Magnesium boride nanosheet / inulin composite hydrogel and application of magnesium boride nanosheet / inulin composite hydrogel in treatment of inflammatory bowel disease and concurrent mental diseases

Abstract

The invention discloses a magnesium boride nanosheet / inulin composite hydrogel and application thereof in treating inflammatory bowel diseases and complicated mental diseases, and the composite hydrogel is synthesized by doping magnesium boride nanosheets into inulin and carrying out hot melting and cooling to form gel. The composite hydrogel has good oxidation resistance and biocompatibility, reducing hydrogen can be generated through a hydrolysis reaction to be used for reducing active oxygen and inflammatory factors of the enteritis part, intestinal flora can be adjusted, meanwhile, the composite hydrogel can be prevented from being affected by gastric acid and reach the intestinal part through the addition of inulin, and the enteritis treatment effect is improved. Therefore, the inflammatory bowel disease and related anxiety and depression symptoms thereof are effectively treated.

Description

Technical Field

[0001] The invention belongs to the field of nano material preparation and biomedicine, and specifically relates to a magnesium boride nanosheet / inulin composite hydrogel and application thereof in treating inflammatory bowel disease and concurrent mental illness. Background Art

[0002] Inflammatory bowel disease (IBD), mainly including Crohn's disease (CD) and ulcerative colitis (UC), is a chronic autoimmune disease with recurrent, refractory and uncontrolled inflammation in the ileum, rectum and colon. More and more studies have shown that excessive oxidative stress and intestinal microbial disorders are key factors in inducing IBD. With the deepening of understanding of IBD, people are increasingly aware that it is not just an intestinal disease, and its extraintestinal symptoms and complications are also receiving more and more attention. Compared with the general population, IBD patients are more susceptible to anxiety, depression and other psychiatric disorders. However, the exact mechanism between IBD and concurrent psychiatric disorders is currently poorly understood, and effective therapeutic interventions are severely lacking. In recent years, studies have found that bidirectional signaling between the gastrointestinal tract (mainly microbiota) and the central nervous system (CNS) suggests that the connection between IBD and depression or anxiety may be bidirectional. At the same time, anxiety and depression are common in IBD patients, which seriously affects their quality of life and social function. Therefore, it is very necessary to develop new and effective therapeutic strategies for the treatment of IBD and its related concurrent psychiatric symptoms.

[0003] As an emerging nanomaterial, magnesium boride (MgB2) has been studied for its applications in anti-inflammatory, antibacterial, and anti-tumor applications. It has stable chemical properties and can produce hydrogen and borohydrides through hydrolysis reactions. Boron hydrides can be used to capture endotoxins (LPS) released by bacteria to form stable boron ester bonds, thereby achieving anti-inflammatory effects. Since oral drugs have to face the challenge of gastric acid, how to pass through gastric juice smoothly and reach the treatment site is one of the key issues currently faced in the study of metal nanomedicines. Inulin is widely present in plants as a reserve carbohydrate, especially in Asteraceae plants. Due to its structural characteristics, it will not be digested or absorbed in the small intestine. At the same time, inulin can be made into gel by a simple heating-cooling process. At present, many studies have shown that inulin gel is not only used to increase bioadhesion and prolong colon retention, but also regulate intestinal flora, restore intestinal barriers and maintain intestinal homeostasis. The present invention successfully developed a composite hydrogel with antioxidant, gastric acid and intestinal retention, which effectively treats IBD and related concurrent anxiety and depression. Summary of the invention

[0004] The purpose of the present invention is to provide a magnesium boride nanosheet / inulin composite hydrogel with antioxidant properties that can pass through gastric juice and be retained in the intestine, thereby solving the refractory and uncontrollable inflammation of inflammatory bowel disease and alleviating the emotional anxiety and depression symptoms caused by it.

[0005] In order to solve the technical problem, the present invention adopts the following technical solution:

[0006] The present invention first discloses a magnesium boride nanosheet / inulin composite hydrogel, wherein the composite hydrogel is formed by mixing polyethylene glycol-modified magnesium boride nanosheets with inulin. The preparation method thereof comprises the following steps:

[0007] Step 1, adding magnesium boride powder to anhydrous ethanol and ultrasonically crushing it, centrifuging it, and taking the supernatant; adding polyethylene glycol to the supernatant, continuing to ultrasonically crush it, centrifuging it, and freeze-drying the obtained 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 evenly, and cool naturally to obtain a magnesium boride nanosheet / inulin composite hydrogel.

[0009] Furthermore, in step 1, the mass ratio of magnesium boride powder to polyethylene glycol is 10 to 20:1. The ultrasonic power of the ultrasonic crushing is 480 to 600 W, the time is 0.5 to 2 hours, the speed of the centrifugation is 3000 to 12000 rpm, the centrifugation time is 15 to 30 minutes, and the process is carried out at room temperature. The obtained polyethylene glycol-modified magnesium boride nanosheets have a thickness of 1 to 3 nm and a diameter of 200 to 300 nm.

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

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

[0012] The composite hydrogel of the present invention is used to treat inflammatory bowel disease and its related concurrent mental illnesses such as anxiety and depression. The mechanism of realization is: the reducing gas H2 produced by the hydrolysis of magnesium boride can effectively remove excessive ROS in the colitis intestine and reduce proinflammatory cytokines, and the boron dihydroxy bonds produced at the same time can minimize the harm of LPS and intestinal leakage. At the same time, inulin hydrogel can effectively protect magnesium boride from gastric acid erosion and prolong intestinal retention time. In addition, magnesium boride nanosheets / inulin hydrogel can effectively reduce neuroinflammation and anxiety and depression symptoms caused by proinflammatory cytokines and LPS spreading through the blood and then crossing the blood-brain barrier to reach the brain. And oral treatment of magnesium boride nanosheets / inulin hydrogel can repair the imbalance of IBD intestinal flora by regulating the abundance and diversity of intestinal microbial communities, while maintaining the integrity of the intestinal barrier, thereby maintaining intestinal microbial and metabolic homeostasis.

[0013] The beneficial effects of the present invention are embodied in:

[0014] 1. The composite hydrogel of the present invention has good antioxidant properties, biocompatibility and stability. Magnesium boride can undergo hydrolysis to produce reducing safe gas hydrogen to remove excessive reactive oxygen and inflammatory factors in the intestinal inflammatory site. The boron hydroxyl generated at the same time can reduce the content of lipopolysaccharide; inulin, as a natural dietary fiber, is protected from gastric acid degradation while improving the safety of the drug; at the same time, inulin can achieve the effect of intestinal retention and enhance the efficacy of the drug.

[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, the conditions are mild, and it is possible to produce on a large scale, and has the potential for industrial and practical applications.

[0017] 4. The materials used in the present invention have good biocompatibility, have no direct or indirect toxic effects on the human body, and have no potential toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the synthesis of composite hydrogel.

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

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

[0021] Figure 4 This is a Fourier infrared absorption spectrum of the polyethylene glycol modified magnesium boride nanosheets, magnesium boride powder, and polyethylene glycol raw material prepared in Example 1.

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

[0023] Figure 6 This is a scanning electron microscope image of the composite hydrogel prepared in Example 1.

[0024] Figure 7 Rheological diagrams of the composite hydrogel prepared in Example 1 and the pure inulin hydrogel.

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

[0026] Fig. 9 Biocompatibility diagram of magnesium boride nanosheets modified with different concentrations of polyethylene glycol.

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

[0028] Fig.11 The following are pictures of the colons of mice in each group after dissection in the evaluation of colitis prevention performance in Example 1.

[0029] Fig.12 This is a statistical chart of the colon length of each group of mice after dissection in the evaluation of colitis prevention performance in Example 1.

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

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

[0032] Fig.15 This is a picture of the colon after dissection of each group of mice in the evaluation of colitis therapeutic performance in Example 1.

[0033] Fig.16 This is a statistical chart of the colon length after dissection of each group of mice in the evaluation of colitis treatment performance in Example 1.

[0034] Fig.17 The abundance of intestinal flora was measured by collecting feces from each group of mice after autopsy in the evaluation of colitis treatment performance in Example 1, where (a) is observed features analysis and (b) is Shannon index analysis.

[0035] Fig.18The evaluation results of each group of mice in the treatment evaluation of anxiety and depression symptoms caused by colitis in Example 1, wherein: (a) is a mine field experiment diagram, (b) is a movement distance diagram and (c) is a central movement time diagram. DETAILED DESCRIPTION

[0036] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0037] Example 1

[0038] like Figure 1 As shown, the composite hydrogel is 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, ultrasonically treat it for 2 h using an ultrasonic crusher (power 480 W), and then centrifuge it at room temperature (3000 rpm, 15 min) to take the supernatant: add 20 mg of polyethylene glycol to the obtained supernatant and continue to ultrasonically crush it for 30 min (power 480 W), and then centrifuge it at room temperature (12000 rpm, 15 min), and freeze-dry the obtained precipitate to obtain polyethylene glycol-modified magnesium boride nanosheets (denoted as MgB2@PEG).

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

[0041] The morphology and properties of the obtained products were characterized as follows:

[0042] 1. Morphology characterization

[0043] Figure 2 This is a transmission electron microscope image of the polyethylene glycol-modified magnesium boride nanosheet obtained in this example. From the image, it can be seen that the diameter of the nanosheet is 200-300 nm.

[0044] Figure 3 This is an atomic force microscope image of the polyethylene glycol-modified magnesium boride nanosheet obtained in this example. From the image, it can be seen that the thickness of the nanosheet is 1 to 3 nm.

[0045] Figure 4 The Fourier transform infrared spectra of the polyethylene glycol modified magnesium boride nanosheets obtained in this example are compared with those of magnesium boride powder and polyethylene glycol raw materials. It can be seen from the figure that the polyethylene glycol and magnesium boride have a peak at 1104 cm -1A peak was formed at , indicating successful modification with polyethylene glycol.

[0046] Figure 5 The XRD spectra of the polyethylene glycol modified magnesium boride nanosheets obtained in this example before and after hydrolysis are compared. The hydrolysis method is to add the polyethylene glycol modified magnesium boride nanosheets to water at a concentration of 2 mg / mL, react at room temperature for 12 hours, and then centrifuge and freeze-dry to powder. It can be seen from the figure that after magnesium boride reacts with water for 12 hours, the peaks at various locations significantly decrease or disappear, so it can be shown that magnesium boride can react with water.

[0047] Figure 6 This is a scanning electron microscope image of the composite hydrogel obtained in this example. From the image, it can be seen that the composite hydrogel has a typical porous structure of a hydrogel.

[0048] Figure 7 The rheological diagrams (loss modulus and storage modulus) of the composite hydrogel and pure inulin hydrogel obtained in this example, from which it can be seen that the storage modulus (G') of the pure inulin hydrogel and the composite hydrogel are slightly larger than the loss modulus (G") and are relatively close, which is consistent with the rheological characteristics of the hydrogel, so the synthesized product is a hydrogel.

[0049] 2. Hydrogen production performance

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

[0051] 3. Biocompatibility

[0052] Fig. 9The biocompatibility diagram of polyethylene glycol modified magnesium boride nanosheets with different concentrations is shown in Figure 2. The characterization method is as follows: dilute the aqueous dispersion of polyethylene glycol modified magnesium boride nanosheets to 62.5μg / mL, 125μg / mL, 250μg / mL, 500μg / mL, and 1000μg / mL, and mix 0.2mL of the above solutions with different concentrations with 0.2mL of treated blood (500μL of fresh blood is added to 4.5mL of normal saline and centrifuged for 5 to 8 times, with a centrifugal speed of 3000rpm and a centrifugal time of 10min. When the blood supernatant is clear and transparent, the supernatant is discarded and the volume is fixed to 5mL with normal saline) and 0.6mL of normal saline, and incubate at 37°C for 4h, then centrifuge at 3000rpm for 10min, aspirate the supernatant to measure the absorbance at OD541nm, and calculate the hemolysis rate. Fig. 9 It can be seen that the hemolysis rate of polyethylene glycol-modified magnesium boride nanosheets with different concentrations is less than 5%, indicating that the material has good biocompatibility.

[0053] 3. Colitis prevention performance

[0054] The evaluation method is as follows: 6-week-old female Balb / c mice were housed in cages of 8 mice and adapted for 1 week before inclusion in the study. In the preventive 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., pentaaminosalicylic acid group, dosage 20 mg / kg), (4) MgB2@PEG group (dosage 20 mg / kg), (5) Iinulin 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 of each group are as follows:

[0055] The control group was fed with normal drinking water, and 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 normal drinking water was used instead of DSS-containing drinking water on day 7. The mice were gavaged on days 1, 3, 5, and 7, with groups (1) and (2) being gavaged with 100 μL of PBS solution, and groups (3) to (8) being gavaged with 100 μL of PBS solution containing the drug according to the corresponding dosage. Body weight, visible stool consistency, and fecal bleeding were assessed daily during the 9-day experimental period. The colon was collected after the mice were euthanized on day 9, and the colon length was calculated.

[0056] Fig.10The weight changes of each group during the experiment show that DSS caused the mice to lose weight continuously, but after the composite hydrogel treatment, the weight loss trend was alleviated. After the treatment with 10 mg / kg magnesium boride nanosheets / inulin composite hydrogel, the weight was restored to normal level and the treatment effect was better than that of pentaaminosalicylic acid. Fig.11 Colon pictures and Fig.12 From the colon length statistics, it can be seen that DSS caused a significant shortening of the mice's colon. The colon length of the mice in the treatment group was relieved to different degrees. After treatment with 10 mg / kg of magnesium boride nanosheets / inulin composite hydrogel, the colon length basically returned to normal levels. Fig.13 The disease activity index of each group of mice is shown. It can be seen that DSS causes a significant increase in the disease activity index of mice. After the composite hydrogel treatment, the disease activity index of mice is significantly restored. The above results show that the composite hydrogel obtained in this example can achieve a therapeutic effect on inflammatory bowel disease.

[0057] 4. Colitis treatment performance

[0058] The evaluation method is as follows: 6-week-old female Balb / c mice were housed in cages of 8 mice and adapted for 1 week before inclusion in the study. In the preventive 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., pentaaminosalicylic acid group, dosage 20 mg / kg), (4) MgB2@PEG group (dosage 20 mg / kg), (5) Iinulin 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 of each group are as follows:

[0059] The control group was fed with normal drinking water, and 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 normal drinking water was used instead of drinking water containing DSS on day 7. The mice were gavaged on days 7, 9, 11, and 13, with groups (1) and (2) being gavaged with 100 μL of PBS solution, and groups (3) to (8) being gavaged with 100 μL of PBS solution containing the drug according to the corresponding dosage. Body weight changes were recorded every 2 days. The colon was collected after the mice were euthanized on day 15, and the colon length was calculated. The feces were collected for intestinal flora analysis.

[0060] Fig.14The weight changes of each group during the experiment show that DSS caused the mice to lose weight continuously in the first 7 days, and the weight loss of untreated mice was not alleviated. However, after the composite hydrogel treatment, the weight loss trend was alleviated and returned to normal levels. It can also be seen that the treatment effect is better than that of pentaaminosalicylic acid. Fig.15 Colon pictures and Fig.16 The colon length statistics show that DSS caused the colon of mice to shorten significantly. The colon length of mice in the treatment group was relieved to different extents. After treatment with 10 mg / kg magnesium boride nanosheets / inulin composite hydrogel, the colon length basically returned to normal levels. Fig.17 It can be seen that the richness of intestinal microorganisms in mice induced by DSS was significantly reduced, while the richness of intestinal microorganisms in the treatment group of mice rebounded, which was basically consistent with the control group, indicating that the composite hydrogel synthesized by inulin can still exert the ability of inulin to regulate intestinal flora. The above results show that the composite hydrogel obtained in this example can achieve a therapeutic effect on inflammatory bowel disease.

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

[0062] The evaluation method was as follows: 6-week-old male C57BL / 6J mice were housed in cages with 10 mice and adapted for 1 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., pentaaminosalicylic acid group, administered at 20 mg / kg), (4) MgB2@PEG group (administered at 20 mg / kg), (5) Iinulin gel group (i.e., pure inulin gel group, administered at 20 mg / kg), (6) MgB2@Inulin gel group (administered at 20 mg / kg). The treatment methods of each group were as follows: The construction of DSS-induced chronic colitis involved the administration of three cycles of DSS, each cycle consisting of one week of drinking 2% DSS and then two weeks of drinking water, for a total of 9 weeks. During the drinking water period of the second and third cycles, mice were gavaged every other day for a total of 14 times. The control group drank normal water throughout the treatment period. During the administration period, groups (1) and (2) were gavaged with 100 μL of PBS solution, and groups (3) to (6) were gavaged with 100 μL of PBS solution containing the drug according to the corresponding dosage.

[0063] After the establishment of DSS-induced chronic colitis, the open-ended test was used to quantify anxiety behavior. Each mouse was placed in the center of a box with a length and width of 50 cm and a height of 40 cm. Their activities were recorded for 10 minutes, and the distance and duration covered by the mice in each different area (corner, periphery and center) were evaluated using a camera system in a dim environment.

[0064] Fig.18 (a) shows the movement trajectory of each group of mice. From the movement trajectories of different groups, it can be seen that DSS caused a significant reduction in the movement trajectory and range of mice, and their motor ability also decreased significantly. At the same time, the mice spent significantly less time in the central area, which indicated that the motor cognitive ability of mice was reduced due to colitis and they developed anxious behavior. After treatment with magnesium boride nanosheets / inulin composite hydrogel, the motor ability of mice was significantly restored, and the total movement distance of mice was basically the same as that of the control group (e.g. Fig.18 (b)), the time mice stayed in the central area (as shown in Fig.18 The results in Table 1 show that the composite hydrogel obtained in this example can achieve a therapeutic effect on the anxiety and depression caused by 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 in the protection scope of the present invention.

Claims

1. A magnesium boride nanosheet / inulin composite hydrogel, characterized in that: The composite hydrogel is formed by mixing polyethylene glycol-modified magnesium boride nanosheets and inulin.

2. The magnesium boride nanosheet / inulin composite hydrogel according to claim 1, characterized in that: The mass ratio of the polyethylene glycol-modified magnesium boride nanosheets to inulin is 1:100-200.

3. A method for preparing the magnesium boride nanosheet / inulin composite hydrogel according to any one of claims 1 to 2, characterized in that: The steps include: Step 1, adding magnesium boride powder to anhydrous ethanol and ultrasonically crushing it, centrifuging it, and taking the supernatant; adding polyethylene glycol to the supernatant, continuing to ultrasonically crush it, centrifuging it, and freeze-drying the obtained precipitate to obtain polyethylene glycol-modified magnesium boride nanosheets; Step 2: Mix the polyethylene glycol-modified magnesium boride nanosheets obtained in step 1 with inulin, add deionized water, heat and stir evenly, and cool naturally to obtain a magnesium boride nanosheet / inulin composite hydrogel.

4. The preparation method according to claim 3, characterized in that: In step 1, the mass ratio of magnesium boride powder to polyethylene glycol is 10-20:

1.

5. The preparation method according to claim 3, characterized in that: In step 1, the ultrasonic power of the ultrasonic disruption is 480-600 W, the time is 0.5-2 h, the speed of the centrifugation is 3000-12000 rpm, and the centrifugation time is 15-30 min.

6. The preparation method according to claim 3, characterized in that: In step 1, the obtained polyethylene glycol-modified magnesium boride nanosheets have a thickness of 1 to 3 nm and a diameter of 200 to 300 nm.

7. The preparation method according to claim 4, characterized in that: In step 2, the dosage ratio of inulin to deionized water is 0.5-1.5 g / mL.

8. The preparation method according to claim 4, characterized in that: In step 2, the heating temperature is 60-80° C., the heating time is 10-15 min, and the cooling time is 8-12 h.

9. Use of the magnesium boride nanosheet / inulin composite hydrogel according to any one of claims 1 to 2 in the preparation of a drug for treating inflammatory bowel disease and its concurrent mental illness.

10. The use according to claim 9, characterized in that: The comorbid psychiatric disorder refers to anxiety and / or depression symptoms caused by inflammatory bowel disease.

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