A multi-layer microsphere based on a hollow porous yeast carrier, its preparation method and application

Abstract

The present invention discloses a multi-layer microsphere based on a hollow porous yeast carrier, its preparation method and application, belonging to the field of biomedical technology. The material comprises a hollow porous yeast carrier; the interior of the hollow porous yeast carrier is loaded with traditional Chinese medicine active molecules; the surface of the hollow porous yeast carrier is coated with a cross-linking agent layer; lactic acid bacteria are loaded on the cross-linking agent layer. The hollow porous yeast carrier is obtained by treating yeast successively with alkali, acid and organic solvent; the yeast is Saccharomyces cerevisiae; the traditional Chinese medicine active molecule is paeoniflorin; the cross-linking agent layer is formed by configuring a cross-linking agent into a solution at a pH value of 4.9 - 5.0 and cross-linking; the cross-linking agent is chitosan and sodium tripolyphosphate; the lactic acid bacteria are Lactobacillus paracasei. The microspheres of the present invention can not only inhibit the growth of Staphylococcus aureus, but also play an anti-inflammatory and repair role, reduce drug resistance, and improve the stability and targeting of drugs in the vaginal environment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to a hollow porous yeast vector-based multilayer microsphere and its preparation method and application. Background Art

[0002] Aerobic vaginitis (AV) is a vaginal inflammatory disease caused by aerobic bacteria infection. Common pathogenic bacteria include Escherichia coli, Streptococcus, and Staphylococcus aureus, etc. In recent years, the incidence of AV has increased, especially in sexually active women, those using antibiotics, or groups with low immune function. Data shows that the prevalence of AV varies depending on region, age, and health status. Among women in developed countries, the incidence is about 10 - 20%, while it may be higher in developing countries. This kind of inflammation has a serious impact on women's health. If not effectively treated for a long time, it may cause a series of complications and reproductive health problems.

[0003] The cause of AV is not fully understood, and it is mainly related to vaginal flora imbalance and immune system disorders. The normal vaginal flora forms a natural barrier in the weakly acidic environment maintained by lactobacilli to inhibit the growth of aerobic bacteria. When certain incentives such as antibiotic use, immunosuppression, or poor hygiene habits disrupt the flora balance, resulting in a decrease in lactobacilli and the proliferation of aerobic bacteria, which leads to infection. AV is also related to local immune response disorders. At the infected site, white blood cells aggregate and secrete a large number of inflammatory factors, such as IL-1β, IL-6, etc., further damaging the epithelial barrier and forming chronic inflammation. The treatment of AV mainly relies on the combined application of antibiotics, lactobacilli preparations, and local treatment. Antibiotics (such as metronidazole, clindamycin) can quickly control aerobic bacteria infection and are suitable for acute-phase patients, but long-term use may lead to flora imbalance and drug resistance; lactobacilli preparations reduce the recurrence risk by restoring the normal vaginal flora, have few side effects, and are often used in combination with antibiotics; local treatment (such as antibacterial ointments, lotions) acts on the infected area concentratedly, reducing systemic side effects, but usually needs to be combined with systemic treatment to achieve better efficacy.

[0004] Active ingredients of traditional Chinese medicines such as paeoniflorin, berberine, and baicalin all have anti-inflammatory and antibacterial effects and are commonly used components in gynecological drugs. However, due to the broad-spectrum antibacterial effect of traditional Chinese medicine components, it is difficult for them to act together with lactobacilli to inhibit pathogenic bacteria on the one hand and restore the normal vaginal flora on the other hand. AV has received increasing attention in gynecological diseases, but its complex etiology and high recurrence rate pose certain challenges to treatment. Early prevention and standardized treatment of it are of great significance for the protection of women's health. Therefore, how to design a drug that can not only anti-inflammatory and antibacterial but also restore the normal vaginal flora, so as to cope with the complex environment in the vagina and improve the treatment effect. Summary of the Invention

[0005] In view of the above prior art, the object of the present invention is to provide a multi-layer microsphere based on a hollow porous yeast carrier, its preparation method and application. The present invention introduces probiotics and anti-inflammatory traditional Chinese medicine active molecules into the imbalanced vaginal microenvironment, and with the help of the hollow porous yeast capsule, it targets the lesion site, inhibits the growth of Staphylococcus aureus, reduces drug resistance, and improves the stability and targeting of drugs in the vaginal environment.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, a multi-layer microsphere based on a hollow porous yeast carrier is provided, including a hollow porous yeast carrier; the interior of the hollow porous yeast carrier is loaded with traditional Chinese medicine active molecules; the surface of the hollow porous yeast carrier is coated with a cross-linking agent layer; and lactic acid bacteria are loaded on the cross-linking agent layer.

[0008] The hollow porous yeast carrier is obtained by sequentially treating yeast with alkali, acid and organic solvent; the yeast is Saccharomyces cerevisiae.

[0009] The traditional Chinese medicine active molecule is paeoniflorin; the cross-linking agent layer is prepared by cross-linking a cross-linking agent configured into a solution in an environment with a pH value of 4.9 - 5.0; the cross-linking agent is chitosan and sodium tripolyphosphate; and the lactic acid bacteria are Lactobacillus paracasei.

[0010] In the second aspect of the present invention, a preparation method of a multi-layer microsphere based on a hollow porous yeast carrier is provided, including the following steps:

[0011] (1) Yeast is sequentially treated with an alkali solution, an acid solution and an organic solvent, and then freeze-dried to obtain a hollow porous structure yeast carrier;

[0012] (2) The hollow porous structure yeast carrier is added to an acidic solution with a pH value of 4.9 - 5.0 containing traditional Chinese medicine active molecules, and stirred at room temperature to obtain a solution of a hollow porous yeast carrier with traditional Chinese medicine active molecules loaded inside;

[0013] (3) The pH value of an aqueous solution containing sodium tripolyphosphate is adjusted to 4.9 - 5.0 and mixed evenly with lactic acid bacteria to obtain a mixed solution; the pH value of an acetic acid solution containing chitosan is adjusted to 4.9 - 5.0 and dropped into the solution of the hollow porous yeast carrier with traditional Chinese medicine active molecules prepared in step (2), stirred at room temperature, then the mixed solution is dropped, allowed to stand and react, washed and freeze-dried to obtain a multi-layer microsphere based on a hollow porous yeast carrier.

[0014] Preferably, in step (1), the yeast is Saccharomyces cerevisiae.

[0015] The yeast is selected from Saccharomyces cerevisiae purchased from the China General Microbiological Culture Collection Center, with the preservation number of CGMCC 2.3888.

[0016] Preferably, in step (1), the alkaline solution is selected from sodium hydroxide solution, potassium hydroxide solution or calcium hydroxide solution, and the concentration of the alkaline solution is 1 M; the acidic solution is selected from sulfuric acid solution, hydrochloric acid solution or nitric acid solution, and the pH value of the acidic solution is 4.2; the organic solvent is at least one of isopropanol, acetone or ether.

[0017] Preferably, in step (2), the mass ratio of the hollow porous structure yeast carrier to the traditional Chinese medicine active molecule is 1-4:4-1; the stirring time is 12 h.

[0018] Preferably, the traditional Chinese medicine active molecule is paeoniflorin.

[0019] Preferably, in step (3), the mass ratio of the hollow porous yeast carrier internally loaded with the traditional Chinese medicine active molecule, chitosan and sodium tripolyphosphate is 0.8:30:1.

[0020] Preferably, in step (3), the concentration of the solution of the hollow porous yeast carrier internally loaded with the traditional Chinese medicine active molecule is 7.2-10.2 mg / mL; the concentration of sodium tripolyphosphate in the mixed solution is 0.1% w / v; the concentration of lactic acid bacteria in the mixed solution is 1×10 4 ~1×10 9 CFU / mL; the concentration of chitosan in the glacial acetic acid solution containing chitosan is 1.5% w / v, and the concentration of the glacial acetic acid solution is 1% v / v.

[0021] Preferably, in step (3), the lactic acid bacteria is Lactobacillus paracasei; the temperature of the static reaction is 4°C, and the static reaction time is 12 h.

[0022] Lactobacillus paracasei is purchased from the China General Microbiological Culture Collection Center, and the preservation number is CGMCC1.2744.

[0023] In the third aspect of the present invention, there is provided the use of the multilayer microspheres based on the hollow porous yeast carrier in the preparation of a drug for treating aerobic vaginitis. The multilayer microspheres based on the hollow porous yeast carrier can not only inhibit aerobic bacteria by releasing traditional Chinese medicine active molecules, but also release lactic acid bacteria to improve the vaginal environment.

[0024] The beneficial effects of the present invention:

[0025] (1) The present invention uses the screened Lactobacillus paracasei, combines with the traditional Chinese medicine active molecule paeoniflorin, and synergistically anti-inflames through the multifunctional yeast carrier.

[0026] (2) The yeast carrier of the present invention has a porous structure, which provides a sustained release platform for the release of paeoniflorin, and the composite layer of chitosan and lactic acid bacteria enhances antibacterial and stability.

[0027] (3) The hollow porous yeast vector-based multilayer microspheres of the present invention have multiple effects of antibacterial, anti-inflammatory and tissue repair, can replace traditional antibiotic treatment, and reduce the risk of drug resistance. Description of the Drawings

[0028] Figure 1 : Schematic diagram of the preparation process of Pae / L.p@YGPs;

[0029] Figure 2 : Transmission electron microscope image of the preparation process of Pae / L.p@YGPs, scale bar is 2 μm;

[0030] Figure 3 : Dynamic light scattering diagram of the preparation process of Pae / L.p@YGPs, where (a) particle size measurement, (b) potential measurement;

[0031] Figure 4 : Antibacterial properties of paeoniflorin against Staphylococcus aureus and Lactobacillus, where (a) antibacterial properties of paeoniflorin against Staphylococcus aureus, (b) antibacterial properties of paeoniflorin against Lactobacillus;

[0032] Figure 5 : Pae release curves of Pae / L.p@YGPs, Pae / L.p@YGPs-4.0, Pae / L.p@YGPs-6.0 and Pae / L.p@SiO2;

[0033] Figure 6 : Transmission electron microscope images of Pae / L.p@YGPs at 4 h, 12 h, 36 h, scale bar is 2 μm;

[0034] Figure 7 : Cytotoxicity study of Pae / L.p@YGPs;

[0035] Figure 8 : Biocompatibility of Pae / L.p@YGPs;

[0036] Figure 9 : Gram staining diagram of the main flora in vaginal lavage fluid after modeling of aerobic vaginitis, scale bar is 10 μm;

[0037] Figure 10 : Comparison of secretions after sampling with sterile cotton swabs after modeling of aerobic vaginitis;

[0038] Figure 11 : Colony counts of Staphylococcus aureus and Lactobacillus before and after treatment of the animal model of aerobic vaginitis, where (a) number of Staphylococcus aureus before treatment, (b) number of Lactobacillus before treatment, (c) number of Staphylococcus aureus after treatment, (d) number of Lactobacillus after treatment;

[0039] Figure 12 : Appearance and histopathological examination of the vagina of rats. The scales are 100 μm and 50 μm respectively. Inflammatory cell infiltration (black arrow ↗), angiogenesis (red arrow ↗);

[0040] Figure 13 : Statistics on the thickness of vaginal epithelial tissue. Specific embodiments

[0041] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0042] As introduced in the background art section, many traditional Chinese medicine active ingredients have anti-inflammatory and antibacterial effects. However, due to the antibacterial effect of traditional Chinese medicine ingredients, which have broad-spectrum antibacterial properties, aerobic bacteria in the vagina will be inhibited, and lactic acid bacteria with facultative anaerobic characteristics will also be affected. Therefore, it is difficult for traditional Chinese medicine ingredients to act together with lactic acid bacteria to achieve the goal of inhibiting pathogenic bacteria on the one hand and restoring the normal vaginal flora on the other hand.

[0043] Based on this, the object of the present invention is to provide a hollow porous yeast carrier-based multilayer microsphere and its preparation method and application. The present invention discovers through research that the hollow porous yeast carrier-based multilayer microsphere uses hollow porous yeast as a carrier, which can control the release rate of traditional Chinese medicine active ingredients, so that the drug concentration fluctuation of traditional Chinese medicine active ingredients will not be too large, avoiding the phenomena of "blood drug concentration peak" and "low value". It can not only kill the pathogenic bacterium Staphylococcus aureus but also maintain the activity of lactic acid bacteria. Compared with other carriers, hollow porous yeast is an ideal carrier for traditional Chinese medicine active ingredients and lactic acid bacteria. In the present invention, after the release of traditional Chinese medicine active ingredients, it can inhibit the growth of the pathogenic bacterium Staphylococcus aureus; by using the "occupation effect" of lactic acid bacteria in the vagina, aerobic bacteria cannot proliferate in the vagina, thereby regulating the balance of the vaginal environment. Therefore, traditional Chinese medicine active ingredients can inhibit the proliferation of aerobic bacteria, and lactic acid bacteria occupy the position of aerobic bacteria on the vaginal epithelium, regulating the balance of the vaginal flora; both methods are used simultaneously to achieve the treatment of aerobic bacterial vaginitis.

[0044] It was found during the preparation process that chitosan, sodium tripolyphosphate, and lactic acid bacteria must be configured into solutions and cross-linked in an environment with a pH value of 4.9 - 5.0; to avoid the leakage of traditional Chinese medicine active ingredients during subsequent preparation processes such as centrifugation and improve the drug loading capacity; and in the vaginal environment, chitosan gradually dissolves and traditional Chinese medicine active ingredients are gradually released. Beyond this range, the cross-linking effect is weak, and even a cross-linked structure cannot be formed.

[0045] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with specific embodiments.

[0046] The test materials used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels.

[0047] Example 1: Preparation of Multilayer Microspheres Based on Hollow Porous Yeast Carriers

[0048] (1) Preparation of Hollow Porous Structure Yeast Carriers (YGPs)

[0049] Disperse 150 g of dry Saccharomyces cerevisiae in 600 mL of preheated 1 M sodium hydroxide solution, heat it under magnetic stirring at 90 °C for 1 h, and cool it to room temperature for 10 - 15 min. Subsequently, centrifuge at 12000 rpm for 3 min, discard the supernatant, and repeat this step twice, for a total of 3 times. Then, mix the alkali-insoluble solid with 100 mL of preheated hydrochloric acid solution (pH = 4.2), disperse it with a glass rod and vortex to make it uniform, heat it under magnetic stirring at 75 °C for 2 h, and centrifuge again at 12000 rpm for 3 min. The insoluble solid is washed successively in deionized water (6 times), isopropanol (8 times), and acetone (4 times). After each washing step, disperse it with a glass rod and vortex to make it uniform, then sonicate at 100 W for 5 min and centrifuge at 12000 rpm for 3 min to discard the supernatant. Resuspend the YGPs with deionized water and pour it into a plate, and place the prepared sample in a -20 °C refrigerator for pre-freezing. The final product is freeze-dried in a vacuum freeze-dryer for 2 days. After freeze-drying is completed, store it in a -20 °C refrigerator for later use. After treatment, most of the cytoplasm in the content has been removed, leaving only the β-glucan layer. The β-glucan layer has pores with a diameter of about 20 - 60 nm and usually has good permeability.

[0050] (2) Preparation of Hollow Porous Yeast Carriers with Chinese Medicine Active Molecules Loaded Inside (Pae@YGPs)

[0051] Use hydrochloric acid to configure deionized water into an acidic solution with a pH value of 4.9 - 5.0, add paeoniflorin (Pae) to 2 mL of the acidic solution to obtain a paeoniflorin solution with a concentration of 4 mg / mL. Add 8 mg of YGPs to the paeoniflorin solution and stir magnetically at room temperature for 12 h to load Pae into YGPs to obtain a Pae@YGPs solution.

[0052] (3) Preparation of Multilayer Microspheres Based on Hollow Porous Yeast Carriers (Pae / L.p@YGPs)

[0053] 2 mL of 1.5% w / v chitosan solution in glacial acetic acid (pH 4.9 - 5.0) was added dropwise to 2 mL of the Pae@YGPs solution prepared in step (2), and magnetically stirred at room temperature for 12 h. 1 mL of sodium tripolyphosphate (TPP) solution (0.1% w / v, adjusted to pH 4.9 - 5.0) was mixed evenly with 10 4 CFU / mL of Lactobacillus paracasei (L.p), and then added dropwise to the reaction system, and magnetically stirred in an ice bath for 30 min. It was left standing at 4 °C for 12 h, centrifuged at 2000 rpm for 10 min, and then the supernatant was discarded. The precipitate was washed 3 times with 0.5 mL of sodium tripolyphosphate (TPP) solution (0.01% w / v, pH 4.9 - 5.0). Finally, vacuum freeze-drying was carried out, and the obtained microcapsules were stored in a -20 °C refrigerator for later use. Finally, YGPs microcapsules with Lactobacillus on the outer layer and Pae loaded inside were obtained, and the finally obtained product was denoted as Pae / L.p@YGPs. The schematic diagram of the preparation process is shown in Figure 1 .

[0054] Comparative Example 1: Preparation of Pae@YGPs CS

[0055] The difference from Example 1 is that Lactobacillus paracasei was not added, and finally Pae@YGPs CS was obtained.

[0056] Example 2: Characterization

[0057] (1)Transmission electron micrograph of Pae / L.p@YGPs

[0058] Samples were observed using a Hitachi HT7700 transmission electron microscope (Hitachi, Tokyo, Japan). A small amount of Saccharomyces cerevisiae (Yeast) fixed with 4% paraformaldehyde, YGPs prepared in step (1) of Example 1, Pae@YGPs prepared in step (2) of Example 1, Pae@YGPs CS prepared in Comparative Example 1, and the Pae / L.P@YGPs solution prepared in step (3) of Example 1 were respectively dropped onto a 200-mesh copper grid. After standing for 3 min, the excess sample solution was blotted with filter paper. Subsequently, it was stained with 3% uranyl acetate for 5 min, the excess staining solution was blotted, and the sample was dried at room temperature for 24 h. Finally, TEM was used for observation and photography to record the morphology of the samples. The obtained results are shown in Figure 2 .

[0059] According to Figure 2It can be seen that after treatment with alkali, acid, and organic solvents, the cytoplasmic components of Yeast were significantly lost, resulting in a decrease in the contrast of YGPs. After loading Pae, the contrast increased significantly. A network structure was formed by crosslinking chitosan and sodium tripolyphosphate and attached to the surface of Pae@YGPs, thus providing a support site for L.p, and Pae / L.p@YGPs was successfully prepared.

[0060] (2)Dynamic light scattering diagram of Pae / L.p@YGPs

[0061] The prepared Saccharomyces cerevisiae (Yeast), YGPs prepared in step (1) of Example 1, Pae@YGPs prepared in step (2) of Example 1, Pae@YGPs CS prepared in Comparative Example 1, and Pae / L.P@YGPs prepared in step (3) of Example 1 were measured for hydrodynamic diameter and Zeta potential using a Malvern Zetasizer Nano ZS90 laser scattering particle size analyzer (MalvernPanalytical, Malvern, UK).

[0062] According to Figure 3 It can be seen that under the layer-by-layer encapsulation of Pae / L.P@YGPs, the particle size increased. The negative charge of YGPs decreased after treatment of Yeast, and further decreased after loading Pae. After encapsulating chitosan, the Zeta potential turned positive, and after adding L.p, the positive potential decreased slightly.

[0063] Comparative Example 2: Preparation of multilayer microspheres based on hollow porous yeast carriers

[0064] The difference from Example 1 is that in step (3), the pH was adjusted to 4.0, and the final product was denoted as Pae / L.p@YGPs-4.0.

[0065] Comparative Example 3: Preparation of multilayer microspheres based on hollow porous yeast carriers

[0066] The difference from Example 1 is that in step (3), the pH was adjusted to 6.0, and the final product was denoted as Pae / L.p@YGPs-6.0.

[0067] Comparative Example 4: Preparation of multilayer microspheres based on hollow porous silica carriers

[0068] The difference from Example 1 is that the hollow porous yeast carrier was replaced with an equal amount of hollow porous silica carrier, and the final product was denoted as Pae / L.p@SiO 2 。

[0069] The preparation method of the hollow porous silica support is as follows: Add 2.5 - 5 g of Pluronic F127 into 50 mL of ethanol: deionized water with a volume ratio of 1:1. Stir in a water bath at 40 - 45 °C for 2 h to ensure that Pluronic F127 is completely dissolved to form a transparent solution. Cool down to 25 °C and continue stirring for 30 min to ensure uniform distribution of micelles. Dropwise add 1 - 3 mL of ammonia water (25 - 28%) as a catalyst, control the pH between 9 - 11, and the hydrolysis products further condense to form SiO 2 sol. Continuously stir for 24 h. Dropwise add tetraethyl orthosilicate (TEOS) at a rate of 1 mL / 10 min, and magnetically stir at room temperature for 6 - 12 h to ensure uniform deposition of the SiO 2 shell. After centrifuging at 12000 rpm for 10 min, wash with ethanol: deionized water with a volume ratio of 1:1 for 3 - 5 times to remove unreacted substances. Stir in 10 - 20 mL of 0.1 M HCl / ethanol solution for 12 h. Ultrasonic for 10 - 15 min to disperse the particles, stir at 40 °C for 6 - 8 h, after centrifuging at 12000 rpm for 10 min, discard the supernatant, wash with ethanol: deionized water with a volume ratio of 9:1 for 3 - 5 times to remove residual hydrochloric acid. Place in a drying oven at 60 - 120 °C and dry for more than 12 h to obtain a hollow porous silica support with a particle size of 500 nm - 10 μm.

[0070] Experimental Example 1: Antibacterial properties of Pae

[0071] To investigate the inhibitory effects of paeoniflorin (Pae) on Staphylococcus aureus and Lactobacillus, the antibacterial properties of Pae against Staphylococcus aureus ( S. aureus ) and Lactobacillus paracasei ( L. paracasei ) were determined. Pae was configured with deionized water into Pae solutions with different concentrations (25.6, 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, and 0.0125 mg / mL, 50 μL). The Pae concentration of 0 mg / mL was used as the control group; they were respectively co-cultured with S. aureus (1 × 10 6 CFU / mL, 50 μL) and L. paracasei (1 × 10 5 CFU / mL, 50 μL) in a 96-well plate for 12 h. After the culture ended, the absorbance at 600 nm was measured with an iMark microplate reader. The obtained results are shown in Figure 4 .

[0072] According to Figure 4It can be seen that within a certain low concentration range of Pae, the growth of Staphylococcus aureus can be inhibited. On the contrary, high-concentration Pae promotes the growth of Staphylococcus aureus, and high-concentration Pae shows inhibition of the growth of Lactobacillus. This indicates that the effects of Pae on different microorganisms are different, showing different biological activities. Pae needs to be maintained within a low concentration range to inhibit Staphylococcus aureus without affecting the growth of Lactobacillus. Therefore, the release concentration of Pae should be within the range of 0.0125 - 0.4 mg / mL. By slowly releasing Pae, the low-concentration level of Pae in the vagina can be maintained to specifically inhibit the growth of pathogenic bacteria without affecting the growth of Lactobacillus.

[0073] Experimental Example 2: Release Test of Active Ingredients

[0074] The paeoniflorin release curves of Pae / L.p@YGPs prepared in Example 1 and the microspheres prepared in Comparative Examples 2 - 4 were plotted. The specific method was as follows: 50 mg of the above microspheres were respectively dispersed in 7 mL of vaginal simulating fluid (SVF, composition: 3.51 g NaCl, 1.4 g KOH, 0.222 g Ca(OH) 2 , 2.00 g lactic acid, 0.018 g bovine serum albumin, 1.00 g acetic acid, 0.016 g glycerol, 5.0 g glucose, 0.4 g urea, 1000 mL deionized water, adjusted to pH 5.0 ± 0.2); then placed in a 15 mL centrifuge tube on a shaker at 37 °C and 100 rpm, and 300 μL of the release solution was collected at regular intervals. After centrifuging the release solution at 2000 rpm for 10 min, 200 μL of the supernatant was collected, and the absorbance of Pae at a wavelength of 231 nm was measured using a YoMim full-wavelength reader. 200 μL of fresh SVF was added to the remaining 100 μL of the release solution, and after mixing evenly, it was returned to the 7 mL release system. The absorbance of each well at each time point was recorded, and the cumulative release amount of Pae at different times was calculated. The experiment was repeated three times, and the results are shown in Figure 5 . The Pae / L.p@YGPs were photographed at 4 h, 12 h, and 36 h using a Hitachi HT7700 transmission electron microscope (Hitachi, Tokyo, Japan), and the results are shown in Figure 6 .

[0075] According to Figure 5 It can be seen that compared with Comparative Examples 2 - 4, after 36 h, Pae / L.p@YGPs obtained a larger release amount compared with other groups. The release amounts of Pae / L.p@YGPs - 4.0 and Pae / L.p@YGPs - 6.0 were less, and Pae / L.p@SiO 2The rapid release in the early stage results in too high a concentration of Pae in the vagina, which is likely to inhibit the growth of lactic acid bacteria and promote the growth of Staphylococcus aureus. However, Pae / L.p@YGPs can encapsulate more Pae and has a sustained-release effect, maintaining Pae within a low concentration range, thereby inhibiting Staphylococcus aureus without harming lactic acid bacteria.

[0076] According to Figure 6 It can be seen that the contrast of Pae / L.p@YGPs prepared in Example 1 gradually decreased within 36 h, indicating that the loaded Pae had been gradually released.

[0077] Test Example 3: Cytotoxicity test

[0078] The CCK-8 experiment was used to test the cytotoxicity of Pae / L.p@YGPs prepared in Example 1 on HUVEC cells. After taking out the cells from liquid nitrogen, they were immediately placed in a 37 °C water bath and shaken rapidly to melt them quickly, so as to reduce the damage of ice crystals to the cell structure. Immediately afterwards, the cell suspension was quickly transferred to 2 mL of complete DMEM medium and centrifuged at 1200 rpm for 5 min. After discarding the supernatant, the cells were resuspended with 1 mL of complete DMEM medium, gently pipetted and mixed evenly, and then the cells were transferred to 7 mL of complete DMEM medium. Subsequently, the cells were cultured in an incubator at 37 °C and 5% CO 2 After the cells adhered well, they were digested and counted. The counted HUVEC cells were diluted with DMEM medium to 2.5 × 10 4 cell / mL, 100 µL per well, and placed in a 96-well plate for culture for 24 h. Subsequently, after preparing a solution of Pae / L.p@YGPs with a concentration of 1 µg / mL - 50 µg / mL using DMEM medium, it was sterilized by ultraviolet irradiation for 24 h. 10 µL of the above-mentioned solution to be tested was added to each well, and they were co-incubated in an incubator at 37 °C and 5% CO 2 for 24 h. Subsequently, 10 µL of CCK-8 solution was added to each well. After culturing for 1 - 4 h, the absorbance at 450 nm was measured using an iMark microplate reader.

[0079] The cell survival rate was calculated according to the following formula: Cell survival rate = [ (As - Ab) / (Ac - Ab) ] × 100%. Among them, As is the absorbance of the experimental well, Ac is the absorbance of the control well, and Ab is the absorbance of the blank well; only HUVEC cells were added to the control well without drug treatment; in the blank group, neither HUVEC cells nor drug treatment was added, and only DMEM medium was added). The obtained results are shown in Figure 7 .

[0080] According to Figure 7It can be seen that as the concentration of Pae / L.p@YGPs increases, the cell viability decreases. Pae / L.p@YGPs at lower concentrations has little significant effect on cells, and the cell number shows a slight increase. However, at a higher concentration of 50.0 μg / mL, the cell viability decreases significantly, indicating that Pae / L.p@YGPs at a concentration of 25.0 μg / mL and below has no significant effect on cells.

[0081] Experimental Example 4: Biocompatibility Experiment

[0082] The hemolysis experiment of red blood cells was used to detect the biosafety of Pae / L.p@YGPs prepared in Example 1. Blood was collected from the marginal ear vein of New Zealand white rabbits (10 weeks old, purchased from Jinan Jinfeng Experimental Animal Co., Ltd.) and placed in an anticoagulant tube containing sodium heparin. After centrifugation at 3000 rpm for 10 min, the plasma and white blood cell layer were discarded, and the red blood cells were collected. The red blood cells were washed 3 times with 0.9% physiological saline, centrifuged for 5 min each time. The washed red blood cells were resuspended in 0.9% physiological saline to prepare a 2% red blood cell suspension. Then, Pae / L.p@YGPs solutions with concentrations of 1 μg / mL to 8 μg / mL were prepared using 0.9% physiological saline. After adding 100 μL of the red blood cell suspension respectively, they were incubated at 37 °C for 1 to 4 h. After the incubation, they were centrifuged at 3000 rpm for 10 min, and images were taken and the supernatant was collected. Finally, the absorbance value of the supernatant was measured at 570 nm using an iMark microplate reader.

[0083] The calculation formula for the hemolysis rate is: HR (%) = OD t – OD n / (OD p – OD n ) × 100%. Among them, OD t represents the absorbance of the experimental group, OD n represents the absorbance of the negative control group, and OD p is the absorbance of the positive control group (using 0.9% physiological saline and 1% Triton X-100 as the negative control group and the positive control group). All experiments were repeated three times. HR < 5%: Complies with biocompatibility, HR 5 - 10%: There is a certain hemolysis risk, HR > 10%: Obvious hemolysis, not suitable as a biomaterial or drug. The obtained results are shown in Figure 8 .

[0084] According to Figure 8 It can be seen that compared with the positive control group (Triton X-100), the hemolytic activity of Pae / L.p@YGPs is less than 5%, showing a lower hemolytic activity, indicating that the Pae / L.p@YGPs composite microspheres have good biocompatibility.

[0085] Experimental Example 5: Animal Experiment

[0086] The experiment was divided into 6 groups: normal group, model group, Pae / L.p@YGPs group, YGPs group, Pae group, and L.p group, with 5 rats in each group. Sprague-Dawley rats (6 weeks old, purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd.) were used as experimental subjects, and a rat vaginal model infected with Staphylococcus aureus was established.

[0087] The specific method was as follows: 2.5 mg / kg β-estradiol was intraperitoneally injected daily for 3 days before infection to promote estrus in Sprague-Dawley rats and make them more susceptible to infection. Subsequently, 50 μL / day of 1×10 8 CFU / mL Staphylococcus aureus (ATCC6538) was inoculated into the vagina of Sprague-Dawley rats for 7 consecutive days to establish an aerobic vaginitis model infected with Staphylococcus aureus. After the model was established, the experimental rats showed typical AV symptoms, including increased vaginal secretions, vulvar swelling and local inflammatory reactions, indicating that the model was successfully constructed; the normal group rats were not treated. The experimental rats were divided into 5 groups. The model group was given 0.9% normal saline, the Pae / L.p@YGPs group was given Pae / L.p@YGPs prepared in Example 1, the YGPs group was given YGPs prepared in step (1) of Example 1, the Pae group was given paeoniflorin, and the L.p group was given lactic acid bacteria for treatment. The dosage of each group was 0.25 mg / kg. After continuous administration for 7 days, the rats were euthanized, and vaginal tissues were taken for histopathological observation.

[0088] Seven days after modeling, the Gram staining method was used to observe the main vaginal flora, and the results are shown in Figure 9 . It can be seen that the main bacteria in the vaginal secretions were purple Staphylococcus aureus.

[0089] Vaginal secretion samples were collected using sterile cotton swabs, and the results are shown in Figure 10 . It can be seen that compared with the normal group, the model group showed a significant increase in yellow vaginal secretions, indicating that the modeling was successful.

[0090] Before and after treatment, vaginal samples were collected using sterile cotton swabs, resuspended with 2 mL of normal saline, diluted, and then inoculated onto mannitol medium and MRS medium for selective culture and colony counting. The results are shown in Figure 11It can be seen that the number of Staphylococcus aureus in the vagina of rats after modeling increased significantly, while the number of Lactobacillus decreased significantly. After 7 days of continuous treatment, the number of Staphylococcus aureus in the Pae / L.p@YGPs group, YGPs group, Pae group, and L.p group all decreased. Among them, the number of Staphylococcus aureus in the Pae / L.p@YGPs group decreased significantly, and at the same time, the number of Lactobacillus increased significantly. Although the Pae group had an inhibitory effect on Staphylococcus aureus, the number of Lactobacillus did not increase, which was not beneficial for restoring the probiotic Lactobacillus in the vagina.

[0091] After 7 days of treatment, the rats were anesthetized and euthanized, and then the vaginal tissues were dissected and fixed in 4% paraformaldehyde solution for histopathological analysis. The results are shown in Figure 12 It can be seen that the redness and swelling of the vagina and tissue inflammation in the rats of the Pae / L.p@YGPs group were alleviated, the morphology of epithelial cells gradually returned to normal, the cell arrangement became more compact, the morphology was regular, and angiogenesis was promoted.

[0092] The thickness of the tissue epithelium was measured, and the results are shown in Figure 13 It can be seen that the thickness of the tissue epithelium in the rats of the Pae / L.p@YGPs group increased significantly.

[0093] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, the present application can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A multilayer microsphere based on a hollow porous yeast carrier, characterized in that: It comprises a hollow porous yeast carrier; the interior of the hollow porous yeast carrier is loaded with active molecules of traditional Chinese medicine; the surface of the hollow porous yeast carrier is coated with a crosslinking agent layer; and lactic acid bacteria are loaded on the crosslinking agent layer; The hollow porous yeast carrier is obtained by treating yeast with alkali, acid and organic solvent in sequence; the yeast is Saccharomyces cerevisiae; The active molecule of traditional Chinese medicine is paeoniflorin; the crosslinking agent layer is prepared by preparing a solution of the crosslinking agent under a pH value of 4.9 to 5.0 and crosslinking; the crosslinking agent is chitosan and sodium tripolyphosphate; the lactic acid bacteria is Lactobacillus paracasei; The hollow porous yeast carrier multilayer microspheres are prepared by the following method: (1) The yeast is treated with alkali solution, acid solution and organic solvent in sequence, and then freeze-dried to obtain a hollow porous structure yeast carrier; (2) The hollow porous yeast carrier is added into an acidic solution with a pH value of 4.9 to 5.0 containing active molecules of traditional Chinese medicine, and stirred at room temperature to obtain a hollow porous yeast carrier solution loaded with active molecules of traditional Chinese medicine; The mass ratio of the hollow porous structure yeast carrier to the active molecules of traditional Chinese medicine is 1-4:4-1; (3) The pH value of the aqueous solution containing sodium tripolyphosphate is adjusted to 4.9-5.0, and lactic acid bacteria are added and mixed uniformly to obtain a mixed solution; the pH value of the glacial acetic acid solution containing chitosan is adjusted to 4.9-5.0 and added dropwise to the hollow porous yeast carrier solution loaded with active Chinese medicine molecules prepared in step (2), stirred at room temperature, and then the mixed solution is added dropwise, allowed to react, washed, and freeze-dried to obtain multilayer microspheres based on hollow porous yeast carriers; the mass ratio of the hollow porous yeast carrier loaded with active Chinese medicine molecules, chitosan, and sodium tripolyphosphate is 0.8:30:

1.

2. The multilayer microsphere based on hollow porous yeast carrier according to claim 1, characterized in that: In step (1), the alkali solution is selected from sodium hydroxide solution, potassium hydroxide solution or calcium hydroxide solution, and the concentration of the alkali solution is 1 M; the acid solution is selected from sulfuric acid solution, hydrochloric acid solution or nitric acid solution, and the pH value of the acid solution is 4.2; and the organic solvent is at least one of isopropanol, acetone or ether.

3. The multilayer microsphere based on hollow porous yeast carrier according to claim 1, characterized in that: In step (2), the stirring time is 12 h.

4. The multilayer microsphere based on hollow porous yeast carrier according to claim 1, characterized in that: In step (3), the concentration of the hollow porous yeast carrier solution loaded with the active molecules of traditional Chinese medicine is 7.2-10.2 mg / mL; the concentration of sodium tripolyphosphate in the mixed solution is 0.1% w / v; the concentration of lactic acid bacteria in the mixed solution is 1×10 4 ~1×10 9 CFU / mL; the concentration of chitosan in the glacial acetic acid solution containing chitosan is 1.5% w / v, and the concentration of the glacial acetic acid solution is 1% v / v.

5. The multilayer microsphere based on hollow porous yeast carrier according to claim 1, characterized in that: In step (3), the temperature of the static reaction is 4° C., and the static reaction time is 12 h.

6. Use of the hollow porous yeast carrier multilayer microspheres according to any one of claims 1 to 5 in the preparation of a drug for treating aerobic bacterial vaginitis, characterized in that: The multilayer microspheres based on the hollow porous yeast carrier can not only inhibit aerobic bacteria by releasing active molecules of traditional Chinese medicine, but also release lactic acid bacteria to improve the vaginal environment.

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