Composition capable of penetrating intestinal barrier during oral administration and application thereof

Abstract

The invention provides a composition capable of penetrating an intestinal barrier by oral administration. The composition contains a carrier or an auxiliary material of a targeted Microflod cell Dectin-1 receptor, and one or / and a plurality of medicinal components / components, wherein the carrier or the auxiliary material of the target Microflood cell Dectin-1 receptor is beta-glucan, and the carrier or the auxiliary material of the target Microflood cell Dectin-1 receptor is beta-glucan. The effective components / components are traditional Chinese medicine effective components / components, or / and probiotics, or / and chemical medicine components, or / and biological medicines. The invention relates to a composition capable of realizing oral administration and penetrating through an intestinal barrier, which is based on the discovery of the inventor that the composition reaches colon through gastrointestinal tracts, targets colon Microflood cells, is taken by the Microflood cells, penetrates through the intestinal barrier and enters immune tissue Colonic Patches of a colon submucosa. Based on the research, the composition disclosed by the invention can be used for treating various diseases, such as ulcerative colitis, Crohn's disease, colon cancer, rheumatoid arthritis or bacterial pneumonia after being orally taken and absorbed by rectum.

Description

Technical Field

[0001] The present invention belongs to the field of drugs and biochemical preparations, and particularly relates to an oral drug delivery system through the intestinal barrier, and a preparation method and use of a drug for treating ulcerative colitis by combining probiotics and rhein as a representative. Background Art

[0002] Ulcerative colitis (UC) is a common inflammatory disease of the colon, and its symptoms include diarrhea, bloody mucus, repeated abdominal pain, etc. In severe cases, it may lead to massive bleeding and toxic megacolon, and even develop into cancer. Some studies have shown that the risk of colorectal cancer in UC patients is 2-3 times higher than that in healthy people. In addition, in 2023, the global estimate of UC was 5 million cases. Currently, the main clinical drugs for UC are immunosuppressants, such as 5-aminosalicylic acid and glucocorticoids, which can relieve symptoms, but their action pathways are single and the side effects are obvious. In 2020, the latest consensus on the treatment of UC in the world is the repair of the colonic multi-barrier (including biological, mechanical, chemical, and immune barriers). Among them, the biological barrier, mechanical barrier, and chemical barrier exist in the superficial layer of the colon, and there are currently some treatment options. However, since the submucosa where the immune barrier is located is in the deep layer of the colon, existing delivery systems are difficult to reach, which greatly limits the effectiveness of multi-layer co-repair. At the same time, some mechanisms of UC treatment (such as immune hyperactivity, bacterial invasion, brain-gut linkage, etc.) cannot be realized due to the lack of a suitable delivery system. Therefore, how to overcome the complex barrier structure and deliver drugs to the submucosa has become the key to UC treatment.

[0003] Microfold cells (M cells), as the "bridge" between the mechanical barrier and the immune barrier, are potential targets for drugs to enter the submucosa. M cells are specialized small intestine and colonic epithelial cells that allow macromolecules to pass through them and enter the deep layer of the colon. The research group of this project selected yeast cell wall (YPs) as the basis to construct a nano-micro drug delivery system with M cells as the target. YPs is a natural polysaccharide component extracted from yeast, which has good gastric stability and colonic targeting, and can help drugs reach the colon directly and improve bioavailability. In addition, a new mechanism that may be used for intestinal mucosa repair was also discovered: calcitonin gene-related peptide (CGRP)-related nerves (CGRP-RN) are distributed in the submucosa, produce CGRP under the stimulation of intestinal flora, and then activate goblet cells to secrete mucus to repair the colonic mucosa.

[0004] Bacillus subtilis (BS) has been widely recognized in the treatment of UC and may be a suitable probiotic for stimulating CGRP-RN, showing promise in mucosal repair. However, the application of BS still faces two problems: one is to deliver BS to the submucosa through oral administration; the aforementioned YPs can deliver BS to the submucosa due to their gastric stability and the ability to target M cells through β-glucan. The other is to ensure the colonization of Bacillus subtilis. Summary of the Invention

[0005] The inventors found that Colonic Patches (CPs) located in the submucosa are a type of colonic immune tissue similar to Peyer's patches in the intestine, which aggregate a large number of immune cells. If the migration of immune cells during disease occurrence is utilized, drugs can be delivered to sites that are difficult to reach by oral administration to treat certain diseases. In addition to treating ulcerative colitis, it can also be used to treat other diseases such as rheumatoid arthritis.

[0006] The present invention provides a composition that can achieve oral penetration through the intestinal barrier, and the present invention also provides the use of this composition.

[0007] A composition that can achieve oral penetration through the intestinal barrier, which contains a carrier or excipient targeting the Dectin-1 receptor of Microflod cells, and one or more pharmaceutically active ingredients / components;

[0008] Among them, the carrier or excipient targeting the Dectin-1 receptor of Microflod cells is β-glucan;

[0009] The pharmaceutically active ingredients / components are effective ingredients / components of traditional Chinese medicine, or / and probiotics, or / and chemical drug ingredients, or / and biological drugs.

[0010] Among them, the β-glucans are yeast cell walls and their related polysaccharides, Candida albicans cell walls and their related polysaccharides, Aspergillus fumigatus cell walls and their related polysaccharides, lentinan, ganoderma polysaccharide, coriolus versicolor polysaccharide, oat polysaccharide, seaweed polysaccharide, gellan gum, bacterial exopolysaccharide, lichenan or low molecular weight β-glucan fragments;

[0011] The probiotic is one of Bacillus subtilis, Lactobacillus rhamnosus, Akkermansia muciniphila, Escherichia coli, lactic acid bacteria or Clostridium butyricum;

[0012] The effective ingredients / components of traditional Chinese medicine are rhein, emodin, chlorogenic acid or berberine, tannic acid.

[0013] It combines the effective ingredients / components of traditional Chinese medicine with β-glucans, and then encapsulates probiotics therein to prepare a composition that can achieve oral penetration through the intestinal barrier.

[0014] The present invention also provides the use of the composition capable of achieving oral penetration of the intestinal barrier in the preparation of a medicament for treating ulcerative colitis or rheumatoid arthritis.

[0015] The present invention provides a colonic submucosal drug delivery system, which uses yeast cell wall (YPs) as a raw material and prepares a novel drug delivery system (Rh-YBS) co-encapsulating Bacillus subtilis (BS) and Rhein (Rh) through chemical modification and extrusion method; wherein, the dosage of yeast cell wall, Bacillus subtilis, and Rhein per mL is:

[0016] Yeast cell wall 1 - 10 mg, Bacillus subtilis 10 8-10 CFUs, Rhein 0.1 - 1 mg.

[0017] Preferably, the dosage of yeast cell wall, Bacillus subtilis, and Rhein per mL is:

[0018] Yeast cell wall 2 mg, Bacillus subtilis 10 9 CFUs, Rhein 0.5 mg.

[0019] Specifically, it includes the following steps:

[0020] a. Prepare yeast cell wall by lysis method and freeze-drying method;

[0021] b. Combine Rhein with yeast cell wall to prepare Rh-YPs intermediate by electrostatic adsorption - vacuum extrusion - electrostatic distribution - hydration rearrangement method;

[0022] c. Load Bacillus subtilis into Rh-YPs intermediate by extrusion method to prepare Rh-YBS.

[0023] Preferably, it includes the following steps:

[0024] a. Prepare Rh-YBS:

[0025] Dissolve Rh in DMSO, add YPs and deionized water, stir at room temperature for 24 h, transfer the reaction mixture into a 1000 Da dialysis bag after reaction, dialyze with deionized water, change the water every 4 h, and continue for 2 - 3 days; then, centrifuge at 7000 rpm for 5 min, collect the precipitate, pre-treat at -20 °C for 12 h and then freeze-dry to obtain Rh-YPs;

[0026] b. Encapsulate BS:

[0027] Take the Rh-YPs prepared in step a, suspend it in cold PBS, ultrasonically treat it, and then mix it with BS culture solution (2×10 9Mix with (CFUs / mL), culture with shaking at 37 °C and 150 rpm for 1 h; then, transfer the mixture to a micro extruder and extrude it through a polycarbonate porous membrane with a pore size of 5 μm to obtain the product.

[0028] The present invention also provides the use of the colon submucosal drug delivery system described above in the preparation of a drug for treating ulcerative colitis; preferably, the pharmaceutical preparation is an oral colon-specific drug delivery preparation.

[0029] The present invention provides a berberine-loaded Bacillus subtilis composition encapsulated with tannic acid-dextran, which is prepared by combining tannic acid, berberine with Bacillus subtilis and then adding a dextran solution for inclusion.

[0030] The present invention also provides a polydopamine-coated Lactobacillus rhamnosus and emodin nanoplatform, which is prepared by encapsulating emodin with lactoferrin to obtain positively charged emodin nanoparticles, adsorbing the nanoparticles onto the surface of probiotic Lactobacillus rhamnosus (LGG) through electrostatic adsorption force, and self-aggregating and coating the surface through the oxidation of dopamine (LENs) under alkaline conditions to form a ROS-sensitive "bacteria-drug combination" micron system.

[0031] The present invention also provides a tannic acid (TA)-iron ion (Fe 3+ )-binding mucin-coated Akkermansia muciniphila nanoplatform, which is prepared by complexing tannic acid with iron ions and then coating mucin-coated Akkermansia muciniphila.

[0032] The present invention also provides an intestinal Peyer's patches (PPs)-targeted preparation for treating rheumatoid arthritis, which is prepared by combining lactoferrin, emodin, yeast cell wall and then encapsulating Bacillus subtilis therein to obtain NPs-YPs@BS.

[0033] The present invention also provides an oral transintestinal barrier drug delivery technology system, comprising the following steps:

[0034] a. Prepare the composition capable of achieving oral transintestinal barrier through micro-nanoparticle preparation technology;

[0035] b. The drug delivery system should reach the colon through the gastrointestinal tract;

[0036] c. The drug delivery system needs to target colon Microflod cells;

[0037] d. The drug delivery system is taken up by Microflod cells and penetrates the intestinal barrier; enter the colonic submucosal immune tissue Colonic Patches.

[0038] The present invention provides a schematic diagram of the oral transintestinal barrier technology system as Figure 1 shown:

[0039] The composition of the present invention that can achieve oral penetration of the intestinal barrier is based on the inventor's discovery that it can pass through the gastrointestinal tract, reach the colon directly, target colonic Microflod cells, be taken up by Microflod cells and penetrate the intestinal barrier, and enter the colonic submucosal immune tissue Colonic Patches. Based on this research, the composition involved in this application can be used to treat various diseases, such as ulcerative colitis and rheumatoid arthritis, after oral administration and colonic absorption. Among them, rhein is mixed with yeast cell wall by the method of "electrostatic adsorption - hydration rearrangement", and then encapsulated with BS to form the delivery system Rh - YBS. The administration system was characterized, the therapeutic effect of Rh - YBS on UC was verified, the roles of YPs and Rh were proved, especially the role of Rh - YBS in the submucosa, and the mechanism of action through the CGRP - related pathway was emphasized. Description of the Drawings

[0040] Figure 1 : Schematic diagram of the oral intestinal barrier penetration technology system;

[0041] Figure 2 : Schematic diagram of the preparation method of Rh - YBS;

[0042] Figure 3 : Spore - TB@GLU under transmission electron microscope;

[0043] Figure 4 : LEG under transmission electron microscope PDA ;

[0044] Figure 5 : LEG under scanning electron microscope and laser confocal electron microscope; PDA ;

[0045] Figure 6 : AKK@TA@MUC under transmission electron microscope and scanning electron microscope;

[0046] Figure 7 : Schematic diagram of the idea of PPs targeting "micro - recruitment" of macrophages to "hitchhike" for drug delivery to treat rheumatoid arthritis;

[0047] Figure 8 : Preparation of the first preparation "EMO - NYPs";

[0048] Figure 9 : Preparation of the second preparation "EMO - NYPs";

[0049] Figure 10:Preventive effect of Rh-YBS on UC (a) Schematic diagram of the treatment process of different preparations on DSS mice. b) Line graph of the effect of different preparations on the body weight of mice (n = 6). c) Line graph of the change in DAI score of each group over time (n = 6). d) Images of the colon in different groups. e) Histogram evaluation of colon length (n = 6). f) H&E staining of colon sections in each group. Scale bar: 100 μm. g)-i) Histogram analysis of the changes in colonic inflammatory factors (IL-6, TNF-α, IFN-γ, IL-1β), oxidative factors (MPO), and anti-inflammatory factors (IL-10) in each group (n = 6). Data are mean ± SEM. *p < 0.05; **p < 0.01);

[0050] Figure 11 :Therapeutic effect of Rh-YBS on UC (a) Schematic diagram of the treatment process of different preparations on DSS mice. b) Line graph of the effect of different preparations on the body weight of mice (n = 6). c) Line graph of the change in DAI score of each group over time (n = 6). d) Images of the colon in different groups. e) Histogram evaluation of colon length (n = 6). Data are mean ± SEM. *p < 0.05; **p < 0.01);

[0051] Figure 12 :H&E staining of colon and tissue sections in each group;

[0052] Figure 13 :Analysis of fecal microbiota in each group of mice (a) Alpha diversity analysis of the microbiota in each group. b) Beta diversity analysis of each group. c) Histogram stacking at the family level. d) Cluster heatmap at the species level. e)-h) Relative abundance levels of Reteri, Muciniphila, Gordonii, and Ovatus);

[0053] Figure 14 :Effect of Rh-YBS on the content of short-chain fatty acids in the feces of mice (n = 5); Data are mean ± SEM. *p < 0.05; **p < 0.01;

[0054] Figure 15 :LEG PDA In vivo Colonic Patches targeting experiment;

[0055] Figure 16 :In vitro verification of the "micro-recruitment" ability of the preparation;

[0056] Figure 17 :In vivo verification of the "micro-recruitment" ability of the preparation;

[0057] Figure 18 :Evaluation of rat body weight, paw thickness, ankle joint width, arthritis index, and foot swelling;

[0058] Figure 19 : Results of Micro-CT, HE staining, and safranin-fast green (SO-FG) staining;

[0059] Figure 20 : Evaluation of the anti-inflammatory effects of the preparation in vitro and in vivo. Specific implementation mode

[0060] Example 1: Preparation of yeast cell wall (YPs)

[0061] Suspend 100 g of brewer's yeast in 1000 mL of deionized water containing 1 Mol of sodium hydroxide. Heat the suspension to 80 °C for 1 h, then centrifuge at 3000 rpm for 10 min. Rinse twice with deionized water, resuspend in hydrochloric acid at pH = 4, and incubate at 60 °C for 1.5 h. After centrifuging the sample at 3000 rpm for 10 minutes, wash it thoroughly with deionized water twice. Subsequently, rinse with isopropanol four times and acetone twice. Centrifuge at 3000 rpm for 10 min, collect the precipitate, and freeze-dry to obtain YPs.

[0062] Example 2: Preparation of Rh-YPs

[0063] Rh and YPs are combined to form Rh-YPs by the method of "electrostatic adsorption - hydration rearrangement". Dissolve 15 mg of Rh in 5 mL of DMSO, add 300 mg of YPs and 15 mL of deionized water, stir at room temperature for 24 h, transfer the reaction mixture into a 1000 Da dialysis bag after the reaction, dialyze with deionized water, and completely replace the dialysis fluid every 4 h for 2 - 3 days. Subsequently, centrifuge at 7000 rpm for 5 min, collect the precipitate, and freeze-dry to obtain Rh-YPs.

[0064] Example 3: Cultivation of Bacillus subtilis (BS)

[0065] Inoculate the BS strain stored at -80 °C on an LB solid medium, incubate at 37 °C for 12 h, pick colonies with correct characteristics, inoculate them into an LB liquid medium, and incubate under the conditions of shaking at 150 rpm and 37 °C for 12 h. Transfer 10 μL of the culture solution into 10 mL of LB liquid medium, incubate at 37 °C for 6 h, centrifuge at 7000 rpm for 10 min, and collect BS.

[0066] Example 4: Preparation of Rh-YBS

[0067] Prepare Rh-YBS by the extrusion method. Take 2 mg of Rh-YPs and suspend it in 1 mL of cold PBS, sonicate for 15 min, and then mix it with 1 mL of BS culture solution (2×10 9Mix with (CFUs / mL), incubate at 37 °C with shaking at 150 rpm for 1 h. Then, transfer the mixture to a micro extruder and extrude it 20 times through a polycarbonate porous membrane with a pore size of 5 μm.

[0068] The preparation method of Rh-YBS provided by the present invention is shown in Figure 2 :

[0069] Example 5: Berberine-loaded Bacillus subtilis spores encapsulated with tannic acid-dextran

[0070] Dilute the purified BS spores to 1×10 8 CFUs / mL with 960 μL of sterile PBS buffer, sequentially add 20 μL of tannic acid solution (TA, 30 mg / mL) and 10 μL of berberine solution (BBR, 4 mg / mL), and then add 10 μL of iron chloride solution (FeCl 3 , 10 mg / mL) to trigger the formation of the metal polyphenol coating. Vortex the mixture vigorously for 1 min after each addition to form a firm Fe-TA layer on the spore surface. Wash twice with sterile PBS to remove unreacted TA, BBR, and iron ions (Fe 3+ ). Then, disperse the sediment of the Spore-TB system in 950 μL of PBS buffer, add 50 μL of dextran solution (GLU, 8 mg / mL), vortex vigorously for 1 min, and wash three times with sterile PBS to obtain Spore-TB@GLU (see Figure 3 ).

[0071] Example 6: Lactobacillus rhamnosus and emodin nanoparticles coated with polydopamine

[0072] Use colon-targeted lactoferrin (LF) to encapsulate emodin (EMO) to prepare positively charged emodin nanoparticles, and adsorb the nanoparticles onto the surface of probiotic Lactobacillus rhamnosus (LGG) through electrostatic adsorption force. To improve its gastric stability, oxidize dopamine (LENs) under alkaline conditions and self-aggregate and coat it on the surface of the probiotic to form a ROS-sensitive "bacteria-drug combination" micron system (LEG PDA ) (see Figure 4 , Figure 5 ).

[0073] Example 7: Akkermansia muciniphila (AKK) coated with mucin by binding tannic acid (TA) and iron ions (Fe3+)

[0074] Through TA and Fe 3+Complexation to form a uniform coating on the surface of the probiotic Akkermansia muciniphila, and then utilize the interaction between TA and mucin to modify the acid-resistant biomaterial mucin on the outermost layer. The specific method is as follows: Take 1 mL of AKK bacterial solution, centrifuge to remove the culture medium, resuspend the precipitated probiotics in PBS, and then make up the volume to 980 μL with PBS. Then, add 10 μL of TA solution and 10 μL of FeCl 3 solution in sequence, stir for 30 minutes to trigger the metal coating. Then wash AKK@TA twice in PBS to remove free FeCl 3 and TA, and then coat with mucin (10 mg / mL) at a constant rotation speed for 1 h to prepare the coated probiotic AKK@TA@MUC (see Figure 6 ).

[0075] Example 8: Two gut Peyer's patches (PPs) targeting preparations for "micro-recruiting" macrophages "hitchhiking" for the treatment of rheumatoid arthritis (see Figure 7 )

[0076] EMO-NPs were prepared by dialysis and were modified to a certain extent. Dissolve 50 mg of lactoferrin (Lf) in 20 mL of ultrapure water, and at the same time dissolve 5 mg of EMO in 1 mL of DMSO to prepare the Lf solution. Then, drop 1 mL of the EMO solution into 20 mL of the Lf solution. To improve the solubility of EMO, use a probe sonicator to oscillate for 5 minutes, and then dialyze the emulsion in ultrapure water for 6 hours (dialysis bag molecular weight value: 1000 Da) to remove DMSO. Filter through a 0.45 μm syringe filter to remove dust and free drugs to obtain EMO-NPs. To prepare NYPs, we adopted the electrostatic-driven solvent hydration / lyophilization self-deposition technique. Dissolve 100 mg of dried YPs in 10 mL of ultrapure water and incubate at 37 °C for 30 min. Then, drop 10 mL of the EMO-NPs into the YPs suspension to prepare EMO-NPs. After incubation for 1 h, centrifuge the suspension at 4000 rpm for 10 min, and then perform two freeze-drying / hydration cycles. Use the capillary action of water to push the NPs attached to the surface into the core of YPs. After the second cycle, thoroughly rinse the suspension with clear water to remove free NPs. Finally, collect EMO-NYPs by freeze-drying ( Figure 8 ).

[0077] Prepare BS@YPs by the extrusion method. Take 2 mg of YPS and suspend it in 1 mL of PBS, sonicate for 15 min, and then mix it with 1 mL of BS culture solution (1×10 9Mix with (CFUs / mL), shake well at 37 °C and 150 rpm, and incubate for 1 h. Then, transfer the mixture to a micro extruder and extrude it 20 times through a polycarbonate porous membrane with a pore size of 5 μm. PEI-RH NPs were prepared by dialysis. First, RH (5 mg) and PEI (20 mg) were dissolved in DMSO. Next, the solution was transferred to a dialysis bag (MWCO: 1000 Da) and dialyzed in ultrapure water for 24 h. Add laminaria japonica solution (0.1 mg / mL) to the collected solution and sonicate for 5 min. Remove free RH by filtration through a 0.8-μm microporous membrane to obtain LA / PEI-RH NPs. NPs-YPs@BS was obtained based on BS@YPs. Add BS@YPs to the NPs solution and stir at room temperature for 20 min to obtain a mixture of NPs-YPs@BS ( Figure 9 ).

[0078] The beneficial effects of the present invention are demonstrated by the following pharmacodynamic experiments

[0079] Test Example 1: Effect of Rh-YBS prepared in Example 4 on ulcerative colitis in mice

[0080] (1) Prophylactic administration

[0081] Take 30 balb / c mice and randomly divide them into 5 groups. Give deionized water containing 3% DSS for 7 consecutive days. At the same time, the mice were orally administered 0.2 mL PBS (model group), 0.2 mL BS (BS group), 0.2 mL YBS (YBS group), 0.2 mL 0.1% laminarin + 0.2 mL Rh-YBS (laminarin + Rh-YBS group), and 0.2 mL Rh-YBS (Rh-YBS group) every 2 days. In addition, take 6 balb / c mice as the blank group and orally administer 0.2 mL PBS every 2 days. Monitor the health status of each group of mice and sacrifice them on the 10th day. Collect the colon, serum, feces, and main organs (heart, liver, spleen, lung, kidney). The results are as Figure 10, compared with the model group, the YBS group, the laminarin + Rh-YBS group, and the Rh-YBS group all showed satisfactory effects (p < 0.01), and there was no significant difference between them (p > 0.05). The weight loss caused by DSS started on the 3rd day, and the curves of the YBS group, the laminarin + Rh-YBS group, and the Rh-YBS group quickly returned to stability, and the end points were significantly higher than those of the model group (p < 0.05). The DAI score showed that the inflammatory levels of the Rh-YBS group, the laminarin + Rh-YBS group, and the Rh-YBS group were always lower than those of the model group and the BS group. The colon conditions and colon lengths of each group showed that the colon lengths of the YBS group, the laminarin + Rh-YBS group, the laminarin + Rh-YBS group, and the Rh-YBS group were similar, all about 8.2 cm (p > 0.05), and the colon lengths of the model group were longer than 6.0 cm (p < 0.01). H&E staining showed that compared with the model group, the colon tissue damage in the YBS group, the laminarin + Rh-YBS group, and the Rh-YBS group was improved. In addition, the H&E staining results of other organs also proved that this administration method did not cause obvious organ damage. In addition, under the action of the YBS group, the laminarin + Rh-YBS group, and the Rh-YBS group, the levels of MPO and pro-inflammatory factors decreased, while the levels of anti-inflammatory factors increased under their action. Observing the serum cytokine levels of each group, the results were similar to those of the colon. The results showed that there was no significant difference between the YBS group, the laminarin + Rh-YBS group, and the Rh-YBS group (p > 0.05).

[0082] (2) Therapeutic administration

[0083] Thirty balb / c mice were randomly divided into 5 groups and given 3% DSS in drinking water for 7 days, and then 0.2 mL of PBS (model group), 0.2 mL of BS (BS group), 0.2 mL of YBS (YBS group), 0.2 mL of 0.1% laminarin + 0.2 mL of Rh-YBS (laminarin + Rh-YBS group), and 0.2 mL of Rh-YBS (Rh-YBS group) were orally administered every 2 days. The BS concentration in each group was 10 8 CFUs / mL. In addition, six balb / c mice were given 0.2 mL of PBS by gavage every two days as the blank group, and the mice were sacrificed on the 15th day after DSS treatment.

[0084] 1. During this period, the changes in the body weight (0 - 4), fecal consistency (0 - 4), and fecal bleeding level (0 - 4) of the mice were recorded. At the time of sacrifice, the colon length was measured. Some colon tissues and major organs (heart, liver, spleen, lung, kidney) were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned at 5 μm, and stained with H&E and PAS. The other colon samples, together with serum and feces, were collected for further experiments. The results are as Figure 11As shown, the body weight of mice continuously decreased on the 5th day after DSS administration, while the body weight of mice improved on the 8th day after Rh-YBS administration. The final body weight of mice in the Rh-YBS group was closest to that of the blank group and was significantly higher than that of other groups (p < 0.05 for the YBS group and p < 0.01 for other groups). The DAI score indicated that severe enteritis symptoms appeared in the DSS group on the 6th day, and the symptoms in the Rh-YBS group began to relieve on the 9th day. At the end point, the enteritis symptoms in the Rh-YBS group were significantly milder than those in other DSS treatment groups (p < 0.05 for the YBS group and p < 0.01 for other groups). The colon length of mice in the model group was significantly shorter than that of the blank group (p < 0.01), and the colon length returned to normal after feeding with the treatment formula. The colon length in the Rh-YBS group was significantly longer than that of other treatment groups (p < 0.01). These results indicate that Rh-YBS has a therapeutic effect on UC and is more effective than BS or YBS.

[0085] 2. Hematoxylin-eosin staining (H&E) was used to verify the therapeutic effect of Rh-YBS on UC and its toxicity to organs. The results are as Figure 12 shown. Compared with the blank group, DSS caused severe mucosal damage, including focal influx of inflammatory cells, crypt loss, and necrosis of colon tissue. The colon crypts and tissues in the Rh-YBS group were more intact, indicating that Rh-YBS improved the signs and symptoms of colon inflammation. In addition, no evidence of histopathological damage was found in the heart, liver, spleen, lung, and kidney in each group. These results further verified the therapeutic effect of Rh-YBS on UC and indicated that Rh-YBS had no obvious organ toxicity.

[0086] 3. ELISA was used to measure the levels of cytokines, MPO, and CGRP in the colon or serum. 1 mg of colon tissue was weighed and homogenized in an ice-water bath at a ratio of 1:9 (m / v) with homogenization medium (recommended 0.9% normal saline). Then, the homogenate was centrifuged at 2500 - 3000 rpm for 10 minutes. Then, the supernatant was used to detect the levels of IL-6, TNF-α, INF-γ, IL-1β, MPO, and IL-10 using ELISA kits respectively. At the same time, the levels of IL-6, TNF-α, IFN-γ, IL-1β, MPO, and IL-10 in the serum were measured.

[0087] 4. Microbiota sequencing analysis of feces was performed. Total genomic DNA was extracted from feces and submucosal samples using the CTAB method. Samples were immediately transferred to sterile tubes after isolation from mice and stored at -80 °C. DNA concentration and purity were determined on 1% agarose gels. According to the concentration, DNA was diluted to 1 ng / μL with sterile water. After extraction, the V3-V4 variable region of the 16S rRNA gene was amplified by PCR using primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GACTACNNGGGTATCTAAT-3′). The reaction used 15 μL High-fidelity PCR Master Mix. The sequencing library was constructed using the NEB Ultra DNA Library Prep Kit. Library quality was evaluated on an Agilent 5400 and sequenced on an Illumina NovaSeq platform, generating 250 bp paired-end reads. The results are as Figure 13 shown. The fecal microbiota diversity in the model group was lower than that in the blank group, and the microbiota diversity increased after treatment with different treatment formulations. The microbiota diversity in the Rh-YBS group was closest to that in the blank group. It was found that the population of Bacteroides increased significantly after DSS treatment, while it decreased after treatment with Rh-YBS and laminarin + Rh-YBS. The microbiota composition in the submucosa was different from that in feces, but still showed the growth and reduction of Bacteroides. These results indicate that Rh-YBS can inhibit harmful bacteria and promote probiotics.

[0088] 5. Analysis of fecal SCFAs content. 200 mg of feces was homogenized with 1% HCl for 1 min, and the homogenate was centrifuged at 3000 rpm for 1 min. The supernatant was acidified with HCl (pH = 0), and each sample was extracted with an equal volume of ether at 4 °C. The samples were treated with 1-tert-butyldimethylsilyl-imidazole at 60 °C for 30 min and analyzed by gas chromatography-mass spectrometry. The results are as Figure 14 shown. Compared with the blank group, DSS decreased the content of short-chain fatty acids in mouse feces. Under the action of Rh-YBS, the short-chain fatty acid level increased significantly (p < 0.01). It is worth mentioning that the increase in butyric acid content in the Rh-YBS group compared with other groups may reflect the proliferation of butyrate-producing bacteria, which is a well-known probiotic, indicating that Rh-YBS has the ability to regulate the microbiota. Rh-YBS can improve the colonic microenvironment, regulate the colonic microbiota, and is beneficial to the improvement of UC.

[0089] Experimental Example 2: Target verification of the Colonic Patches of LEGPDA prepared in Example 6

[0090] LGG was fluorescently labeled with CY5-NHS and used for the preparation of a formulation. Colitis mice induced by 3% DSS were orally administered for 4 h. The colon was collected, and the co-localization of LGG and T cells was observed by immunofluorescence. Confocal images showed that T cells were concentrated in Colonic Patches and LGG was present, indicating that LGG could reach and remain in Colonic Patches( Figure 15 ).

[0091] Experiment on the ability of BS@YPs prepared in Example 8 of Test Example 3 to recruit macrophages

[0092] Drugs for treating RA (EMO or RH) deliver macrophages recruited by BS microsomes to the RA lesion site, thus exerting therapeutic effects. To verify whether BS@YPs can recruit macrophages, we conducted in vitro experiments. Macrophages were inoculated on the upper layer of Transwell chambers. After incubating with different BS preparations and BS metabolites outside the chambers for 24 h, crystal violet staining was used, and the cells on the inner side of the chambers were gently wiped off. Then, the number of cells migrated to the lower layer of the chambers was observed under a microscope. The results showed that both BS and BSYPs had good recruitment effects compared with the Control group, while the number of cell transfers in the YPs group was very small, indicating that BS in BSYPs was responsible for the recruitment effect. Interestingly, BS metabolites showed the strongest recruitment ability, suggesting that the mechanism of BS and BSYPs in recruiting macrophages might be that BS stimulates macrophages through the metabolites produced, causing macrophages to migrate from the upper layer to the lower layer, indicating that BSBYPs have the ability to recruit macrophages in vitro( Figure 16 ).

[0093] To further verify whether BSYPs can recruit macrophages in vivo in PPs, we conducted in vivo experiments. After orally administering different BS preparations for 12 h, PPs were taken, and paraffin sections of rat PPs tissues were stained with DAPI (labeling cell nuclei) and CY3-labeled CD68 antibody (labeling macrophages) to evaluate the ability of BSYPs to recruit macrophages in vivo in PPs. As Figure 17 shown, the content of macrophages in PPs of the Control group was extremely low. In the BS group and the group treated with LA, the number of macrophages in PPs increased slightly, while the number of macrophages in the BSYPs group increased significantly. This indicates that BSYPs have good ability to recruit macrophages in vivo, and we can use BS to "recruit" macrophages, break through the bottleneck that the proportion of macrophages in PPs is less than 5%, and greatly improve the efficiency of targeted drug delivery through the macrophage vehicle pathway in PPs.

[0094] Therapeutic ability of BS@YPs prepared in Example 8 of Test Example 4 for rheumatoid arthritis

[0095] The therapeutic effects of different preparations on AIA rats were evaluated in terms of body weight, ankle joint diameter, sole thickness, and joint inflammation index.

[0096] As Figure 18 shown, the body weight of rats in the Model group increased slowly. Compared with other groups, the body weight gain trend of rats treated in the BS-YPs+EMO-NYPs treatment group was closest to that of the Normol group, indicating that BS-YPs+EMO-NYPs has a certain alleviating effect on RA. Similarly, compared with the Model group, the ankle joint diameter and sole thickness in the BS-YPs+EMO-NYPs group decreased significantly (p<0.01). In addition, the change in arthritis index is a key parameter for evaluating the anti-inflammatory effect during the progression of arthritis. Compared with the Model group, the arthritis index score in the BS-YPs+EMO-NYPs group gradually decreased over time (p<0.01) and tended to be close to that of the Normol group. In addition, as Figure 18 can be seen, the paws of rats in the Model group were most severely swollen, and the paw swelling in the BSPYs+NYPs group was significantly reduced, which was consistent with the changes in ankle joint diameter and sole thickness and the evaluation results of joint inflammation index. In addition, compared with the LA+BS-YPs+EMO-NYPs group, the BS-YPs+EMO-NYPs group had a better therapeutic effect, highlighting the important role of YPs in specifically targeting M cells on PPs, thus successfully carrying drugs into PPs, and then targeting macrophages, enabling the drugs to hitchhike and target the inflammatory site smoothly. More importantly, the BS-YPs+EMO-NYPs group was superior to the EMO-NYPs group and the CCL+BS-YPs+EMO-NYPs group in terms of therapeutic effects on paw swelling, ankle joint diameter, and joint inflammation index, which highlighted the advancement of using BS-YPs to recruit macrophage hitchhikers in PPs to achieve high-density targeted treatment of RA inflammatory sites by macrophage populations carrying emodin microspheres and exerting therapeutic effects.

[0097] Since bone damage, cartilage destruction, and synovial infiltration are the main characteristics of RA, we used micro-CT, hematoxylin and eosin (H&E) staining, and safranin O-fast green staining to verify the therapeutic effect of BS-YPs+EMO-NYPs. Micro-CT analysis showed ( Figure 19) The Model group showed rough bone surfaces in the ankle joints with severe bone erosion. Compared with the Model group, the BS-YPs+EMO-NYPs treatment group had smoother bone surfaces, closer to those of the normal group, indicating that BS-YPs+EMO-NYPs effectively reversed bone erosion. Notably, the bone surfaces of the ankle joints in the LA+BS-YPs+EMO-NYPs and CCL+BS-YPs+EMO-NYPs treatment groups were still relatively rough, highlighting the importance of the targeting effect of YPs and the macrophage hitchhiking system in the treatment of RA. The results of joint H&E and safranin-fast green staining showed that the articular surfaces of the rats in the Normol group were smooth and intact, with no inflammatory cell infiltration or cartilage damage seen around. In addition, the interface between bone and cartilage could be clearly distinguished by its morphology. However, the rats in the Model group showed hyperplasia of synovial tissue accompanied by a large number of inflammatory cell infiltrations, and the joint cavity was significantly narrowed or even disappeared. In addition, due to bone erosion, the articular cartilage was completely damaged, and the interface between bone and cartilage was difficult to distinguish. After treatment with EMO-NYPs and BS+EMO-NYPs, synovial inflammation and cartilage erosion were significantly reduced, but the treatment effect of the BS-YPs+EMO-NYPs group was significantly better. The articular surfaces of the rats in this group were relatively smooth and intact, the joint cavity showed no obvious changes, the degree of inflammatory cell infiltration around was significantly reduced, and the cartilage boundary was clearer, and its tissue morphology was almost the same as that of the normal group. Moreover, compared with the LA+BS-YPs+EMO-NYPs group or the CCL+BS-YPs+EMO-NYPs group, the therapeutic benefits of the BS-YPs+EMO-NYPs group for RA were clearly demonstrated. The results of synovial H&E showed that the synovial tissue of the Normol group was flat, the fibrous stroma in the synovium was arranged loosely, the cells were arranged regularly, there was no inflammatory cell infiltration, and the number of synovial blood vessels was small and arranged sparsely. The synovium of the Model group showed stratified hyperplasia, with a large number of inflammatory cell infiltrations and fibrous tissue hyperplasia inside, and obvious hyperplasia of synovial blood vessels or pannus formation. In the BS-YPs+EMO-NYPs treatment group, there was no obvious hyperplasia in the synovium, the number of synovial inflammatory cells was significantly reduced, and the hyperplasia of synovial blood vessels was greatly improved compared with the model group.

[0098] All in all, these results indicate that all treatment groups alleviated joint inflammation in rats to varying degrees, among which BS-YPs+EMO-NYPs was the most significant in reducing synoviocyte proliferation, inhibiting inflammatory cell infiltration and improving bone damage in AIA rats.

[0099] To evaluate the anti-inflammatory effect of EMO-NYPs, we conducted experiments both in vitro and in vivo. First, we studied their effects on LPS-induced RAW264.7 macrophages in vitro and detected the expression levels of pro-inflammatory factors TNF-α, IL-1β and anti-inflammatory factors IL-4, IL-10. The results were as Figure 20As shown. On the one hand, compared with the Control group, LPS-induced macrophages significantly increased the expression of inflammatory factors TNF-α and IL-1β (p<0.01). Compared with the Model group, the EMO-NYPs group significantly reduced the expression of these pro-inflammatory factors (p<0.01), indicating its anti-inflammatory activity. On the other hand, compared with the Control group, the levels of IL-4 and IL-10 in the Model group were significantly decreased (p<0.01), while EMO-NYPs significantly increased the levels of these anti-inflammatory factors compared with the Model group. Then in the in vivo study, we used the Elisa method to measure the contents of inflammatory cytokines and anti-inflammatory cytokines in rat serum. The levels of pro-inflammatory cytokines TNF-α and IL-1β in the serum of the Model group were significantly higher than those in the Normol group (p<0.01). After treatment with BS-YPs+EMO-NYPs, the levels of TNF-α and IL-1β were significantly lower than those in the EMO-NYPs group and the BS+EMO-NYPs group (p<0.01). In addition, the anti-inflammatory cytokines IL-4 and IL-10 showed an opposite trend of change to TNF-α and IL-1β. To further explore the effect of BS-YPs+EMO-NYPs on synovial inflammatory cytokines in AIA rats in vivo, we used IHC to detect the expression of TNF-α, IL-1β, IL-4 and IL-10 in rat joint synovial tissue. As Figure 20 shown, compared with the Model group, the expression of pro-inflammatory cytokines TNF-α and IL-1β decreased in each treatment group, while the anti-inflammatory cytokines IL-4 and IL-10 showed an opposite expression trend, and the BS-YPs+EMO-NYPs treatment group showed a better therapeutic effect. These results illustrate that EMO-NYPs has anti-inflammatory effects, and BS-YPs+EMO-NYPs shows the best anti-inflammatory effect in vivo.

Claims

1. A composition that can be orally administered to penetrate the intestinal barrier, characterized in that: It contains a carrier or excipient targeting the Dectin-1 receptor of Microflod cells, and one or / and multiple active ingredients / components; Among them, the carrier or excipient targeting the Dectin-1 receptor of Microflod cells is β-glucan; The medicinal ingredients / components are effective ingredients / components of traditional Chinese medicine, or / and probiotics, or / and chemical medicine ingredients, or / and biological medicines.

2. The composition capable of achieving oral permeation through the intestinal barrier according to claim 1, characterized in that: The β-glucan is yeast cell wall and related polysaccharides, Candida albicans cell wall and related polysaccharides, Aspergillus fumigatus cell wall and related polysaccharides, Lentinan, Ganoderma lucidum polysaccharide, Versicolor polysaccharide, oat polysaccharide, seaweed polysaccharide, curdlan, bacterial extracellular polysaccharide, lichen polysaccharide or low molecular weight β-glucan fragment; The probiotic is one of Bacillus subtilis, Lactobacillus rhamnosus, Akkermansia muciniphila, Escherichia coli, lactic acid bacteria or Clostridium butyricum; The effective ingredients / components of the traditional Chinese medicine are rhein, emodin, chlorogenic acid or berberine, and tannic acid.

3. The composition capable of achieving oral permeation through the intestinal barrier according to claim 1 or 2, characterized in that: It combines the effective ingredients / components of traditional Chinese medicine with β-glucans, and then encapsulates probiotics therein to prepare a composition that can be orally taken through the intestinal barrier.

4. Use of the composition capable of orally penetrating the intestinal barrier according to any one of claims 1 to 3 in the preparation of a medicament for treating ulcerative colitis or rheumatoid arthritis.

5. A colon submucosal drug delivery system, characterized in that: It is a novel drug delivery system (Rh-YBS) co-encapsulated with Bacillus subtilis (BS) and Rhein (Rh) using yeast cell walls (YPs) as raw materials through chemical modification and extrusion method; wherein the amount of yeast cell walls, Bacillus subtilis and Rhein per mL is: Yeast cell wall 1-10mg, Bacillus subtilis 10 8-10 CFUs, rhein 0.1-1mg.

6. The colon submucosal drug delivery system according to claim 5, characterized in that: Each mL contains yeast cell wall, Bacillus subtilis, and rhein in the following amounts: Yeast cell wall 2mg, Bacillus subtilis 10 9 CFUs, rhein 0.5mg.

7. The method for preparing the colon submucosal drug delivery system according to claim 5 or 6, characterized in that: It includes the following steps: a. Preparation of yeast cell wall by lysis and freeze-drying; b. The Rh-YPs intermediate was prepared by combining rhein with yeast cell wall through electrostatic adsorption-vacuum extrusion-electrostatic distribution-hydration rearrangement method; c. Bacillus subtilis was loaded into the Rh-YPs intermediate by extrusion to prepare Rh-YBS.

8. Use of the colonic submucosal drug delivery system according to any one of claims 5 or 6 in the preparation of a drug for treating ulcerative colitis; preferably, the drug preparation is an oral colonic drug delivery preparation.

9. A berberine-loaded Bacillus subtilis nanoformulation encapsulated by tannic acid-glucan, characterized in that: It is made by combining tannic acid, berberine and Bacillus subtilis, and then adding glucan solution for inclusion.

10. A polydopamine-coated Lactobacillus rhamnosus and rhamnosin nanoformulation, characterized in that: It encapsulates rhein in lactoferrin to produce positively charged rhein nanoparticles, which are then adsorbed to the surface of the probiotic Lactobacillus rhamnosus (LGG) through electrostatic adsorption. The nanoparticles are oxidized under alkaline conditions by dopamine (LENs) and self-aggregated and wrapped on the surface to form a "bacteria-drug combination" micron system that is sensitive to ROS.

11. A tannic acid (TA)-iron ion (Fe3+) combined with mucin-coated Akkermansia muciniphila nanoformulation, characterized in that: The preparation method is to chelate tannic acid with iron ions and then coat the Akkermansia muciniphila with mucin.

12. A targeted preparation for Peyer's patches (PPs) in the intestine for treating rheumatoid arthritis, characterized in that: It combines lactoferrin, rhamnosin, and yeast cell wall, and then encapsulates Bacillus subtilis in them to prepare NPs-YPs@BS.

13. An oral intestinal barrier drug delivery technology system, characterized in that: The following steps are involved: a. preparing the composition capable of oral permeation through the intestinal barrier according to any one of claims 1 to 3 by micro-nano formulation technology; b. The drug delivery system should reach the colon through the gastrointestinal tract; c. The drug delivery system needs to target colonic Microflod cells; d. The drug delivery system is taken up by Microflod cells and penetrates the intestinal barrier; it enters the Colonic Patches, the immune tissue in the submucosal layer of the colon.

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