CuS / PGS-PSBMA-calcium alginate hydrogel microsphere for treating inflammatory bowel disease and preparation method of CuS / PGS-PSBMA-calcium alginate hydrogel microsphere

Abstract

The invention discloses a CuS / PGS-PSBMA-calcium alginate hydrogel microsphere for treating inflammatory bowel disease and a preparation method thereof, and the preparation method of the oral hydrogel microsphere comprises the following steps: (1) crosslinking CuS / PGS and PSBMA, and preparing the CuS / PGS-PSBMA hydrogel microsphere by adopting an air flow control technology; and (2) performing calcium ion diffusion cross-linking to obtain a core-shell structure hydrogel microsphere with calcium alginate as a shell and CuS / PGS-PSBMA as a core, thereby obtaining the CuS / PGS-PSBMA-calcium alginate hydrogel microsphere for treating the inflammatory bowel disease. The core-shell structure hydrogel microsphere for treating the inflammatory bowel disease is prepared by utilizing an air flow control technology and an ion diffusion method, can be accurately dissolved and release hydrogel at the colon part after being orally taken, and is adhered to the local intestinal wall to promote polarization of intestinal M2 macrophages, so that the inflammatory bowel disease is treated, and the curative effect is good. The medicine is a safe and multi-effect enteritis treatment medicine with clinical transformation significance.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, in particular to a medicine for treating inflammatory bowel disease and a preparation method thereof. Background Art

[0002] Inflammatory bowel disease (IBD) consists of ulcerative colitis (UC) and Crohn's disease (CD), affecting approximately 6 to 8 million people worldwide. As a chronic, progressive, and relapsing intestinal disease, IBD seriously affects patients' quality of life and daily life, increasing the medical burden. Although it is generally believed that IBD is caused by an abnormal immune response to microorganisms in genetically susceptible individuals, its exact pathogenesis is largely unknown.

[0003] Currently available IBD treatments include non-targeted therapies (such as aminosalicylic acid, glucocorticoids, and immunomodulators) and targeted therapies (such as anti-TNF, anti-IL-12 / IL-23, and anti-α4β7 integrin). Although biological targeted therapies are effective for many patients, up to 30% of patients still do not respond to initial treatment, and up to 50% of patients lose their response over time. Therefore, a deep understanding of the operating mechanism of the intestinal immune system and the immune pathogenesis of IBD will help us develop immunotherapeutic drugs for IBD and provide new immunotherapy methods for IBD.

[0004] The human gut microbiota is composed of trillions of microorganisms, including fungi, unicellular animals, viruses, archaea, and the vast majority of bacteria. The gut microbiota plays a vital role in the pathogenesis of IBD by regulating the activation of the innate immune system, affecting host energy metabolism, immune homeostasis and maturation, and maintaining mucosal integrity.

[0005] There is increasing evidence that the composition of the intestinal microbiota is altered in patients with IBD. For example, Escherichia coli increases in the intestine as a pathogen, has the ability to survive and replicate in macrophages, and induces TNF-α secretion and inflammatory responses in IBD. In addition, Faecalibacterium prausnitzii, as a probiotic, can stimulate DCs to secrete the anti-inflammatory cytokine IL-10 and inhibit the production of IL-12 and INF-γ in the intestine, which are significantly reduced in the intestine of patients with IBD.

[0006] Oral administration is the preferred route of administration for chronic gastrointestinal diseases, with the advantages of simplicity, safety, and good patient compliance, and can produce local treatment effects on the gastrointestinal mucosa. However, most oral drugs are difficult to withstand the destruction of gastric acid and digestive enzymes, and non-targeted drugs have high systemic exposure rates and low bioavailability after administration, resulting in the effect of oral administration not meeting expectations. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention provides a CuS / PGS-PSBMA-calcium alginate hydrogel microsphere for treating inflammatory bowel disease and a preparation method thereof, which can effectively pass through the gastric environment, better regulate intestinal bacteria and play a role in inflammatory tissues in intestinal mucosal ulcers.

[0008] To achieve the above object, the present invention provides a method for preparing CuS / PGS-PSBMA-calcium alginate hydrogel microspheres for treating inflammatory bowel disease, characterized in that it comprises the following steps: (1) preparing CuS / PGS, allowing CuS / PGS to cross-link with PSBMA, and then using gas flow control technology to prepare CuS / PGS-PSBMA hydrogel microspheres; (2) Through calcium ion diffusion crosslinking, core-shell structured hydrogel microspheres with calcium alginate as the shell and CuS / PGS-PSBMA as the core were obtained, namely, CuS / PGS-PSBMA-calcium alginate hydrogel microspheres for the treatment of inflammatory bowel disease were obtained.

[0009] Furthermore, in step (1), an ultrasonic disruptor is used to disperse CuS / PGS particles, and then the CuS / PGS and PSBMA solutions are uniformly mixed, and then the mixed solution is uniformly injected into a coaxial needle using a microinjection pump, and then nitrogen is sprayed into the mixed solution. The shear force generated by the nitrogen is used to cut the solution into uniform droplets, and finally the droplets fall into a collection dish containing a Ca(NO3)2 solution below, and the mixed solution and the Ca(NO3)2 solution are mixed, and then allowed to stand in an oven, and finally rinsed with ultrapure water to obtain CuS / PGS-PSBMA hydrogel microspheres.

[0010] Furthermore, in step (2), the CuS / PGS-PSBMA hydrogel microspheres are collected, washed twice to remove residual substances on the surface, and then added to a sodium alginate solution with a mass percentage concentration of 0.5%, so that the calcium ions in the microspheres gradually diffuse to the surface of the microspheres and cross-link the sodium alginate to form a calcium alginate hydrogel shell, thereby obtaining CuS / PGS-PSBMA-calcium alginate hydrogel microspheres with a core-shell structure.

[0011] Furthermore, the preparation steps of the CuS / PGS particles in step (1) are as follows: 1. Add PGS into a beaker, then add ultrapure water into the beaker, then crush the agglomerated PGS with a glass rod and stir; 2. Place the beaker containing the mixed solution of PGS into an ultrasonic cleaner for ultrasonic dispersion; 3. Add CuCl2•H2O into the beaker of the mixture and stir it magnetically; 4. Add sodium citrate into the mixture beaker and stir magnetically; 5. Place Na2S•9H2O in a new beaker, add ultrapure water to dissolve it, and drip the solution into the beaker in the previous step at a uniform rate with magnetic stirring; 6. Move the stirred beaker to a magnetic stirrer (heating pot) for reaction, and then cool in an ice bath; 7. Divide the cooled mixed solution evenly into multiple plastic centrifuge tubes, centrifuge them, and remove the supernatant; 8. Add ultrapure water to each tube, centrifuge, and remove the supernatant. Repeat several times; 9. Write the label, open the lid and put it in the oven to dry.

[0012] Furthermore, the preparation of the CuS / PGS-PSBMA hydrogel microspheres in step (1) can also be carried out by the following steps: dissolving SBMA and APS in ultrapure water, then adding PEGDMA solution, and stirring at room temperature to obtain a gel compound.

[0013] Furthermore, the size of the CuS / PGS-PSBMA hydrogel microspheres in step (1) is 225.23±10.21 μm.

[0014] The advantage of adopting the above technical scheme is that by preparing CuS / PGS-PSBMA hydrogel microspheres and mixing them with calcium alginate, CuS / PGS-PSBMA-calcium alginate hydrogel microspheres for treating inflammatory bowel disease are obtained. The microspheres have a core-shell structure and can pass smoothly through the gastric acid environment through the external calcium alginate. After reaching the intestinal environment, the calcium alginate is dissolved, so that the CuS / PGS-PSBMA hydrogel microspheres can better play the role of regulating intestinal bacteria or inflammatory tissues in intestinal mucosal ulcers and play a dual role of anti-inflammatory and flora regulation. The microspheres are a safe, multi-effective enteritis treatment drug with clinical translational significance.

[0015] The present invention prepares a core-shell hydrogel microsphere that can treat inflammatory bowel disease. The core-shell hydrogel microsphere can be well used for the treatment of enteritis. The CuS / PGS-PSBMA hydrogel inside the core-shell hydrogel microsphere can adhere to the intestinal mucosa and play an anti-inflammatory and flora-regulating role. It is a safe, multi-effective enteritis treatment drug with clinical translational significance and is an ideal candidate for the treatment of IBD. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the preparation process of CuS / PGS-PSBMA-calcium alginate hydrogel microspheres; Figure 2 Schematic diagram of CuS / PGS-PSBMA-calcium alginate hydrogel microspheres for the treatment of inflammatory bowel disease; Figure 3 HE staining results of PSBMA hydrogel microspheres in the treatment of IBD; Figure 4 Figure 3 shows the Western Blot results of the effect of PSBMA hydrogel microspheres on macrophage differentiation. DETAILED DESCRIPTION

[0017] The present invention provides a method for preparing CuS / PGS-PSBMA-calcium alginate hydrogel microspheres for treating inflammatory bowel disease. Figure 1-4 As shown: It includes the following steps: (1) Weigh 288 mg of PGS, add 100 ml of ultrapure water, crush the agglomerated PGS with a glass rod, and stir; (2) Place the beaker containing the mixed solution in an ultrasonic cleaner for 20 minutes at 200W; (3) Weigh 256.5 mg CuCl2·H2O, add it into a beaker, and stir magnetically for 4 h at 420 rpm. (4) Weigh 300 mg of sodium citrate into a beaker and stir magnetically for 2 h at 420 rpm. (5) Weigh 360 mg of Na2S·9H2O into a 100 ml beaker, add 30 ml of ultrapure water to dissolve (Na2S·9H2O concentration is 50 mmol / L), and drip the solution into the beaker in the previous step at a uniform rate. Stir magnetically for 5 min at 420 rpm. (6) Move the stirred beaker to a magnetic stirrer (heating pot) and react for 17 min at 90°C and 650 rpm. Then cool in an ice bath. (7) Divide the cooled mixture evenly into four 50 ml plastic centrifuge tubes, centrifuge for 5 min at 9000 rpm, and remove the supernatant; (8) Add 15 ml of ultrapure water to each tube, centrifuge for 5 min at 9000 rpm, and remove the supernatant. Repeat 3 times; (9) Write a label, open the lid, put it in an oven and dry it for 2 hours, then obtain CuS / PGS particles; (10) Dissolve 0.56 g SBMA and 0.0056 g APS in 1.8 ml ultrapure water, then add 5.6 μl PEGDMA solution and stir at room temperature for 5 min to obtain a viscous PSBMA solution; (11) Disperse CuS / PGS with an ultrasonic crusher for 15 min, mix it evenly with the PSBMA solution, and then use a microinjection pump to uniformly inject the mixed solution into a coaxial needle. The shear force generated by 0.4 L / min nitrogen gas is used to cut the solution into uniform droplets. The droplets fall into a collection dish containing 0.1 mol / L Ca(NO3)2 solution below. Then, place it in a 70 °C oven for 30 min and rinse it with ultrapure water to obtain CuS / PGS-PSBMA hydrogel microspheres. (12) After collecting the CuS / PGS-PSBMA hydrogel microspheres and washing them twice to remove the residual substances on the surface, they were added to a sodium alginate solution with a mass percentage concentration of 0.5%. The calcium ions in the microspheres gradually diffused to the surface of the microspheres and cross-linked the sodium alginate to form a calcium alginate hydrogel shell, thereby obtaining core-shell structured hydrogel microspheres; In this embodiment, Figure 1 2 is a schematic diagram of the preparation steps and application of the CuS / PGS-PSBMA-calcium alginate hydrogel microspheres for treating inflammatory bowel disease of the invention, Figure 1 The process in the figure shows that the uniform size CuS / PGS-PSBMA hydrogel microspheres are prepared by gas flow control technology, and then calcium ions diffuse outward from the inside of the microspheres and cross-link alginate on the surface of the microspheres, thereby forming a calcium alginate hydrogel shell on the surface of CuS / PGS-PSBMA to obtain hydrogel microspheres with a core-shell structure. Figure 2 The process in the figure illustrates that the CuS / PGS-PSBMA-calcium alginate hydrogel microspheres for treating inflammatory bowel disease reach the colon and release CuS / PGS-PSBMA hydrogel. The CuS / PGS-PSBMA hydrogel accumulates in the inflamed colon mucosa, regulates intestinal inflammation by inducing the differentiation of M2 macrophages, and reduces mucosal damage.

[0018] In this embodiment, CuS is copper sulfide; PGS is attapulgite; SBMA is methacrylate sulfobetaine; PSBMA is polymethacrylate sulfobetaine; PEGDMA is polyethylene glycol dimethacrylate; CuCl2·H2O is cupric chloride monohydrate; Na2S·9H2O is sodium sulfide nonahydrate; Ca(NO3)2 is calcium nitrate; APS is an ammonium persulfate substitute; DSS is dextran sulfate sodium salt.

[0019] In this experimental example, a DSS-induced colitis mouse model was constructed to detect the therapeutic effect of CuS / PGS-PSBMA-calcium alginate hydrogel microspheres in vivo. DSS-induced colitis is the most commonly used IBD animal model. DSS dissolved in drinking water can damage epithelial cells and intestinal barriers after being consumed by mice, leading to the invasion of intestinal microbiota and subsequent inflammation.

[0020] The experimental method is as follows: The mice were randomly divided into control group, PSBMA group, DSS group, and DSS+PSBMA group. The first two groups were given normal drinking water, and the other groups were given drinking water containing 3% w / v DSS. Other treatments were given by gavage once a day. Figure 3 ), and found that CuS / PGS-PSBMA hydrogel microspheres treatment significantly reduced inflammatory cell infiltration and colon mucosal destruction. At the same time, Western blot was used to detect the protein levels of inos and arginase 1 (M1 and M2 markers) in colon tissue, and it was found that CuS / PGS-PSBMA could promote the differentiation of M2 macrophages ( Figure 4 ), while M2 is characterized by anti-inflammation and tissue repair, therefore, CuS / PGS-PSBMA hydrogel microspheres can significantly promote anti-inflammation and colon mucosal repair.

[0021] The above example is only one preferred specific example of the present invention. Common changes and substitutions made by those skilled in the art within the scope of the technical solution of the present invention are all included in the protection scope of the present invention.

Claims

1. A method for preparing CuS / PGS-PSBMA-calcium alginate hydrogel microspheres for treating inflammatory bowel disease, characterized in that: The steps include: (1) preparing CuS / PGS, allowing CuS / PGS to cross-link with PSBMA, and then using gas flow control technology to prepare CuS / PGS-PSBMA hydrogel microspheres; (2) Through calcium ion diffusion crosslinking, core-shell structured hydrogel microspheres with calcium alginate as the shell and CuS / PGS-PSBMA as the core were obtained, namely, CuS / PGS-PSBMA-calcium alginate hydrogel microspheres for the treatment of inflammatory bowel disease were obtained.

2. The method for preparing CuS / PGS-PSBMA-calcium alginate hydrogel microspheres for treating inflammatory bowel disease according to claim 1, characterized in that: In step (1), an ultrasonic crusher is used to disperse CuS / PGS particles, and then the CuS / PGS and PSBMA solutions are uniformly mixed. The mixed solution is then uniformly injected into a coaxial needle using a microinjection pump, and nitrogen is then sprayed into the mixed solution. The shear force generated by the nitrogen is used to cut the solution into uniform droplets, and finally the droplets fall into a collection dish containing a Ca(NO3)2 solution below. The mixed solution and the Ca(NO3)2 solution are mixed, and then allowed to stand in an oven, and finally rinsed with ultrapure water to obtain CuS / PGS-PSBMA hydrogel microspheres.

3. The method for preparing CuS / PGS-PSBMA-calcium alginate hydrogel microspheres for treating inflammatory bowel disease according to claim 1, characterized in that: In step (2), the CuS / PGS-PSBMA hydrogel microspheres are collected, washed twice to remove residual substances on the surface, and then added to a sodium alginate solution with a mass percentage concentration of 0.5%, so that the calcium ions in the microspheres gradually diffuse to the surface of the microspheres and cross-link the sodium alginate to form a calcium alginate hydrogel shell, thereby obtaining CuS / PGS-PSBMA-calcium alginate hydrogel microspheres with a core-shell structure.

4. The method for preparing CuS / PGS-PSBMA-calcium alginate hydrogel microspheres for treating inflammatory bowel disease according to claim 1, characterized in that: The preparation steps of the CuS / PGS particles in step (1) are as follows:

1. Add PGS into a beaker, then add ultrapure water into the beaker, then crush the agglomerated PGS with a glass rod and stir; 2. Place the beaker containing the mixed solution of PGS into an ultrasonic cleaner for ultrasonic dispersion; 3. Add CuCl2•H2O into the beaker of the mixture and stir it magnetically; 4. Add sodium citrate into the mixture beaker and stir magnetically; 5. Place Na2S•9H2O in a new beaker, add ultrapure water to dissolve it, and drip the solution into the beaker in the previous step at a uniform rate with magnetic stirring; 6. Move the stirred beaker to a magnetic stirrer (heating pot) for reaction, and then cool in an ice bath; 7. Divide the cooled mixed solution evenly into multiple plastic centrifuge tubes, centrifuge them, and remove the supernatant; 8. Add ultrapure water to each tube, centrifuge, remove the supernatant, and repeat several times; 9. Write the label, open the lid and put it in the oven to dry.

5. The method for preparing CuS / PGS-PSBMA-calcium alginate hydrogel microspheres for treating inflammatory bowel disease according to claim 1, characterized in that: For the PSBMA solution described in step (1), SBMA and APS are dissolved in ultrapure water, and then the PEGDMA solution is added and stirred at room temperature to obtain a PSBMA solution.

6. The method for preparing CuS / PGS-PSBMA-calcium alginate hydrogel microspheres for treating inflammatory bowel disease according to claim 1, characterized in that: The size of the CuS / PGS-PSBMA hydrogel microspheres described in step (1) is 225.23±10.21 μm.

7. A CuS / PGS-PSBMA-calcium alginate hydrogel microsphere for treating inflammatory bowel disease, characterized in that: The invention is prepared by the method according to any one of claims 1 to 6.

Images