A selenium-chitosan functionalized bismuth-based metal compound, a preparation method and application thereof

Abstract

The application discloses application of selenized chitosan CS-Se in preparation of a drug for increasing gastric residence time of Bi-MOF, and simultaneously discloses a selenized chitosan functionalized bismuth-based metal compound, belonging to the technical field of Helicobacter pylori treatment, and the compound is Bi-MOF@CS-Se; the application synthesizes a bismuth-based metal framework Bi-MOF and selenized chitosan CS-Se respectively, and prepares a Bi-MOF solution with a concentration of 2 mg / mL; then, Se-CS is added into a 1% glacial acetic acid solution, and is ultrasonically dissolved until completely dissolved; then, the above solution is added into the Bi-MOF solution prepared in step 1), and is ultrasonically dispersed for 30 min; the solution is centrifuged and washed with ultrapure water for three times, and is freeze-dried to obtain Bi-MOF@CS-Se.

Description

Technical Field

[0001] The present invention relates to the technical field of Helicobacter pylori treatment, and more particularly to a selenized chitosan functionalized bismuth-based metal compound, a preparation method and an application thereof. Background Art

[0002] Helicobacter pylori (H. pylori), a chronic infectious pathogen, is a major cause of chronic gastritis, peptic ulcers, and even gastric cancer. Urease produced by H. pylori neutralizes gastric acid, raising the pH around the bacterium. Urease adheres to the gastric mucosa via various adhesins, such as blood group antigen-binding adhesin (BabA) and sialic acid-binding adhesin (SabA), thereby ensuring its long-term colonization in the stomach. The virulence factors subsequently produced stimulate mucosal cells, neutrophils, and macrophages to secrete large amounts of inflammatory chemokines, inducing a severe mucosal inflammatory response. Simultaneously, activated inflammatory cells produce large amounts of reactive oxygen species through respiration, disrupting the balance between oxidative and antioxidant functions in the body and leading to oxidative stress-mediated gastric mucosal damage.

[0003] Currently, the primary approach to eradicating Helicobacter pylori is through drug therapy, including multiple antibiotics. However, the emergence of drug-resistant bacteria has led to a decline in the eradication rate of antibiotic therapy. Furthermore, the therapeutic efficacy of antibiotics is further compromised by their low mucus permeability, poor mucosal retention, and inability to exert immunomodulatory effects. Furthermore, long-term antibiotic use can lead to intestinal dysbiosis, which can induce intestinal diseases. Therefore, the development of new therapeutic strategies to address the shortcomings of clinical antibiotic therapy is urgently needed.

[0004] Metal-organic frameworks (MOFs) are a class of porous crystalline materials with a network-like structure assembled through coordination bonds between metal ions or metal oxide clusters and organic ligands. In recent years, MOFs have been widely used in the diagnosis and treatment of various diseases, particularly in the antibacterial field, due to their excellent biosafety, degradability, and ease of functionalization. Bismuth and its compounds are among the few metallo-antimicrobial agents that exhibit excellent antibacterial activity against Helicobacter pylori without developing drug resistance. Bismuth agents have also been clinically used as adjuncts to antibiotic therapy to eradicate H. pylori. Studies have shown that bismuth agents inhibit H. pylori primarily through the following pathways: downregulating the expression of the virulence proteins CagA and VacA; disrupting the flagellar structure responsible for bacterial colonization; inhibiting antioxidant enzymes produced by H. pylori; and disrupting multiple metabolic pathways involved in the translation of growth RNA. Furthermore, they can reverse the sensitivity of drug-resistant bacteria to antibiotics. Therefore, constructing bismuth-based metal-organic frameworks with bismuth ions as metal nodes and trimesic acid as organic ligands holds promise for the efficient elimination of H. pylori and its resistant strains. However, regular gastric emptying and the replacement of the mucus layer make it difficult for Bi-MOF to remain in the stomach for a long time, and it is unable to achieve immune regulation and protective effects on the intestinal flora. Therefore, the functionalization of Bi-MOF is crucial for its efficient therapeutic effect against Helicobacter pylori.

[0005] Chitosan, the most widely studied natural cationic polysaccharide, exhibits positive charges that can interact with negatively charged cells or mucins, allowing it to adhere to the gastric mucosa and enhance gastric retention. Modification of chitosan with selenium further imparts its anti-inflammatory and antioxidant properties.

[0006] Therefore, how to functionalize Bi-MOF by selenized chitosan to achieve multi-effect treatment of Helicobacter pylori infection is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a bismuth-based metal compound functionalized with selenized chitosan, which prolongs the retention time of Bi-MOF in the stomach and improves the efficiency of combating Helicobacter pylori.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] Application of selenized chitosan CS-Se in the preparation of a drug for increasing the gastric retention time of Bi-MOF, wherein the main component of the drug is Bi-MOF@CS-Se, and the structural formula of selenized chitosan CS-Se is shown below;

[0010]

[0011] Selenium is one of the essential trace elements for the human body and can participate in regulating biological processes such as oxidative stress, endoplasmic reticulum stress, antioxidant defense, immunity and inflammatory response, including antioxidant, anti-inflammatory, anti-apoptosis, and immune response regulation. Various selenium-containing compounds can be used as antioxidants to directly remove ROS, or they can enhance antioxidant capacity by activating antioxidant enzymes in cells and promoting the removal of ROS. In addition, it can also inhibit induced cellular inflammation by regulating the NF-KB signaling pathway. Chitosan is the most studied natural cationic polysaccharide polymer. The positive charge it exhibits can interact with negatively charged cells or mucins, thereby adhering to the gastric mucosa and enhancing the retention time in the stomach. Therefore, the present invention uses selenized chitosan to encapsulate Bi-MOF, which increases the retention time of Bi-MOF in the stomach and thus improves the anti-inflammatory and antioxidant effects.

[0012] As an inventive concept identical to the above technical solution, the present invention also seeks to protect a method for preparing a selenized chitosan-functionalized bismuth-based metal compound, wherein the selenized chitosan-functionalized bismuth-based metal compound is Bi-MOF@CS-Se, comprising the following steps:

[0013] 1) Synthesize bismuth-based metal framework Bi-MOF and selenized chitosan CS-Se, respectively, and prepare a Bi-MOF solution with a concentration of 2 mg / mL;

[0014] 2) The Se-CS obtained in step 1) was added to a 1% glacial acetic acid solution and sonicated until completely dissolved. The above solution was then added to the Bi-MOF solution prepared in step 1) and sonicated for 30 minutes. The mixture was washed three times by centrifugation with ultrapure water and freeze-dried to obtain Bi-MOF@CS-Se powder; wherein the ratio of Se-CS: 1% glacial acetic acid solution: Bi-MOF solution = 20 mg: 3 ml: 10 ml.

[0015] Preferably, the synthesis process of Bi-MOF is:

[0016] (11) Dissolve bismuth nitrate pentahydrate Bi(NO3)3 and trimesic acid H3BTC in dimethylformamide (DMF). After fully dissolving, heat in an oil bath at 120°C for 12 h, wash twice with DMF by centrifugation, and soak for 2 h.

[0017] (12) The mixture was centrifugally washed with methanol or acetone three times, and finally dried under vacuum at 100 °C for 8 h to obtain Bi-MOF powder; wherein, Bi(NO3)3:H3BTC:DMF:=150 mg:750 mg:60 mL.

[0018] Preferably, the rotation speed during the centrifugal washing is 8000 rpm.

[0019] Preferably, the CS-Se synthesis process is:

[0020] (13) Chitosan CS was placed in ultrapure water, and after sufficient swelling, glacial acetic acid was added and magnetic stirring was performed to form a transparent solution;

[0021] (14) Then, sodium selenite was dissolved in ultrapure water, and after being fully dissolved, it was slowly added to the transparent solution, and the reaction was carried out for 2 h. An excess of anhydrous ethanol was added, and the white precipitate was collected by centrifugation and washed, and freeze-dried to obtain a sample; wherein, CS: ultrapure water: glacial acetic acid: sodium selenite: ultrapure water: = 1.0 g: 49 mL: 1 mL: 1.33 g: 2 ml.

[0022] Preferably, in step (14), the centrifugal washing is performed twice with anhydrous ethanol and twice with ultrapure water.

[0023] As an inventive concept identical to the above technical solution, the present invention also seeks to protect the use of the selenized chitosan-functionalized bismuth-based metal compound prepared by the above preparation method in the preparation of a drug for inhibiting the expression of inflammatory factor-related mRNA in gastric epithelial cells.

[0024] Helicobacter pylori produces large amounts of lipopolysaccharide (LPS), which stimulates gastric epithelial cells to secrete proinflammatory mediators (such as IL-1, IL-6, IL-8, and TNF-α), initiating an inflammatory response and leading to gastric epithelial cell damage and necrosis. Furthermore, the inflammatory response induced by H. pylori infection can induce macrophages, neutrophils, and gastric mucosal cells to release large amounts of oxygen free radicals (ROS). These ROS further cause cell damage and inflammatory responses. Therefore, eliminating H. pylori-induced inflammation and ROS accumulation is crucial for the prevention and treatment of H. pylori-related diseases.

[0025] As an inventive concept identical to the above technical solution, the present invention also seeks to protect the use of the selenized chitosan-functionalized bismuth-based metal compound prepared by the above preparation method in the preparation of drugs for inhibiting Helicobacter pylori.

[0026] The above technical solution demonstrates that, compared to existing technologies, the present inventors have prepared a selenized chitosan-functionalized Bi-MOF (Bi-MOF@CS-Se), which can adhere to the gastric mucosa through charge interaction and respond to degradation by gastric acid and pepsin. The released Bi-MOF exhibits excellent antibacterial properties against a variety of clinically drug-resistant strains. It can also alleviate the inflammatory response and excessive oxidative stress caused by HP infection by regulating the expression of inflammatory factors and the production of reactive oxygen species. Importantly, Bi-MOF@CS-Se does not affect the homeostasis of the intestinal flora, providing a promising strategy for the safe and effective treatment of Helicobacter pylori. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0028] Figure 1 The attached figure shows the structural analysis of CS-Se and the analysis diagram of Bi-MOF@CS-Se;

[0029] Figure 2 The accompanying figure is a structural analysis diagram of Bi-MOF;

[0030] Figure 3 The attached figure shows the effect of Bi-MOF on inhibiting Helicobacter pylori in vitro;

[0031] Figure 4 The attached figure shows the drug resistance results of Hp532 to levofloxacin, clarithromycin and metronidazole;

[0032] Figure 5 The attached figure shows the drug resistance results of Hp536 to levofloxacin, clarithromycin and metronidazole;

[0033] Figure 6 The attached figure shows the drug resistance results of Hp533 to levofloxacin, clarithromycin and metronidazole;

[0034] Figure 7 The attached figure shows the structural changes of Bi-MOF@CS-Se in PBS buffer;

[0035] Figure 8 The attached figure shows the effect of Bi-MOF@CS-Se on the structure of Helicobacter pylori in different treatment solutions;

[0036] Figure 9 The attached figure shows the effect of Bi-MOF@CS-Se on the mRNA expression of inflammatory factors and ROS levels in gastric mucosal cells;

[0037] Figure 10 The attached figure shows the effect of Bi-MOF@CS-Se on ROS levels in GES-1 cells;

[0038] Figure 11 The attached figure shows the fluorescence residual images of Bi-MOF and Bi-MOF@CS-Se at different time in the stomach;

[0039] Figure 12 The attached figure is a schematic diagram of Bi-MOF and Bi-MOF@CS-Se being excreted from the stomach tissue through the intestine;

[0040] Figure 13The attached figure is a schematic diagram of Bi-MOF@CS-Se eradicating Helicobacter pylori and alleviating inflammation in vivo;

[0041] Figure 14 The attached figure is a schematic diagram of the results of Bi-MOF and Bi-MOF@CS-Se inhibiting drug-resistant strains;

[0042] Figure 15 The attached figure shows the abundance, diversity and colony structure of intestinal microorganisms in the feces of Hp-infected mice treated with PBS, Bi-MOF, Bi-MOF@CS-Se, OAC (omeprazole + amoxicillin + clarithromycin), and OA + Bi-MOF@CS-Se by gavage. DETAILED DESCRIPTION

[0043] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0044] Example 1 Preparation of Bismuth-Based Metal Compounds Functionalized with Selenized Chitosan

[0045] 1) Synthesis of Bi-MOF: 150 mg of bismuth nitrate pentahydrate (Bi(NO3)3)3 and 750 mg of trimesic acid (H3BTC) were dissolved in 60 mL of dimethylformamide (DMF). After complete dissolution, the mixture was heated in an oil bath at 120°C for 12 h. The mixture was washed twice with DMF by centrifugation and then soaked for 2 h. The mixture was then washed three times with methanol or acetone at 8000 rpm to displace the DMF. Finally, the mixture was dried in a vacuum at 100°C for 8 h. The resulting Bi-MOF powder exhibited a rod-like structure. Scanning electron microscopy images showed that the powder had a rod-like structure. Figure 2 A;

[0046] 2) Synthesis of selenized chitosan (CS-Se): 1.0 g of chitosan CS was placed in 49 mL of ultrapure water. After sufficient swelling, 1 mL of glacial acetic acid was added and magnetic stirring was performed to form a transparent solution. 1.33 g of sodium selenite was dissolved in 2 mL of ultrapure water. After sufficient dissolution, the solution was slowly added to the chitosan solution and reacted for 2 h. An excess of anhydrous ethanol was added to produce a white precipitate. The white precipitate was collected by centrifugation and washed twice with anhydrous ethanol and ultrapure water, and freeze-dried to obtain a sample. Figure 1 As shown in A and 1B, the selenized chitosan showed a looser structure, which made it more water-soluble. The Fourier spectrum showed that compared with the chitosan alone, the selenized chitosan had a higher water solubility at 807.8 cm -1 The Se=O double bond vibration peak appeared at , indicating the successful synthesis of selenized chitosan ( Figure 1 C and 1D), and the structural formula is shown below.

[0047]

[0048] 3) Selenized chitosan modified bismuth-based MOF (Bi-MOF@CS-Se): 20 mg of Se-CS was added to 3 mL of glacial acetic acid solution (1%) and ultrasonicated for 10 min until completely dissolved. The above solution was then added to 10 mL of Bi-MOF solution (2 mg / mL), ultrasonically dispersed for 30 min, washed three times with ultrapure water by centrifugation, and freeze-dried to obtain Bi-MOF@CS-Se powder. SEM and elemental energy spectrum more intuitively show the distribution and content of characteristic elements such as Bi and Se. Figure 2 B- Figure 2 D). The crystal structure of Bi-MOF@CS-Se was determined by X-ray diffractometer. The crystal peaks of Bi-MOF@CS-Se were consistent with the characteristic peaks of Bi-MOF alone ( Figure 2 E). At the same time, the Fourier infrared spectrum of Bi-MOF@CS-Se is at 1650cm -1 and 807.8cm -1 The stretching vibration peaks of CO and Se=O bonds are shown at Figure 2 F, Figure 2 G), which is consistent with the characteristic peaks of Bi-MOF and CS-Se. In order to determine the valence state of Bi and Se elements in Bi-MOF@CS-Se, XPS analysis was performed ( Figure 1 E, Figure 1 F, Figure 1 G,), the spectrum results show that Bi and Se elements exist in trivalent and tetravalent forms, respectively. The above results all indicate the successful preparation of Bi-MOF@CS-Se.

[0049] Example 2 In vitro antibacterial activity of Bi-MOF

[0050] Eradication of Helicobacter pylori is a prerequisite for resolving infection. This example evaluated for the first time the inhibitory effect of rod-shaped Bi-MOF on Helicobacter pylori in vitro.

[0051] The specific process is as follows:

[0052] The virulence protein CagA-positive strain HpPMSS1 (from Massachusetts Institute of Technology, USA) was selected for testing. The activated bacteria were scraped off and resuspended in fresh Brucella broth. The OD of the bacterial solution was adjusted. 600=0.1. Then 0.625mg Bi-MOF was added to 10ml of resuspended bacterial solution and cultured overnight in a three-gas incubator. The co-cultured bacterial solution was further diluted and 100μl was spread on Brucella agar plates and cultured for another 48 hours. From the colony pictures and colony count results on the agar plates ( Figure 3 A), the number of colonies in the Bi-MOF group was significantly lower than that in the control group, and its antibacterial effect was even better than that of amoxicillin at the same concentration. In addition, the same volume of bacterial solution ( Figure 3 B), colony counts showed that the 250ug / ml, 125ug / ml and 62.5ug / ml concentration groups could reduce the original viable bacterial count by more than three orders of magnitude, while the 31.25ug / ml concentration group failed to reduce the original viable bacterial count by more than three orders of magnitude ( Figure 3 C), so the minimum bactericidal concentration (MBC) of Bi-MOF is considered to be 62.5ug / ml.

[0053] At the same time, in order to determine the minimum inhibitory concentration (MIC) of Bi-MOF, the OD of the bacterial solution before and after treatment with different concentrations of Bi-MOF was measured. 600 value( Figure 3 D), it was observed that there was no significant change in bacterial concentration before and after incubation in the Bi-MOF group with a concentration greater than or equal to 31.25ug / ml, so the MIC of Bi-MOF was considered to be 31.25ug / ml.

[0054] To evaluate the therapeutic effect of Bi-MOF on drug-resistant bacteria, this experiment isolated H. pylori strains (numbers: 532, 533, and 536) from the gastric tissue of patients who failed to eradicate the disease with second-line clinical treatment. By co-culturing with antibiotics at the above critical resistance concentrations, it was found that the proliferation of clinical strains H. pylori 532 / 536 was not affected by clarithromycin, metronidazole, and levofloxacin, and was a triple-drug-resistant bacteria ( Figure 4 , 6). Levofloxacin can effectively inhibit the proliferation of Hp533, but clarithromycin and metronidazole have no significant effect on its proliferation, making it a dual-drug resistant bacterium ( Figure 5 After determining the drug resistance of the isolates, Bi-MOF was used to treat the above isolates. The results showed that Bi-MOF was not affected by drug resistance. The number of colonies formed in the treated group was significantly lower than that in the untreated group. Its antibacterial effect was comparable to or slightly lower than that of amoxicillin. Figure 3 E, 3F, 3G).

[0055] Example 3 In vitro acid-responsive antibacterial activity of Bi-MOF@CS-Se

[0056] Chitosan was modified with selenium and then coated on the Bi-MOF surface, giving the system functions such as gastric mucosal targeting and acid-responsive drug release. Scanning electron microscopy revealed that in an environment of simulated gastric fluid (pH = 2), the chitosan nanocoating of the system degraded and the organic framework structure collapsed. However, the Bi-MOF@CS-Se structure in PBS solution was intact and did not undergo structural changes ( Figure 7 The Bi content in the supernatant was further measured by ICP-OES. The results showed that the Bi content in the supernatant of the simulated gastric fluid treatment group was higher than that in the PBS group (Table 1). This suggests that gastric acid can promote the structural changes of Bi-MOF@CS-Se, expose Bi-MOF, and further release Bi.

[0057] Table 1

[0058]

[0059] Furthermore, to study the in vitro anti-Helicobacter pylori effect of Bi-MOF@CS-Se, Bi-MOF@CS-Se treated with simulated gastric fluid or PBS solution was co-cultured with HpPMSS1 for 24 hours. The results of agar plate culture showed that only the colony count of the Bi-MOF@CS-Se group treated with simulated gastric fluid was significantly less than that of the group treated with gastric acid alone, while there was no difference between the Bi-MOF@CS-Se group treated with PBS solution and the group treated with PBS alone ( Figure 8 A, 8B).

[0060] To explore the mechanism of Bi-MOF@CS-Se inhibiting H. pylori, the bacterial morphology of different groups was observed using scanning electron microscopy. It was found that the integrity of the bacterial membrane structure of Helicobacter pylori in the Bi-MOF@CS-Se group treated with simulated gastric acid was destroyed, and the bacteria also underwent disintegration and necrosis. However, there was no significant effect on the bacterial morphology of the Bi-MOF@CS-Se group treated with PBS compared with the simple PBS and simulated gastric acid treatments ( Figure 8 D). We further tested the protein content of the culture supernatant and found that the acidified Bi-MOF@CS-Se treated group had a higher protein content, while the other three groups had no significant differences ( Figure 8 C).

[0061] To explore the effect of drug resistance on the antibacterial effect of Bi-MOF@CS-Se, clinical drug-resistant strains Hp532 / 533 / 536 were co-cultured with acidified Bi-MOF@CS-Se. The agar plate images and counts showed that the number of colonies in the treated groups was significantly lower than that in the control group ( Figure 8E, 8F, and 8G). Therefore, it can be assumed that in simulated gastric fluid, the outer chitosan layer of Bi-MOF@CS-Se degrades, causing the organic metal framework to collapse, releasing Bi nanoparticles that disrupt the biofilm structure, leading to the outflow of bacterial contents and promoting bacterial necrosis. Furthermore, the antibacterial properties of Bi-MOF@CS-Se are not affected by drug resistance.

[0062] Example 4 In vitro biocompatibility, anti-inflammatory and antioxidant activities of Bi-MOF@CS-Se

[0063] First, the effect of 250ug / mlBi-MOF or Bi-MOF@CS-Se on the viability of gastric epithelial cells HF E145 or GES-1 in vitro was evaluated. HFE-145 or GES-1 cells were cultured in a culture medium containing 10% FBS and 1% penicillin at 37°C and 5% CO2 incubator for 24 hours, and then seeded in a 96-well plate at a concentration of 2000 cells / well and incubated for another 24 hours. Subsequently, the complete culture medium was replaced with a 250μg / mL mixture of Bi-MOF or Bi-MOF@CS-Se culture medium and incubated for 24h, 48h and 72h, and then 20μL of cell counting kit (Cell couting kit-8, CCK8) test solution was added to the above solution, and the absorbance was measured by a multifunctional microplate reader after incubation in the dark for 2 hours. The results showed that no decrease in cell viability was observed in the Bi-MOF group and the Bi-MOF@CS-Se group ( Figure 9 A, 9B) shows that it has good biosafety.

[0064] Then, we explored whether Bi-MOF@CS-Se has anti-inflammatory ability. The process was as follows: H. pylori P MSS1 was used to infect gastric epithelial cells HFE-145 and GES-1 cells for 12 hours to induce an in vitro H. pylori gastritis cell model, and then Bi-MOF@CS-Se was given or not. The reaction system was constructed according to Tables 2 and 3, and the mRNA levels of the above inflammatory factors in each group of cells were detected by q-PCR (reaction conditions: 1 cycle of pre-denaturation (50℃ 2min; 95℃ 2min), 40 cycles of PCR reaction (95℃ 15s; 55℃ 15s; 72℃ 1min), annealing (95℃ 15s; 60℃ 1min; 95℃ 15s)) (primers and amplification system are shown in Tables 2 and 3). The results showed that Hp infection could induce the expression of IL-1, IL-6, IL-8 and TNF-α mRNA levels in both gastric epithelial cells. However, after treatment with Bi-MOF@CS-Se, the mRNA levels of the above inflammatory factors in HFE-145 cells were reduced ( Figure 9 C), reduced the mRNA levels of IL-1, IL-6 and IL-8 in GES-1 cells ( Figure 9 D).

[0065] Table 2

[0066]

[0067]

[0068] Table 3

[0069]

[0070] The DCFH-DA fluorescent probe was further used to evaluate the ability of Bi-MOF@CS-Se to scavenge oxygen free radicals in vitro. GES-1 cells were infected with HPPMSS1 for 12 hours and then treated with or without Bi-MOF@CS-Se for 12 hours. Fluorescence imaging showed that compared with the control group, the fluorescence signal in the cells of the Hp infection group increased significantly, while the fluorescence of the Bi-MOF@CS-Se treatment group was significantly quenched and had no significant difference from the control group, indicating that Bi-MOF@CS-Se can significantly scavenge ROS induced by Hp infection. Figure 9 E. Figure 9 G). In addition, in order to exclude the possibility that the effect of Bi-MOF@CS-Se on intracellular ROS levels is related to the clearance of Hp infection, positive reference reagents H2O2 and Rosup reagent were used to stimulate and induce ROS levels in GES-1 cells. The results showed that compared with the group stimulated by the positive reagent alone, the percentage of positive cells in the Bi-MOF@CS-Se treatment group was significantly lower than that in the untreated group, indicating that Bi-MOF@CS-Se can clear ROS induced by the positive reagents ( Figure 9 F. Figure 9 H, Figure 10 ).

[0071] Example 5 Gastric Retention Time, Antibacterial and Anti-inflammatory Activities of Bi-MOF and Bi-MOF@CS-Se

[0072] In recent years, the complex physiological environment in the stomach has brought major challenges to drug delivery vehicles. Chitosan, due to its chemical structure characteristics, has the effect of increasing mucus permeability and can target drugs to the gastric mucosal layer. We used Fluorescein 5-isothiocyanate (FITC) fluorescent dye to label Bi-MOF and Bi-MOF@CS-Se, respectively, and then gavaged C57BL / 6 mice with FITC-Bi-MOF or FITC-Bi-MOF@CS-Se, respectively. The mice were killed at 4h, 8h, 12h and 24h, and the stomach and small intestinal tissues were removed. The fluorescence intensity at a wavelength of 525nm was detected using an animal live imaging instrument. It was found that Bi-MOF was emptied faster in the gastric tissue, and no fluorescence signal was detected in the gastric and small intestinal tissues 24h after gavage. However, in the Bi-MOF@CS-Se group, the fluorescence signal in the gastric tissue decayed more slowly, and residual fluorescence could still be detected in the gastric tissue ( Figure 11 Similarly, 24 hours after oral gavage, fluorescence could still be detected in the intestine of the Bi-MOF@CS-Se group, indicating that Bi-MOF@CS-Se was continuously excreted from the stomach tissue through the intestine ( Figure 12 ). Therefore, it is believed that Bi-MOF@CS-Se has stronger gastric retention than Bi-MOF.

[0073] To investigate the ability of Bi-MOF@CS-Se to eradicate Helicobacter pylori and alleviate inflammation in vivo, we orally administered Helicobacter pylori PMSS1 to mice for four weeks and then killed them. By HE staining of gastric tissue and Helicobacter pylori-specific silver staining, we detected a large number of Helicobacter pylori colonization in the mucus layer of gastric tissue ( Figure 13 A, 13B), and we also performed Gram staining on tissue culture bacteria to confirm that the bacteria were Gram-negative rod-shaped bacteria. Figure 13 C). After confirming successful H. pylori colonization, I treated the animals with PBS, Bi-MOF, Bi-MOF@CS-Se, OAC (omeprazole + amoxicillin + clarithromycin), and OA + Bi-MOF@CS-Se for one week before killing them. First, we evaluated the antibacterial activity in vivo. In vitro tissue culture results showed that the colony count in the Bi-MOF@CS-Se group was significantly reduced compared to the PBS group. Although slightly more than the OAC group, there was no significant difference between the OAC group and the Bi-MOF@CS-Se group after replacing clarithromycin. Figure 11 C, 11D). RT-PCR detection of Helicobacter specific sequences also yielded similar results. Compared with the PBS group, the abundance of Helicobacter in the Bi-MOF@CS-Se group was significantly reduced, but the Helicobacter in the Bi-MOF@CS-Se+OA group was less than that in the OAC group ( Figure 11 E).

[0074] HE staining of gastric tissue revealed that inflammatory cells aggregated in the gastric lamina propria of the PBS group. However, after Bi-MOF@CS-Se treatment, the aggregation of inflammatory cells was reduced ( Figure 11 F). RT-PCR analysis of gastric tissue ( Figure 11 G, 11H, 11I, 11J, 11K), also found that compared with the uninfected group, the H. pylori-infected group had higher IL-1β mRNA (proinflammatory cytokine) levels and lower IL-10 mRNA (anti-inflammatory cytokine) levels; infection also promoted an increase in IL-8, TNF-α, and IL-18 mRNA (proinflammatory cytokine) levels (the differences were not statistically significant). However, compared with the infected group, Bi-MOF@CS-Se treatment reduced IL-1β and TNF-α mRNA levels; it was also observed that the IL-8 and IL-18 mRNA levels in the treated group decreased, while IL-10 mRNA increased (the differences were not statistically significant).

[0075] In addition, I also administered the clinically isolated drug-resistant bacteria Hp536 to C57BL / 6 mice for gavage to establish a model. One month after the last gavage, HE staining and silver staining of gastric tissues revealed that Hp had successfully colonized the mucus layer of the gastric tissues of mice ( Figure 14 A, 14B). We then treated the mice with Bi-MOF or Bi-MOF@CS-Se for one week and then killed them. We then took gastric tissue for quantitative tissue culture. The results showed that Bi-MOF and Bi-MOF@CS-Se were not affected by drug resistance and could significantly reduce the content of drug-resistant Hp in the stomach of mice ( Figure 14 Therefore, based on the above results, it can be considered that Bi-MOF@CS-Se can not only eliminate H. pylori (including clinically drug-resistant bacteria) in the body, but also alleviate gastric inflammation caused by H. pylori infection.

[0076] Example 6 Biosafety of Bi-MOF and Bi-MOF@CS-Se

[0077] 16s rRNA sequencing was used to evaluate the abundance, diversity, and colony structure of intestinal microorganisms in the feces of Hp-infected mice treated with PBS, Bi-MOF, Bi-MOF@CS-Se, OAC (omeprazole + amoxicillin + clarithromycin), and OA + Bi-MOF@CS-Se by gavage. Figure 15 As shown in A, 15B, and 15C, there was no significant difference in Chao1 index between the Bi-MOF@CS-Se group and the control group. There were differences in Shannon and Simpson indices, but the differences were small (but their values ​​were still significantly higher than those of the OAC group). The α diversity of the OAC group was significantly reduced. This indicates that compared with antibiotics, Bi-MOF@CS-Se has no significant effect on intestinal flora. At the same time, through β diversity analysis ( Figure 15D), it was also found that OAC treatment led to significant differences in intestinal flora, while the difference in the Bi-MOF@CS-Se treatment group was small. Then, the relative abundance of the colony structure in the intestinal flora was further analyzed. As can be seen from the figure ( Figure 15 E), the number of Proteobacteria (rich in pathogenic bacteria) in the intestines of mice in the OAC treatment group increased significantly, while the number of Bacteroidetes (rich in beneficial bacteria) decreased significantly; while there was no significant change in the bacterial genera in the Bi-MOF@CS-Se group, further indicating that Bi-MOF@CS-Se treatment had little effect on the colony structure. Finally, the liver, lungs, and kidneys of mice were taken and the histopathological changes were detected by Hematoxylinandeosin (H&E) Kit staining. The results suggest that Bi-MOF@CS-Se has good compatibility in vivo ( Figure 15 F). Finally, the biochemical laboratory of the hospital tested the serum ALT, ALP, cre and BUN, which reflect the changes of liver and kidney function ( Figure 15 I, 15J, 15K, and 15L), the Bi-MOF@CS-Se treatment group also showed no significant difference compared with the PBS group, which also suggested that Bi-MOF@CS-Se can be used safely in vivo.

[0078] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0079] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of selenized chitosan CS-Se in the preparation of a drug for increasing the gastric retention time of Bi-MOF, wherein the main component of the drug is Bi-MOF@CS-Se.

2. A method for preparing a bismuth-based metal compound functionalized with selenized chitosan, wherein the bismuth-based metal compound is Bi-MOF@CS-Se, characterized in that: The following processes are included: 1) Synthesize bismuth-based metal framework Bi-MOF and selenized chitosan CS-Se, respectively, and prepare a Bi-MOF solution with a concentration of 2 mg / mL; 2) The Se-CS obtained in step 1) was added to a 1% glacial acetic acid solution and sonicated until completely dissolved. The above solution was then added to the Bi-MOF solution prepared in step 1) and sonicated for 30 minutes. The mixture was washed three times by centrifugation with ultrapure water and freeze-dried to obtain Bi-MOF@CS-Se powder; wherein the ratio of Se-CS: 1% glacial acetic acid solution: Bi-MOF solution = 20 mg: 3 ml: 10 ml.

3. The method for preparing a bismuth-based metal compound functionalized with selenized chitosan according to claim 2, characterized in that: The synthesis process of Bi-MOF is: (11) Dissolve bismuth nitrate pentahydrate Bi(NO3)3 and trimesic acid H3BTC in dimethylformamide (DMF). After fully dissolving, heat in an oil bath at 120°C for 12 h, wash twice with DMF by centrifugation, and soak for 2 h. (12) The mixture was centrifugally washed with methanol or acetone three times and finally dried under vacuum at 100 °C for 8 h to obtain Bi-MOF powder; wherein, Bi(NO3)3:H3BTC:DMF=150 mg:750 mg:60 mL.

4. The method for preparing a bismuth-based metal compound functionalized with selenized chitosan according to claim 3, characterized in that: The rotation speed during the centrifugal washing is 8000 rpm.

5. The method for preparing a bismuth-based metal compound functionalized with selenized chitosan according to claim 4, characterized in that: The CS-Se synthesis process is: (13) Chitosan CS was placed in ultrapure water, and after sufficient swelling, glacial acetic acid was added and magnetic stirring was performed to form a transparent solution; (14) Sodium selenite was then dissolved in ultrapure water. After fully dissolved, it was slowly added to the transparent solution and reacted for 2 h. Excess anhydrous ethanol was added, and the white precipitate was collected by centrifugation and washed, and freeze-dried to obtain the sample. in, CS: Ultrapure water: glacial acetic acid: sodium selenite: ultrapure water = 1.0g:49mL:1mL:1.33g:2ml.

6. The method for preparing a bismuth-based metal compound functionalized with selenized chitosan according to claim 5, characterized in that: In step (14), the centrifugal washing is performed twice with anhydrous ethanol and twice with ultrapure water.

7. Use of the selenized chitosan-functionalized bismuth-based metal compound prepared by the preparation method according to any one of claims 2 to 6 in the preparation of a drug for inhibiting Helicobacter pylori, wherein the bismuth-based metal compound has a prolonged residence time in the stomach, thereby combating Helicobacter pylori.

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