A composite probiotic microsphere and a preparation method and application thereof

Abstract

The application discloses a kind of composite probiotic microspheres and its preparation method and application.The method of the present application first uses the dehydration condensation of-COO- in sodium alginate and-NH2 in aminated beta-glucan, to obtain stable Alg-betaG crosslinking material, then using O / W complex emulsion desolventizing microfluidic device to prepare double crosslinking structure composite probiotic microspheres, the microspheres prepared by the method of the present application are uniform in particle size, about 50-100 μm, the particle size distribution is monodisperse, with good microsphere morphology.Animal experiment results prove that the microspheres can significantly enhance the intestinal colonization activity of probiotics, are beneficial to the long-term treatment of anti-in-stent restenosis, and have a certain therapeutic effect on atherosclerosis.

Description

Technical Field

[0001] This invention relates to the field of cardiovascular disease treatment, and in particular to a compound probiotic microsphere, its preparation method, and its application. Background Technology

[0002] Cardiovascular and cerebrovascular diseases (CVD) are the leading cause of death worldwide, with coronary atherosclerotic heart disease (CHD), primarily caused by acute myocardial infarction, being the main reason for its high mortality rate. According to the latest data, the number of CHD deaths worldwide has reached 17.9 million, accounting for 30% of all deaths globally. Atherosclerosis (AS), as the main pathological basis of CVD, is the preferred treatment for acute coronary artery stenosis or occlusion caused by unstable AS plaque rupture, salvaging dying myocardium, and reducing CHD mortality.

[0003] PCI procedures mainly include coronary angioplasty and stent implantation. Because stent implantation avoids the elastic recoil of blood vessels after simple balloon dilation and offers better coronary recanalization stability, it has gradually become the primary procedure for treating coronary heart disease (CHD). While stent implantation provides CHD patients with an immediate "window of opportunity," in-stent restenosis (ISR) remains a major challenge, significantly reducing long-term prognosis and leading to CHD recurrence. However, current strategies for preventing and treating ISR are insufficient. ISR without rapid and effective intervention can cause irreversible cardiac damage, ultimately leading to acute cardiac death. Research data indicates that the incidence of ISR after stent implantation exceeds 10%, and approximately 25% of ISR patients suffer acute myocardial infarction due to a lack of effective treatment, ultimately resulting in death. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a composite probiotic microsphere.

[0005] Another objective of this invention is to provide a method for preparing the above-mentioned composite probiotic microspheres by providing acid protection and precise intestinal release characteristics of the cross-linked shell.

[0006] Another objective of this invention is to significantly improve the ability of probiotics to regulate the homeostasis of the gut-plaque axis and their biosafety, and to provide the application of the above-mentioned compound probiotic microspheres. The objective of this invention is achieved through the following technical solution: A method for preparing compound probiotic microspheres includes the following steps: (1) The sodium alginate solution and the ethylenediamine-ammoniated β-glucan solution were mixed and stirred to carry out the cross-linking reaction. After the reaction was completed, the mixture was dialyzed and dried to obtain Alg-βG cross-linked material; (2) Add Lactobacillus reuteri, 3,3-dimethyl-1-butanol, inulin, and Alg-βG crosslinking material to water to prepare a dispersed phase solution. Add Span 80 and calcium iodide to liquid paraffin to prepare a continuous phase. Synthesize O / W microspheres using a microfluidic device. After washing, obtain composite probiotic microspheres.

[0007] The concentration of the sodium alginate solution in step (1) is 1-4 wt%.

[0008] The concentration of the ethylenediamine-amylated β-glucan solution in step (1) is 0.5 to 2 wt%.

[0009] The volume ratio of sodium alginate solution and ethylenediamine-ammonia-modified β-glucan solution in step (1) is 3 to 7:1.

[0010] The conditions for the crosslinking reaction in step (1) are: stirring at 300-600 rpm and reacting at 2-6°C for 3-5 hours.

[0011] The dialysis described in step (1) is dialysis in deionized water using a dialysis bag with a capacity of 3000-4000 Da.

[0012] The final concentration of *Lactobacillus reuteri* in the dispersed phase in step (2) is 1–2 × 10⁻⁶. 9 CFU / mL.

[0013] The final concentration of 3,3-dimethyl-1-butanol in the dispersed phase in step (2) is 0.5-2 wt%.

[0014] The final concentration of inulin in the dispersed phase in step (2) is 1-3 wt%.

[0015] The final concentration of the Alg-βG crosslinking material in the dispersed phase in step (2) is 1 to 3 wt%.

[0016] The mass ratio of liquid paraffin, Span 80 and calcium iodide in step (2) is 90-96:3-10:0.1-0.3.

[0017] The conditions for the synthesis of the microfluidic device described in step (2) are a flow rate of 2 to 5 μL / min for the dispersed phase and a flow rate of 300 to 400 μL / min for the continuous phase.

[0018] A compound probiotic microsphere was prepared by the above-described preparation method.

[0019] The above-mentioned compound probiotic microspheres are used in the preparation of drugs for the treatment of atherosclerosis.

[0020] The above-mentioned compound probiotic microspheres are used in the preparation of drugs to alleviate in-stent restenosis.

[0021] The above-mentioned compound probiotic microspheres are used in the preparation of drugs to alleviate intestinal leakage. The present invention has the following advantages and effects compared with the prior art: (1) The method of the present invention first uses the dehydration condensation of -COO- in sodium alginate and -NH2 in amino-β-glucan to obtain a stable Alg-βG crosslinked material. Then, an O / W double emulsion desolventizing microfluidic device is used to prepare composite probiotic microspheres with double crosslinked structure. The microspheres prepared by the method of the present invention have uniform particle size, about 50-100 μm, and the particle size distribution is monodisperse, with good microsphere morphology. (2) In the microspheres of this invention, *Lactobacillus reuteri* extensively inhibits the growth of harmful bacteria and the secretion of LPS by secreting reuterin, which is beneficial for repairing intestinal barrier function and regulating microbial homeostasis. On the other hand, it promotes M2-type MΦ polarization and reduces systemic inflammatory response by producing SCFAs after fermenting dietary fiber, which is beneficial for improving the inflammatory immune microenvironment within the scaffold and provides a strong guarantee for the comprehensive regulation of the "gut-plaque axis" that induces ISR. 3,3-Dimethyl-1-butanol (DMB), as a newly proposed targeted inhibitor of TMAO synthase, enhances the effect of *Lactobacillus reuteri* on the microecological remodeling of the "gut-plaque axis" of ISR and the clearance of its harmful metabolites in a "strong combination" manner. DMB can competitively block the catalytic decomposition of choline-containing foods by the key enzyme choline trimethylamine lyase (CutC) in TMAO synthesis, significantly reducing the generation and circulation levels of TMAO in the intestine. DMB can not only reduce the damage to the cardiovascular system by blocking the source synthesis pathway of TMAO, but also inhibit foam cell formation by reducing circulating cholesterol levels, thus shrinking AS plaques in the aortic root to some extent. Attached Figure Description

[0022] Figure 1 An optical microscope image of the microspheres prepared in Example 1 of this invention; Figure 2 The images show SEM images of the microspheres prepared in Example 1 of this invention; the left image shows the microspheres prepared in Example 1, and the right image shows the microspheres prepared in Comparative Example 1. Figure 3 The image shows an energy diffusion X-ray spectrometer image of the microspheres prepared in Example 1 of this invention. Figure 4 The microspheres prepared in Example 1 of this invention are effective against Lactobacillus reuteri (… Lac.r Encapsulation efficiency of 3,3-dimethyl-1-butanol (DMB) and inulin; Figure 5The release efficiency of Lactobacillus reuteri (Lac.r) from the microspheres prepared in Example 1 of the present invention under alkaline simulated intestinal fluid (SIF) conditions containing β-glucanase (β-G); Figure 6 The release efficiency of 3,3-dimethyl-1-butanol (DMB) in the microspheres prepared in Example 1 of the present invention under alkaline simulated intestinal fluid (SIF) conditions containing β-glucanase (β-G); Figure 7 The release efficiency of inulin in the microspheres prepared in Example 1 of this invention under alkaline simulated intestinal fluid (SIF) conditions containing β-glucanase (β-G); Figure 8 This is an IVIS live imaging image from Example 4 of the present invention; Figure 9 This is an image showing the results of oil red staining of the aorta in Example 4 of the present invention; Figure 10 This invention provides an example of using carotid CTA imaging in real time to detect in-stent restenosis in Embodiment 5 of the present invention. Figure 11 This is an H&E staining image of the New Zealand rabbit in-stent restenosis model in Example 5 of the present invention. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0024] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available. Example 1: Preparation method of compound probiotic microspheres (1) Mix 1.5wt% sodium alginate solution and 1wt% ethylenediamine-amylated β-glucan solution at a volume ratio of 5:1 and carry out cross-linking reaction under stirring conditions (300~600r / min). The cross-linking reaction temperature is 4℃, the pH value is 7.5, and the reaction time is 4h. After the reaction is completed, the reactants are transferred to a 3500Da dialysis bag and dialyzed in plasma water at 4℃ to remove unreacted substances. The dialysis water needs to be changed multiple times during the dialysis process until the pH value of the dialysis water is stable (pH=6.5~7). The cross-linked material after dialysis is freeze-dried to obtain Alg-βG cross-linked material. (2) The final concentration is 1×10 9CFU / mL of Lactobacillus reuteri, 1 wt% of 3,3-dimethyl-1-butanol, 2 wt% of inulin, and 1 wt% of the Alg-βG crosslinking material prepared in step (1) were added to deionized water to prepare a dispersed phase solution. A continuous phase was prepared according to the ratio of 94.8 wt% liquid paraffin, 5 wt% Span 80 and 0.2 wt% calcium iodide. The synthesis was carried out using a microfluidic device. The flow rate of the dispersed phase was 3.5 μL / min and the flow rate of the continuous phase was 350 μL / min. The dispersed phase (aqueous phase) was sheared by the continuous phase (oil phase) to form monomolecular droplets, and O / W (water-in-oil) composite probiotic microspheres were obtained. The microspheres were cleaned with a mixture of 95% isopropanol and 5% n-hexane, collected and stored at 4℃.

[0025] The preparation and application of the microfluidic chip were described in the reference “Chen M, Guo X, Shen L, et al. Monodisperse CaCO3-loaded gelatin microspheres for reversing lactic acid-induced chemotherapy resistance during TACE treatment. [J]. International Journal of Biological Macromolecules, 2023:, 123160. DOI:10.1016 / j.ijbiomac.2023.123160. Comparative Example 1 The composite probiotic microspheres were synthesized according to the method of Example 1, except that the sodium alginate solution added in step (1) was adjusted to 1 wt%. Comparative Example 2 The composite probiotic microspheres were synthesized according to the method of Example 1, except that the sodium alginate solution added in step (1) was adjusted to 4 wt%. Example 2 Characterization of probiotic microspheres 2.1 Observation under an optical microscope The microspheres synthesized in Example 1 were imaged under a 10× optical microscope. The experimental results are as follows: Figure 1 As shown in the figure, the microspheres prepared by the present invention have uniform particle size, ranging from 50 to 100 μm, and exhibit monodispersity in particle size distribution, thus possessing good microsphere morphology.

[0026] 2.2 SEM characterization After the microspheres synthesized in Example 1 and Comparative Examples 1-2 were dropped onto a silicon mesh and air-dried, the particle size of the microspheres was examined using a scanning electron microscope (SEM) at 1 kV, and the elemental composition and distribution of the microspheres were examined using an energy dispersive spectroscopy (EDS) instrument.

[0027] SEM photos such as Figure 2 As shown, the energy diffusion X-ray spectrometer image is as follows: Figure 3 As shown. From Figure 2 and 3 As can be seen, the surface of the microspheres prepared in Example 1 shows a sodium alginate-β-glucan cross-linked structure and contains elements such as C, Ca, and O, confirming the successful preparation of monodisperse microspheres. The microspheres prepared in Comparative Example 1 are softer, more prone to collapse and breakage, and cannot achieve effective encapsulation. The microspheres in Comparative Example 2 showed obvious cracking and leakage, also failing to achieve effective encapsulation, demonstrating that the concentration of sodium alginate significantly affects the formation of the microspheres.

[0028] 2.3 Encapsulation efficiency test 2.3.1 Determination of encapsulation efficiency of Lactobacillus reuteri First, a 2% sodium citrate solution was prepared as the release medium. 1 g of dried microspheres was weighed and placed in 9 mL of the release solution, and the mixture was incubated at 37 °C and 100 rpm until the microspheres were completely lysed. Then, 200 μL of the lysis buffer was evenly spread onto MRS solid medium, and the number of viable *Lactobacillus reuteri* encapsulated in the microspheres was determined by plate counting. The encapsulation efficiency was calculated using the following formula: Encapsulation rate of Lactobacillus reuteri (%) = (N / N0) × 100% In the formula: N is the number of viable Lactobacillus reuteri encapsulated in the microspheres, and N0 is the total number of viable Lactobacillus reuteri initially added to the system.

[0029] 2.3.2 Determination of DMB encapsulation efficiency (gas chromatography) The encapsulation efficiency of DMB in microspheres was determined by gas chromatography. First, a standard curve was plotted: DMB stock solution was gradually diluted with deionized water to prepare a series of standard solutions with concentrations of 0, 5, 10, 20, 50, 100, 200, and 300 ppm. Using the external standard method, a standard curve was established with peak area as the ordinate and DMB concentration as the abscissa.

[0030] Take 2 mL of the microsphere lysis buffer prepared in section 2.3.1 and perform GC quantitative analysis. The encapsulation efficiency is calculated using the following formula: DMB encapsulation rate (%) = (W1 / W2) × 100% In the formula: W1 is the mass of DMB encapsulated in the microspheres (mg), and W2 is the total mass of DMB initially added to the system (mg).

[0031] 2.3.3 Determination of inulin encapsulation efficiency (enzyme-linked immunosorbent assay) The encapsulation efficiency of inulin in microspheres was analyzed using enzyme-linked immunosorbent assay (ELISA). The experiment was conducted strictly in accordance with the instructions for the inulin ELISA kit. (1) Use the series of standards provided in the kit to plot a standard curve; (2) Take 2 mL of the microsphere lysis buffer prepared in 2.3.1 as the sample to be tested; (3) Add the standard and the sample to be tested (with duplicates) to the antibody-coated wells in sequence, add enzyme-labeled reagent, incubate at 37 ℃ for 60 min and wash 5 times; (4) Add color developer A and B in sequence, and develop color at 37 ℃ in the dark for 15 min; (5) Add a stop solution to terminate the reaction; (6) Within 15 minutes after the reaction is terminated, the absorbance of each well is measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The inulin content in the sample is calculated based on the standard curve.

[0032] The formula for calculating the inulin encapsulation efficiency is the same as the formula for the DMB encapsulation efficiency in 2.3.2.

[0033] Experimental results are as follows Figure 4 As shown, the microspheres prepared in Example 1 were effective against Lactobacillus reuteri (… Lac.r The encapsulation efficiency of 3,3-dimethyl-1-butanol (DMB) and inulin was determined, and the results are as follows: Figure 4 As shown, the encapsulation efficiency of Lactobacillus reuteri (Lac.r) was 86.7%, that of 3,3-dimethyl-1-butanol (DMB) was 81.7%, and that of inulin was 79.2%. Example 3: Simulated Gastric Fluid Stability Test This experiment tested the release efficiency of the microspheres prepared in Example 1 in detecting Lactobacillus reuteri (Lac.r), 3,3-dimethyl-1-butanol (DMB), and inulin under simulated gastric juice (SGF), alkaline simulated intestinal juice (SIF), and alkaline simulated intestinal juice (SIF) containing β-glucanase (β-G). The specific steps are as follows: Preparation of simulated gastric juice (SGF): Dissolve 16.4 mL of 0.1 mol / L HCl solution and 10 g of pepsin in deionized water, and finally bring the volume to 1000 mL and adjust the pH to 1.2 to obtain simulated gastric juice.

[0034] Preparation of simulated intestinal fluid (SIF): Dissolve 6.8g of potassium dihydrogen phosphate in 500ml of water, adjust the pH to 7.4 with 0.1mol / sodium hydroxide solution, and dissolve 10g of pancreatic enzyme in an appropriate amount of water. Mix the two solutions and dilute with water to 1000ml to obtain simulated intestinal fluid.

[0035] Preparation of simulated intestinal fluid containing β-glucanase (β-G): Add 50 μg / mL of β-glucanase to the simulated intestinal fluid prepared above to obtain simulated intestinal fluid containing β-glucanase (β-G).

[0036] The microspheres prepared in Example 1 were added to the three simulated solutions prepared above, with an addition amount of 1 g / L. The solutions were then treated with constant temperature shaking (100 rpm) in a 37°C water bath for 24 h. During this period, samples were taken periodically and the release of components was detected by HPLC (the detection method here is the same as the experimental method for detecting each component of the encapsulation efficiency).

[0037] like Figures 5-7 As shown. It can be seen that *Lactobacillus reuteri* (… Lac.r The rapid response and decomposition of 3,3-dimethyl-1-butanol (DMB) and inulin demonstrate that the sodium alginate-β-glucan cross-linked microspheres co-loaded with Lac.r, DMB, and inulin prepared in this invention possess characteristics such as reasonable design, stable process, and excellent performance. Example 4: Animal experiments with intestinal colonization 4.1 Animal Culture The experimental animals used in this study were all 4-6 weeks old, male ApoE - / - Mice, C57BL / 6J mice, were purchased from Shrek Animal Co., Ltd. in Shanghai, China. All experimental procedures complied with the requirements of the Medical Ethics Committee of Lishui University School of Medicine.

[0038] 4.2 Experimental Procedure This experiment verifies the effect of the microspheres prepared in Example 1 on probiotics. Lac.r To investigate the effect of post-release intestinal colonization activity, *Lactobacillus reuteri* was cultured to OD=1.0, and 1 mL of 1×10⁻⁶ cells was collected. 8 CFU / mL bacterial suspension was mixed with 15µL Cy5.5 working solution (100μM), incubated at 37 ℃ in the dark for 60 min, and washed twice by centrifugation with PBS to obtain Cy5.5-labeled Lactobacillus reuteri. The compound probiotic microspheres carrying Cy5.5 dye labeling were then prepared according to the steps in Example 1.

[0039] The mice were divided into two groups of six each: Control group: Lac.rGroup 1, which consists of only Lactobacillus reuteri, using 1 mL of 1×10 8 Lactobacillus reuteri suspension labeled with Cy5.5 dye was administered by gavage; Experimental group: The microspheres prepared in Example 1 were administered by gavage with an equal volume of compound probiotic microspheres prepared from a suspension of Lactobacillus reuteri labeled with Cy5.5 dye, approximately 300 μL 2 wt%.

[0040] After gavage, in vivo imaging was performed using an IVIS in vivo imaging system at time points of 2h, 4h, 24h, 48h, and 96h. After the experiment, mice were euthanized, and the aorta was dissected and stained with Oil Red chromatograph.

[0041] 4.3 Experimental Results Experimental results are as follows Figures 8-9 As shown, where Figure 8 As shown in the in vivo imaging results, it can be seen that in vivo imaging with equal amounts of Cy5.5 fluorescent dye... Lac.r Under the premise of [specific conditions], the microspheres prepared in Example 1 [were observed] in the intestines at all time points 4 hours after oral administration. Lac.r The fluorescence intensity was significantly stronger than that of other groups, which further confirms that the novel composite probiotic microspheres prepared in this invention can significantly enhance the intestinal colonization activity of probiotics, which is beneficial for long-term treatment against in-stent restenosis. Figure 9 The results of oil red staining of the aorta showed that the number of vascular plaques in the experimental group mice was significantly reduced compared with that in the control group, proving that the preparation has a certain therapeutic effect on atherosclerosis. Example 5: Animal Model Experiment of Stent Restenosis 5.1 Animal Culture The experimental rabbits used in this study were all male New Zealand White rabbits (2.5-3 kg) purchased from Shrek Animal Co., Ltd. in Shanghai, China.

[0042] 5.2 Experimental Procedure This study used 12 healthy male New Zealand white rabbits (approximately 3 months old, weighing 2.5–3 kg) for in vivo vascular stent implantation experiments. The experimental animals were randomly divided into two groups (n=6 in each group): the experimental group was administered a suspension of compound probiotic microspheres prepared in Example 1 at a concentration of 2 wt% by gavage after modeling was completed, at a dose of 10 mL / kg each time, while the control group was administered an equal volume of physiological saline by gavage.

[0043] The stent implantation experiment was performed under general anesthesia via intravenous injection of sodium pentobarbital (25 mg / ml, 0.7 ml / kg). The common carotid artery (CCA) and its branches (internal and external carotid arteries) were dissected and exposed. A WE43 magnesium alloy stent was delivered to the target segment of the CCA via a balloon catheter through the external carotid artery (ECA) route. The stent was inflated at 8 atm and maintained for 40 seconds to allow apposition. After catheter withdrawal, the ECA was ligated, and ampicillin sodium solution (10 mg / ml, 1 ml / kg) was administered intramuscularly to prevent infection. Postoperatively, dual antiplatelet therapy with aspirin (100 mg / kg) and clopidogrel (3 mg / kg) was administered for 3 consecutive days.

[0044] Carotid CTA follow-up examinations were performed regularly on days 0, 7, 14, and 28 after stent implantation. The imaging data were analyzed using RadiAntDICOM Viewer (version 2020.2.3). After the experiment, the patient was euthanized by sodium pentobarbital overdose, and intestinal tissue and stent restenosis plaque tissue were harvested for H&E staining.

[0045] 5.3 Experimental Results In vivo restenosis in different experimental groups was as follows: Figure 10 As shown, the results indicate that oral administration of the microspheres prepared in Example 1 can significantly reduce the occurrence of in-situ in-stent restenosis.

[0046] H&E staining was performed on intestinal tissue and stent restenosis plaque tissue from an orthotopic New Zealand rabbit stent restenosis model. The results are as follows: Figure 11 As shown in the figure, the microsphere treatment of the present invention can effectively reduce the formation of restenosis plaques while improving intestinal leakage. The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing compound probiotic microspheres, characterized in that... Includes the following steps: (1) The sodium alginate solution and the ethylenediamine-ammoniated β-glucan solution were mixed and stirred to carry out the cross-linking reaction. After the reaction was completed, the mixture was dialyzed and dried to obtain Alg-βG cross-linked material; (2) Add Lactobacillus reuteri, 3,3-dimethyl-1-butanol, inulin, and Alg-βG crosslinking material to water to prepare a dispersed phase solution. Add Span 80 and calcium iodide to liquid paraffin to prepare a continuous phase. Synthesize O / W microspheres using a microfluidic device. After washing, obtain composite probiotic microspheres.

2. The preparation method according to claim 1, characterized in that: The concentration of the sodium alginate solution in step (1) is 1–4 wt%. The concentration of the ethylenediamine-amylated β-glucan solution in step (1) is 0.5–2 wt%; The volume ratio of sodium alginate solution and ethylenediamine-ammonia-modified β-glucan solution in step (1) is 3 to 7:

1.

3. The preparation method according to claim 1, characterized in that: The conditions for the crosslinking reaction in step (1) are: stirring at 300-600 rpm and reacting at 2-6°C for 3-5 hours.

4. The preparation method according to claim 1, characterized in that: The dialysis described in step (1) is dialysis in deionized water using a dialysis bag with a capacity of 3000-4000 Da.

5. The preparation method according to claim 1, characterized in that: The final concentration of *Lactobacillus reuteri* in the dispersed phase in step (2) is 1–2 × 10⁻⁶. 9 CFU / mL; The final concentration of 3,3-dimethyl-1-butanol in the dispersed phase in step (2) is 0.5–2 wt%. The final concentration of inulin in the dispersed phase in step (2) is 1–3 wt%; The final concentration of the Alg-βG crosslinking material in the dispersed phase in step (2) is 1 to 3 wt%.

6. The preparation method according to claim 1, characterized in that: The mass ratio of liquid paraffin, Span 80 and calcium iodide in step (2) is 90-96:3-10:0.1-0.

3.

7. The preparation method according to claim 1, characterized in that: The conditions for the synthesis of the microfluidic device described in step (2) are a flow rate of 2 to 5 μL / min for the dispersed phase and a flow rate of 300 to 400 μL / min for the continuous phase.

8. A compound probiotic microsphere, characterized in that... It is prepared by any of the preparation methods described in claims 1 to 7.

9. The use of the composite probiotic microspheres according to claim 8 in the preparation of drugs for the treatment of atherosclerosis.

10. The use of the composite probiotic microspheres according to claim 8 in the preparation of a drug for relieving in-stent restenosis.

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