Preparation method of molybdenum carbide nanomedicine for treating oxidative stress inflammatory bowel disease

Abstract

The invention relates to the field of biomedicine, and discloses a method for preparing a molybdenum carbide nano drug for treating oxidative stress inflammatory bowel disease. The method comprises the following steps: S1: taking original Mo2C powder, putting it into a centrifuge tube with a 0.22 μm filter membrane, adding a 20% tetrabutylammonium hydroxide (TBAOH) aqueous solution, stirring it magnetically at room temperature for 48 hours, and then centrifuging (8500 rpm, 5 minutes, 22°C) to remove TBAOH, S2: washing it with ultrapure water for multiple times until the lower layer of liquid is clear, S3: after washing, transferring the precipitate to a freeze dryer for freeze drying at -80°C, S4: after completing the above operations, the obtained product is in the form of black powder, i.e., a Mo2C nano drug with a diameter of about 500-600 nm, S5: storing it in a sealed bottle away from light to reduce unnecessary oxidation and decomposition, and S6: resuspending it in a phosphate buffered saline (PBS) solution and ultrasonicating it for 10 minutes before use to obtain a Mo2C nano drug suspension. The present invention emphasizes the in vitro / extracellular ROS scavenging effect of Mo2C. The preferred Mo2C nanomedicine has a diameter of about 500-600nm and is in the form of a sheet with a thickness of about 11nm.

Description

Technical Field

[0001] The present invention relates to the technical field of speech signal processing, and in particular to a method for preparing a molybdenum carbide nanomedicine for treating oxidative stress inflammatory bowel disease. Background Art

[0002] Inflammatory Bowel Disease (IBD) is a chronic, recurrent intestinal inflammatory disease. There are two main types: ulcerative colitis (UC) and Crohn's disease (CD). Ulcerative colitis mainly affects the colon and rectal mucosa, with symptoms such as diarrhea, blood in the stool, abdominal pain, etc. The lesions are usually limited to the colon mucosa and are characterized by continuous distribution. Crohn's disease can affect the entire digestive tract, but is most common in the terminal ileum and colon, with symptoms including abdominal pain, diarrhea, loss of appetite, weight loss, etc. The lesions are distributed discontinuously and may be accompanied by inflammation and scar formation in the deep tissues of the intestinal wall. The cause of inflammatory bowel disease is not yet fully understood and may be related to multiple factors such as genetics, immune abnormalities, and environmental factors. Its treatment strategies include drug therapy and surgical treatment. Drug therapy mainly includes anti-inflammatory drugs, immunomodulators, and biological agents, which aim to control inflammation and relieve symptoms. Patients with severe lesions or who are not responsive to drug therapy require surgical treatment. In general, the current treatment effect of inflammatory bowel disease is not ideal, and the quality of life of patients with severe conditions is greatly affected, and the risk of cancer is much higher than that of the normal population.

[0003] In the pathological process of IBD, inflammatory stimulation triggers the activation of immune cells, such as macrophages and neutrophils, which release a large amount of reactive oxygen species (ROS). Excessive ROS causes direct damage to the intestinal mucosa, triggers lipid peroxidation, affects the integrity of the mucosal barrier, and leads to cell membrane damage and increased permeability, which allows harmful substances and bacteria to penetrate into the intestinal mucosa, activate the immune system, and further aggravate the inflammatory response. Therefore, ROS accumulation leads to oxidative stress damage, which is an important inducing and promoting factor of inflammatory bowel disease (Front Endocrinol (Lausanne), 2023; 14: 1217165). Antioxidant intervention has gradually become an important auxiliary treatment for inflammatory bowel disease. Existing treatment methods are mostly oral antioxidants (including synthetic antioxidants and antioxidant natural products), but these compounds (especially synthetic antioxidants) themselves are reactants and are easily absorbed into the blood, and their metabolic outcomes are very uncertain. The safety of antioxidants has always been a controversial topic, and many studies have demonstrated the correlation between long-term consumption of synthetic antioxidants and certain health problems, such as gastrointestinal diseases and increased susceptibility to cancer. Therefore, formulating new antioxidant treatment strategies that specifically target the intestine and developing related new drugs are urgent issues to be addressed in the treatment of IBD today.

[0004] Molybdenum carbide (Mo2C) is a new type of nanomedicine, and its application prospects in the medical field have attracted much attention. The special structure of this drug gives it good biocompatibility, tissue compatibility and many excellent physical and chemical properties. In recent years, Mo2C nanomedicine has shown great application potential in tumor treatment, photodynamic therapy, bioimaging and other aspects. This material has good biocompatibility and tissue compatibility, which is beneficial to reduce the immune response and toxicity of the human body to it (Analyst, 2020, 145 (23): 7609-15.). A previous study pointed out that Mo2C nanomedicine simulates the catalytic activity of various reactive oxygen species (ROS) scavenging enzymes in the body (such as SOD and CAT), effectively removes ROS in animal tissues and plasma, and significantly inhibits oxidative stress damage in tissues (Biomaterials, 2022, 287.). At the same time, molybdenum carbide has extremely strong biological stability and is almost not degraded in the human environment. Although this property greatly limits its use in blood vessels and tissues, it is extremely beneficial for the application of this nanomedicine in the digestive tract. Molybdenum carbide does not degrade in the intestines or absorb into the blood. After fully contacting the intestinal mucosa, it only acts as a catalyst to remove ROS and is then excreted with feces. It has high safety and has good therapeutic or adjuvant potential for inflammatory bowel disease. In addition, Mo2C nanomedicine also has good surface modification properties, and more applications can be achieved by changing its surface chemical structure and functional modification. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing molybdenum carbide nanomedicines for treating oxidative stress inflammatory bowel disease, in order to allow antioxidants to act more specifically on the intestines and solve the problem that oral antioxidants are easily decomposed or absorbed into the blood by the small intestine. The present invention uses molybdenum carbide nanozymes with extremely high biological stability as ROS scavengers. Molybdenum carbide nanozymes are hardly degraded in the human environment. Although this property greatly limits the scenarios of its use in blood vessels and tissues, it is extremely beneficial for the application of the nanomedicine in the digestive tract. Molybdenum carbide is not degraded in the intestines, is not absorbed into the blood, and only acts as a catalyst to remove ROS after full contact with the intestinal mucosa, and is then excreted with feces. It has high safety and reflects a good therapeutic or adjuvant potential for inflammatory bowel disease. Not only that, Mo2C nanomedicines also have good surface modification properties, and more applications are achieved by changing their surface chemical structure and functional modification.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for preparing a molybdenum carbide nanomedicine for treating oxidative stress inflammatory bowel disease, the preparation method comprising the following steps:

[0007] S1: Take the original Mo2C powder, put it into a centrifuge tube with a 0.22μm filter membrane, add 20% tetrabutylammonium hydroxide (TBAOH) aqueous solution, stir magnetically at room temperature for 48h, and then centrifuge (8500rpm, 5min, 22℃) to remove TBAOH;

[0008] S2: Wash with ultrapure water several times until the lower liquid is clear;

[0009] S3: After washing, the precipitate is transferred to a freeze dryer for freeze drying at -80°C;

[0010] S4: After completing the above operation, the product obtained is a black powder, i.e., a Mo2C nanodrug with a diameter of about 500-600 nm;

[0011] S5: Store in a sealed bottle away from light to reduce unnecessary oxidation and decomposition;

[0012] S6: Resuspend in phosphate buffered saline (PBS) and sonicate for 10 min before use to obtain Mo2C nanodrug suspension.

[0013] Preferably, the drug is in the form of a sheet with a thickness of about 11 nm and a diameter of 200-800 nm, preferably 500-600 nm.

[0014] Preferably, the drug will not be degraded and absorbed in the intestine.

[0015] Preferably, the drug effectively removes reactive oxygen species (ROS) in the intestinal tract including the large intestine and the small intestine.

[0016] Preferably, the drug effectively alleviates intestinal oxidative stress caused by sleep deprivation and reduces inflammatory response.

[0017] Preferably, the drug can treat ulcerative colitis, relieve inflammatory symptoms, and protect the intestinal barrier structure.

[0018] Preferably, the drug can be used in combination with other therapeutic drugs such as Changyanning, and can also be used as an auxiliary drug to enhance the therapeutic effect on ulcerative colitis.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention emphasizes the in vitro / extracellular ROS scavenging effect of Mo2C. The preferred Mo2C nanomedicine has a diameter of about 500-600 nm and a thickness of about 11 nm. Compared with the size of zero-valent Mo nanodots (<100 nm), it is larger and can easily adhere to the tissue surface without being internalized by the intestinal villus cells, which greatly reduces the potential cytotoxicity of the nanomedicine.

[0021] (2) The carbon element introduced into Mo2C nanomedicine can reduce the percentage of heavy metal Mo per unit mass, effectively reduce the effect of Mo on cell activity, and reduce the cost of raw materials;

[0022] (3) The carbon element added to Mo2C nanomedicine can effectively increase the efficiency of electron transfer and better improve the catalytic removal efficiency of ROS;

[0023] (4) Compared with zero-valent Mo nanodots, the addition of carbon elements provides potential covalent modification sites for nanomedicines, giving them good surface modification properties and enabling more applications by changing their surface chemical structure and functional modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 DHE staining of the small intestine and large intestine after Mo2C nanomedicine treatment;

[0025] Figure 2 This is the evaluation result of biochemical indexes of small intestine and large intestine after Mo2C nanomedicine treatment;

[0026] Figure 3 Immunohistochemical staining of inflammatory factors in the small intestine and large intestine after Mo2C nanomedicine treatment;

[0027] Figure 4 This is a graph showing the weight changes of mice after the treatment of UC with Mo2C nanomedicine and Changyanning;

[0028] Figure 5 This is the length change of the large intestine after Mo2C nanomedicine and Changyanning treated UC;

[0029] Figure 6 This is the HE staining result of the large intestine after Mo2C nanomedicine and Changyanning treated UC. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work are within the scope of protection of the present invention.

[0031] Example 1

[0032] The therapeutic effect of Mo2C nanomedicine on intestinal oxidative stress induced by sleep deprivation (SD)

[0033] Alexandra V. proved that sleep deprivation can effectively cause the accumulation of reactive oxygen species (ROS) in the intestines of animals (Cell. 2020Jun 11; 181(6): 1307-1328), thereby inducing intestinal oxidative stress. Therefore, in this embodiment, 21 C57BL / 6J mature male commercial mice aged 8-10 weeks and weighing 22-25g were selected and kept in standard cages. The ambient temperature was 22-24°C, the ventilation conditions were good, the relative humidity was about 50±5%, and commercial mouse feed and drinking water (ultrapure water) were provided, and the supply was not restricted. After adapting to the above conditions for 7 days, the mice were evenly divided into three groups: a control group (Control), a sleep deprivation model group (SD), and a sleep deprivation + Mo2C treatment group (SD+Mo2C). Sleep deprivation was achieved by a small animal sleep deprivation instrument, which had a stainless steel rotating rod with a diameter of 40 cm, fixed in the center of a plastic cage box with a height of 18 cm and a diameter of 40 cm. The rotating rod rotated at a speed of 3 rpm and rotated continuously for 12 hours from 8 am to 8 pm every day to prevent the mice from sleeping, and stopped rotating from 8 pm to 8 am the next day to give the mice a rest; this method effectively disrupted the normal daily routine of mice, during which the mice had free access to water and food, and the sleep deprivation period lasted for 5 days. The control group was given normal food and water, and 200 μL of PBS was given daily by gavage; the model group was given normal food and water, and 200 μL of PBS was given daily by gavage and sleep deprivation; the sleep deprivation + Mo2C treatment group was given normal food and water, and 200 μL of 20 mg / mL Mo2C nanodrug was given daily by gavage while receiving sleep deprivation. The SD group and SD+Mo2C group were both evaluated behaviorally to confirm the successful SD modeling, and then the mice were killed under general anesthesia, and the small and large intestine tissues were collected for biochemical and histopathological evaluations.

[0034] First, in order to observe the distribution of ROS in the small intestine and large intestine tissues of the three groups of mice, the intestinal tissue samples were quickly frozen in liquid nitrogen and made into 4 μm frozen sections. Dihydroethidium (DHE, ROS-specific fluorescent probe) and 4',6-diamidino-2-phenylindole (4',6-diamidino-2-phenylindole, DAPI) fluorescent probes were used for simultaneous staining. Fluorescence was observed under a confocal microscope. The fluorescence staining results are shown in Figure 1 It can be seen that in the large and small intestine samples of the SD group, significantly stronger DHE fluorescence appeared than that of the control group, while the DHE fluorescence of the large and small intestine samples of the SD+Mo2C group was close to that of the control group, indicating that the intervention of Mo2C significantly inhibited the accumulation of intestinal ROS mediated by SD.

[0035] In order to further prove the mitigating effect of Mo2C on SD-mediated intestinal ROS accumulation, intestinal tissue homogenate extracts were used to evaluate four biochemical indicators: reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx). The corresponding kits were used and tested according to the instructions provided by the manufacturer. The results are shown in Figure 2 As shown in the results, SD treatment significantly increased the oxidative stress indicators (ROS and MDA) in the intestine of mice, while the indicators that inhibit oxidative stress effects (SOD and GPx) were significantly downregulated; however, Mo2C intervention greatly promoted the correction of these indicators to normal levels.

[0036] Finally, the small intestine and large intestine tissues were stained using immunohistochemistry to reveal the different distribution of four inflammatory factors, tumor necrosis factor α (TNF-α), serum interleukin 6 (IL-6), serum interleukin 10 (IL-10), and serum interleukin 1β (IL-1β), in the small intestine and large intestine of the three groups of mice. The staining was performed using the corresponding kits according to the instructions provided by the manufacturer. The results are shown in Figure 3 As shown in the data, SD treatment can upregulate the four inflammatory factors in the intestine to varying degrees, while Mo2C intervention can significantly alleviate the upregulation of these inflammatory factors.

[0037] The results of this example demonstrate that Mo2C effectively alleviates intestinal oxidative stress and related intestinal inflammation caused by sleep deprivation.

[0038] Example 2

[0039] The therapeutic effect of Mo2C nanomedicine on ulcerative colitis (UC)

[0040] A total of 21 C57BL / 6J mature male commercial mice aged 8-10 weeks and weighing 22-25g were used in this study. The mice were housed in standard cages (7 mice per cage) with an ambient temperature of 22-24°C, good ventilation, and a relative humidity of approximately 50±5%. Commercial mouse feed and drinking water (ultrapure water) were provided with an unlimited supply. After adapting to the above conditions for 7 days, the mice were evenly divided into five groups: control group (Con), model group (DSS), CYN treatment group (CYN), Mo2C treatment group (MC), and CYN and Mo2C combined treatment group (CM).

[0041] The mouse ulcerative colitis model was established by daily gavage of 3g / kg dextran sulfate sodium salt (DSS) for 5 days; the control group mice were gavaged with 200μL sterile water daily; the treatment group was given drug treatment 6 hours after daily gavage of 3g / kg DSS, and the Changyanning treatment group was given 1.01292g / kg

[0042] The mice were gavaged with aqueous solution of Changyanning granules, the Mo2C treatment group was gavaged with Mo2C suspension at 100 mg / kg, and the Changyanning and Mo2C combined treatment group was gavaged with a mixed suspension of Changyanning 1.0 g / kg and Mo2C 100 mg / kg. The treatment intervention lasted for 7 days. The feeding conditions of mice in each group remained unchanged, and they were given normal food and water. The daily body weight of mice was recorded. After the 7-day treatment, the mice were killed, the colon tissues were collected, and the colon length of each group of mice was measured and recorded. The colon tissues were used for biochemical and histopathological evaluations.

[0043] Experimental results: Figure 1 As shown in the figure, on the seventh day, the body weight of the DSS model group was significantly lower than that of the control group by about 30% (***p<0.001), while the three treatment groups all showed some improvement, among which the body weight of mice in the CYN group and the MC group was about 17% lower than that of the Con group (about 14% relative to the DSS model group), and the combined drug CM group had the best effect, which was 12% lower than the Con group (about 21% relative to the DSS model group, and #p<0.05), indicating that both Changyanning and Mo2C can inhibit the weight loss of DSS-induced ulcerative colitis mice to a certain extent, but the combination of the two drugs has a better effect. The colon length of mice in each group is shown in the figure. Figure 2 As shown in the figure: compared with the Con group, the colon of mice in the DSS model group was shortened by about 40% (***p<0.001), while the mice in the Changyanning CYN group and the Mo2C MC group showed a certain therapeutic effect, and the shortening of the colon length was reduced by about 20% (*p<0.05); while the colon of mice in the combined drug CM group was only shortened by about 15%, which was significantly longer than that in the DSS model group (37%, #p<0.05), showing the best therapeutic effect. Further histopathological analysis results are shown in the figure. Figure 3 As shown: Compared with the Con group, the DSS group showed obvious glandular destruction, inflammatory cell infiltration, mucosal damage and ulcers; compared with the DSS group, the three drug-treated groups could reduce the destruction of colon glandular structure and inflammatory cell infiltration, among which the colon tissue structure of the combined drug CM group was the most complete.

[0044] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0045] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of molybdenum carbide nanomedicine in the preparation of a drug for treating oxidative stress inflammatory bowel disease, characterized in that: Preparation method of the molybdenum carbide nanomedicine The following steps are involved: S1: Take the original Mo2C powder, put it into a centrifuge tube with a 0.22μm filter membrane, add 20% tetrabutylammonium hydroxide, i.e. TBAOH aqueous solution, stir magnetically at room temperature for 48h and then centrifuge, including 8500rpm, 5min, 22℃, to remove TBAOH; S2: Wash with ultrapure water several times until the lower liquid is clear; S3: After washing, the precipitate is transferred to a freeze dryer for freeze drying at -80°C; S4: After completing the above operation, the product obtained is a black powder, i.e., a Mo2C nanodrug with a diameter of about 500-600 nm; S5: Store in a sealed bottle away from light to reduce unnecessary oxidation and decomposition; S6: Before use, resuspend in phosphate buffered saline (PBS) and sonicate for 10 min to obtain Mo2C nanodrug suspension.

2. The use of the molybdenum carbide nanomedicine according to claim 1 in the preparation of a drug for treating oxidative stress inflammatory bowel disease, characterized in that: The drug is in the form of a sheet, with a thickness of about 11 nm and a diameter of 200-800 nm.

3. The use of the molybdenum carbide nanomedicine according to claim 1 in the preparation of a drug for treating oxidative stress inflammatory bowel disease, characterized in that: The drug will not be degraded and absorbed in the intestine.

4. The use of the molybdenum carbide nanomedicine according to claim 1 in the preparation of a drug for treating oxidative stress inflammatory bowel disease, characterized in that: The drug effectively removes reactive oxygen species, or ROS, in the intestinal tract, including the large intestine and the small intestine.

5. The use of the molybdenum carbide nanomedicine according to claim 1 in the preparation of a drug for treating oxidative stress inflammatory bowel disease, characterized in that: The drug effectively alleviates intestinal oxidative stress caused by sleep deprivation and reduces inflammatory response.

6. The use of the molybdenum carbide nanomedicine according to claim 1 in the preparation of a drug for treating oxidative stress inflammatory bowel disease, characterized in that: The oxidative stress inflammatory bowel disease is ulcerative colitis, and the drug can relieve inflammatory symptoms and protect the intestinal barrier structure.

7. The use of the molybdenum carbide nanomedicine according to claim 6 in the preparation of a drug for treating oxidative stress inflammatory bowel disease, characterized in that: The drug can be used in combination with other therapeutic drugs, including Changyanning, and can also be used as an auxiliary drug to enhance the therapeutic effect on ulcerative colitis.

8. The use of the molybdenum carbide nanomedicine according to claim 2 in the preparation of a drug for treating oxidative stress inflammatory bowel disease, characterized in that: The drug diameter is selected to be 500-600nm.

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