Hollow porous yeast carrier-based multilayer microsphere as well as preparation method and application thereof

Abstract

The invention discloses a multilayer microsphere based on a hollow porous yeast carrier as well as a preparation method and application of the multilayer microsphere, and belongs to the technical field of biological medicines. The material comprises a hollow porous yeast carrier, traditional Chinese medicine active molecules are loaded in the hollow porous yeast carrier; 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 with alkali, acid and an organic solvent in sequence; the yeast is saccharomyces cerevisiae; the traditional Chinese medicine active molecule is paeoniflorin; the cross-linking agent layer is obtained by preparing a cross-linking agent into a solution in an environment with the pH value of 4.9-5.0 and performing cross-linking; the cross-linking agent is prepared from chitosan and sodium tripolyphosphate; the lactic acid bacteria are lactobacillus paracasei. The microspheres provided by the invention can inhibit the growth of staphylococcus aureus, also can play an anti-inflammatory repairing role, reduce the drug resistance, and improve the stability and targeting property of the drug in the vagina environment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a multilayer microsphere based on a hollow porous yeast carrier and a preparation method and application thereof. Background Art

[0002] Aerobic vaginitis (AV) is an inflammatory disease of the vagina caused by aerobic bacteria. Common pathogens include Escherichia coli, Streptococcus, and Staphylococcus aureus. In recent years, the incidence of AV has increased, especially in sexually active women, those who use antibiotics, or those with weakened immune systems. Data show that the prevalence of AV varies by region, age, and health status. The incidence rate is about 10-20% among women in developed countries, and may be higher in developing countries. This inflammation has a serious impact on women's health, and long-term lack of effective treatment may lead to a series of complications and reproductive health problems.

[0003] The etiology of AV is not fully understood, and it is mainly related to the imbalance of vaginal flora and immune system disorders. The normal vaginal flora is mainly composed of a weakly acidic environment maintained by lactic acid bacteria, which forms a natural barrier to inhibit the growth of aerobic bacteria. When certain factors such as antibiotic use, immunosuppression or poor hygiene habits disrupt the balance of the flora, lactic acid bacteria decrease, aerobic bacteria proliferate and cause infection. AV is also related to the disorder of local immune response. White blood cells gather and secrete a large number of inflammatory factors such as IL-1β and IL-6 in the infected area, which further damage the epithelial barrier and form chronic inflammation. The treatment of AV mainly relies on the combined use of antibiotics, lactic acid bacteria preparations and local treatments. Antibiotics (such as metronidazole and clindamycin) can quickly control aerobic infections and are suitable for patients in the acute phase, but long-term use may lead to flora imbalance and drug resistance; lactic acid bacteria preparations restore the normal vaginal flora, reduce the risk of recurrence, have few side effects, and are often used in combination with antibiotics; local treatments (such as antibacterial ointments and lotions) focus on the infected area to reduce systemic side effects, but usually need to be combined with systemic treatment to achieve better efficacy.

[0004] Active ingredients of traditional Chinese medicines such as peony root, berberine, and baicalin all have anti-inflammatory and antibacterial effects and are commonly used ingredients in gynecological drugs. However, due to the broad-spectrum antibacterial effects of traditional Chinese medicine ingredients, it is difficult for them to work together with lactic acid bacteria to achieve the goal of inhibiting pathogenic bacteria on the one hand and restoring normal vaginal flora on the other. AV has received more and more attention in gynecological diseases, but its complex etiology and high recurrence rate make its treatment challenging. Early prevention and standardized treatment are of great significance for the protection of women's health. Therefore, how to design a drug that can both fight inflammation and inhibit bacteria and restore 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-mentioned prior art, the purpose of the present invention is to provide a multilayer microsphere based on a hollow porous yeast carrier and a preparation method and application thereof. The present invention introduces probiotics and anti-inflammatory Chinese medicine active molecules to treat the imbalance of the vaginal microenvironment, and uses the hollow porous yeast capsule to target the lesion site, inhibit the growth of Staphylococcus aureus, reduce drug resistance, and improve the stability and targeting of drugs in the vaginal environment.

[0006] To achieve the above object, the present invention adopts the following technical solution: In a first aspect, the present invention provides a multilayer microsphere based on a hollow porous yeast carrier, comprising 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 4.9-5.0 and crosslinking; the crosslinking agent is chitosan and sodium tripolyphosphate; and the lactic acid bacteria is Lactobacillus paracasei.

[0007] The second aspect of the present invention provides a method for preparing multilayer microspheres based on hollow porous yeast carriers, comprising the following steps: (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; (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 evenly 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 traditional Chinese medicine active 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 the hollow porous yeast carrier.

[0008] Preferably, in step (1), the yeast is Saccharomyces cerevisiae.

[0009] The yeast was selected from Saccharomyces cerevisiae purchased from China General Microbiological Culture Collection Center with the collection number of CGMCC2.3888.

[0010] Preferably, 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.

[0011] Preferably, in step (2), the mass ratio of the hollow porous structure yeast carrier to the active molecule of traditional Chinese medicine is 1-4:4-1; and the stirring time is 12 h.

[0012] Preferably, the active molecule of traditional Chinese medicine is paeoniflorin.

[0013] Preferably, in step (3), the mass ratio of the hollow porous yeast carrier loaded with traditional Chinese medicine active molecules, chitosan and sodium tripolyphosphate is 0.8:30:1.

[0014] Preferably, in step (3), the concentration of the hollow porous yeast carrier solution loaded with traditional Chinese medicine active molecules 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.

[0015] 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.

[0016] Lactobacillus paracasei was purchased from China General Microbiological Culture Collection Center with the collection number of CGMCC1.2744.

[0017] The third aspect of the present invention provides the use of multilayer microspheres based on hollow porous yeast carriers in the preparation of drugs for treating aerobic bacterial vaginitis. The multilayer microspheres based on hollow porous yeast carriers 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.

[0018] Beneficial effects of the present invention: (1) The present invention utilizes the screened Lactobacillus paracasei, combined with the active molecule paeoniflorin from traditional Chinese medicine, to achieve synergistic anti-inflammatory effects through a multifunctional yeast carrier.

[0019] (2) The yeast carrier of the present invention has a porous structure that provides a sustained-release platform for the release of paeoniflorin, and the chitosan and lactic acid bacteria composite layer enhances antibacterial and stability.

[0020] (3) The hollow porous yeast carrier multilayer microspheres of the present invention have multiple functions of antibacterial, anti-inflammatory and tissue repair, which can replace traditional antibiotic treatment and reduce the risk of drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Schematic diagram of the preparation process of Pae / Lp@YGPs; Figure 2 : Transmission electron microscopy image of the preparation process of Pae / Lp@YGPs, the scale bar is 2 μm; Figure 3 : Dynamic light scattering diagram of the preparation process of Pae / Lp@YGPs, including (a) particle size measurement, (b) potential measurement; Figure 4 : The antibacterial properties of paeoniflorin against Staphylococcus aureus and lactic acid bacteria, including (a) the antibacterial properties of paeoniflorin against Staphylococcus aureus, (b) the antibacterial properties of paeoniflorin against lactic acid bacteria; Figure 5 : Pae release curves of Pae / Lp@YGPs, Pae / Lp@YGPs-4.0, Pae / Lp@YGPs-6.0 and Pae / Lp@SiO2; Figure 6 : Transmission electron microscopy images of Pae / Lp@YGPs at 4 h, 12 h, and 36 h, the scale bar is 2 μm; Figure 7 :Cytotoxicity study of Pae / Lp@YGPs; Figure 8 :Biocompatibility of Pae / Lp@YGPs; Fig. 9 : Gram staining of the main flora of vaginal lavage fluid after aerobic bacterial vaginitis modeling, scale bar is 10 μm; Fig.10 :Comparison of secretions after sterile cotton swab sampling after modeling of aerobic bacterial vaginitis; Fig.11 : Colony counts of Staphylococcus aureus and lactic acid bacteria before and after treatment in an animal model of aerobic vaginitis, including (a) the number of Staphylococcus aureus before treatment, (b) the number of lactic acid bacteria before treatment, (c) the number of Staphylococcus aureus after treatment, and (d) the number of lactic acid bacteria after treatment; Fig.12 :Appearance and histopathological examination of the rat vagina, scale bars are 100 μm and 50 μm, respectively, inflammatory cell infiltration (black arrow ↗), angiogenesis (red arrow ↗); Fig.13 : Statistics of vaginal epithelial tissue thickness. DETAILED DESCRIPTION

[0022] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0023] As introduced in the background technology section, many active ingredients of traditional Chinese medicine have anti-inflammatory and antibacterial effects. However, due to the antibacterial effect of traditional Chinese medicine ingredients, they have a broad spectrum of antibacterial properties, which will inhibit aerobic bacteria in the vagina, and lactic acid bacteria with facultative anaerobic characteristics are also not immune. Therefore, it is difficult for traditional Chinese medicine ingredients to work together with lactic acid bacteria to achieve the goal of inhibiting pathogenic bacteria on the one hand and restoring normal vaginal flora on the other.

[0024] Based on this, the purpose of the present invention is to provide a multilayer microsphere based on a hollow porous yeast carrier and a preparation method and application thereof. The present invention has found through research that the multilayer microsphere based on a hollow porous yeast carrier uses a hollow porous yeast as a carrier to control the release rate of the active ingredients of traditional Chinese medicine, so that the concentration fluctuation of the active ingredients of traditional Chinese medicine will not be too large, and the "blood drug concentration peak" and "underestimation" phenomenon can be avoided. It can kill the pathogenic bacteria Staphylococcus aureus and maintain the activity of lactic acid bacteria. Compared with other carriers, hollow porous yeast is an ideal carrier for active ingredients of traditional Chinese medicine and lactic acid bacteria. In the present invention, the active ingredients of traditional Chinese medicine can inhibit the growth of pathogenic bacteria Staphylococcus aureus after release; the "occupying effect" of lactic acid bacteria in the vagina is utilized to prevent aerobic bacteria from proliferating in the vagina, thereby regulating the balance of the vaginal environment. Therefore, the active ingredients of traditional Chinese medicine can inhibit the proliferation of aerobic bacteria, and lactic acid bacteria occupy the position of aerobic bacteria in the vaginal epithelium to regulate the balance of the flora in the vagina; a two-pronged approach is used to achieve the treatment of aerobic bacterial vaginitis.

[0025] During the preparation process, it was found that chitosan, sodium tripolyphosphate and lactic acid bacteria must be prepared into solutions and cross-linked at a pH of 4.9 to 5.0 to avoid leakage of active ingredients of traditional Chinese medicine due to centrifugation and other operations during the subsequent preparation process, and to increase the drug loading; when entering the vaginal environment, chitosan gradually dissolves and the active ingredients of traditional Chinese medicine are gradually released. Beyond this range, the cross-linking effect is weak, and even a cross-linking structure cannot be formed.

[0026] 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 in conjunction with specific embodiments.

[0027] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0028] Example 1: Preparation of multilayer microspheres based on hollow porous yeast carriers (1) Preparation of hollow porous yeast carriers (YGPs) 150 g of dry Saccharomyces cerevisiae was dispersed in 600 mL of preheated 1 M sodium hydroxide solution, heated at 90 °C with magnetic stirring for 1 h, and cooled at room temperature for 10-15 min. Subsequently, the mixture was centrifuged at 12,000 rpm for 3 min, the supernatant was discarded, and this step was repeated twice for a total of 3 times. Next, the alkali-insoluble solid was mixed with 100 mL of preheated hydrochloric acid solution (pH = 4.2), broken up with a glass rod and vortexed to disperse evenly, heated at 75 °C with magnetic stirring for 2 h, and centrifuged again at 12,000 rpm for 3 min. The insoluble solid was washed in deionized water (6 times), isopropanol (8 times), and acetone (4 times) in turn. After each washing step, the solid was broken up with a glass rod and vortexed to disperse evenly, followed by ultrasonication at 100 W for 5 min, centrifuged at 12,000 rpm for 3 min, and the supernatant was discarded. YGPs were resuspended in deionized water and poured into a plate, and the prepared samples were placed in a -20 °C refrigerator for pre-freezing. The final product was freeze-dried in a vacuum freeze dryer for 2 days, and after freeze-drying, it was stored in a -20°C refrigerator for later use. After treatment, most of the cytoplasm of the contents has been removed, leaving only the β-glucan layer, which has a pore size of about 20-60 nm and generally has good permeability.

[0029] (2) Preparation of hollow porous yeast carriers (Pae@YGPs) loaded with active molecules of traditional Chinese medicine Deionized water was prepared into an acidic solution with a pH value of 4.9-5.0 using hydrochloric acid, and paeoniflorin (Pae) ​​was added to 2 mL of the acidic solution to obtain a paeoniflorin solution with a concentration of 4 mg / mL. 8 mg of YGPs were added to the paeoniflorin solution and magnetically stirred for 12 h at room temperature to load Pae into YGPs to obtain a Pae@YGPs solution.

[0030] (3) Preparation of multilayer microspheres based on hollow porous yeast carriers (Pae / Lp@YGPs) 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 stirred magnetically at room temperature for 12 h. 1 mL of sodium tripolyphosphate (TPP) solution (0.1% w / v, pH 4.9-5.0) was added to 10 4After mixing evenly with Lactobacillus paracasei (Lp) at CFU / mL, add it dropwise to the reaction system and stir it magnetically in an ice bath for 30 min. Let it stand at 4°C for 12 h, centrifuge it at 2000 rpm for 10 min, and discard the supernatant. Wash the precipitate three times with 0.5 mL of sodium tripolyphosphate (TPP) solution (0.01% w / v, pH 4.9~5.0). Finally, vacuum freeze-dry it, and the obtained microcapsules are stored in a -20°C refrigerator for later use. Finally, YGPs microcapsules with lactobacilli wrapped on the outer layer and Pae loaded on the inside are obtained, and the final product is recorded as Pae / Lp@YGPs. See the schematic diagram of the preparation process. Figure 1 .

[0031] Comparative Example 1: Preparation of Pae@YGPs CS The difference from Example 1 is that Lactobacillus paracasei is not added, and Pae@YGPs CS is finally obtained.

[0032] Example 2: Characterization (1) Transmission electron microscopy image of Pae / Lp@YGPs The samples were observed using a Hitachi HT7700 transmission electron microscope (Hitachi, Tokyo, Japan). A small amount of dried 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 Pae / LP@YGPs prepared in step (3) of Example 1 were respectively dropped on a 200-mesh copper grid. After standing for 3 minutes, excess sample solution was removed with filter paper. Subsequently, the samples were stained with 3% uranyl acetate for 5 minutes, the excess staining solution was removed, and the samples were dried at room temperature for 24 hours. Finally, TEM was used for observation and photography to record the morphology of the samples. The results are shown in Figure 2 .

[0033] according to Figure 2 It can be seen that after being treated with alkali, acid and organic solvent, the cytoplasmic components of Yeast were significantly lost, resulting in a decrease in the contrast of YGPs. After loading Pae, the contrast increased significantly. The network structure formed by cross-linking chitosan and sodium tripolyphosphate was attached to the surface of Pae@YGPs, thereby providing a supporting site for Lp, and Pae / Lp@YGPs was successfully prepared.

[0034] (2) Pae / Lp@YGPs dynamic light scattering graph The hydrodynamic diameter and zeta potential of the prepared dry brewer's yeast (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 / LP@YGPs prepared in step (3) of Example 1 were measured using a Malvern Zetasizer Nano ZS90 laser scattering particle size analyzer (Malvern Panalytical, Malvern, UK).

[0035] according to Figure 3 It can be seen that the particle size increased under the layer-by-layer encapsulation of Pae / LP@YGPs. The negative charge of YGPs after Yeast treatment decreased, and the negative charge decreased further after loading Pae. After encapsulation of chitosan, the Zeta potential turned positive, and after adding Lp, the positive potential decreased slightly.

[0036] Comparative Example 2: Preparation of multilayer microspheres based on hollow porous yeast carriers The difference from Example 1 is that in step (3), the pH is adjusted to 4.0, and the final product is recorded as Pae / Lp@YGPs-4.0.

[0037] Comparative Example 3: Preparation of multilayer microspheres based on hollow porous yeast carriers The difference from Example 1 is that in step (3), the pH is adjusted to 6.0, and the final product is recorded as Pae / Lp@YGPs-6.0.

[0038] Comparative Example 4: Preparation of Hollow Porous Silica Carrier Multilayer Microspheres The difference from Example 1 is that the hollow porous yeast carrier is replaced by an equal amount of hollow porous silica carrier, and the final product is recorded as Pae / Lp@SiO2.

[0039] The preparation method of the hollow porous silica carrier is as follows: 2.5~5 g of Pluronic F127 is added to 50 mL of ethanol: deionized water in a volume ratio of 1:1. Stir in a 40~45℃ water bath for 2 h to ensure that Pluronic F127 is completely dissolved to form a transparent solution. Cool to 25℃ and continue stirring for 30 min to ensure uniform distribution of micelles. 1~3 mL of ammonia water (25-28%) is added dropwise as a catalyst, and the pH is controlled between 9-11. The hydrolysis product is further condensed to form SiO2 sol, and stirring is continued for 24 h. Tetraethoxysilane (TEOS) is added dropwise at a rate of 1 mL / 10 min, and magnetic stirring is performed at room temperature for 6~12 h to ensure uniform deposition of the SiO2 shell. After centrifugation at 12000 rpm for 10 min, wash 3~5 times with ethanol: deionized water in a volume ratio of 1:1 to remove unreacted products. Stir in 10-20 mL 0.1 M HCl / ethanol solution for 12 h. Ultrasonicate for 10-15 min to disperse the particles, stir at 40°C for 6-8 h, centrifuge at 12000 rpm for 10 min, discard the supernatant, wash 3-5 times with ethanol: deionized water = volume ratio 9:1 to remove residual hydrochloric acid. Dry in a drying oven at 60-120°C for more than 12 h to obtain a hollow porous silica carrier with a particle size of 500 nm-10 μm.

[0040] Test Example 1: Antibacterial properties of Pae In order to investigate the inhibitory effect of paeoniflorin (Pae) ​​on Staphylococcus aureus and lactic acid bacteria, the inhibitory effect of Pae on Staphylococcus aureus ( S. aureus ) and Lactobacillus paracasei ( L. paracasei ) were used to determine the antibacterial properties. Pae was mixed with deionized water to prepare Pae solutions of 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), with the Pae concentration of 0 mg / mL as the control group; S. aureus (1 × 10 6 CFU / mL, 50 µL) and L. paracasei (1 × 10 5 CFU / mL, 50 µL) were co-cultured in a 96-well plate for 12 h. After the culture, the absorbance at 600 nm was measured using an iMark microplate reader. The results are shown in Figure 4 .

[0041] according to Figure 4It can be seen that within a certain low concentration range of Pae, the growth of Staphylococcus aureus can be inhibited, while high concentration Pae promotes the growth of Staphylococcus aureus. High concentration Pae inhibits the growth of lactic acid bacteria. This shows that Pae has different effects on different microorganisms and exhibits different biological activities. Pae needs to be maintained in a low concentration range to inhibit Staphylococcus aureus without affecting the growth of lactic acid bacteria. Therefore, the release concentration of Pae should be in the range of 0.0125~0.4 mg / mL. By slowly releasing Pae to maintain a low concentration level of Pae in the vagina, the growth of pathogenic bacteria can be specifically inhibited without affecting the growth of lactic acid bacteria.

[0042] Test Example 2: Active ingredient release test The Pae / Lp@YGPs prepared in Example 1 and the microspheres prepared in Comparative Examples 2 to 4 were plotted for the release curve of paeoniflorin. The specific method was as follows: 50 mg of the above microspheres were dispersed in 7 mL of vaginal simulated fluid (SVF, composed of: 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, pH adjusted to 5.0±0.2); then placed in a 15 mL centrifuge tube at 37°C and 100 rpm shaker, and 300 μL of the release solution was collected at regular intervals. After the release solution was centrifuged 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. Add 200 μL of fresh SVF to the remaining 100 μL release solution, mix well and return to the 7 mL release system. Record the absorbance of each well at each time point and calculate the cumulative release of Pae at different times. The experiment was repeated three times and the results are shown in Figure 5 The Pae / Lp@YGPs were photographed at 4 h, 12 h, and 36 h using a Hitachi HT7700 transmission electron microscope (Hitachi, Tokyo, Japan). Figure 6 .

[0043] according to Figure 5It can be seen that, compared with Comparative Examples 2-4, after 36 h, Example 1 has a higher release of Pae / Lp@YGPs than other groups, while Pae / Lp@YGPs-4.0 and Pae / Lp@YGPs-6.0 have a lower release. Pae / Lp@SiO2 is released faster in the early stage, which makes the Pae concentration in the vagina too high, which easily inhibits the growth of lactic acid bacteria and promotes the growth of Staphylococcus aureus. Pae / Lp@YGPs can encapsulate more Pae and has a sustained release effect, which keeps Pae in a low concentration range, inhibiting Staphylococcus aureus without harming lactic acid bacteria.

[0044] according to Figure 6 It can be seen that the contrast of Pae / Lp@YGPs prepared in Example 1 gradually decreased within 36 h, indicating that the loaded Pae has been gradually released.

[0045] Test Example 3: Cytotoxicity Test The Pae / Lp@YGPs prepared in Example 1 were tested for HUVEC cytotoxicity using the CCK-8 experiment. After taking the cells out of the liquid nitrogen, they were immediately placed in a 37°C water bath and shaken rapidly to melt them quickly to reduce the damage of ice crystals to the cell structure. The cell suspension was then quickly transferred to 2 mL of DMEM complete medium and centrifuged at 1200 rpm for 5 min. After discarding the supernatant, the cells were resuspended in 1 mL of DMEM complete medium, slowly blown to mix, and then the cells were transferred to 7 mL of DMEM complete medium. Subsequently, the cells were cultured in a 37°C, 5% CO2 incubator. After the cells were well attached to the wall, they were digested and counted. The counted HUVEC cells were diluted to 2.5 × 10 with DMEM medium. 4 cell / mL, 100 µL per well, placed in a 96-well plate and cultured for 24 h. Subsequently, Pae / Lp@YGPs was prepared with DMEM medium to a concentration of 1 µg / mL~50 µg / mL and sterilized by ultraviolet irradiation for 24 h. 10 µL of the above test solution was added to each well and incubated in a 5% CO2 incubator at 37°C for 24 h. Subsequently, 10 µL of CCK-8 solution was added to each well, and after culturing for 1~4 h, the absorbance at 450 nm was measured using an iMark microplate reader.

[0046] The cell survival rate was calculated according to the following formula: Cell survival rate = [ (As - Ab) / (Ac - Ab) ] × 100%. Where 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; the blank group was not added with HUVEC cells or drug treatment, but only DMEM culture medium was added). The results are shown in Figure 7 .

[0047] according to Figure 7 It can be seen that with the increase of Pae / Lp@YGPs concentration, the cell survival rate decreased. The lower concentration of Pae / Lp@YGPs had almost no significant effect on the cells, and the cell number increased slightly, but at a higher concentration of 50.0 μg / mL, the cell survival rate decreased significantly, indicating that the concentration of Pae / Lp@YGPs at 25.0 μg / mL and below had no significant effect on the cells.

[0048] Test Example 4: Biocompatibility Test The biosafety of Pae / Lp@YGPs prepared in Example 1 was tested by red blood cell hemolysis experiment. Blood was collected from the 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. Centrifuge at 3000 rpm for 10 min, discard the plasma and white blood cell layer, and collect the red blood cells. Wash the red blood cells 3 times with 0.9% saline, centrifuge for 5 min each time. Resuspend the washed red blood cells in 0.9% saline to prepare a 2% red blood cell suspension. Next, 1 µg / mL~8 µg / mL Pae / Lp@YGPs solution was prepared using 0.9% saline, and 100 µL of red blood cell suspension was added to each solution and incubated at 37°C for 1~4 h. After the incubation, centrifuge at 3000 rpm for 10 min, take a photo to collect images, and collect the supernatant. Finally, the absorbance value of the supernatant was measured at 570 nm using an iMark microplate reader.

[0049] The formula for calculating 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, OD p is the absorbance of the positive control group (0.9% saline and 1% Triton X-100 were used as negative and positive controls). All experiments were repeated three times. HR < 5%: biocompatibility is met, HR 5-10%: there is a certain risk of hemolysis, HR > 10%: obvious hemolysis, not suitable as a biomaterial or drug. The results are shown in Figure 8 .

[0050] according to Figure 8It can be seen that compared with the positive control group (Triton X-100), the hemolytic activity of Pae / Lp@YGPs was less than 5%, showing lower hemolytic activity, indicating that the Pae / Lp@YGPs composite microspheres had good biocompatibility.

[0051] Test Example 5: Animal Test The experiment was divided into 6 groups: normal group, model group, Pae / Lp@YGPs group, YGPs group, Pae group, and Lp group, with 5 rats in each group. SD rats (6 weeks old, purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.) were used as experimental subjects to establish a rat vaginal model of Staphylococcus aureus infection.

[0052] The specific method is: before infection, 2.5 mg / kg β-estradiol was injected intraperitoneally every day for 3 consecutive days to promote estrus in SD rats, which is more likely to cause infection. Subsequently, 1×10 8 CFU / mL Staphylococcus aureus (ATCC6538) 50 μL / day for 7 consecutive days to establish an aerobic bacterial vaginitis model infected with Staphylococcus aureus. After the model was established, the rats in the experimental group showed typical AV symptoms, including increased vaginal discharge, vulvar redness and swelling, and local inflammatory reaction, indicating that the model was successfully constructed; the rats in the normal group did not receive any treatment. The rats in the experimental group were divided into 5 groups. The model group was given 0.9% normal saline, the Pae / Lp@YGPs group was given Pae / Lp@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 Lp group was given lactic acid bacteria for treatment. The dosage of each group was 0.25 mg / kg. After 7 consecutive days of administration, the rats were euthanized and the vaginal tissues were taken for histopathological observation.

[0053] Seven days after modeling, Gram staining was used to observe the main flora in the vagina. The results are shown in Fig. 9 It can be seen that the vaginal secretions are mainly composed of purple Staphylococcus aureus.

[0054] Use a sterile cotton swab to collect a sample of vaginal discharge. The results are shown in Fig.10 It can be seen that compared with the normal group, the model group has a significant increase in yellow vaginal secretions, indicating that the modeling is successful.

[0055] Before and after treatment, vaginal samples were collected using sterile cotton swabs, resuspended in 2 mL of saline, and diluted and inoculated into mannitol medium and MRS medium for selective culture and colony counting. The results are shown in Fig.11. It can be seen that the number of Staphylococcus aureus in the vagina of rats after modeling increased significantly, while the number of lactic acid bacteria decreased significantly. After 7 days of continuous treatment, the number of Staphylococcus aureus in the Pae / Lp@YGPs group, YGPs group, Pae group and Lp group decreased, among which the number of Staphylococcus aureus in the Pae / Lp@YGPs group decreased significantly, while the number of lactic acid bacteria increased significantly. Although the Pae group had an inhibitory effect on Staphylococcus aureus, the number of lactic acid bacteria did not increase, which is not conducive to the recovery of probiotic lactobacilli in the vagina.

[0056] After 7 days of treatment, the rats were anesthetized and euthanized, and then the vaginal tissues were obtained by dissection and fixed in 4% paraformaldehyde solution for histopathological analysis. The results are shown in Fig.12 It can be seen that the redness and swelling of the vaginal appearance and tissue inflammation of the rats in the Pae / Lp@YGPs group were alleviated, the morphology of epithelial cells gradually returned to normal, the cells were arranged more closely, the morphology was regular, and angiogenesis was promoted.

[0057] The thickness of the epithelium was measured and the results are shown in Fig.13 It can be seen that the thickness of the epithelial tissue in the rats in the Pae / Lp@YGPs group increased significantly.

[0058] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. 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 4.9-5.0 and crosslinking; the crosslinking agent is chitosan and sodium tripolyphosphate; and the lactic acid bacteria is Lactobacillus paracasei.

2. The method for preparing multilayer microspheres based on hollow porous yeast carriers according to claim 1, characterized in that: The following steps are involved: (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; (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 evenly 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 traditional Chinese medicine active 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 the hollow porous yeast carrier.

3. The preparation method according to claim 2, characterized in that: In step (1), the yeast is Saccharomyces cerevisiae.

4. The preparation method according to claim 2, 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.

5. The preparation method according to claim 2, characterized in that: In step (2), the mass ratio of the hollow porous structure yeast carrier to the active molecule of traditional Chinese medicine is 1-4:4-1; and the stirring time is 12 h.

6. The preparation method according to claim 5, characterized in that: The active molecule of traditional Chinese medicine is paeoniflorin.

7. The preparation method according to claim 2, characterized in that: In step (3), the mass ratio of the hollow porous yeast carrier loaded with traditional Chinese medicine active molecules, chitosan and sodium tripolyphosphate is 0.8:30:

1.

8. The preparation method according to claim 2, 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.

9. The preparation method according to claim 2, characterized in that: 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.

10. Use of the hollow porous yeast carrier multilayer microspheres according to claim 1 in preparing 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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