A magnesium boride nanosheet / inulin composite hydrogel and its application in treating inflammatory bowel disease and concurrent mental illness

Abstract

The application discloses a magnesium boride nanosheet / inulin composite hydrogel and application thereof in treating inflammatory bowel disease and concurrent mental illness, wherein the composite hydrogel is synthesized by doping magnesium boride nanosheets into inulin and then forming a gel through heat melting and cooling. The composite hydrogel has good oxidation resistance and biocompatibility, can generate reducing hydrogen through a hydrolysis reaction to reduce active oxygen and inflammatory factors at an intestinal inflammation site, can regulate intestinal flora, and can reach the intestinal site by being free from the influence of gastric acid due to the addition of inulin, so that the composite hydrogel can effectively treat inflammatory bowel disease and related anxiety and depression symptoms.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterial preparation and biomedicine, specifically relating to a magnesium boride nanosheet / inulin composite hydrogel and its application in the treatment of inflammatory bowel disease and comorbid mental illness. Background Technology

[0002] Inflammatory bowel disease (IBD), primarily including Crohn's disease (CD) and ulcerative colitis (UC), is a chronic autoimmune disease characterized by relapsing, refractory, and uncontrolled inflammation in the ileum, rectum, and colon. A growing body of research indicates that excessive oxidative stress and gut microbiota dysbiosis are key factors inducing IBD. With a deeper understanding of IBD, it is increasingly recognized that it is not merely a gut disease; its extraintestinal symptoms and complications are also receiving increasing attention. Compared to the general population, IBD patients are more susceptible to anxiety, depression, and other mental illnesses. However, the exact mechanisms between IBD and its associated mental disorders are poorly understood, and effective treatment interventions are severely lacking. Recent studies have discovered bidirectional signaling between the gastrointestinal tract (primarily the microbiota) and the central nervous system (CNS), suggesting that the link between IBD and depression or anxiety may be bidirectional. Simultaneous anxiety and depression are prevalent in IBD patients, significantly impacting their quality of life and social functioning. Therefore, developing new and effective treatment strategies for IBD and its associated mental symptoms is crucial.

[0003] Magnesium boride (MgB2), as an emerging nanomaterial, has been studied for its applications in anti-inflammatory, antibacterial, and antitumor activities. Its chemical stability allows it to produce hydrogen gas and boron hydroxyl compounds through hydrolysis. These compounds can be used to capture endotoxins (LPS) released by bacteria, forming stable boron ester bonds, thereby achieving an anti-inflammatory effect. Since oral medications must overcome the challenge of gastric acid, how to successfully pass through gastric juice and reach the therapeutic site is one of the key issues currently facing the research of metal nanomedicines. Inulin, as a storage carbohydrate, is widely found in plants, especially in the Asteraceae family. Due to its structural characteristics, it is not digested or absorbed in the small intestine. Furthermore, inulin can be gelled through a simple heating-cooling process. Current research shows that inulin gels not only increase bioadhesion and prolong colonic retention but also regulate gut microbiota, restore the intestinal barrier, and maintain intestinal homeostasis. This invention successfully developed a composite hydrogel with antioxidant, gastric acid-passing, and intestinal retention properties, effectively treating IBD and related complications such as anxiety and depression. Summary of the Invention

[0004] The purpose of this invention is to provide a magnesium boride nanosheet / inulin composite hydrogel with antioxidant properties that can pass through gastric juice and remain in the intestine, thereby addressing the refractory and uncontrollable inflammation of inflammatory bowel disease and alleviating the resulting anxiety and depression symptoms.

[0005] To solve the technical problem, the present invention adopts the following technical solution:

[0006] This invention first discloses a magnesium boride nanosheet / inulin composite hydrogel, wherein the composite hydrogel is formed by blending polyethylene glycol-modified magnesium boride nanosheets with inulin. Its preparation method includes the following steps:

[0007] Step 1: Add magnesium boride powder to anhydrous ethanol and sonicate it. Centrifuge and take the supernatant. Add polyethylene glycol to the supernatant and continue to sonicate it. Centrifuge and freeze dry the precipitate to obtain polyethylene glycol modified magnesium boride nanosheets.

[0008] Step 2: Mix the polyethylene glycol-modified magnesium boride nanosheets obtained in Step 1 with inulin at a mass ratio of 1:100-200, add deionized water, heat and stir until uniform, and then cool naturally to obtain magnesium boride nanosheet / inulin composite hydrogel.

[0009] Further, in step 1, the mass ratio of magnesium boride powder to polyethylene glycol is 10–20:1. The ultrasonic disruption is performed at a power of 480–600 W for 0.5–2 h, and the centrifugation is carried out at a speed of 3000–12000 rpm for 15–30 min at room temperature. The resulting polyethylene glycol-modified magnesium boride nanosheets have a thickness of 1–3 nm and a diameter of 200–300 nm.

[0010] Further, in step 2: the mass ratio of the polyethylene glycol-modified magnesium boride nanosheets to inulin is 1:100-200. The ratio of inulin to deionized water is 0.5-1.5 g / mL. The heating temperature is 60-80℃, the heating time is 10-15 min, and the cooling time is 6-12 h.

[0011] The composite hydrogel of this invention has the function of treating inflammatory bowel disease, and has no significant toxicity to mammalian cells, exhibiting good biosafety. It can also improve symptoms such as anxiety and depression caused by colitis.

[0012] The mechanism by which the composite hydrogel described in this invention treats inflammatory bowel disease (IBD) and related comorbid mental illnesses such as anxiety and depression is as follows: the reducing gas H2 produced by the hydrolysis of magnesium boride effectively removes excess ROS from the colitis intestinal tract and reduces pro-inflammatory cytokines. Simultaneously, the generated boron dihydroxy bonds minimize the harm of LPS and intestinal leakage. Meanwhile, the inulin hydrogel effectively protects magnesium boride from gastric acid erosion and prolongs its intestinal retention time. Furthermore, the magnesium boride nanosheets / inulin hydrogel effectively reduces neuroinflammation and anxiety / depressive symptoms caused by pro-inflammatory cytokines and LPS spreading through the bloodstream and crossing the blood-brain barrier to reach the brain. Oral treatment with magnesium boride nanosheets / inulin hydrogel can repair the IBD intestinal flora imbalance by regulating the abundance and diversity of the intestinal microbiota, while maintaining the integrity of the intestinal barrier, thereby maintaining intestinal microbial and metabolic homeostasis.

[0013] The beneficial effects of this invention are reflected in:

[0014] 1. The composite hydrogel of this invention exhibits excellent antioxidant properties, biocompatibility, and stability. Magnesium boride undergoes a hydrolysis reaction to produce reducing, safe hydrogen gas, which removes excess reactive oxygen species and inflammatory factors from inflamed areas of the intestine. Simultaneously, the generated boron hydroxyl groups reduce the content of lipopolysaccharides. Inulin, as a natural dietary fiber, is protected from degradation by gastric acid, thus improving drug safety. Furthermore, inulin can remain in the intestine to enhance drug efficacy.

[0015] 2. The composite hydrogel of the present invention is used to treat colitis and can also relieve anxiety and depression caused by chronic colitis.

[0016] 3. The preparation process of the composite hydrogel of the present invention is simple and the conditions are mild, which makes it possible to produce on a large scale and has the potential for industrial and practical applications.

[0017] 4. The materials used in this invention have excellent biocompatibility and have no direct or indirect toxic effects on the human body, and have no potential toxicity. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the synthesis of composite hydrogels.

[0019] Figure 2 This is a transmission electron microscope (TEM) image of the polyethylene glycol-modified magnesium boride nanosheets prepared in Example 1.

[0020] Figure 3 An atomic force microscope image of the polyethylene glycol-modified magnesium boride nanosheets prepared in Example 1.

[0021] Figure 4 Fourier transform infrared absorption spectra of polyethylene glycol-modified magnesium boride nanosheets, magnesium boride powder, and polyethylene glycol raw materials prepared in Example 1.

[0022] Figure 5 The XRD spectra of the polyethylene glycol-modified magnesium boride nanosheets prepared in Example 1 before and after hydrolysis.

[0023] Figure 6 The image shows a scanning electron microscope (SEM) image of the composite hydrogel prepared in Example 1.

[0024] Figure 7 The rheological diagrams are those of the composite hydrogel prepared in Example 1 and the pure inulin hydrogel.

[0025] Figure 8 The diagram shows the generation of hydrogen gas from the composite hydrogel prepared in Example 1 and the polyethylene glycol-modified magnesium boride nanosheets.

[0026] Figure 9 Biocompatibility diagram of polyethylene glycol-modified magnesium boride nanosheets at different concentrations.

[0027] Figure 10 This is a graph showing the weight changes of mice in each group during the performance evaluation of colitis prevention in Example 1.

[0028] Figure 11 This is a anatomical colon image of mice in each group during the colitis prevention performance evaluation in Example 1.

[0029] Figure 12 This is a statistical diagram of the colon length of mice in each group after dissection in Example 1, which evaluated the preventive performance of colitis.

[0030] Figure 13 This is a statistical chart of the disease activity index of mice in each group during the performance evaluation of colitis prevention in Example 1.

[0031] Figure 14 This is a graph showing the weight changes of mice in each group during the performance evaluation of colitis treatment in Example 1.

[0032] Figure 15 This is a anatomical colon image of mice in each group during the performance evaluation of colitis treatment in Example 1.

[0033] Figure 16 This is a statistical diagram of the colon length of mice in each group after dissection in Example 1, which evaluated the therapeutic performance of colitis.

[0034] Figure 17 The abundance of gut-like flora in feces collected after dissection of mice in Example 1 for evaluating the therapeutic performance of colitis is shown in (a) for observed features analysis and (b) for Shannon index analysis.

[0035] Figure 18The following are the evaluation results of mice in each group in the treatment evaluation of anxiety and depression symptoms caused by colitis in Example 1, where: (a) is a plot of the mine field experiment, (b) is a plot of movement distance and (c) is a plot of central movement time. Detailed Implementation

[0036] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0037] Example 1

[0038] like Figure 1 As shown, the composite hydrogel was prepared according to the following steps in this embodiment:

[0039] Step 1: Add 300 mg of magnesium boride powder to 30 mL of anhydrous ethanol, and sonicate for 2 h using an ultrasonic homogenizer (480 W). Then centrifuge at room temperature (3000 rpm, 15 min) and collect the supernatant. Add 20 mg of polyethylene glycol to the supernatant and continue ultrasonic homogenization for 30 min (480 W). Then centrifuge at room temperature (12000 rpm, 15 min) and freeze-dry the precipitate to obtain polyethylene glycol-modified magnesium boride nanosheets (denoted as MgB2@PEG).

[0040] Step 2: Mix polyethylene glycol-modified magnesium boride nanosheets with 1.2g of inulin at a mass ratio of 1:120, add 2mL of deionized water, heat in an 80℃ water bath and stir at 800rpm for 10min, then cool at room temperature for 12h to form magnesium boride nanosheet / inulin composite hydrogel (denoted as MgB2@Inulin gel).

[0041] The obtained product was characterized in the following ways in terms of morphology and properties:

[0042] 1. Morphological characteristics

[0043] Figure 2 The image shows a transmission electron microscope (TEM) image of the polyethylene glycol-modified magnesium boride nanosheets obtained in this embodiment. The image shows that the nanosheets have a diameter of 200–300 nm.

[0044] Figure 3 This is an atomic force microscope image of the polyethylene glycol-modified magnesium boride nanosheets obtained in this embodiment. The image shows that the thickness of the nanosheets is 1–3 nm.

[0045] Figure 4 The Fourier transform infrared spectra of the polyethylene glycol-modified magnesium boride nanosheets, magnesium boride powder, and polyethylene glycol raw material obtained in this embodiment are compared. The figures show that polyethylene glycol and magnesium boride exhibit similar wavelengths at 1104 cm⁻¹. -1The formation of a peak at this location indicates successful modification of polyethylene glycol.

[0046] Figure 5 The XRD spectra of the polyethylene glycol-modified magnesium boride nanosheets obtained in this embodiment are compared before and after hydrolysis. The hydrolysis method involves adding the polyethylene glycol-modified magnesium boride nanosheets to water at a concentration of 2 mg / mL, reacting at room temperature for 12 hours, and then centrifuging and freeze-drying into powder. The figures show that after 12 hours of reaction, the peaks of magnesium boride with water significantly decreased or disappeared, indicating that magnesium boride can react with water.

[0047] Figure 6 The image shows a scanning electron microscope (SEM) image of the composite hydrogel obtained in this embodiment. As can be seen from the image, the composite hydrogel has a typical porous structure of hydrogels.

[0048] Figure 7 The rheological diagrams (loss modulus and storage modulus) of the composite hydrogel and the pure inulin hydrogel obtained in this embodiment are shown. It can be seen from the figure that the storage modulus (G') of both the pure inulin hydrogel and the composite hydrogel is slightly greater than the loss modulus (G”) and is quite close, which is consistent with the rheological characteristics of hydrogels. Therefore, the synthesized hydrogel is a hydrogel.

[0049] 2. Hydrogen production performance

[0050] Figure 8 The figure shows the hydrogen production of the composite hydrogel and polyethylene glycol-modified magnesium boride nanosheets obtained in Example 1. The characterization method is as follows: 3 mg of MgB2@Inulin gel or MgB2@PEG was added to 3 mL of methylene blue solution with a concentration of 10 μg / mL. The reaction was carried out at room temperature, and the ultraviolet spectra were measured at different time points. The amount of hydrogen produced was determined by ultraviolet absorption. It can be seen from the figure that both polyethylene glycol-modified magnesium boride nanosheets and composite hydrogel can continuously produce hydrogen. The addition of bacterial powder did not affect the hydrogen production performance of magnesium boride.

[0051] 3. Biocompatibility

[0052] Figure 9The biocompatibility of polyethylene glycol-modified magnesium boride nanosheets at different concentrations is shown in the figure. The characterization method was as follows: Aqueous dispersions of polyethylene glycol-modified magnesium boride nanosheets were diluted to 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, and 1000 μg / mL. 0.2 mL of each of these solutions was then mixed with 0.2 mL of treated blood (500 μL of fresh blood was added to 4.5 mL of physiological saline and centrifuged 5–8 times at 3000 rpm for 10 min until the blood supernatant was clear and transparent; the supernatant was then discarded, and the volume was adjusted to 5 mL with physiological saline) and 0.6 mL of physiological saline, respectively. The mixture was incubated at 37°C for 4 h, followed by centrifugation at 3000 rpm for 10 min. The absorbance at OD541 nm was measured, and the hemolysis rate was calculated. Figure 9 The results show that the hemolysis rate of polyethylene glycol-modified magnesium boride nanosheets at different concentrations is less than 5%, indicating that the material has good biocompatibility.

[0053] 3. Colitis prevention performance

[0054] The evaluation method was as follows: Eight 6-week-old female Balb / c mice were housed in each cage and acclimatized for one week before inclusion in the study. In the prevention and treatment of UC, healthy mice were randomly divided into 8 groups (n=8): (1) Control group; (2) 3% DSS control group; (3) 5-ASA group (i.e., pentamirrolic acid group, dosage 20 mg / kg); (4) MgB2@PEG group (dosage 20 mg / kg); (5) Inulin gel group (i.e., pure inulin gel group, dosage 20 mg / kg); (6) 5 mg / kg MgB2@Inulin gel group; (7) 10 mg / kg MgB2@Inulin gel group; (8) 20 mg / kg MgB2@Inulin gel group. The treatment methods for each group were as follows:

[0055] The control group was fed with regular drinking water, while the other groups were fed with drinking water containing 3% DSS. The first day of oral administration of DSS was designated as day 0, and regular drinking water was used instead of the DSS-containing water on day 7. Administered the medication by gavage on days 1, 3, 5, and 7. Groups (1) and (2) were administered 100 μL of PBS solution by gavage, while groups (3) through (8) were administered 100 μL of PBS solution containing the medication according to the corresponding dosage. Body weight, visible fecal consistency, and fecal bleeding were assessed daily during the 9-day experiment. The colons were collected after euthanasia on day 9, and colon length was calculated.

[0056] Figure 10The experiment showed that DSS caused a continuous decrease in mouse weight, but the weight loss was alleviated after treatment with the composite hydrogel. After treatment with 10 mg / kg magnesium boride nanosheets / inulin composite hydrogel, the weight returned to normal, and the treatment effect was better than that of pentamisalicylic acid. Simultaneously, through… Figure 11 Colon pictures and Figure 12 The colon length statistics show that DSS caused a significant shortening of the colon in mice. The colon length of mice in the treatment group was alleviated to varying degrees. After treatment with 10 mg / kg magnesium boride nanosheets / inulin composite hydrogel, the colon length basically returned to the normal level. Figure 13 The results show the disease activity index of each group of mice. It can be seen that DSS significantly increased the disease activity index, and after treatment with the composite hydrogel, the disease activity index of the mice showed significant recovery. In summary, the results indicate that the composite hydrogel obtained in this embodiment can achieve a therapeutic effect on inflammatory bowel disease.

[0057] 4. Performance in treating colitis

[0058] The evaluation method was as follows: Eight 6-week-old female Balb / c mice were housed in each cage and acclimatized for one week before inclusion in the study. In the prevention and treatment of UC, healthy mice were randomly divided into 8 groups (n=8): (1) Control group; (2) 3% DSS control group; (3) 5-ASA group (i.e., pentamirrolic acid group, dosage 20 mg / kg); (4) MgB2@PEG group (dosage 20 mg / kg); (5) Inulin gel group (i.e., pure inulin gel group, dosage 20 mg / kg); (6) 5 mg / kg MgB2@Inulin gel group; (7) 10 mg / kg MgB2@Inulin gel group; (8) 20 mg / kg MgB2@Inulin gel group. The treatment methods for each group were as follows:

[0059] The control group was fed with regular drinking water, while the other groups were fed with drinking water containing 3% DSS. The first day of oral administration of DSS was designated as day 0, and regular drinking water was used instead of the DSS-containing water on day 7. Administered the medication by gavage on days 7, 9, 11, and 13. Groups (1) and (2) were administered 100 μL of PBS solution by gavage, while groups (3) through (8) were administered 100 μL of PBS solution containing the corresponding dosage by gavage. Body weight changes were recorded every two days. After euthanasia on day 15, the colons were collected, colon length was calculated, and feces were collected for intestinal flora analysis.

[0060] Figure 14The study showed that DSS caused a continuous decrease in mouse weight during the first 7 days, and the weight loss in untreated mice did not improve. However, after treatment with the composite hydrogel, the weight loss trend was alleviated and the weight returned to normal levels. This treatment was more effective than that with pentamirric acid. Figure 15 Colon pictures and Figure 16 Statistical analysis of colon length showed that DSS significantly shortened the colon in mice. The treatment group experienced varying degrees of relief in colon length, with the colon length essentially returning to normal after treatment with 10 mg / kg magnesium boride nanosheets / inulin composite hydrogel. Figure 17 It was found that the gut microbiota richness in mice induced by DSS was significantly reduced, while the gut microbiota richness in the treatment group recovered and was basically consistent with that in the control group. This indicates that the composite hydrogel synthesized from inulin can still exert the ability of inulin to regulate the gut microbiota. In summary, the results show that the composite hydrogel obtained in this embodiment can achieve a therapeutic effect on inflammatory bowel disease.

[0061] 5. Treatment of anxiety and depression symptoms caused by colitis

[0062] Evaluation method: Ten 6-week-old male C57BL / 6J mice were housed in each cage and acclimatized for one week before inclusion in the study. Healthy mice were randomly divided into 6 groups (n=10): (1) Control group; (2) DSS control group; (3) 5-ASA group (i.e., pentamirrolic acid group, dose 20 mg / kg); (4) MgB2@PEG group (dose 20 mg / kg); (5) Inulin gel group (i.e., pure inulin gel group, dose 20 mg / kg); (6) MgB2@Inulin gel group (dose 20 mg / kg). The treatment methods for each group were as follows: The construction of DSS-induced chronic colitis involved three cycles of DSS administration, each cycle consisting of one week of drinking 2% DSS followed by two weeks of drinking water, for a total of 9 weeks. During the second and third cycles of drinking water, mice were administered the drug by gavage every other day for a total of 14 times. The control group drank plain water throughout the treatment period. During the administration period, groups (1) and (2) were given 100 μL of PBS solution by gavage, and groups (3) to (6) were given 100 μL of PBS solution containing the drug by gavage according to the corresponding dosage.

[0063] After successfully constructing DSS-induced chronic colitis, an open-ended test was used to quantify anxiety behavior. Each mouse was placed in the center of a box 50 cm long and wide and 40 cm high. Their activity was recorded for 10 minutes in a dimly lit environment, and the distance and duration of coverage by the mice in each different area (corner, periphery, and center) were assessed using a camera system.

[0064] Figure 18 (a) shows the movement trajectory diagrams of each group of mice. The movement trajectories of different groups show that DSS significantly reduced the movement trajectory and range of the mice, and also significantly decreased their motor ability. Simultaneously, the mice spent significantly less time in the central region, indicating that their motor cognitive ability was impaired due to colitis and that they exhibited anxious behavior. After treatment with magnesium boride nanosheets / inulin composite hydrogel, the mice's motor ability significantly recovered, and the total distance they moved was essentially the same as the control group (e.g., ...). Figure 18 (b) shows the time the mouse spends in the central region (as shown in the figure). Figure 18 (c) The results also showed that the mice's behavioral and cognitive abilities were significantly higher than those of the DSS group, indicating that the mice's behavior and cognitive abilities were significantly improved after treatment compared to the DSS group. In summary, the results indicate that the composite hydrogel obtained in this embodiment can achieve a therapeutic effect on the anxiety and depression associated with colitis.

[0065] The above are merely exemplary embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a magnesium boride nanosheet / inulin composite hydrogel in the preparation of a drug for treating colitis, wherein the magnesium boride nanosheet / inulin composite hydrogel is composed of polyethylene glycol-modified magnesium boride nanosheets and inulin.

2. The application according to claim 1, characterized in that, The preparation method of the magnesium boride nanosheet / inulin composite hydrogel includes the following steps: Step 1: Add 300 mg of magnesium boride powder to 30 mL of anhydrous ethanol, sonicate for 2 h using an ultrasonic disruptor with an ultrasonic power of 480 W, then centrifuge at 3000 rpm for 15 min at room temperature, and collect the supernatant. Add 20 mg of polyethylene glycol to the supernatant and continue ultrasonic disruption for 30 min with an ultrasonic power of 480 W. Then centrifuge at 12000 rpm for 15 min at room temperature, and freeze-dry the precipitate to obtain polyethylene glycol-modified magnesium boride nanosheets with a diameter of 200-300 nm and a thickness of 1-3 nm. Step 2: Mix polyethylene glycol-modified magnesium boride nanosheets with 1.2g of inulin at a mass ratio of 1:120, add 2 mL of deionized water, heat in an 80℃ water bath and stir at 800 rpm for 10 min, then cool at room temperature for 12 h to form magnesium boride nanosheet / inulin composite hydrogel.

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