An oral MANF microcapsule and its application in preparing a preparation for treating ulcerative colitis

Abstract

The present invention discloses oral MANF microcapsules and their use in the preparation of a preparation for treating ulcerative colitis. Using sodium alginate, hyaluronic acid, and MANF protein as raw materials, the present invention prepares MANF sodium alginate hyaluronic acid composite hydrogel microspheres using a simple and gentle gas shearing method. These microspheres are then emulsified and coated with Eudragit S 100 as an enteric coating material to prepare the oral MANF microcapsules. Animal experiments have confirmed that oral MANF microcapsules have no toxic side effects and that MANF can be effectively delivered to the inflamed colonic tissue of mice with ulcerative colitis. Oral administration of MANF microcapsules significantly alleviates symptoms in mice with ulcerative colitis, improves intestinal flora composition, and demonstrates significantly superior efficacy compared to intravenous injection of the same MANF dose.

Description

Technical Field

[0001] The present invention belongs to the technical field of oral pharmaceutical preparations, and particularly relates to an oral MANF microcapsule and an application thereof in preparing a preparation for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a chronic, relapsing disease of the colon and rectum and a major subtype of inflammatory bowel disease (IBD). IBD has been linked to multiple factors, including immune system dysfunction, genetic susceptibility, dysbiosis, pathogenic infections, and environmental factors. Clinically, many small-molecule medications (such as corticosteroids, aminosalicylates, and immunomodulators) are available for IBD patients. However, systemic use of these drugs often carries the risk of severe side effects and does not prevent relapses.

[0003] In recent decades, the development of many biological therapies has brought new perspectives to the treatment of IBD. In 2005, infliximab (IFX) became the first biological agent approved for UC. Since then, many biological agents such as adalimumab, golimumab, vedolizumab, and ustekinumab have been approved. Mesencephalicastrocyte-derived neurotrophic factor (MANF) is a secretory protein induced by endoplasmic reticulum stress. It has been reported that MANF has an alleviating effect on diseases such as Parkinson's disease, Alzheimer's disease, stroke, retinal regeneration, and diabetes related to endoplasmic reticulum stress. Recently, our research group found that myeloid-specific knockout of MANF can upregulate the expression of BATF2 and promote Ly6C hi CX3CR1 int The recruitment of inflammatory macrophages and the accumulation of Th17 cells further aggravate colonic inflammation. Intravenous injection of recombinant human MANF protein can significantly alleviate acute ulcerative colitis in mice by activating the innate immune inflammatory response. However, all studies on the therapeutic application of recombinant human MANF protein to date have been based on local and intravenous injections.

[0004] Oral administration is convenient, safe, and the preferred route of administration for patients with chronic gastrointestinal diseases. However, the pH of the gastrointestinal tract (GIT) varies widely: from the stomach (1 to 2.5), the small intestine (5.5 to 7), and the large intestine (5.5 to 7.5). The digestive tract is also rich in enzymes and microorganisms. Maintaining normal drug function in such a harsh gastrointestinal environment is extremely challenging, especially for protein-based drugs. Therefore, the development of an oral formulation of MANF is of great significance. Summary of the Invention

[0005] To address the shortcomings of human recombinant MANF protein in the clinical treatment of ulcerative colitis (UC), the present invention provides an oral MANF microcapsule formulation for the treatment of UC, achieving a major breakthrough from intravenous injection to oral administration. By developing an oral MANF microcapsule formulation, the present invention successfully delivers MANF protein to the colon via oral route. Animal studies have demonstrated that this formulation can alleviate dextran sodium sulfate (DSS)-induced ulcerative colitis damage in mice and improve intestinal flora. Furthermore, oral administration is more effective than intravenous injection at the same MANF dose, and even halving the oral MANF dose can achieve similar efficacy to intravenous injection. Specifically, the present invention utilizes the following technical solutions:

[0006] First, the present invention provides an oral MANF microcapsule for treating ulcerative colitis, which includes sodium alginate / hyaluronic acid composite hydrogel microspheres loaded with MANF and an enteric polymer material coated on the surface of the microspheres.

[0007] Secondly, the present invention also provides a method for preparing the above-mentioned oral MANF microcapsules, which can be prepared by the following steps:

[0008] Sodium alginate (SA), hyaluronic acid (HA) and MANF protein solution were mixed evenly in proportion. The mixed solution of sodium alginate, hyaluronic acid and MANF protein solution was dripped into calcium chloride solution using the airflow shear method to control the gas flow rate for full cross-linking to obtain sodium alginate / hyaluronic acid composite hydrogel microspheres loaded with MANF protein. After centrifugation, the microspheres were freeze-dried and further coated with enteric polymer material.

[0009] In the preparation method described above, the sodium alginate solution concentration may be 1-2.5%, the hyaluronic acid solution concentration may be 0.1-1%, preferably 0.1-0.5%, and the calcium chloride solution concentration may be 2-10%, preferably 2-5%. The concentration of the MANF protein in the sodium alginate / hyaluronic acid mixed solution may be no higher than 10 mg / mL, preferably 1-5 mg / mL, and more preferably 1-3 mg / mL. The volume ratio of the sodium alginate / hyaluronic acid mixed solution to the calcium chloride solution may be 1-3:1. The calcium chloride solution concentration may be 2-10%. The shear gas flow rate may be 0.2-0.8 L / min.

[0010] For the preparation method described above, a method that can be selected as a preferred embodiment can be: the preparation method of the oral MANF microcapsules is as follows: 1%-2.5% sodium alginate and 0.1%-1% hyaluronic acid are mixed uniformly, and then MANF protein is added thereto so that the final concentration is not higher than 10 mg / mL, and the mixed solution is dripped into 2-10% calcium chloride using an air flow shear method for full cross-linking; the microspheres are freeze-dried after centrifugation, and 10-50 mg of the freeze-dried microspheres are dispersed in 2-10 mL of a mixed solution of acetone and ethanol (acetone / ethanol volume ratio 2:1) containing 4-6% enteric polymer material; the mixture is transferred to light liquid paraffin containing 0.5-2% span 80, and the solvent is evaporated by stirring at 500-1000 rpm; the mixture is centrifuged and washed with n-hexane and deionized water respectively, finally obtaining sodium alginate / hyaluronic acid composite hydrogel microspheres loaded with MANF protein and wrapped in enteric polymer material, i.e., oral MSH@E microcapsules, which are stored at -20°C. The MSH@E microcapsules obtained in this way have high drug loading capacity (5-8%) and encapsulation efficiency (78-85%).

[0011] Furthermore, as a more preferred embodiment, the oral MANF microcapsules are prepared by uniformly mixing 1.5% sodium alginate and 0.25% hyaluronic acid. MANF protein is then added to the mixture to a final concentration of 2 mg / mL. This mixture is then dripped into a 2% calcium chloride solution using an airflow shear device for thorough crosslinking. After centrifugation, the microspheres are lyophilized. Fifty milligrams of the lyophilized microspheres are dispersed in 10 mL of a mixture of acetone and ethanol (acetone / ethanol volume ratio 2:1) containing 5% Eudragit S 100. This mixture is then transferred to light liquid paraffin containing 1% Span 80 and the solvent is evaporated at 1000 rpm for at least 3 hours. The mixture is then centrifuged and washed with n-hexane and deionized water to obtain MSH@E microcapsules, which are then stored at -20°C. The resulting MSH@E microcapsules achieve both an optimal drug loading (6%) and encapsulation efficiency (80%).

[0012] The oral MANF microcapsules prepared by the present invention feature a simple preparation method and mild conditions, maximally preserving the biological activity of the MANF protein. They are pH-sensitive, enabling minimal drug release in the upper gastrointestinal tract and time-dependent release in the colonic fluid. Hyaluronic acid and sodium alginate, as natural polymers, exhibit excellent biocompatibility. CD44 is highly expressed on the surface of macrophages in areas of colitis inflammation, and hyaluronic acid can specifically target CD44 molecules. MANF protein can bind to these molecules through non-covalent interactions such as hydrogen bonding and electrostatic interactions, leading to enrichment in the inflamed area, thus achieving better targeted therapy.

[0013] Thirdly, the oral MANF microcapsules of the present invention can be used in the preparation of preparations for treating ulcerative colitis. Of course, the oral MANF microcapsules obtained by the above-mentioned preparation method can also be used in the preparation of preparations for treating ulcerative colitis.

[0014] The oral MANF microcapsules prepared by this invention can be used for long-term oral treatment of ulcerative colitis. Animal experiments have confirmed that MANF can be effectively delivered to the inflamed colonic tissues of mice with ulcerative colitis. Results, including body weight, DAI scores, and histopathological staining, demonstrate that the oral MANF microcapsules are significantly more effective than those administered intravenously at the same dose. Repeated high-dose oral administration to experimental animals showed no acute toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Schematic diagram of the preparation of oral MANF microcapsules and their application in the treatment of colitis.

[0016] Figure 2 : Scanning electron microscopy images of MSH and MSH@E.

[0017] Figure 3 : In vitro gastrointestinal simulated release profiles of MSH@E, MECC and MSH (simulated gastric fluid SGF-2 h, simulated small intestinal fluid SIF-3 h and simulated colonic fluid SCF-12 h).

[0018] Figure 4 : Biocompatibility evaluation of MSH@E. (A) Body weight change, (B) HE staining images of major organ tissues.

[0019] Figure 5 : In vivo fluorescence imaging of normal mice and DSS-induced colitis mice after oral administration of MS@E and MSH@E and corresponding gastrointestinal tissue fluorescence imaging.

[0020] Figure 6 : His immunohistochemical staining of colon tissue sections from colitis mice after oral administration of MS@E and MSH@E. The lower image is a partial magnification of the upper image. (A) Negative control, (B) PBS group, (C) MS@E, and (D) MSH@E.

[0021] Figure 7 : Anti-inflammatory effect of MSH@E in vitro. Expression levels of (A) IL-6, (B) IL-1β, (C) TNF-α, and (D) MPO in the cell supernatant of each group after addition of SH@E, MANF, and MSH@E to the LPS-induced RAW 264.6 cell inflammation model.

[0022] Figure 8Evaluation of the efficacy of MSH@E in ulcerative colitis. (A) Schematic diagram of the ulcerative colitis mouse model and treatment process. C57BL / 6J mice were given 3.5% w / v DSS in drinking water for 7 days and then received drug treatment from days 4 to 7. (B) Body weight change, (C) DAI score, (D) colon weight, and (E) colon length.

[0023] Figure 9 HE-stained images of colon tissue sections of mice in each experimental group after treatment with different drugs. The lower image is a partial magnification of the upper image. (A) Healthy mice, (B) PBS group, (C) SH@E group, (D) MS@E group, (E) MANF (iv) group, (F) ½MSH@E group, (G) MSH@E group, and (H) 5-ASA group.

[0024] Figure 10 : Levels of IL-6 (A), IL-1β (B), TNF-α (C) and MPO (D) in the serum of mice in each experimental group after treatment with different drugs.

[0025] Figure 11 16S (rRNA) sequencing analysis of the intestinal microbiota in mice with colitis after oral administration of MSH@E. (A) Chao index, (B) Shannon index, (C) relative abundance of Escherichia-Shigella, and (D) relative abundance of Akkermansia. DETAILED DESCRIPTION

[0026] The following embodiments are further descriptions of the present invention to illustrate the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required by the present invention. Example 1

[0027] Preparation of oral MANF microcapsules (MSH@E):

[0028] (1) Prepare an aqueous solution with a final concentration of 1.5% sodium alginate SA and 0.25% hyaluronic acid HA;

[0029] (2) Add MANF protein to the above solution to a final concentration of 2 mg / mL. After mixing, add the mixed solution to an air shear device, adjust the air flow to 0.6 L / min, and drip it dropwise into a 2% CaCl2 solution. Stir for 10 min.

[0030] (3) Collect the microspheres and wash them twice with deionized water;

[0031] (4) Freeze-dry and store in a sealed container at -20°C for future use;

[0032] (5) Weigh 0.5 g of Eudragit S 100 and dissolve it in 10 mL of a mixed solvent of ethanol and acetone (acetone / ethanol volume ratio 2:1);

[0033] (6) Weigh 50 mg of MANF-loaded sodium alginate / hyaluronic acid microspheres and add them to the solution in (5) above;

[0034] (7) Measure 70 mL of liquid paraffin and add Span 80 to a final concentration of 1%;

[0035] (8) Add solution (5) to (7) and stir at 1000 rpm / min at room temperature until the solvent evaporates completely;

[0036] (9) The microspheres were collected by centrifugation, washed with n-hexane and deionized water, freeze-dried, and sealed and stored at -20 °C until use.

[0037] The morphology of the hydrogel microspheres prepared in Example 1 was observed by scanning electron microscopy. Figure 2 As shown in the figure, the microcapsules prepared by the present invention are spherical. The drug loading capacity of the MSH@E microcapsules obtained by the present invention is 6% and the encapsulation efficiency is 80%.

[0038] Comparative Example 1: Preparation of oral microcapsules (SH@E) without MANF protein

[0039] The difference from Example 1 is that no MANF protein solution is added in step (2).

[0040] Comparative Example 2: Preparation of oral MANF microcapsules (MS@E) without hyaluronic acid HA

[0041] Preparation of MANF-loaded Eudragit S 100-coated sodium alginate hydrogel microspheres (MS@E). The difference from Example 1 is that the addition of hyaluronic acid in step (1) is omitted.

[0042] Comparative Example 3: Preparation of MANF / sodium alginate / hyaluronic acid composite hydrogel microspheres (MSH) without enteric coating.

[0043] The difference from Example 1 is that the Eudragit S 100 coating is not performed in steps (5-9).

[0044] Comparative Example 4: Preparation of MANF Enteric-coated Capsules (MECC)

[0045] The MANF protein was directly encapsulated in commercial enteric-coated capsules (YG00-2, Yuyan Instrument Co., Ltd.). Example 2

[0046] In vitro release experiment of oral MANF microcapsules (MSH@E).

[0047] Appropriate amounts of oral MANF microcapsules (MSH@E), MANF enteric-coated capsules (MECC), and MANF sodium alginate / hyaluronic acid composite hydrogel microspheres (MSH) were placed in artificial gastrointestinal fluid at different pH values ​​and incubated. The release of proteins at different time points was determined using the Coomassie brilliant blue method to investigate the degradation behavior of the microspheres at different pH values ​​in vivo. Figure 3 As shown, protein release from the microspheres in artificial gastric fluid was almost zero within 2 hours. When transferred to artificial small intestinal fluid, the cumulative protein release was less than 5%, while protein release was complete within 12 hours in artificial colonic fluid. The comparative MANF enteric-coated capsules, on the other hand, showed higher release than the oral MANF microcapsules in both artificial gastric and small intestinal fluids, and rapid and substantial release occurred in colonic fluid. Comparative MANF sodium alginate hyaluronic acid composite hydrogel microspheres (MSH), without Eudragit S 100 coating, showed slight release in small intestinal fluid and complete release in colonic fluid within 8 hours, demonstrating that oral MANF microcapsules effectively protect MANF in the upper gastrointestinal tract while providing a good delayed release effect in the colon. Example 3

[0048] Biocompatibility evaluation of oral MANF microcapsules (MSH@E):

[0049] In vivo biocompatibility experiments were conducted on 12 female C57BL / 6 mice divided into two groups and orally administered with PBS or MSH@E. The MSH@E group received a MANF dose of 750 mg / kg for six consecutive days and then were sacrificed after a 24-hour fast. Daily body weights were recorded during the dosing period, and major organ and gastrointestinal tissues (heart, liver, spleen, lung, kidney, and colon) were collected for hematoxylin-eosin staining. Figure 4 As shown, there was no significant difference in the body weight of the experimental group mice compared with the control group ( Figure 4 A), Hematoxylin-eosin staining results showed that the MSH@E hydrogel microspheres group did not cause pathological changes and inflammatory lesions in the organs of mice compared with the control group ( Figure 4 B). Example 4

[0050] Targeting evaluation of oral MANF microcapsules (MSH@E).

[0051] The anthocyanin fluorescent dye Cy 5 was coupled to the MANF protein in the composite hydrogel microspheres. Healthy mice and colitis mice were then orally administered the same dose of PBS, Cy 5, MS@E, and MSH@E (Cy 5: 0.5 mg / kg). Abdominal fluorescence was recorded using an optical imaging system at 2, 6, 12, and 24 hours. Mice were sacrificed 24 hours after administration, and the colons were harvested for fluorescence imaging. Figure 5 As shown, the fluorescence intensity of the Cy5, MS@E and MSH@E groups gradually weakened over time. The Cy5 group had almost no fluorescence after 24 hours, while the fluorescence intensity of the hyaluronic acid HA-modified MSH@E group was still very high. Gastrointestinal tissue imaging showed that the hyaluronic acid-modified MSH@E group was significantly enriched in the colon. In addition, the fluorescence of MSH@E-treated colitis mice was stronger than that of healthy mice. These results indicate that MSH@E retains a longer time in the colon tissue of DSS-induced colitis mice, confirming that the addition of HA increases the in vivo targeting properties of MSH@E. The MANF protein used in this experiment carries a histidine His tag, and immunohistochemical labeling of His shows a brownish-yellow color ( Figure 6 ), compared with the MS@E group, the MSH@E group showed a more obvious brown-yellow color, indicating that a large amount of MANF protein was successfully delivered to the colon, which suggests that HA plays an important role in MSH@E targeting the colon inflammation site. Example 5

[0052] In vitro anti-inflammatory evaluation of oral MANF microcapsules (MSH@E).

[0053] For all cell experiments, RAW 264.7 cells were cultured with MSH@E extract. Typically, MSH@E was incubated in RPMI1640 medium for 72 hours, and the supernatant was collected by centrifugation to obtain the MSH@E extract. Cells were seeded in 6-well plates overnight, co-cultured with LPS (1 µg / mL), and then incubated with MSH@E extract (322 µg / mL, equivalent to 20 µg / mL of MANF protein) for 24 hours. Subsequently, the activity levels of myeloperoxidase (MPO) (Boster, EK0943), IL-6 (Boster, EK0411), IL-1β (Boster, EK0394), and TNF-α (Boster, EK0527) in the cell supernatant were measured by ELISA. The ELISA results showed that LPS activation increased the levels of IL-6, IL-1β, TNF-α, and MPO. After treatment with MANF and MSH@E, the levels of IL-6, IL-1β, TNF-α, and MPO decreased. It is worth noting that when the concentration of MANF in MSH@E was the same as that in the MANF group (20 μg / mL), the anti-inflammatory effect of MSH@E was better than that of the MANF group ( Figure 7AD). Example 6

[0054] Evaluation of the efficacy of oral MANF microcapsules (MSH@E).

[0055] Mice with ulcerative colitis were randomly divided into seven groups: PBS-colitis mice were orally administered with PBS, 5-ASA-colitis mice were orally administered with 5-ASA (1 mg / kg), SH@E-colitis mice were orally administered with SH@E (16 mg / kg), MS@E-colitis mice were orally administered with MS@E (equivalent to MANF 1 mg / kg), MANF(iv)-colitis mice were intravenously administered with MANF (1 mg / kg), MSH@E-colitis mice were orally administered with MS@E (equivalent to MANF 1 mg / kg), and 1 / 2 MSH@E-colitis mice were orally administered with half the dose of MSH@E (equivalent to MANF 0.5 mg / kg). Healthy mice served as the control group. Figure 8 As shown in A, mice in each experimental group were dosed once daily from days 4 to 7. During treatment, body weight, rectal bleeding, and stool density were examined daily, and the disease activity index (DAI) was determined. On day 10, mice were sacrificed, and spleen and colon samples were collected. Spleen weight and colon length were recorded. Colon tissue was histologically sectioned. Orbital blood was collected to measure serum inflammatory cytokine levels. Feces were collected to assess the composition and abundance of the intestinal microbiota using 16S rRNA gene sequencing.

[0056] Weight results as Figure 8 As shown in Figure B, the weight of mice in the DSS model group gradually decreased compared with the healthy control group, with a decrease of approximately 40% on day 10. Compared with the PBS group, the weight of mice in the oral gavage MS@E, 1 / 2 MSH@E, MSH@E groups, and the tail vein injection MANF group all showed a downward trend. However, the weight loss of mice in the oral MS@E, 1 / 2 MSH@E, MSH@E, and tail vein injection MANF groups gradually slowed down, with the weight loss of mice in the MSH@E group being the most significant. Consistent with the weight change results, the disease activity index ( Figure 8 C), colon weight ( Figure 8 D) and colon length ( Figure 8 E) The results also showed that MSH@E had the best therapeutic effect. HE staining results ( Figure 9) showed that the colons of mice in the DSS group showed obvious immune cell infiltration, mucosal tissue loss, and disappearance of goblet cells and crypt structures, while the crypt structures in the MSH@E group were partially restored and tissue damage was alleviated. Elisa results showed that MSH@E treatment significantly reduced the levels of IL-6, IL-1β, TNF-α, and MPO in mice with colitis, indicating that MSH@E can alleviate colitis by regulating the levels of MPO, IL-6, IL-1β, and TNF-α. Figure 10 At the same time, the bacterial community composition of colitis mice was significantly changed compared with that of healthy mice. Oral administration of MSH@E significantly increased Chao 1 and Shannon diversity indices, and improved species richness and microbial community diversity ( Figure 11 A, B). It has been reported that the abundance of Escherichia-Shigella, a highly virulent strain of Proteobacteria, increased significantly in mice with inflammatory bowel disease. Akkermansia, a member of the Verrucomicrobia phylum, plays a crucial role in maintaining the integrity of the intestinal barrier and is considered a beneficial probiotic. MSH@E microcapsules reduced the relative abundance of Shigella and increased the relative abundance of Akkermansia ( Figure 11 C, D). These results indicate that the abundance and diversity of the intestinal flora in colitis mice after MSH@E treatment tend to return to the levels of healthy mice, indicating that ulcerative colitis is effectively improved. The oral MANF microcapsules of the present invention can be used for long-term oral treatment of ulcerative colitis.

[0057] It should be noted that the technical contents of the present invention described above are only for the purpose of explaining and illustrating the technical essence of the present invention to enable those skilled in the art to understand the technical essence of the present invention. Therefore, the technical contents described above are not intended to limit the substantial protection scope of the present invention. The substantial protection scope of the present invention shall be based on the claims. Those skilled in the art should be aware that any modifications, equivalent substitutions, and improvements based on the substantial spirit of the present invention shall fall within the substantial protection scope of the present invention.

Claims

1. An oral MANF microcapsule, comprising sodium alginate / hyaluronic acid composite hydrogel microspheres loaded with MANF, wherein the surface of the composite hydrogel microspheres is coated with an enteric polymer material.

2. The method for preparing the oral MANF microcapsules according to claim 1, characterized in that: Sodium alginate solution, hyaluronic acid solution and MANF protein solution were mixed evenly in proportion, and the mixed solution of sodium alginate solution, hyaluronic acid solution and MANF protein solution was dripped into calcium chloride solution by controlling the gas flow rate using air flow shear method to fully cross-link to obtain sodium alginate / hyaluronic acid composite hydrogel microspheres loaded with MANF protein. After centrifugation, the microspheres were freeze-dried and further coated with enteric polymer material on the surface.

3. The preparation method according to claim 2, wherein The concentration of the sodium alginate solution is 1-2.5%, the concentration of the hyaluronic acid solution is 0.1-1%, and the concentration of the calcium chloride solution is 2-10%.

4. The preparation method according to claim 3, wherein The concentration of the hyaluronic acid solution is 0.1-0.5%.

5. The preparation method according to claim 3, wherein The concentration of the calcium chloride solution is 2-5%.

6. The preparation method according to claim 2, wherein The concentration of the MANF protein in the sodium alginate / hyaluronic acid mixed solution is not higher than 10 mg / mL.

7. The preparation method according to claim 6, wherein The concentration of the MANF protein in the sodium alginate / hyaluronic acid mixed solution is 1-5 mg / mL.

8. The preparation method according to claim 7, wherein The concentration of the MANF protein in the sodium alginate / hyaluronic acid mixed solution is 1-3 mg / mL.

9. The preparation method according to claim 2, wherein The volume ratio of the MANF / sodium alginate / hyaluronic acid mixed solution to the calcium chloride solution is 1-3:

1.

10. The preparation method according to claim 2, wherein The gas flow rate is 0.2-0.8 L / min.

11. The preparation method according to claim 2, wherein The enteric polymer material is Eudragit S 100.

12. The preparation method according to claim 2, wherein The composite hydrogel microspheres are prepared by uniformly mixing a 1-2.5% sodium alginate solution with a 0.1-1% hyaluronic acid solution, then adding MANF protein so that the MANF protein concentration is no higher than 10 mg / mL, then dripping the mixed solution into a 2-10% calcium chloride solution at a controlled gas flow rate for full cross-linking, centrifuging, and freeze-drying the microspheres; dispersing 10-50 mg of the freeze-dried microspheres in 2-10 mL of a mixed solution of acetone and ethanol containing 4-6% enteric polymer material, wherein the acetone / ethanol volume ratio is 2:1; transferring the mixture into light liquid paraffin containing 0.5-2% Span80, and then stirring at 500-1000 rpm to evaporate the solvent; centrifuging and washing with n-hexane and deionized water, respectively, to finally obtain enteric polymer-wrapped sodium alginate / hyaluronic acid composite hydrogel microspheres loaded with MANF, namely, oral MANF microcapsules MSH@E.

13. Use of the oral MANF microcapsules according to claim 1 in preparing a preparation for treating ulcerative colitis.

14. Use of the oral MANF microcapsules obtained by the preparation method according to any one of claims 2 to 10 in the preparation of a preparation for treating ulcerative colitis.

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