Preparation method and application of probiotic-loaded hydrogel precursor solution for treating stomach diseases

Abstract

The invention discloses a preparation method and application of a probiotic-loaded hydrogel precursor solution for treating stomach diseases, and belongs to the field of preparation of medicinal high polymer materials. Comprising the following steps: oxidizing hyaluronic acid with sodium periodate to obtain oxidized hyaluronic acid; reacting the oxidized hyaluronic acid with dopamine to prepare dopamine modified oxidized hyaluronic acid; the preparation method comprises the following steps: carrying out amidation reaction on chitosan and N-acetyl-L-cysteine to prepare thiolated chitosan; the preparation method comprises the following steps: mixing dopamine modified oxidized hyaluronic acid and a thiolated chitosan aqueous solution with probiotics, and carrying out a cross-linking reaction under oxidation and acidic conditions to obtain a hydrogel precursor solution; the hydrogel precursor solution can be taken orally and swallowed, can be further gelatinized in situ under the condition of stomach focus oxidation, has excellent adhesion, and can be effectively retained in the stomach and keep the activity of probiotics. In addition, the hydrogel can continuously remove excessive active oxygen in the stomach, relieve the inflammatory response of gastric mucosa and remarkably improve the microenvironment of the focus.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical polymer material preparation, specifically relating to the preparation method and application of a probiotic-loaded hydrogel precursor solution for treating gastric diseases. Background Technology

[0002] Gastric diseases refer to pathological changes such as inflammation, erosion, or ulceration in the gastric tissue or mucosa. These are mainly caused by irritants such as nonsteroidal anti-inflammatory drugs (NSAIDs) and ethanol, leading to damage to the gastric mucosa and the formation of ulcers in the submucosa and muscularis propria. Under normal circumstances, the gastric mucosa acts as an important protective barrier, effectively resisting the erosion of gastric acid and harmful substances. However, when the body is subjected to long-term chemical or psychological stimulation, excessive gastric acid secretion can damage the integrity of the mucosa, inducing gastritis, ulcers, and other diseases, and in severe cases, even developing into gastric cancer. Current clinical treatments mainly rely on proton pump inhibitors (PPIs), gastric mucosal protectants, and hemostatic agents. While these can relieve symptoms and promote healing, approximately 20% of patients experience side effects. Long-term use of PPIs may also lead to malnutrition, increased risk of infection and fractures, and some antacids and protectants also have potential toxicity. Furthermore, these drugs have poor stability and low bioavailability in the acidic environment of the stomach, significantly affecting their efficacy. The healing process of ulcers is complex, involving physiological processes such as cell regeneration and angiogenesis. Even after the surface wound heals, tissue function may still be abnormal, resulting in a high risk of recurrence.

[0003] Lactobacillus rhamnosus has demonstrated significant protective effects in the treatment of gastric diseases, providing an important direction for exploring novel therapeutic strategies. Gastric diseases are mainly caused by damage to the gastric mucosal barrier, increased oxidative stress, and elevated inflammatory responses, and Lactobacillus rhamnosus can effectively intervene in this process through multiple mechanisms. Studies have shown that Lactobacillus rhamnosus lowers gastric pH and inhibits the proliferation of harmful bacteria by secreting metabolites such as lactic acid, short-chain fatty acids, and antimicrobial peptides, while simultaneously regulating the balance of intestinal flora to restore gastrointestinal microecological homeostasis. Furthermore, Lactobacillus rhamnosus also reduces oxidative stress and the accumulation of reactive oxygen species (ROS) by activating the nuclear factor E2-related factor 2 (Nrf2) signaling pathway, thereby mitigating alcohol-induced damage to gastric mucosal cells. Its anti-inflammatory effect is manifested in alleviating gastric mucosal inflammation by inhibiting the overactivation of the nuclear factor κB (NF-κB) pathway and reducing the expression of pro-inflammatory factors (such as TNF-α, IL-6, and IL-1β). Animal experiments further confirmed that *Lactobacillus rhamnosus* can significantly reduce ulcer area, increase the levels of gastric mucosal defense factors (such as gastric mucus and prostaglandin E2), and promote the repair of damaged tissue. Combined with preclinical research results, *Lactobacillus rhamnosus* provides a safe and effective probiotic intervention method for the treatment of gastric diseases.

[0004] However, despite the significant protective effect of Lactobacillus rhamnosus in the treatment of gastric diseases, it faces a severe challenge in the highly acidic environment of the stomach. After oral administration, a large number of live bacteria are inactivated by gastric acid, resulting in a significant reduction in its survival rate and colonization ability in the stomach, thus limiting the full realization of its therapeutic effect. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing and applying a probiotic-loaded hydrogel precursor solution for treating gastric diseases, thereby solving the problems in the prior art.

[0006] The objective of this invention can be achieved through the following technical solutions: The method for preparing a probiotic-loaded hydrogel precursor solution for treating gastric diseases includes the following steps: Sodium periodate was used to oxidize hyaluronic acid to obtain oxidized hyaluronic acid; then it was reacted with dopamine to prepare dopamine-modified oxidized hyaluronic acid. Thioyl chitosan was prepared by amidation reaction of chitosan with N-acetyl-L-cysteine; Dopamine-modified oxidized hyaluronic acid and thiolated chitosan were dissolved in sterile PBS, then mixed with probiotics, and cross-linked under oxidative acidic conditions to obtain a hydrogel precursor solution.

[0007] Furthermore, the process for preparing oxidized hyaluronic acid is as follows: hyaluronic acid is dissolved in water, and sodium periodate solution is added to react; after the reaction is completed, ethylene glycol is added and stirred to quench any unreacted sodium periodate; then, oxidized hyaluronic acid is obtained by dialysis, freezing, and drying.

[0008] Furthermore, the process for preparing dopamine-modified oxidized hyaluronic acid includes: dissolving oxidized hyaluronic acid in a 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) solution (pH 5.5), then adding dopamine hydrochloride, adjusting the pH of the reaction system to the range of 5.0-5.5, stirring the reaction in the dark for 24 hours, and then dialyzing, removing impurities, and lyophilizing to obtain dopamine-modified oxidized hyaluronic acid.

[0009] Furthermore, the molar ratio of oxidized hyaluronic acid, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) and dopamine hydrochloride is 1:1.5:2.5.

[0010] Further, the process for preparing thiolated chitosan is as follows: chitosan is dispersed in deionized water, 1-hydroxybenzotriazole is added, and after stirring until the solution is clear, N-acetyl-L-cysteine ​​is added to obtain a reaction mixture. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide dissolved in distilled water is added to the reaction mixture, the pH is adjusted to 5, and the reaction is stirred. Then, thiolated chitosan is obtained by dialysis, freezing, and drying.

[0011] Furthermore, the molar ratio of chitosan, 1-hydroxybenzotriazole, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-acetyl-L-cysteine ​​is 1:2.58:10.32:5.16.

[0012] Furthermore, the probiotic is Lactobacillus rhamnosus.

[0013] Furthermore, in the cross-linking reaction, the acidic condition is pH=2~3, and the oxidation condition is the addition of sodium periodate solution.

[0014] A probiotic-loaded hydrogel precursor solution for treating gastric diseases was prepared using the above-described preparation method.

[0015] The above-mentioned hydrogel precursor solution is used in the preparation of drugs for treating gastric diseases.

[0016] The beneficial effects of this invention are: 1. The hydrogel precursor solution prepared by this invention, after being administered orally into the stomach, can form a hydrogel in situ in the oxidative microenvironment of the gastric lesion, effectively isolating gastric acid and harmful substances and reducing further stimulation to ulcer tissue.

[0017] 2. The hydrogel precursor solution of the present invention forms a hydrogel in situ under oxidative conditions in the gastric lesion, which can adhere to the gastric mucosa, achieve long-term retention in the stomach, protect the activity of probiotics and their targeted release in the gastric acid environment, regulate the gastric mucosal microenvironment at the lesion site, and provide an efficient strategy for the prevention and treatment of gastric diseases.

[0018] 3. The hydrogel precursor solution prepared in this invention forms a hydrogel in situ under oxidative conditions in gastric lesions, exhibiting excellent biocompatibility and reactive oxygen species (ROS) responsiveness. After oral administration, it can continuously scavenge excess ROS in the stomach, significantly alleviating oxidative stress and exerting a powerful antioxidant effect. Simultaneously, the hydrogel can inhibit the release of pro-inflammatory factors, reduce gastric mucosal inflammation, and achieve an anti-inflammatory effect, providing a highly efficient strategy for the prevention and treatment of gastric diseases.

[0019] 4. The hydrogel precursor solution of the present invention is in the form of lyophilized powder, which helps to ensure long-term stable storage. In addition, the composition of this type of hydrogel is simple and the preparation process is easy to operate, which is beneficial to its evaluation and detection before clinical translation.

[0020] 5. The dopamine-modified oxidized hyaluronic acid obtained in the preparation process of this invention has good biocompatibility and antioxidant effect. The resulting thiolated chitosan has good biocompatibility and strong tissue adhesion. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The hydrogen nuclear magnetic resonance spectra of hyaluronic acid (HA) and oxidized hyaluronic acid (OHA) in Example 1 are shown. Figure 2 The infrared spectrum of oxidized hyaluronic acid (OHA) in Example 1; Figure 3 The hydrogen nuclear magnetic resonance spectrum of dopamine-modified oxidized hyaluronic acid (OD-CN) in Example 1; Figure 4 The hydrogen nuclear magnetic resonance spectrum of thiolated chitosan (CN-NAC) in Example 2; Figure 5 Images showing the gelation of the hydrogel precursor solution in Example 3 under simulated acidic and oxidative conditions in a gastric lesion; Figure 6 SEM images of the hydrogel obtained in Example 3 and the hydrogel loaded with Bacillus rhamnosus (HL); Figure 7 This is a graph showing the in vitro antioxidant results of the hydrogel obtained in Example 4; Figure 8 This is a graph showing the in vitro anti-inflammatory results of the hydrogel obtained in Example 5; Figure 9 This is a graph showing the in vivo retention results of the hydrogel precursor solution obtained in Example 6. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] The method for preparing a probiotic-loaded hydrogel precursor solution for treating gastric diseases includes the following steps: S1. Hyaluronic acid (HA) was oxidized using sodium periodate to obtain oxidized hyaluronic acid (OHA). The oxidized hyaluronic acid was dissolved in 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) solution (pH 5.5), and then dopamine hydrochloride was added to adjust the pH of the reaction system to the range of 5.0-5.5. The reaction was stirred in the dark for 24 hours. After dialysis, impurity removal, and lyophilization, dopamine-modified oxidized hyaluronic acid (OHA-DA) was obtained. S2, chitosan (CS) was dispersed in deionized water, 1-hydroxybenzotriazole (HOBt) was added, and the mixture was stirred continuously until the solution was completely clear. Then N-acetyl-L-cysteine ​​was added, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) dissolved in distilled water was added dropwise to the reaction mixture. The pH was adjusted to 5, and the mixture was stirred at room temperature for 3 hours. After stepwise dialysis, the mixture was freeze-dried to obtain thiolated chitosan (CS-NAC). S3, dopamine-modified oxidized hyaluronic acid and thiolated chitosan were dissolved separately in sterile PBS to obtain two polymer solutions. These two polymer solutions were then mixed with probiotics and subjected to a cross-linking reaction under oxidative (sodium periodate solution) and acidic (pH=2) conditions to obtain a hydrogel precursor solution. This precursor solution can be further gelled in situ under simulated oxidative conditions of gastric lesions.

[0025] In S1, the synthetic route for dopamine-modified oxidized hyaluronic acid (OHA-DA) is as follows: The structural formula of dopamine-modified oxidized hyaluronic acid (OHA-DA) is as follows: R1 and R2 are selected from Or an aldehyde group, R3 is selected from hydroxyl or .

[0026] In S1, the molecular weight of the hyaluronic acid is 20-400 kDa. In S2, the molecular weight of the chitosan is 10-100 kDa.

[0027] In S3, the probiotic is Lactobacillus rhamnosus (LGG).

[0028] The technical solution of the present invention will be described below through the following embodiments; wherein the sources of the main raw materials are as follows: Hyaluronic acid, manufactured by Maclean Chemicals, specification / purity 99%, CAS number: 9004-61-9; Chitosan, manufactured by Yuanye, specification / purity 90%, CAS number: 9012-76-4; 1-Hydroxybenzotriazole (HOBt), manufactured by Aladdin, specification / purity ≥99%, CAS number: 2592-95-2; 4-(4,6-Dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM), manufactured by Aladdin, specification / purity ≥97%, CAS number: 3945-69-5; 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), manufactured by Aladdin, specification / purity ≥98%, CAS number: 247-361-2; N-hydroxysuccinimide (NHS), manufactured by Aladdin, specification / purity ≥98%, CAS number: 228-001-3; N-acetyl-L-cysteine, manufactured by Merck, with a purity of ≥99.81%, CAS No.: 616-91-1; Sodium periodate, manufactured by Anaiji, with a purity of ≥99%, CAS No.: 7790-28-5; Ethylene glycol, manufactured by Sinopharm Group, specification / purity is analytical grade, CAS number: 64-17-5; Example 1 This embodiment describes the synthesis of dopamine-modified oxidized hyaluronic acid (OHA-DA); (1) Synthesis of oxidized hyaluronic acid, the synthetic route is as follows: The specific process is as follows: HA (1 g, 0.1 mmol) was added to a single-necked round-bottom flask and dissolved in 100 mL of high-purity water. Then, 5 mL of sodium periodate solution (10.7%, w / v) was gently added with stirring. The reaction was carried out at 25°C in the dark for 8 hours, followed by the addition of 0.5 mL of ethylene glycol and stirring for another hour to quench any unreacted sodium periodate. The resulting product was dialyzed against deionized water for 3 days, with the deionized water changed three times a day. Finally, it was freeze-dried to obtain OHA.

[0029] (2) Synthesis of dopamine-modified oxidized hyaluronic acid (OHA-DA), the synthetic route is as follows: DMTMM (72 mg, 0.318 mmol) was added to a solution of oxidized hyaluronic acid (OHA, 20 mg, 0.212 mmol), followed by slow addition of dopamine hydrochloride (DA, 100 mg, 0.53 mmol). The pH was adjusted to 5.5, and the reaction was continued with stirring for 24 hours to ensure complete coupling. After the reaction, the mixture was dialyzed sequentially with acidic phosphate buffer (PBS) and deionized water (TDW) to remove unreacted chemicals and reaction byproducts. After dialysis, the solution was lyophilized to obtain the OHA-DA conjugate, which was stored at 4°C for later use. In this example, to verify the successful preparation of oxidized hyaluronic acid (OHA) and its dopamine-modified product (OHA-DA), a series of spectroscopic characters were performed on the sample. ¹H NMR spectrum (¹H NMR) Figure 1 The results showed that, compared with hyaluronic acid (HA), OHA exhibited three new characteristic peaks in the δ 4.6–5.0 ppm range. These chemical shifts corresponded to the resonance peaks of the aldehyde protons, indicating that part of the sugar ring in HA underwent oxidation to open and form an aldehyde structure. This result was further supported by Fourier transform infrared spectroscopy (FTIR) analysis: [e.g., ...] Figure 2 As shown, OHA exhibited a characteristic absorption peak at 1731 cm⁻¹ for the carbonyl (C=O) stretching vibration, verifying the effective conduct of the oxidation reaction. Subsequently, the ¹H NMR spectrum of the dopamine-modified product OHA-DA was analyzed. Figure 3 Analysis revealed a characteristic peak at δ 6.7 ppm, attributed to the protons of the dopamine benzene ring, indicating that dopamine had been successfully grafted onto the OHA molecule.

[0030] Example 2 This embodiment describes the synthesis of thiolated chitosan (CS-NAC); The synthesis route is as follows: The specific process is as follows: Chitosan (CS, 0.50 g, 1 mmol) was dispersed in deionized water (46 mL). 1-Hydroxybenzotriazole (HOBt, 0.3486 g, 2.58 mmol) was gradually added under magnetic stirring, with stirring continuing until the solution was completely clear. N-acetyl-L-cysteine ​​(NAC, 0.842 g, 5.16 mmol) was added, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC, 0.1978 g, 10.32 mmol) dissolved in distilled water (4 mL) was added dropwise to the reaction mixture. During the reaction, the pH was adjusted to 5, and the mixture was stirred continuously at room temperature for 3 hours. After the reaction was completed, the resulting reaction solution was transferred to a dialysis bag and dialyzed for 3 days in 5 mmol / L HCl solution (containing 2 mmol / L EDTA), 1 day in 5 mmol / L HCl solution (containing 2 mmol / L EDTA and 1% NaCl), and 1 day in 1 mmol / L HCl solution. All dialysis was performed in the dark at 4 °C. After dialysis, the product was freeze-dried (-50 °C) to obtain thiolated chitosan (CS-NAC), and stored at 4 °C for later use.

[0031] In this embodiment, the proton NMR spectrum of thiolated chitosan (CS-NAC) is as follows: Figure 4 As shown, characteristic peaks corresponding to its -CH2SH and -CO-CH3 groups appeared at δ 3.21 ppm and δ 2.15 ppm in the spectrum, indicating that N-acetyl-L-cysteine ​​was successfully grafted onto chitosan.

[0032] Example 3 This embodiment describes the gelation experiment of hydrogel under simulated gastric lesion oxidation conditions in vitro; The two gel components prepared in Examples 1 and 2, 40 mg / ml of OHA-DA and 40 mg / ml of CS-NAC, were dissolved in sterile PBS. The resulting polymer solutions were mixed with Lactobacillus rhamnosus (LGG) at a volume ratio of 1:1 (the final concentration of LGG was 10). 5 -10 6 After (CFU / mL), a cross-linking reaction is carried out under oxidative (sodium periodate solution) and acidic (pH=2) conditions to rapidly form a hydrogel precursor solution with a certain degree of fluidity. This precursor solution further gels in situ under simulated gastric lesion oxidative conditions (sodium periodate solution); such as... Figure 5 As shown, the hydrogel does not flow when inverted, indicating that a stable gel state has been formed; at the same time, the hydrogel can be used to write "CPU" in liquid on the surface of the petri dish.

[0033] SEM images of the hydrogel, such as Figure 6As shown in A and B, where, Figure 6 In the diagram, A represents a low-magnification schematic with a scale bar of 20µm, showing the overall porous network structure of the hydrogel. Figure 6 The high-magnification schematic diagram of the hydrogel in section B, with a scale bar of 2µm, shows the details of individual fibers. SEM image of the hydrogel (HL) loaded with *Rhamnosus rhamnosus*, as shown below. Figure 6 As shown in C and D, where, Figure 6 In the diagram, C represents a low-magnification schematic of the hydrogel with a scale bar of 20µm, showing the overall porous structure. Figure 6 The high-magnification schematic diagram of the hydrogel in D, with a scale bar of 2µm, shows the successful loading of Rhamnosus rhamnosus onto the hydrogel.

[0034] Example 4 In this embodiment, an in vitro antioxidant experiment was conducted on the hydrogel prepared in Example 3; The antioxidant capacity of the hydrogel was systematically studied using free radical scavenging experiments, involving the scavenging performance of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) and hydroxyl radicals (•OH). For the DPPH free radical scavenging experiment, 500 mg of hydrogel was immersed in 5 mL of DPPH solution and allowed to stand in the dark for 24 hours. The optical density (OD) at 517 nm was recorded. The absorbance of the DPPH solution was measured in the wavelength range of 350–650 nm using a UV spectrophotometer. The scavenging performance of hydroxyl radicals (•OH) was evaluated using the same method. The OD value was recorded at 557 nm, and the absorbance of the solution was measured in the ranges of 400–900 nm and 200–900 nm, respectively. The free radical scavenging rate was calculated using the following formula: Clearance rate (%) = [(Ac − As) / Ac] × 100% Where Ac represents the absorbance of the control group and As represents the absorbance of the experimental sample group. This method provides a multi-dimensional evaluation of the antioxidant properties of hydrogel precursor solutions, ensuring the comprehensiveness and accuracy of the data.

[0035] Experimental results are as follows Figure 7 As shown, the hydrogel group (HL) loaded with Lactobacillus rhamnosus achieved scavenging rates of 74% and 46% for hydroxyl radicals (•OH) and 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH), respectively, indicating that the hydrogel has strong antioxidant capacity.

[0036] Example 5 In this embodiment, an in vitro anti-inflammatory experiment was conducted on the hydrogel prepared in Example 3; To verify the in vitro anti-inflammatory properties of the prepared hydrogel, ethanol-induced mouse gastric mucosal cells (GSE-1) were selected as model cells. Cells were seeded in 96-well plates and cultured until adherent. The cells were then divided into a control group (PBS), a group containing *Rhamnosus spp.* (LGG), a hydrogel-treated group (Hydrogel), and a group containing *Rhamnosus spp.* (HL) for co-incubation. Each group was stimulated with a specific concentration of ethanol solution for 4 h to induce an inflammatory response. The cells were then cultured for another 24 h. After culture, the supernatant was collected from each group, and the levels of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and the anti-inflammatory factor interleukin-10 (IL-10) in the cell supernatant were detected using enzyme-linked immunosorbent assay (ELISA). The experimental results are as follows: Figure 8 As shown, compared with the ethanol-induced group, the secretion of TNF-α and IL-6 in the HL group was significantly reduced, while the secretion of IL-10 was significantly increased, and the difference was statistically significant (P < 0.05).

[0037] The above results indicate that the hydrogel loaded with Rhamnosus rhamnosus has good in vitro anti-inflammatory properties, can effectively inhibit the production of pro-inflammatory factors and promote the expression of anti-inflammatory factors, thereby achieving a significant anti-inflammatory effect.

[0038] Example 6 In this embodiment, an in vivo retention experiment was conducted on the hydrogel precursor solution prepared in Example 3; To evaluate the in vivo retention performance of the probiotic-loaded hydrogel precursor solution, an ethanol-induced gastric ulcer animal model was constructed for in vivo retention evaluation. All animals were randomly divided into three groups. Mice in the model group were administered anhydrous ethanol by gavage at a dose of 5 mL / kg to establish a gastric ulcer model. In the experimental group, after establishing the gastric ulcer model by gavage with anhydrous ethanol, mice were administered 200 μL of *Lactobacillus rhamnosus* (LGG, final concentration 10) by gavage. 5 -10 6 A hydrogel precursor solution (CFU / mL) (prepared by mixing 40 mg / mL OHA-DA and 40 mg / mL CS-NAC in a 1:1 volume ratio under oxidative and acidic conditions) was used to further gel in situ under oxidative conditions at the gastric lesion site. The control group consisted of healthy mice that were administered 200 µL of the precursor solution loaded with Lactobacillus rhamnosus by gavage.

[0039] Experimental results are as follows Figure 9 As shown, the hydrogel precursor solution can be gelled in situ under oxidative conditions of gastric ulcers and achieve good retention and adhesion in the stomach.

[0040] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0041] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for preparing a probiotic-loaded hydrogel precursor solution for treating gastric diseases, characterized in that, Includes the following steps: Sodium periodate was used to oxidize hyaluronic acid to obtain oxidized hyaluronic acid; then it was reacted with dopamine to prepare dopamine-modified oxidized hyaluronic acid. Thioyl chitosan was prepared by amidation reaction of chitosan with N-acetyl-L-cysteine; Dopamine-modified oxidized hyaluronic acid and thiolated chitosan were dissolved in sterile PBS, then mixed with probiotics, and cross-linked under oxidative acidic conditions to obtain a hydrogel precursor solution.

2. The method for preparing the probiotic-loaded hydrogel precursor solution for treating gastric diseases according to claim 1, characterized in that, The process for preparing oxidized hyaluronic acid is as follows: hyaluronic acid is dissolved in water, and sodium periodate solution is added to react; after the reaction is completed, ethylene glycol is added and stirred to quench any unreacted sodium periodate; then, oxidized hyaluronic acid is obtained by dialysis, freezing, and drying.

3. The method for preparing the probiotic-loaded hydrogel precursor solution for treating gastric diseases according to claim 1, characterized in that, The process for preparing dopamine-modified oxidized hyaluronic acid includes: reacting oxidized hyaluronic acid with dopamine hydrochloride under the catalysis of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, followed by dialysis, impurity removal, and lyophilization to obtain dopamine-modified oxidized hyaluronic acid.

4. The method for preparing the probiotic-loaded hydrogel precursor solution for treating gastric diseases according to claim 3, characterized in that, The molar ratio of oxidized hyaluronic acid, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride and dopamine hydrochloride is 1:1.5:2.

5.

5. The method for preparing the probiotic-loaded hydrogel precursor solution for treating gastric diseases according to claim 1, characterized in that, The process for preparing thiolated chitosan is as follows: chitosan is dispersed in deionized water, 1-hydroxybenzotriazole is added, and the mixture is stirred continuously until the solution is completely clear. Then, N-acetyl-L-cysteine ​​is added to obtain a mixed reaction product. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide dissolved in distilled water is added dropwise to the reaction mixture. The pH is adjusted to 5 and the mixture is stirred to react. After dialysis, freezing, and drying, thiolated chitosan is obtained.

6. The method for preparing the probiotic-loaded hydrogel precursor solution for treating gastric diseases according to claim 5, characterized in that, The molar ratio of chitosan, 1-hydroxybenzotriazole, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-acetyl-L-cysteine ​​was 1:2.58:10.32:5.

16.

7. The method for preparing the probiotic-loaded hydrogel precursor solution for treating gastric diseases according to claim 1, characterized in that, The probiotic is Lactobacillus rhamnosus.

8. The method for preparing the probiotic-loaded hydrogel precursor solution for treating gastric diseases according to claim 1, characterized in that, In the crosslinking reaction, the acidic condition is pH=2~3, and the oxidation condition is the addition of sodium periodate solution.

9. A probiotic-loaded hydrogel precursor solution for treating gastric diseases, characterized in that, It was prepared using the preparation method described in any one of claims 1-8.

10. The use of the hydrogel precursor solution according to claim 9 in the preparation of a medicament for treating gastric diseases.

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