Preparation method and application of probiotic-drug co-delivery oral self-emulsifying agent

Abstract

The invention relates to a preparation method and application of a probiotic-drug co-delivery oral self-emulsifying agent, which can effectively solve the problems of preparation of the probiotic-drug co-delivery oral self-emulsifying agent and application of the probiotic-drug co-delivery oral self-emulsifying agent in drugs for treating atherosclerosis. Span80 and Cremophor RH40 are used as a compound emulsifier, PEG400 is used as a co-emulsifier, Res is used as a model drug, L.P resuspended by skim milk powder is used as an internal water phase, FA is used as a target head, a water-in-oil type targeted self-emulsifying preparation is constructed, after the water-in-oil type targeted self-emulsifying preparation is orally taken into an intestinal tract, the water-in-oil type targeted self-emulsifying preparation can be subjected to self-emulsifying phase inversion to form an oil-in-water type nano-emulsion, lactobacillus plantarum of the internal water phase is released and colonized in a gastrointestinal tract, different targets are co-delivered, synergistic interaction is achieved, and the water-in-oil type targeted self-emulsifying preparation is prepared. The preparation method is simple, production and preparation are easy, the biocompatibility is good, the medicine is an innovation in the aspect of medicine for treating atherosclerosis, and the economic and social benefits are remarkable.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a preparation method and application of a probiotic-drug co-delivery oral self-emulsifier. Background Art

[0002] Delivering drugs to the lesion site through a drug carrier delivery system to achieve the purpose of treating the disease is a commonly used technical means of treating diseases. Atherosclerosis is a chronic vascular inflammatory disease associated with oxidative stress and endothelial dysfunction, accompanied by lipid metabolism disorders, and is the main cause of cardiovascular disease. Regulating the body's abnormal lipid metabolism by improving inflammatory stimulation at the plaque site is considered an effective measure for treating atherosclerosis. By designing an oral nano-drug delivery system that can target the atherosclerotic site to regulate the plaque inflammatory microenvironment and allow probiotics to colonize the intestine to regulate lipid metabolism disorders, and achieve heterotarget co-delivery, it is possible to effectively break the vicious cycle of inflammatory infiltration and abnormal lipid metabolism and effectively treat atherosclerosis.

[0003] Resveratrol (Res) is a natural polyphenol compound. Studies have shown that it has beneficial therapeutic effects on a variety of cardiovascular diseases, including atherosclerosis, hypertension, stroke, myocardial infarction, and heart failure. Resveratrol can treat atherosclerosis through multiple mechanisms, including inhibiting inflammatory signaling pathways and alleviating oxidative stress, offering the advantage of integrating multiple targets and effects.

[0004] Lactobacillus plantarum (LP) is a common probiotic belonging to the genus Lactobacillus and a facultative anaerobic bacterium. Studies have shown that Lactobacillus plantarum can lower serum cholesterol levels and regulate lipid metabolism through multiple pathways, including cholesterol assimilation, bile salt hydrolase (BSH) production, activation of the LXR signaling pathway, upregulation of ABCG5 / 8 expression in the small intestine, promoting cholesterol efflux from enterocytes into the intestinal lumen, inhibition of the FXR receptor pathway to promote CYP7A1 gene expression, increased bile acid synthesis in the body, and cholesterol breakdown. It also downregulates the liver HMGCR gene to inhibit cholesterol synthesis, thereby inhibiting cholesterol synthesis and lowering serum cholesterol levels. This modulates lipid metabolism and plays a role in the treatment of atherosclerosis, including improving lipid metabolism disorders.

[0005] Folic acid (FA) is a member of the water-soluble B vitamin family. Studies have shown that folate receptors (FRs) are overexpressed on macrophages in atherosclerotic plaques and are also widely present on the surface of intestinal epithelial cells. Therefore, folic acid can specifically bind to FRs, targeting intestinal epithelial cells and increasing the passage of drugs through these cells. This allows the drugs to be targeted to macrophages in the plaque area, potentially treating atherosclerosis.

[0006] So far, there has been no public report on whether it is possible to organically combine Lactobacillus plantarum and folic acid to jointly deliver resveratrol for the precise and effective treatment of atherosclerosis. Summary of the Invention

[0007] In view of the above situation, in order to overcome the shortcomings of the existing technology, the purpose of the present invention is to provide a preparation method and application of a probiotic-drug co-delivery oral self-emulsifier, which can effectively solve the preparation problem of the probiotic-drug co-delivery oral self-emulsifier and realize its application in the treatment (anti-) atherosclerosis drug.

[0008] The technical solution provided by the present invention is: a method for preparing an oral self-emulsifying agent for co-delivery of probiotics and drugs, which uses olive oil as the oil phase, Span80 (Span 80) and Cremophor RH40 (hydrogenated castor oil polyoxyethylene ether) as composite emulsifiers, PEG400 (polyethylene glycol-400) as a co-emulsifier, Res (resveratrol) as a model drug, LP (Lactobacillus plantarum) resuspended in skim milk powder as the internal aqueous phase, and FA (folic acid) as a target head to construct an oil-in-water targeted self-emulsifying preparation. After oral administration into the intestine, the preparation can self-emulsify and transform into an oil-in-water nanoemulsion. The Lactobacillus plantarum in the internal aqueous phase is then released and colonizes in the gastrointestinal tract, achieving heterogeneous co-delivery and synergistic synergy to accurately and effectively treat atherosclerosis. This is achieved by the following steps: (1) Preparation of Res-SEDDS (resveratrol-self-emulsifying drug delivery system): The composite emulsifier and PEG400 were mixed by ultrasonication for 20 min at a mass ratio of 2:1 to obtain a mixture. 1 g of the mixture and olive oil were weighed at a mass ratio of 4:6-6:4, vortexed for 3 min, and placed in an ultrasonic cleaner with a power of 100 W and ultrasonicated for 20 min to fully mix to obtain a blank SEDDS (self-emulsifying drug delivery system without drug). 5-20 mg of Res was then added, vortexed and ultrasonicated to obtain uniform dispersion to obtain Res-SEDDS. The composite emulsifier is Span80 and Cremophor RH40, with an HBL value of 9 (the same below); (2) Preparation of FA-PEG (folic acid-polyethylene glycol): Take 5-20 mg of FA, dissolve it in 1 ml of DMSO (dimethyl sulfoxide), add 6.2 mg of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride), 4.028 mg of NHS (N-hydroxysuccinimide) and 17.4 μl of triethanolamine, activate it in the dark under nitrogen protection for 0.5-2 h, and add NH2-PEG 3350-NH2 (polyethylene glycol derivative with amino groups at both ends) 75-100 mg, stirred for 6-24 h, added 50 mmol / L Na2CO3 2.5 ml, centrifuged, took the supernatant and dialyzed it with ultrapure water (MWCO = 1000 Da), and lyophilized to obtain FA-PEG; (3) Preparation of CHEMS-NHS (Cholesteryl hemisuccinate-N-hydroxysuccinimide): Dissolve 100 mg of CHEMS and 47.5 mg of NHS in 5 ml of tetrahydrofuran to obtain the first solution. Dissolve 125 mg of DCC (N,N'-dicyclohexylcarbodiimide) in 2.5 ml of tetrahydrofuran to obtain the second solution. Mix the first and second solutions and stir for 6-24 hours. Filter and rotary evaporate to remove the tetrahydrofuran. Add a mixture of 3 ml of tetrahydrofuran and 6 ml of isopropanol. Recrystallize in an ice-water bath, filter, and dry in vacuo to obtain CHEMS-NHS. (4) Preparation of FA-Res-SEDDS (folic acid-resveratrol-self-emulsifying drug delivery system): 4 μmol of FA-PEG and 4 μmol of CHEMS-NHS were dissolved in 3-10 ml of chloroform and reacted in the dark at room temperature under nitrogen protection for 6-24 h. The chloroform was removed by rotary evaporation at 35°C and hydrated with 3 ml of 50 mmol / L Na2CO3. The mixture was centrifuged at 12,000 r / min for 10 min. The supernatant was dialyzed against deionized water with a MWCO of 8,000-14 kDa and lyophilized to obtain FA-PEG-CHEMS. FA-PEG-CHEMS was added to blank SEDDS at a mass ratio of FA-PEG-CHEMS to blank SEDDS of 0.5-3:100. The mixture was ultrasonically mixed and then 5-20 mg of resveratrol was added. The mixture was ultrasonically mixed for 20 min to obtain FA-Res-SEDDS. (5) Preparation of FA-Res-LP-SEDDS (probiotic-drug co-delivery oral self-emulsifier): Take 1 mL of LP with an OD value of 0.1-2, centrifuge to obtain the precipitate, wash with PBS buffer at pH 7.4, and resuspend in 75-150 μL of 10% skim milk powder to obtain a resuspension. Vortex and add dropwise to 1 g of FA-Res-SEDDS to obtain FA-Res-LP-SEDDS.

[0009] The probiotic-drug co-delivery oral self-emulsifier prepared by the above method is used in the preparation of drugs for treating atherosclerosis.

[0010] The preparation method of the present invention is simple, easy to operate and produce, and has good biocompatibility. Oral administration can actively deliver resveratrol to the site of atherosclerotic plaques and simultaneously colonize Lactobacillus plantarum in the intestine, thereby achieving heterotarget co-delivery of oral preparations, improving the bioavailability of oral drugs, accurately regulating the inflammatory microenvironment, and improving lipid metabolism disorders. The multi-pronged approach greatly improves the therapeutic effect, is an innovation in the treatment of atherosclerosis drugs, and has significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Figure 3 Changes in particle size and PDI of FA-Res-SEDDS of the present invention after incubation in (a) SGF and (b) SIF (n=3); Figure 2 Release curves of FA-Res-SEDDS of the present invention in simulated gastric fluid (a) and simulated intestinal fluid (b) (n=3); Figure 3 Colony counts of Free LP, Res-LP-SEDDS, and FA-Res-LP-SEDDS of the present invention after incubation in gastric fluid for different times; Figure 4 Growth curves of Free LP, Res-LP-SEDDS, and FA-Res-LP-SEDDS of the present invention in intestinal fluid (a) and colony counts after 2 and 8 h of incubation (b) (n=3). DETAILED DESCRIPTION

[0012] The specific implementation methods of the present invention are described in detail below with reference to examples and specific situations.

[0013] The present invention is specifically implemented by the following examples. Example

[0014] The present invention provides a method for preparing a probiotic-drug co-delivery oral self-emulsifier, which is achieved by the following steps: (1) Preparation of Res-SEDDS: The composite emulsifier and PEG400 were mixed by ultrasonication for 20 min at a mass ratio of 2:1 to obtain a mixture. 1 g of the mixture and olive oil were weighed at a mass ratio of 4:6, vortexed for 3 min, and placed in an ultrasonic cleaner with a power of 100 W and ultrasonicated for 20 min to fully mix to obtain blank SEDDS. 5-20 mg of Res was then added, vortexed and ultrasonicated to uniformly disperse it to obtain Res-SEDDS. (2) Preparation of FA-Res-SEDDS: Take 5 mg of FA, dissolve it in 1 ml of DMSO, add 6.2 mg of EDC, 4.028 mg of NHS and 17.4 μl of triethanolamine, activate it in the dark under nitrogen protection for 0.5, and add NH2-PEG 3350 -NH2 75 mg, stirred for 8 h, added 50 mmol / L Na2CO3 2.5 ml, centrifuged, took the supernatant and dialyzed it with ultrapure water, MWCO = 1000 Da, and lyophilized to obtain FA-PEG; (3) Preparation of CHEMS-NHS (Cholesteryl hemisuccinate-N-hydroxysuccinimide): Dissolve 100 mg of CHEMS and 47.5 mg of NHS in 5 ml of tetrahydrofuran to obtain the first solution. Dissolve 125 mg of DCC (N,N'-dicyclohexylcarbodiimide) in 2.5 ml of tetrahydrofuran to obtain the second solution. Mix the first and second solutions and stir for 6-24 hours. Filter and rotary evaporate to remove the tetrahydrofuran. Add a mixture of 3 ml of tetrahydrofuran and 6 ml of isopropanol. Recrystallize in an ice-water bath, filter, and dry in vacuo to obtain CHEMS-NHS. (4) Preparation of FA-Res-SEDDS (folic acid-resveratrol-self-emulsifying drug delivery system): 4 μmol of FA-PEG and 4 μmol of CHEMS-NHS were dissolved in 3 ml of chloroform and reacted in the dark at room temperature under nitrogen protection for 24 h. The chloroform was removed by rotary evaporation at 35°C and hydrated with 3 ml of 50 mmol / L Na2CO3. The mixture was centrifuged at 12000 r / min for 10 min. The supernatant was dialyzed with deionized water (MWCO = 8000-14 kDa) and freeze-dried to obtain FA-PEG-CHEMS. FA-PEG-CHEMS was added to blank SEDDS at a mass ratio of 1:100. After ultrasonic mixing, 5 mg of resveratrol was added and ultrasonicated for 20 min to obtain FA-Res-SEDDS. (5) Preparation of FA-Res-LP-SEDDS (probiotic-drug co-delivery oral self-emulsifier): Take 1 mL of LP with an OD value of 0.5, centrifuge to obtain the precipitate, wash with PBS buffer with a pH value of 7.4, take 75-150 μL of 10% skim milk powder to resuspend, vortex and add dropwise to 1 g of FA-Res-SEDDS to obtain FA-Res-LP-SEDDS. Example

[0015] The present invention provides a method for preparing a probiotic-drug co-delivery oral self-emulsifier, which is achieved by the following steps: (1) Preparation of Res-SEDDS: The composite emulsifier and PEG400 were mixed at a mass ratio of 2:1 and ultrasonically mixed for 20 min to obtain a mixture. 1 g of the mixture and olive oil were weighed at a mass ratio of 5:5, vortexed for 3 min, and placed in an ultrasonic cleaner with a power of 100 W and ultrasonicated for 20 min to fully mix to obtain blank SEDDS. 10 mg of Res was then added, vortexed and ultrasonically dispersed to obtain Res-SEDDS. (2) Preparation of FA-PEG: Take 20 mg of FA, dissolve it in 1 ml of DMSO, add 6.2 mg of EDC, 4.028 mg of NHS and 17.4 μl of triethanolamine, activate it in the dark under nitrogen protection for 2 h, and add NH2-PEG 3350 -NH2 100 mg, stirred for 18 h, added 50 mmol / L Na2CO3 2.5 ml, centrifuged, took the supernatant and dialyzed it with ultrapure water, MWCO = 1000 Da, and lyophilized to obtain FA-PEG; (3) Preparation of CHEMS-NHS: Dissolve 100 mg of CHEMS and 47.5 mg of NHS in 5 ml of tetrahydrofuran to obtain the first solution. Dissolve 125 mg of DCC in 2.5 ml of tetrahydrofuran to obtain the second solution. Mix the first and second solutions and stir for 8 h. Filter and evaporate to remove the tetrahydrofuran. Add a mixture of 3 ml of tetrahydrofuran and 6 ml of isopropanol, recrystallize in an ice-water bath, filter, and dry in vacuo to obtain CHEMS-NHS. (4) Preparation of FA-Res-SEDDS: 4 μmol of FA-PEG and 4 μmol of CHEMS-NHS were dissolved in 10 ml of chloroform and reacted in the dark at room temperature under nitrogen protection for 24 h. The chloroform was removed by rotary evaporation at 35°C and hydrated with 3 ml of 50 mmol / L Na2CO3. The mixture was centrifuged at 12000 r / min for 10 min. The supernatant was dialyzed with deionized water (MWCO = 8000-14 kDa) and freeze-dried to obtain FA-PEG-CHEMS. FA-PEG-CHEMS was added to blank SEDDS at a mass ratio of FA-PEG-CHEMS to blank SEDDS of 3:100. After ultrasonic mixing, 10 mg of resveratrol was added and ultrasonicated for 20 min to obtain FA-Res-SEDDS. (5) Preparation of FA-Res-LP-SEDDS: Take 1 mL of LP with an OD value of 1.5, centrifuge to obtain the precipitate, wash with PBS buffer with a pH value of 7.4, take 150 μL of 10% skim milk powder to resuspend, vortex and add dropwise to 1 g of FA-Res-SEDDS to obtain FA-Res-LP-SEDDS. Example

[0016] The present invention provides a method for preparing a probiotic-drug co-delivery oral self-emulsifier, which is achieved by the following steps: (1) Preparation of Res-SEDDS: The composite emulsifier and PEG400 were mixed by ultrasonication at a mass ratio of 2:1 for 20 min to obtain a mixture. 1 g of the mixture and olive oil were weighed at a mass ratio of 6:4, vortexed for 3 min, and placed in an ultrasonic cleaner with a power of 100 W and ultrasonicated for 20 min to fully mix to obtain blank SEDDS. 15 mg of Res was then added, vortexed and ultrasonicated to uniformly disperse it to obtain Res-SEDDS. (2) Preparation of FA-PEG: Take 13 mg of FA, dissolve it in 1 ml of DMSO, add 6.2 mg of EDC, 4.028 mg of NHS and 17.4 μl of triethanolamine, activate it in the dark under nitrogen protection for 1 h, and add NH2-PEG 3350 -NH2 82.1 mg, stirred for 12 h, added 50 mmol / L Na2CO3 ml, centrifuged, took the supernatant and dialyzed it with ultrapure water, MWCO = 1000 Da, and lyophilized to obtain FA-PEG; (3) Preparation of CHEMS-NHS: Dissolve 100 mg of CHEMS and 47.5 mg of NHS in 5 ml of tetrahydrofuran to obtain a first solution. Dissolve 125 mg of DCC in 2.5 ml of tetrahydrofuran to obtain a second solution. Mix the first and second solutions and stir for 12 h. Filter and rotary evaporate to remove tetrahydrofuran. Add a mixture of 3 ml of tetrahydrofuran and 6 ml of isopropanol, recrystallize in an ice-water bath, filter, and dry in vacuo to obtain CHEMS-NHS. (4) Preparation of FA-Res-SEDDS: 4 μmol of FA-PEG and 4 μmol of CHEMS-NHS were dissolved in 5 ml of chloroform and reacted in the dark at room temperature under nitrogen protection for 12 h. The chloroform was removed by rotary evaporation at 35 °C and hydrated with 3 ml of 50 mmol / L Na2CO3. The mixture was centrifuged at 12000 r / min for 10 min. The supernatant was dialyzed with deionized water (MWCO = 8000-14 kDa) and freeze-dried to obtain FA-PEG-CHEMS. FA-PEG-CHEMS was added to blank SEDDS at a mass ratio of FA-PEG-CHEMS to blank SEDDS of 2:100. After ultrasonic mixing, 15 mg of resveratrol was added and ultrasonicated for 20 min to obtain FA-Res-SEDDS. (5) Preparation of FA-Res-LP-SEDDS: Take 1 mL of LP with an OD value of 1.0, centrifuge to obtain the precipitate, wash with PBS buffer with a pH value of 7.4, take 120 μL of 10% skim milk powder to resuspend, vortex and add dropwise to 1 g of FA-Res-SEDDS to obtain FA-Res-LP-SEDDS.

[0017] The particle size of the probiotic-drug co-delivery oral self-emulsifier prepared by the above method is 20-100 nm.

[0018] The probiotic-drug co-delivery oral self-emulsifier prepared by the above method is used in the preparation of drugs for treating atherosclerosis.

[0019] The preparation method of the present invention is simple, easy to produce and prepare, and has good biocompatibility. FA-SEDDS is used as a matrix material, Res is used as a model drug, and LP is used as an adjuvant to construct a nano drug delivery system with atherosclerotic plaque targeting and probiotic synergy. This multi-pronged approach is used to treat atherosclerosis, and is an innovation in the treatment of atherosclerosis. Experiments have shown that very good and beneficial technical effects have been achieved. The relevant experimental data are as follows (taking Example 3 as an example): Experimental study on the stability of FA-Res-SEDDS preparation under simulated gastrointestinal fluid conditions The prepared FA-Res-SEDDS and Res-SEDDS were diluted 100-fold with artificial gastric juice and artificial intestinal juice and incubated at 37°C with shaking at 100 rpm. Samples were taken at different time points after incubation (artificial gastric juice: 0, 0.5, 1.0, 2.0, 3.0 h; artificial intestinal juice: 0, 0.5, 1, 2, 4, 6, 8, 10, 12 h). The particle size and PDI changes were measured by laser particle size analyzer to investigate the gastrointestinal stability of FA-Res-SEDDS and Res-SEDDS.

[0020] The results are as follows Figure 1 As shown in the figure, the particle size and PDI of FA-Res-SEDDS remained almost unchanged under the conditions of incubation in simulated gastrointestinal fluid, indicating that it has strong resistance to enzymatic degradation in the gastrointestinal tract and has good stability.

[0021] In vitro release behavior of FA-Res-SEDDS preparations The release of FA-Res-SEDDS and Res-SEDDS in artificial gastrointestinal fluid was investigated by dialysis. 2 ml of FA-Res-SEDDS and Res-SEDDS were added to dialysis bags (MWCO=3500 Da) and placed in a certain volume of artificial gastrointestinal fluid. The samples were incubated at 37°C and 100 rpm with shaking. 2 ml of release medium was removed at different time points after incubation (artificial gastric fluid: 0, 0.5, 1.0, 2.0, 3.0 h; artificial intestinal fluid: 0, 0.5, 1, 2, 4, 6, 8, 10, 12 h), and 2 ml of fresh medium was added. The concentration of Res in the samples was determined by UV spectrophotometry, and the cumulative release rate of the drug was calculated.

[0022] The results are as follows Figure 2 As shown in the data, the cumulative release rate of FA-Res-SEDDS in simulated gastric fluid was less than 8%; in simulated intestinal fluid, the cumulative release rate of FA-Res-SEDDS was 20.9077±0.53%, indicating that FA-Res-SEDDS has good stability in the gastrointestinal environment.

[0023] Experiment on the survival activity of Lactobacillus plantarum under simulated gastric fluid conditions in vitro Three equal aliquots of LP (OD ≈ 0.5) were prepared as previously described, and FA-Res-LP-SEDDS, Res-LP-SEDDS, and Free LP were prepared. These aliquots were added to 2 mL of artificial gastric fluid (SGF) and incubated in a shaker at 37°C. 500 μL of each aliquot was collected at 0.5 h, 1 h, and 2 h, centrifuged, and washed twice with pre-chilled PBS. The bacterial pellet was resuspended in 500 μL of pre-chilled PBS and serially diluted to the appropriate concentration. After 100 μL was plated, the pellet was anaerobically incubated for 48 h and colony counted.

[0024] The results showed that compared with the unencapsulated LP, the number of viable bacteria in the FA-Res-LP-SEDDS group and the Res-LP-SEDDS group was much higher than that in the free LP after incubation in simulated gastric fluid for 2 h, indicating that the encapsulation of the emulsion had a certain resistance to gastric fluid and played a certain role in protecting the probiotics. 3350-CHEMS modification, the FA-Res-LP-SEDDS group has a higher number of viable bacteria than the Res-LP-SEDDS group and a stronger ability to resist gastric juice erosion. Figure 3 .

[0025] Survival activity experiment of Lactobacillus plantarum under simulated intestinal fluid conditions in vitro Three equal amounts of LP (OD ≈ 0.5) were taken and FA-Res-LP-SEDDS, Res-LP-SEDDS, and Free LP were prepared according to the previously described method. They were added simultaneously to 4 mL of artificial intestinal fluid containing MRS medium and cultured in a constant temperature shaker at 37°C. 200 μL of the solution was taken at 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h, respectively. The solution was centrifuged and washed twice with pre-cooled PBS buffer. The bacterial pellet was resuspended in 200 μL PBS buffer and added to a 96-well plate. The OD value of the bacteria was measured using a microplate reader, and a curve of time and bacterial OD value was plotted. The bacterial solution taken at 2 h and 8 h was gradiently diluted to the appropriate concentration, and 100 μL was taken for bacterial plate coating. The colonies were counted after anaerobic incubation for 48 h.

[0026] The results showed that FA-Res-LP-SEDDS and Res-LP-SEDDS incubated in intestinal fluid had similar growth trends to Free LP, indicating that the emulsion coating can gradually fall off during the emulsification process, providing strong evidence for the colonization of probiotics in the intestine; at the same time, through the investigation of the number of Lactobacillus plantarum, the number of Lactobacillus plantarum increased significantly when the incubation time was from 2 h to 8 h, indicating that the number of bacteria increased with the removal of the coating, as shown in Figure 2. Figure 4 .

[0027] In addition to the above four repeated experiments, the present invention also conducted the same experiments on other embodiments, and obtained the same or similar results, which are not listed here one by one.

[0028] Experiments have shown that the method of the present invention is stable and reliable, and the obtained product is of good quality. Compared with the prior art, it has the following beneficial technical effects: (1) After entering the gastrointestinal tract, the preparation will self-emulsify into O / W nanoemulsion and release Lactobacillus plantarum. The O / W nanoemulsion targets intestinal epithelial cells and AS plaques through folic acid, achieving the targeted and efficient release of resveratrol. Lactobacillus plantarum will colonize in the gastrointestinal tract, ultimately achieving hetero-targeted co-delivery of drugs.

[0029] (2) Resveratrol targeted to the plaque site can regulate the plaque inflammatory microenvironment by inducing macrophage phenotypic remodeling, and Lactobacillus plantarum colonizing the intestine can reduce the level of harmful cholesterol in the blood. The two work together to effectively break the vicious cycle between vascular inflammation and dyslipidemia, and treat atherosclerosis in a two-pronged approach.

Claims

1. A method for preparing a probiotic-drug co-delivery oral self-emulsifier, characterized in that: Olive oil is used as the oil phase, Span80 and Cremophor RH40 are used as composite emulsifiers, PEG400 is used as a co-emulsifier, Res is used as a model drug, LP resuspended in skimmed milk powder is used as the internal aqueous phase, and FA is used as the target head to construct an oil-in-water targeted self-emulsifying preparation. After oral administration into the intestine, it can self-emulsify and transform into an oil-in-water nanoemulsion. The plant lactobacillus in the internal aqueous phase is then released and colonizes in the gastrointestinal tract. Different targets are co-delivered and synergistically enhanced to accurately and effectively treat atherosclerosis. This is achieved by the following steps: (1) Preparation of Res-SEDDS: The composite emulsifier and PEG400 were mixed by ultrasonication for 20 min at a mass ratio of 2:1 to obtain a mixture. 1 g of the mixture and olive oil were weighed at a mass ratio of 4:6-6:4, vortexed for 3 min, and placed in an ultrasonic cleaner with a power of 100 W and ultrasonicated for 20 min to fully mix to obtain blank SEDDS. 5-20 mg of Res was then added, vortexed and ultrasonicated to uniformly disperse it to obtain Res-SEDDS. The composite emulsifier is Span80 and Cremophor RH40, with an HBL value of 9; (2) Preparation of FA-PEG: Take 5-20 mg FA, dissolve it in 1 ml DMSO, add 6.2 mg EDC, 4.028 mg NHS and 17.4 μl triethanolamine, activate it in the dark under nitrogen protection for 0.5-2 h, add NH2-PEG 3350 -NH2 75~100 mg, stirred for 6~24 h, added 50mmol / L Na2CO3 2.5 ml, centrifuged, took the supernatant and dialyzed it with ultrapure water (MWCO=1000 Da), and lyophilized to obtain FA-PEG; (3) Preparation of CHEMS-NHS: Dissolve 100 mg of CHEMS and 47.5 mg of NHS in 5 ml of tetrahydrofuran to obtain the first solution. Dissolve 125 mg of DCC (N,N'-dicyclohexylcarbodiimide) in 2.5 ml of tetrahydrofuran to obtain the second solution. Mix the first and second solutions and stir for 6-24 hours. Filter and rotary evaporate to remove the tetrahydrofuran. Add a mixture of 3 ml of tetrahydrofuran and 6 ml of isopropanol. Recrystallize in an ice-water bath, filter, and dry in vacuo to obtain CHEMS-NHS. (4) Preparation of FA-Res-SEDDS: 4 μmol of FA-PEG and 4 μmol of CHEMS-NHS were dissolved in 3-10 ml of chloroform and reacted in the dark at room temperature under nitrogen protection for 6-24 h. The chloroform was removed by rotary evaporation at 35°C and hydrated with 3 ml of 50 mmol / L Na2CO3. The mixture was centrifuged at 12,000 r / min for 10 min. The supernatant was dialyzed against deionized water with a MWCO of 8,000-14 kDa and lyophilized to obtain FA-PEG-CHEMS. FA-PEG-CHEMS was added to blank SEDDS at a mass ratio of FA-PEG-CHEMS to blank SEDDS of 0.5-3:

100. The mixture was ultrasonically mixed and then 5-20 mg of resveratrol was added. The mixture was ultrasonically mixed for 20 min to obtain FA-Res-SEDDS. (5) Preparation of FA-Res-LP-SEDDS: Take 1 mL of LP with an OD value of 0.1-2, centrifuge to obtain the precipitate, wash with PBS buffer at pH 7.4, and resuspend in 75-150 μL of 10% skim milk powder to obtain a resuspension. Vortex and add dropwise to 1 g of FA-Res-SEDDS to obtain FA-Res-LP-SEDDS.

2. The method for preparing the probiotic-drug co-delivery oral self-emulsifier according to claim 1, characterized in that: This is accomplished by the following steps: (1) Preparation of Res-SEDDS: The composite emulsifier and PEG400 were mixed by ultrasonication for 20 min at a mass ratio of 2:1 to obtain a mixture. 1 g of the mixture and olive oil were weighed at a mass ratio of 4:6, vortexed for 3 min, and placed in an ultrasonic cleaner with a power of 100 W and ultrasonicated for 20 min to fully mix to obtain blank SEDDS. 5-20 mg of Res was then added, vortexed and ultrasonicated to uniformly disperse it to obtain Res-SEDDS. (2) Preparation of FA-Res-SEDDS: Take 5 mg of FA, dissolve it in 1 ml of DMSO, add 6.2 mg of EDC, 4.028 mg of NHS and 17.4 μl of triethanolamine, activate it in the dark under nitrogen protection for 0.5, and add NH2-PEG 3350 -NH2 75 mg, stirred for 8 h, added 50 mmol / L Na2CO3 2.5 ml, centrifuged, took the supernatant and dialyzed it with ultrapure water, MWCO = 1000 Da, and lyophilized to obtain FA-PEG; (3) Preparation of CHEMS-NHS: Dissolve 100 mg of CHEMS and 47.5 mg of NHS in 5 ml of tetrahydrofuran to obtain a first solution. Dissolve 125 mg of DCC in 2.5 ml of tetrahydrofuran to obtain a second solution. Mix the first and second solutions and stir for 6-24 hours. Filter and evaporate to remove the tetrahydrofuran. Add a mixture of 3 ml of tetrahydrofuran and 6 ml of isopropanol, recrystallize in an ice-water bath, filter, and dry in vacuo to obtain CHEMS-NHS. (4) Preparation of FA-Res-SEDDS: 4 μmol of FA-PEG and 4 μmol of CHEMS-NHS were dissolved in 3 ml of chloroform and reacted in the dark at room temperature under nitrogen protection for 24 h. The chloroform was removed by rotary evaporation at 35°C and hydrated with 3 ml of 50 mmol / L Na2CO3. The mixture was centrifuged at 12000 r / min for 10 min. The supernatant was dialyzed with deionized water (MWCO = 8000-14 kDa) and freeze-dried to obtain FA-PEG-CHEMS. FA-PEG-CHEMS was added to blank SEDDS at a mass ratio of 1:

100. After ultrasonic mixing, 5 mg of resveratrol was added and ultrasonicated for 20 min to obtain FA-Res-SEDDS. (5) Preparation of FA-Res-LP-SEDDS: Take 1 mL of LP with an OD value of 0.5, centrifuge to obtain the precipitate, wash with PBS buffer with a pH value of 7.4, take 75-150 μL of 10% skim milk powder to resuspend, vortex and add dropwise to 1 g of FA-Res-SEDDS to obtain FA-Res-LP-SEDDS.

3. The method for preparing the probiotic-drug co-delivery oral self-emulsifier according to claim 1, characterized in that: This is accomplished by the following steps: (1) Preparation of Res-SEDDS: The composite emulsifier and PEG400 were mixed at a mass ratio of 2:1 and ultrasonically mixed for 20 min to obtain a mixture. 1 g of the mixture and olive oil were weighed at a mass ratio of 5:5, vortexed for 3 min, and placed in an ultrasonic cleaner with a power of 100 W and ultrasonicated for 20 min to fully mix to obtain blank SEDDS. 10 mg of Res was then added, vortexed and ultrasonically dispersed to obtain Res-SEDDS. (2) Preparation of FA-PEG: Take 20 mg of FA, dissolve it in 1 ml of DMSO, add 6.2 mg of EDC, 4.028 mg of NHS and 17.4 μl of triethanolamine, activate it in the dark under nitrogen protection for 2 h, and add NH2-PEG 3350 -NH2 100 mg, stirred for 18 h, added 50 mmol / L Na2CO3 2.5 ml, centrifuged, took the supernatant and dialyzed it with ultrapure water, MWCO = 1000 Da, and lyophilized to obtain FA-PEG; (3) Preparation of CHEMS-NHS: Dissolve 100 mg of CHEMS and 47.5 mg of NHS in 5 ml of tetrahydrofuran to obtain the first solution. Dissolve 125 mg of DCC in 2.5 ml of tetrahydrofuran to obtain the second solution. Mix the first and second solutions and stir for 8 h. Filter and evaporate to remove the tetrahydrofuran. Add a mixture of 3 ml of tetrahydrofuran and 6 ml of isopropanol, recrystallize in an ice-water bath, filter, and dry in vacuo to obtain CHEMS-NHS. (4) Preparation of FA-Res-SEDDS: 4 μmol of FA-PEG and 4 μmol of CHEMS-NHS were dissolved in 10 ml of chloroform and reacted in the dark at room temperature under nitrogen protection for 24 h. The chloroform was removed by rotary evaporation at 35°C and hydrated with 3 ml of 50 mmol / L Na2CO3. The mixture was centrifuged at 12000 r / min for 10 min. The supernatant was dialyzed with deionized water (MWCO = 8000-14 kDa) and freeze-dried to obtain FA-PEG-CHEMS. FA-PEG-CHEMS was added to blank SEDDS at a mass ratio of FA-PEG-CHEMS to blank SEDDS of 3:

100. After ultrasonic mixing, 10 mg of resveratrol was added and ultrasonicated for 20 min to obtain FA-Res-SEDDS. (5) Preparation of FA-Res-LP-SEDDS: Take 1 mL of LP with an OD value of 1.5, centrifuge to obtain the precipitate, wash with PBS buffer with a pH value of 7.4, take 150 μL of 10% skim milk powder to resuspend, vortex and add dropwise to 1 g of FA-Res-SEDDS to obtain FA-Res-LP-SEDDS.

4. The method for preparing the probiotic-drug co-delivery oral self-emulsifier according to claim 1, characterized in that: This is accomplished by the following steps: (1) Preparation of Res-SEDDS: The composite emulsifier and PEG400 were mixed by ultrasonication at a mass ratio of 2:1 for 20 min to obtain a mixture. 1 g of the mixture and olive oil were weighed at a mass ratio of 6:4, vortexed for 3 min, and placed in an ultrasonic cleaner with a power of 100 W and ultrasonicated for 20 min to fully mix to obtain blank SEDDS. 15 mg of Res was then added, vortexed and ultrasonicated to uniformly disperse it to obtain Res-SEDDS. (2) Preparation of FA-PEG: Take 13 mg of FA, dissolve it in 1 ml of DMSO, add 6.2 mg of EDC, 4.028 mg of NHS and 17.4 μl of triethanolamine, activate it in the dark under nitrogen protection for 1 hour, and add NH2-PEG 3350 -NH2 82.1mg, stirred for 12h, added 50mmol / L Na2CO3ml, centrifuged, took the supernatant and dialyzed it with ultrapure water, MWCO = 1000Da, and lyophilized to obtain FA-PEG; (3) Preparation of CHEMS-NHS: Dissolve 100 mg of CHEMS and 47.5 mg of NHS in 5 ml of tetrahydrofuran to obtain a first solution. Dissolve 125 mg of DCC in 2.5 ml of tetrahydrofuran to obtain a second solution. Mix the first and second solutions and stir for 12 h. Filter and rotary evaporate to remove tetrahydrofuran. Add a mixture of 3 ml of tetrahydrofuran and 6 ml of isopropanol, recrystallize in an ice-water bath, filter, and dry in vacuo to obtain CHEMS-NHS. (4) Preparation of FA-Res-SEDDS: 4 μmol of FA-PEG and 4 μmol of CHEMS-NHS were dissolved in 5 ml of chloroform and reacted in the dark at room temperature under nitrogen protection for 12 h. The chloroform was removed by rotary evaporation at 35 °C and hydrated with 3 ml of 50 mmol / L Na2CO3. The mixture was centrifuged at 12000 r / min for 10 min. The supernatant was dialyzed with deionized water (MWCO = 8000-14 kDa) and freeze-dried to obtain FA-PEG-CHEMS. FA-PEG-CHEMS was added to blank SEDDS at a mass ratio of FA-PEG-CHEMS to blank SEDDS of 2:

100. After ultrasonic mixing, 15 mg of resveratrol was added and ultrasonicated for 20 min to obtain FA-Res-SEDDS. (5) Preparation of FA-Res-LP-SEDDS: Take 1 mL of LP with an OD value of 1.0, centrifuge to obtain the precipitate, wash with PBS buffer with a pH value of 7.4, take 120 μL of 10% skim milk powder to resuspend, vortex and add dropwise to 1 g of FA-Res-SEDDS to obtain FA-Res-LP-SEDDS.

5. The method for preparing a probiotic-drug co-delivery oral self-emulsifier according to any one of claims 1 to 4, characterized in that: The particle size of the probiotic-drug co-delivery oral self-emulsifier is 20-100 nm.

6. Use of the probiotic-drug co-delivery oral self-emulsifier prepared by the method according to any one of claims 1 to 5 in the preparation of a drug for treating atherosclerosis.

Images