyanine green to bismuth silver sulfide hollow nanospheres is 1:20.

[0049] As a preferred embodiment of the present invention, in step 4, the mass ratio of the composite nanospheres to the bacterial outer membrane vesicles is 20:1.

[0050] As a preferred embodiment of the present invention, in steps 1 to 3, the rotation speed of the centrifugal separation is 6000-9000 rpm / min.

[0051] As a preferred embodiment of the present invention, in step 4, the rotation speed of the centrifugal separation is 6000 rpm / min.

[0052] As a preferred embodiment of the present invention, the wavelength of the near infrared light used is 808 nm.

[0053] Another embodiment of the present invention provides a nanomedicine, which partially or completely contains the above-mentioned targeted multifunctional nanospheres, and the above-mentioned nanomedicine is used to prepare a drug for treating targeted harmful bacteria, or the above-mentioned nanomedicine is used to prepare a drug for treating periodontitis.

[0054] Several specific examples are provided below to verify the effectiveness of this method.

[0055] Example 1: Step a, preparation of zinc sulfide template: 1 mmol zinc acetate and 0.01 mmol polyvinyl pyrrolidone were dissolved in 100 ml deionized water and stirred for 15 minutes until it became a clear solution. At the same time, 5 mmol thioacetamide was added to 50 ml deionized water and stirred for 15 minutes. Then, the thioacetamide aqueous solution was quickly added to the flask and the mixed solution was stirred at 100 o The mixture was stirred in an oil bath at 400 °C for 2 hours. The mixed solution was cooled to room temperature and centrifuged at 9000 rpm / min for 15 minutes to separate the product, which was then washed several times with deionized water and anhydrous ethanol. o C in a vacuum oven for more than 3 hours to obtain zinc sulfide solid.

[0056] Step b, preparation of hollow bismuth silver sulfide spheres: 60 mg of zinc sulfide solid and 41.1 mg of thiourea were weighed on an electronic balance and added to 30 mL of ethylene glycol, and stirred vigorously to produce a milky white dispersion. The mixed solution was stirred in an oil bath at 130°C for 10 minutes. At the same time, 10 mL of ethylene glycol solution containing 0.25 mmol of silver nitrate and 0.25 mmol of bismuth nitrate was prepared, and added dropwise to the above milky white dispersion, and stirred at a constant temperature for 2 hours. The mixed solution was cooled to room temperature, centrifuged at 9000 rpm / min for 15 minutes to separate the product, and washed several times with deionized water and anhydrous ethanol. The product was centrifuged at 60 o C in a vacuum oven for more than 3 hours to obtain bismuth silver sulfide solid (AgBiS2).

[0057] Step c, preparation of composite nanospheres loaded with photosensitizer: an aqueous solution of indocyanine green (0.1 mg mL −1 ,4 mL) was added dropwise into the aqueous suspension of bismuth sulfide silver hollow nanospheres (1 mg mL −1 , 10 mL) and stirred for 24 hours. Then let it stand for 48 hours. Centrifuge with deionized water for 15 minutes and collect the final product three times. Centrifuge at 9000 rpm / min for 15 minutes to separate the product, and wash it with deionized water several times. o C in a vacuum oven for more than 3 hours to obtain bismuth silver sulfide / indocyanine green composite nanosphere solid (AgBiS2 / ICG).

[0058] Step d, extraction of bacterial outer membrane vesicles: Cultivate Fusobacterium nucleatum to the logarithmic phase, centrifuge at 6000 rpm / min for 15 minutes, collect the supernatant, and repeat several times. Filter using a 0.45 μm vacuum filter. Subsequently, centrifuge at 200000 g for 2 hours, collect the precipitate, which is the bacterial outer membrane vesicles. Dissolve the precipitate in PBS and store at -80o C is for backup.

[0059] Step e: Preparation of targeted multifunctional nanospheres: The bacterial outer membrane vesicle PBS suspension (0.025 mg mL -1 ) and bismuth silver sulfide / indocyanine green composite nanospheres PBS suspension (0.5 mg mL -1 ) were mixed with equal volumes and vortexed for 5 minutes. Subsequently, the mixture was placed in a 100 W ultrasound for 5 minutes. The product (OMV-AgBiS2 / ICG) was separated by low-temperature centrifugation at 6000 g for 15 minutes and washed several times with PBS. The product was freeze-dried and stored at low temperature.

[0060] Example 2: Compared with Example 1, except that the amount of polyvinyl pyrrolidone added in step a is replaced with 0.005 mmol, the rest is the same as Example 1.

[0061] Example 3: Compared with Example 1, except that the amount of polyvinyl pyrrolidone added in step a is replaced with 0.02 mmol, the rest is the same as Example 1.

[0062] Example 4: Compared with Example 1, except that the amount of silver nitrate and bismuth nitrate added in step b is replaced by 0.01 mmol, the rest is the same as Example 1.

[0063] Example 5: Compared with Example 1, except that the amount of silver nitrate and bismuth nitrate added in step b is replaced by 0.02 mmol, the rest is the same as Example 1.

[0064] Example 6: Compared with Example 1, except that the oil bath temperature in step b is changed to 110 o Except for C, the rest is the same as Example 1.

[0065] Example 7: Compared with Example 1, except that the oil bath temperature in step b is changed to 150 o Except for C, the rest is the same as Example 1.

[0066] Example 8: Compared with Example 1, except that the constant temperature stirring in step b is changed to 6 hours, the rest is the same as Example 1.

[0067] Example 9: Compared with Example 1, except that the constant temperature stirring in step b is changed to 12 hours, the rest is the same as Example 1.

[0068] Example 10: Compared with Example 1, except that the constant temperature stirring in step b is changed to 24 hours, the rest is the same as Example 1.

[0069] Example 11: Compared with Example 1, except that the amount of indocyanine green added in step c is replaced with 0.05 mg, the rest is the same as Example 1.

[0070] Example 12: Compared with Example 1, except that the amount of indocyanine green added in step c is replaced with 0.2 mg, the rest is the same as Example 1.

[0071] Example 13: Compared with Example 1, except that the "60 o C vacuum oven for more than 3 hours" is replaced by "freeze-dried for more than 24 hours", and the rest is the same as Example 1.

[0072] Example 14: Compared with Example 1, except that the Fusobacterium nucleatum described in step d is replaced by Streptococcus sanguis and Streptococcus gordonii, the rest is the same as Example 1.

[0073] Example 15: Compared with Example 1, except that the concentration of the bacterial outer membrane vesicle PBS suspension in step e was replaced with 0.01 mg mL -1 Others are the same as in Example 1.

[0074] Example 16: Compared with Example 1, except that the concentration of the bacterial outer membrane vesicle PBS suspension in step e was replaced with 0.05 mg mL -1 Others are the same as in Example 1.

[0075] Example 17: The AgBiS2 sample prepared in Example 1 was characterized by transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM), respectively. It can be seen that AgBiS2 has a hollow spherical structure with an average particle size of 141.1 nm (see Figure 1 a and 1b). The TEM image of OMV-AgBiS2 / ICG shows a thin layer of OMV structure on the surface of the nanospheres, with an average particle size of ~171.4 nm (see Figure 1 c).

[0076] Example 18: The AgBiS2 sample prepared in Example 1 was subjected to X-ray diffraction (XRD) analysis. The results are as follows: Figure 2 As shown, the results show that the peak positions are consistent with the standard card (JCPDS No.11-1178), confirming the AgBiS2 crystal structure.

[0077] Example 19: The AgBiS2 / ICG sample prepared in Example 1 was subjected to elemental analysis. The results are as follows: Figure 3 As shown, the results showed that AgBiS2 / ICG nanospheres contained Ag, Bi, S and Na elements, confirming that ICG was successfully loaded into AgBiS2.

[0078] Example 20: The OMV, AgBiS2 / ICG and OMV-AgBiS2 / ICG samples prepared in Example 1 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Coomassie brilliant blue staining. Figure 4 As shown, the same protein bands were observed for pure OMVs and OMV-AgBiS2 / ICG samples, demonstrating that OMVs were successfully coated on the AgBiS2 / ICG surface.

[0079] Example 21: The AgBiS2 / ICG sample prepared in Example 1 was subjected to a photothermal performance test. Figure 5 As shown, the power densities used were 0.5 W, 0.75 W, and 1 W cm -2 After irradiation with near-infrared laser for only 3 minutes, the concentration was 50 μg mL -1 The temperatures of the AgBiS2 / ICG nanospheres can reach 38.6°C, 43.3°C and 48.3°C, respectively. This indicates that the AgBiS2 / ICG nanospheres synthesized in this embodiment have good photothermal effects.

[0080] Example 22: The AgBiS2 and AgBiS2 / ICG samples prepared in Example 1 were subjected to photodynamic performance tests. The generated ROS were tested using 1,3-diphenylisobenzofuran (DPBF). Figure 6 As shown, the power density is 1 Wcm -2 After irradiation with near-infrared laser for 1 minute, the DPBF absorption peak intensity of AgBiS2 sample decreased by nearly 60%. After loading ICG, the DPBF absorption peak intensity of AgBiS2 / ICG sample decreased by nearly 98%. This indicates that the AgBiS2 / ICG nanospheres synthesized in this example have good photodynamic properties.

[0081] Example 23: The OMV-AgBiS2 / ICG sample prepared in Example 1 was suspended in complete culture medium by ultrasonic dispersion to form a concentration of 300 μg mL -1 of colloidal suspension.

[0082] Example 24: The colloidal suspension obtained in Example 23 was prepared by gradient dilution to a concentration of 150 μg mL -1 Up to 25 μg mL -1 The samples were divided into concentration gradients, and no drug was added to the control group. After L929 fibroblasts and human gingival fibroblasts were co-cultured with drugs for 72 hours, the cell activity was detected using the CCK-8 assay. Figure 7 As shown, it was found that both cells-1 No significant cytotoxicity was shown at these concentrations.

[0083] Example 25: The OMV-AgBiS2 / ICG sample with a concentration gradient prepared in Example 24 was measured by the red blood cell lysis assay. Specifically, 150 μg mL -1 Up to 25 μg mL -1 The samples with different concentrations were compatible with rabbit red blood cells. Triton-X 100 and phosphate buffer were used as positive and negative controls, respectively. Figure 8 As shown, the hemolysis rate corresponding to all tested drug concentrations did not exceed 2%, demonstrating good blood compatibility.

[0084] Example 26: The AgBiS2, AgBiS2 / ICG and OMV-AgBiS2 / ICG samples prepared in Example 1 were dispersed in a bacterial tryptone soy broth medium by ultrasonication to form a concentration of 100 μg mL -1 of colloidal suspension.

[0085] Example 27: Establishing a single-strain biofilm, respectively, 8 CFU mL -1 Streptococcus gordonii, Porphyromonas gingivalis, Fusobacterium nucleatum and nanomedicine were cultured in a 24-well plate for 72 hours and then treated. In this example, there were five experimental groups, namely, a control group, an indocyanine green group, an AgBiS2 sample group, an AgBiS2 / ICG sample group and an OMV-AgBiS2 / ICG sample group. The final concentration of the sample suspension in this example was 50 μg mL -1 , which is obtained by diluting the suspension of Example 26. The illumination conditions of the experiment are a daily power density of 1 W cm -2 The samples were irradiated with 808 nm near-infrared laser for 3 minutes each time, for a total of 3 times.

[0086] In this example, the antibacterial properties of the multifunctional nanospheres were evaluated by the colony forming unit (CFU) counting method. The specific operation was as follows: the bacterial biofilms after treatment in each sample group were resuspended in 1 mL of culture medium, and then the resuspended bacterial solution was diluted by gradient dilution method and titrated on blood agar plates. Fig. 9As shown in the figure, the CFU of the single bacterial biofilm in the AgBiS2 / ICG group decreased by about 3 log, and after OMV encapsulation, the CFU count of the OMV-AgBiS2 / ICG group decreased by an additional 1 log. The experimental results reflect the good killing effect of OMV-AgBiS2 / ICG nanospheres on biofilm.

[0087] Example 28: Establishment of a multi-species bacterial biofilm by using a bacterial biofilm in the logarithmic phase with a concentration of 1.0×10 8 CFU mL -1 S. gordonii was cultured in a 24-well plate for 24 hours. Then, the bacteria were cultured in the logarithmic phase at a concentration of 1.0×10 8 CFU mL -1 Porphyromonas gingivalis, 1.0×10 8 CFU mL -1 Fusobacterium nucleatum and the nano drug were added to the culture system and cultured for 72 hours, followed by treatment. The experimental grouping and lighting conditions of this example were the same as those of Example 27.

[0088] In this example, bacterial TEM evaluation was used to evaluate bacterial targeting. The specific operation was as follows: the bacterial biofilms of each sample group were collected by centrifugation and resuspended in an electron microscope fixative for 2 hours. Subsequently, a series of treatments were performed to prepare bacterial samples for TEM image acquisition. Fig.10 As shown, damaged bacteria can be observed in all treatment groups, especially in the AgBiS2, AgBiS2 / ICG, and OMV-AgBiS2 / ICG sample groups. In addition, it can be seen that in the AgBiS2 and AgBiS2 / ICG groups, the nanospheres exist in a free state. However, in the OMV-AgBiS2 / ICG group, the OMV-AgBiS2 / ICG nanospheres are anchored on the bacterial surface, which fully proves its ability to target bacteria.

[0089] Example 29: To further evaluate the effect of targeting harmful bacteria, fluorescence in situ hybridization (FISH staining) was used. The specific operation was as follows: 8 CFU mL -1 S. gordonii was cultured in a 24-well plate for 24 hours. Then, the bacteria were cultured in the logarithmic phase at a concentration of 1.0×10 8 CFU mL -1 Porphyromonas gingivalis, 1.0×10 6 CFUmL -1Lactobacillus reuteri and nanomedicine were added to the culture system and cultured for 72 hours, followed by treatment. The experimental grouping and lighting conditions of this embodiment were the same as those of Example 27. In this embodiment, Lactobacillus reuteri is a probiotic that can be taken from food, and an increase in its proportion is beneficial to inhibiting pathogens.

[0090] In the present embodiment, the bacterial biofilm samples after the above treatment are collected. The sample is placed in 4% cold paraformaldehyde for fixation, digested with lysozyme to achieve permeability, followed by pre-hybridization, hybridization and other steps, and finally the image of each sample is collected by laser confocal scanning fluorescence microscopy. As shown in Figure 11, in the control group, Streptococcus gordonii and Porphyromonas gingivalis significantly inhibited the growth of Lactobacillus reuteri. The ratio of Lactobacillus reuteri in the indocyanine green group, AgBiS2 / ICG group and OMV-AgBiS2 / ICG sample group increased. Wherein, the ratio of Lactobacillus reuteri is the highest in the OMV-AgBiS2 / ICG sample group.

[0091] Example 30: In order to verify the electron transfer activity, the AgBiS2 / ICG and OMV-AgBiS2 / ICG samples prepared in Example 1 were measured in bacterial medium for linear sweep voltammetry (LSV) curves and electrochemical impedance spectroscopy (EIS). From the results of the LSV curve, the OMV-AgBiS2 / ICG sample showed a larger saturation current (as shown in Figure 12a). The EIS curve results showed that the OMV-AgBiS2 / ICG sample group had a lower electrochemical impedance and stronger electron transfer ability than the AgBiS2 / ICG sample group (as shown in Figure 12b). The above results show that the OMV-AgBiS2 / ICG sample accelerated the electron transfer of bacteria.

[0092] Example 31: In this example, in order to further verify the interference of OMV-AgBiS2 / ICG samples on bacterial transfer behavior, bacterial transcriptome was used for verification. The specific operation method is: use the method in Example 27 to culture a single-species biofilm of Porphyromonas gingivalis and carry out treatment. The samples were then collected by low-temperature centrifugation, treated with liquid nitrogen, and sent for sample testing. As shown in Figure 13, the results of GO enrichment analysis show that the bacterial electron transfer behavior is significantly upregulated. Combined with the results of Example 30, it can be seen that OMV-AgBiS2 / ICG nanospheres not only accelerate the electron transfer behavior at the molecular level, but also accelerate the electron transfer behavior at the genetic level, which is beneficial to enhance the bactericidal effect on bacteria.

[0093] Example 32: The AgBiS2, AgBiS2 / ICG and OMV-AgBiS2 / ICG samples prepared in Example 1 were dispersed in phosphate buffer by ultrasonication to form a concentration of 100 μg mL -1 of colloidal suspension.

[0094] Example 33: The colloidal suspension obtained in Example 32 was prepared to a final concentration of 50 μg mL -1 AgBiS2, AgBiS2 / ICG and OMV-AgBiS2 / ICG samples were used in animal experiments. Specifically, the periodontitis model was established in the molar area of ​​Wistar male rats by injecting bacterial solution and ligating with silk thread. After 10 days of treatment, the model was established. The light conditions of the experiment were 1 W cm -2 The samples were irradiated with 808 nm near-infrared laser for 3 minutes each time, for a total of 3 times.

[0095] In this example, MicroCT was used to evaluate the alveolar bone resorption caused by periodontitis. Fig.14 As shown, the OMV-AgBiS2 / ICG sample group showed significant differences in mesial and distal bone loss as well as buccal and palatal bone loss compared with the inflammatory control group. Compared with the blank control group, the inflammatory control group caused nearly four-fifths of alveolar bone loss. In contrast, the bone volume fraction (BV / TV) value of the OMV-AgBiS2 / ICG sample group was second only to the blank control group, which was approximately 70% of the remaining bone volume of the blank control group. These results indicate that OMV-AgBiS2 / ICG nanospheres can effectively control bacteria-induced inflammatory alveolar bone resorption.

[0096] In this embodiment, the inflammation of periodontal soft and hard tissues was evaluated by Masson staining. As shown in Figure 15, in the inflammation control group, most of the collagen fibers in the gingival tissue showed a degraded state of red staining. The AgBiS2 / ICG sample group and the OMV-AgBiS2 / ICG sample group retained more blue fibers. In addition, the gingival soft tissue of the inflammation control group was significantly damaged, the alveolar bone was absorbed in large quantities, and the distance between the alveolar ridge top and the gingival margin was long. In the OMV-AgBiS2 / ICG sample group, the gingival soft tissue was less damaged, the alveolar bone absorption was not obvious, and the distance between the alveolar ridge top and the gingival margin was short. The above results show that OMV-AgBiS2 / ICG nanospheres can effectively inhibit periodontitis inflammation and reduce damage to soft and hard tissues.

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing multifunctional nanospheres targeting bacteria, characterized in that: The specific steps include: Step 1: Mix a certain amount of zinc acetate and polyvinyl pyrrolidone aqueous solution and stir to obtain a clear solution; quickly add thioacetamide solution, increase the system temperature, and stir at a constant temperature; Cool and centrifuge to obtain the product, wash it several times, and dry it to obtain the template material zinc sulfide; Step 2: Add a certain amount of zinc sulfide and thiourea to ethylene glycol, and stir vigorously to obtain a milky white solution; increase the system temperature, add a mixed solution of silver nitrate and bismuth nitrate prepared with ethylene glycol to the above solution, and stir at a constant temperature; Cool and centrifuge to obtain the product, wash it several times, and dry it to obtain bismuth silver sulfide hollow nanospheres; Step 3: dropping the indocyanine green aqueous solution into the bismuth silver sulfide hollow nanosphere dispersion, stirring at room temperature, then standing, centrifuging to obtain the product, washing several times, and drying to obtain the composite nanosphere; Step 4: Cultivate Fusobacterium nucleatum to the logarithmic phase, and use ultracentrifugation to separate and extract bacterial outer membrane vesicles; Step 5: The dispersion of the product in step 3 and the dispersion of bacterial outer membrane vesicles are mixed and vortexed, and then the bacterial outer membrane vesicles are encapsulated by ultrasound, and the targeted multifunctional nanospheres are collected by low-temperature centrifugation.

2. The method for preparing multifunctional nanospheres targeting bacteria according to claim 1, characterized in that: In step 1, the molar ratio of zinc acetate, polyvinyl pyrrolidone and thioacetamide is 100:(0.5-2):500; the reaction temperature is 100° C. to 120° C.; and the reaction time is 2 hours.

3. The method for preparing multifunctional nanospheres targeting bacteria according to claim 2, characterized in that: In step 2, the molar ratio of zinc sulfide to thiourea is 1.14:1; the molar ratio of silver nitrate to bismuth nitrate is 1:1; the reaction temperature is 110° C. to 150° C.; and the reaction time is 2 hours.

4. The method for preparing multifunctional nanospheres targeting bacteria according to claim 3, characterized in that: In step 3, the molar ratio of indocyanine green to bismuth silver sulfide hollow nanospheres is 1:

20.

5. The method for preparing multifunctional nanospheres targeting bacteria according to claim 2, characterized in that: In step 4, the mass ratio of the composite nanospheres to the bacterial outer membrane vesicles is 20:

1.

6. The method for preparing multifunctional nanospheres targeting bacteria according to claim 1, characterized in that: In step 1 to step 3, the rotation speed of centrifugal separation is 6000-9000 rpm / min.

7. The method for preparing multifunctional nanospheres targeting bacteria according to claim 1, characterized in that: In step 4, the rotation speed of the centrifugal separation is 6000 rpm / min.

8. A nanomedicine, a multifunctional nanosphere prepared by the method for preparing multifunctional nanospheres targeting bacteria according to any one of claims 1 to 7, characterized in that: Part or all of the above-mentioned targeted multifunctional nanospheres are included.

9. The nano drug according to claim 8, characterized in that: The nanomedicine is used in preparing medicine for treating targeted harmful bacteria.

10. The nano drug according to claim 8, characterized in that The nano drug is used in preparing drugs for treating periodontitis.

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