Multi-component inulin hydrogel as well as preparation method and application thereof

Abstract

The invention discloses multi-component inulin hydrogel as well as a preparation method and application thereof, and belongs to the technical field of nano materials and medicines.The multi-component inulin hydrogel is characterized in that a medicine L-alanyl-L-glutamine contributing to intestinal mucosa repair is entrapped in the inulin hydrogel; the multi-component inulin hydrogel has the advantages that the multi-component inulin hydrogel is prepared from the inulin and composite nanoparticles (namely mesoporous polydopamine-gallium composite nanoparticles) composed of an active oxygen scavenger polydopamine and an antibacterial agent gallium ions, the repairing capacity on intestinal barriers is further enhanced, and the prepared multi-component inulin hydrogel can be used for effectively treating inflammatory bowel diseases and non-alcoholic steatohepatitis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nanomaterials and medicine, and particularly relates to a multi-component inulin hydrogel as well as a preparation method and application thereof. BACKGROUND

[0002] Inflammatory Bowel Disease (IBD) is a group of chronic and recurrent intestinal inflammatory diseases with unknown etiology. Its onset is affected by the interaction of multiple factors such as genetics, immunity, environment and intestinal flora. Unhealthy eating habits (such as high-fat and high-sugar diet), long-term intake of alcohol and food additives often cause damage to intestinal cells, destruction of intestinal mucus layer and disorder of intestinal flora, which in turn leads to an increase in intestinal permeability, becoming an important inducement of IBD. The intestinal mucosa not only allows the absorption of nutrients, but also has immune sensing function, which can limit the transport of potentially harmful antigens and microorganisms, forming a direct barrier between the external environment and the internal environment of the human body. In the development of IBD, the inflammatory response mediated by pro-inflammatory cytokines not only reduces the synthesis of intestinal tight junction proteins, and the oxidative stress generated thereby also has a negative impact on the repair function of normal intestinal cells, thereby further exacerbating the damage to the intestinal barrier. Therefore, the damage to the intestinal barrier and the occurrence of IBD form a vicious cycle of "damage-inflammation-re-damage".

[0003] Nonalcoholic Fatty Liver Disease (NAFLD) is a chronic liver disease closely related to metabolic abnormalities, which is prone to progress from simple steatosis to more severe nonalcoholic steatohepatitis (NASH), and eventually develop into fibrosis, leading to cirrhosis and liver cancer. Studies have found that patients with nonalcoholic steatohepatitis have increased intestinal permeability, and the severity of liver disease is closely related to the degree of intestinal barrier damage. Changes in the intestinal barrier and subsequent translocation of small amounts of bacteria or bacterial products are now considered an important mechanism leading to the inflammatory features of metabolic diseases. Therefore, repairing the damaged intestinal barrier not only has important significance for the treatment of inflammatory bowel disease, but also has great potential to become an effective means of treating nonalcoholic steatohepatitis and preventing its progression.

[0004] At present, the commonly used drugs for treating intestinal inflammation include 5-aminosalicylic acid drugs, glucocorticoids, immunosuppressants and some biological agents, etc. The main purpose of these drugs is to treat the disease by inhibiting inflammation, which may indirectly help the intestinal barrier repair in the process. However, it is also extremely important to focus on the key role of the treatment strategy of intestinal barrier repair in the treatment of intestinal diseases and other related metabolic diseases. Inulin is a natural soluble dietary fiber that can promote the proliferation of intestinal probiotics, regulate the balance of the flora, and at the same time metabolize short-chain fatty acids (such as butyric acid, propionic acid, etc.), which can enhance the expression of intestinal epithelial tight junction proteins, repair the physical barrier function of the intestine, and reduce the intestinal permeability; and can stimulate the secretion of mucin by intestinal epithelial cells to enhance the chemical barrier function. In addition, short-chain fatty acids can also promote the differentiation of regulatory T cells, maintain intestinal immune tolerance, and relieve intestinal inflammation. Inulin aqueous solution has a gel-forming property under certain conditions, but this property is closely related to the concentration of inulin. If the concentration is too low, it cannot form an ideal gel, and if the concentration is too high, the gel structure will be poor, the hardness will be abnormally high, and the elasticity will decrease due to intermolecular repulsion or competition for water. SUMMARY

[0005] To solve the above technical problems, the present application provides a multi-component inulin hydrogel and its preparation method and application. The multi-component inulin hydrogel of the present application is an inulin hydrogel loaded with L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticles, which can be applied to intestinal barrier protection and has therapeutic effects on both inflammatory bowel disease and non-alcoholic steatohepatitis.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0007] The present application provides a multi-component inulin hydrogel, which comprises L-alanyl-L-glutamine (ALG), mesoporous polydopamine-gallium composite nanoparticles (MPDA-Ga) and inulin.

[0008] The present application loads the drug L-alanyl-L-glutamine which helps to repair the intestinal mucosa in the inulin hydrogel, and the composite nanoparticles composed of the active oxygen scavenger polydopamine and the antibacterial agent gallium ion (i.e. mesoporous polydopamine-gallium composite nanoparticles), which further strengthens the repair ability of the intestinal barrier. The prepared multi-component inulin hydrogel can achieve effective treatment of inflammatory bowel disease and non-alcoholic steatohepatitis.

[0009] The present application selects an appropriate inulin concentration to prepare an injectable inulin hydrogel with excellent rheological properties. The inulin is administered in the form of an injectable gel, which not only exerts the intestinal barrier repair effect of inulin itself, but also prolongs the retention time of inulin in the intestinal tract due to the increased viscosity of the gel compared with the aqueous solution. In addition, the inulin gel system prepared by the present application still does not affect the structural stability and rheological properties of the gel after loading a small amount of drug, has good accommodation for the loaded drug, and can be used as an effective drug delivery carrier. Therefore, the present application adds the intestinal mucosa repair drug L-alanyl-L-glutamine to the inulin gel to further strengthen the barrier repair, and the nanoparticles composed of an active oxygen scavenger polydopamine and an antibacterial agent gallium ion can not only alleviate the damage of oxidative stress to the intestinal barrier, but also inhibit the invasion of harmful pathogenic bacteria and regulate the flora. The synergistic effect of the above components can strengthen the repair of the damaged intestinal barrier in multiple dimensions including physical, chemical, immune, and microbial, and achieve effective treatment of inflammatory bowel disease and non-alcoholic steatohepatitis.

[0010] Further, the mass ratio of the inulin, L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticles is 6: (0.5-1): (0.002-0.005), preferably 6:1:0.005.

[0011] The present application also provides a preparation method of the above-mentioned multi-component inulin hydrogel, comprising the following steps:

[0012] L-alanyl-L-glutamine is added to the inulin solution and stirred uniformly to obtain a mixed solution;

[0013] The aqueous dispersion of mesoporous polydopamine-gallium composite nanoparticles is added to the mixed solution and stirred uniformly, and then the multi-component inulin hydrogel is obtained by standing at room temperature.

[0014] Further, the preparation method of the inulin solution is as follows: inulin is added to water, heated in a water bath, and stirred uniformly to obtain the inulin solution.

[0015] Further, the preparation method of the mesoporous polydopamine-gallium composite nanoparticles comprises the following steps: a water-soluble gallium salt solution is added to an aqueous dispersion of mesoporous polydopamine nanoparticles, stirred and reacted, the product is collected, centrifuged, and washed with water to obtain the mesoporous polydopamine-gallium composite nanoparticles. Further, the mass ratio of the water-soluble gallium salt to the mesoporous polydopamine nanoparticles is 5:2.

[0016] Illustratively, the water-soluble gallium salt is gallium nitrate (Ga(NO3)3).

[0017] Further, the preparation method of the mesoporous polydopamine nanoparticles comprises the following steps:

[0018] The poloxamer 407 is added into a mixed solvent composed of anhydrous ethanol and water, stirred until the solution becomes clear and transparent, dopamine is added, and stirred uniformly; 1,3,5-trimethylbenzene is added under stirring, stirred uniformly, to obtain a mixed solution; then, an ammonia solution is added into the mixed solution under stirring, and the reaction is stirred at room temperature; after the reaction is completed, the solution after the reaction is centrifuged at room temperature, washed, to obtain the mesoporous polydopamine nanoparticle.

[0019] Further, the mass ratio of the poloxamer 407 to the dopamine is 1:0.5.

[0020] Further, the volume ratio of the 1,3,5-trimethylbenzene to the ammonia solution is 2:5.

[0021] The application further provides application of the above-mentioned multi-component inulin hydrogel in preparation of a medicine for treating inflammatory bowel disease.

[0022] The application further provides application of the above-mentioned multi-component inulin hydrogel in preparation of a medicine for treating non-alcoholic steatohepatitis.

[0023] Compared with the prior art, the application has the following advantages and technical effects:

[0024] (1) In the multi-component inulin hydrogel loaded with L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticles provided by the application, L-alanyl-L-glutamine is added into the inulin hydrogel, which further promotes the important role of inulin in repairing the intestinal barrier; the mesoporous polydopamine-gallium composite nanoparticles can not only remove excess active oxygen in the intestinal tract to protect the intestinal barrier from oxidative stress damage, but also can assist in strengthening the regulation of intestinal flora by the prebiotic inulin.

[0025] (2) The inulin hydrogel loaded with L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticles provided by the application has a multi-component synergistic effect, has the ability to repair the intestinal barrier, and further has the ability to treat inflammatory bowel disease and non-alcoholic steatohepatitis, and effectively relieves the inflammatory bowel disease induced by dextran sulfate sodium salt and the non-alcoholic steatohepatitis induced by choline-deficient amino acid high-fat diet.

[0026] (3) The multi-component inulin hydrogel loaded with L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticles provided by the application has a simple preparation process, mild preparation conditions, and low production cost; the components used have good biological safety, and have certain clinical application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0027] The accompanying drawings, which form a part of this application, are included to provide a further understanding of the application and are incorporated in and constitute a part of this application. The drawings illustrate embodiments of the present application and, together with the description, serve to explain the application. In the drawings:

[0028] Figure 1 Transmission electron micrograph of mesoporous polydopamine in Example 1 (scale bar 100 nm).

[0029] Figure 2 Transmission electron micrograph of mesoporous polydopamine-gallium composite nanoparticles in Example 1 (scale bar 100 nm).

[0030] Figure 3 X-ray photoelectron spectroscopy of mesoporous polydopamine-gallium composite nanoparticles in Example 1.

[0031] Figure 4 Scanning electron micrograph of inulin hydrogel loaded with L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticles in Example 2 (scale bar 20 μιη).

[0032] Figure 5 Rheological profile of inulin hydrogel loaded with L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticles in Example 2.

[0033] Figure 6 ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging rate of mesoporous polydopamine-gallium composite nanoparticles in Example 1.

[0034] Figure 7 Cytotoxicity profile of mesoporous polydopamine-gallium composite nanoparticles in Example 1 on human normal colonic epithelial cells.

[0035] Figure 8 Detection of intestinal permeability in mice after treatment with inulin hydrogel loaded with L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticles in Example 2.

[0036] Figure 9 Body weight of mice after treatment with blank inulin hydrogel in Comparative Example 1, inulin hydrogel loaded with L-alanyl-L-glutamine in Comparative Example 2, inulin hydrogel loaded with mesoporous polydopamine-gallium composite nanoparticles in Comparative Example 3, inulin hydrogel loaded with mesoporous polydopamine nanoparticles in Comparative Example 4, and inulin hydrogel loaded with L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticles in Example 2.

[0037] Figure 10Photographs of the colon of mice after treatment with the blank inulin hydrogel of Comparative Example 1, the L-alanyl-L-glutamine loaded inulin hydrogel of Comparative Example 2, the mesoporous polydopamine-gallium composite nanoparticle loaded inulin hydrogel of Comparative Example 3, the mesoporous polydopamine nanoparticle loaded inulin hydrogel of Comparative Example 4, and the L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticle loaded inulin hydrogel of Example 2.

[0038] Figure 11 Bar graph of the colon length of mice after treatment with the blank inulin hydrogel of Comparative Example 1, the L-alanyl-L-glutamine loaded inulin hydrogel of Comparative Example 2, the mesoporous polydopamine-gallium composite nanoparticle loaded inulin hydrogel of Comparative Example 3, the mesoporous polydopamine nanoparticle loaded inulin hydrogel of Comparative Example 4, and the L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticle loaded inulin hydrogel of Example 2.

[0039] Figure 12 Graph of the alanine aminotransferase level of mice after treatment with the L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticle loaded inulin hydrogel of Example 2 in mice with non-alcoholic steatohepatitis.

[0040] Figure 13 Graph of the aspartate aminotransferase level of mice after treatment with the L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticle loaded inulin hydrogel of Example 2 in mice with non-alcoholic steatohepatitis. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present application will now be described in detail, with reference to the figures. The detailed description is not intended to limit the present application, but rather to explain certain aspects, features, and embodiments of the present application.

[0042] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of a parameter, unless otherwise indicated, each intervening value by each intervening value, or any other stated value or implicitly supported value by every stated value or implicitly supported value within the stated range is expressly contemplated. The above description is intended to be illustrative, and not restrictive. Many other embodiments will be apparent to those of skill in the art upon reviewing the above description. The scope of the application should, therefore, be determined not with reference to the above description, but instead with reference to the appended claims, along with their full scope of equivalents.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described therein. In case of conflict, the content of the present specification will control.

[0044] Many modifications and variations of this application of the application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0045] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional, unrecited elements or method steps.

[0046] The embodiments of the present application provide a multi-component inulin hydrogel, raw materials of which include L-alanyl-L-glutamine (ALG), mesoporous polydopamine-gallium composite nanoparticles (MPDA-Ga) and inulin.

[0047] In the preferred embodiments of the present application, the mass ratio of inulin, L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticles is 6: (0.5-1): (0.002-0.005), preferably 6:1:0.005.

[0048] The embodiments of the present application also provide a preparation method of the above multi-component inulin hydrogel, comprising the following steps:

[0049] L-alanyl-L-glutamine is added into the inulin solution and stirred uniformly to obtain a mixed solution;

[0050] The aqueous dispersion of mesoporous polydopamine-gallium composite nanoparticles is added into the mixed solution and stirred uniformly, and the multi-component inulin hydrogel is obtained after standing at room temperature.

[0051] In the preferred embodiments of the present application, the preparation method of the inulin solution is as follows: inulin is added into water, heated in a water bath, and stirred uniformly to obtain an inulin solution.

[0052] For example, when preparing the inulin solution, the temperature of water bath heating is 60℃, the stirring speed is 1000 rpm, and the stirring time is 15 min.

[0053] In a preferred embodiment of the present application, the preparation method of the mesoporous polydopamine-gallium composite nanoparticles comprises the following steps: adding an aqueous solution of a soluble gallium salt into an aqueous dispersion of mesoporous polydopamine nanoparticles, stirring and reacting, collecting the product, centrifuging, and washing with water to obtain the mesoporous polydopamine-gallium composite nanoparticles.

[0054] In a preferred embodiment of the present application, the mass ratio of the soluble gallium salt to the mesoporous polydopamine nanoparticles is 5:2.

[0055] Illustratively, the soluble gallium salt is gallium nitrate (Ga(NO3)3).

[0056] Illustratively, the preparation method of the aqueous solution of Ga(NO3)3 is as follows: 50 mg of Ga(NO3)3 is dissolved in 500 μL of deionized water to obtain the aqueous solution of Ga(NO3)3.

[0057] Illustratively, in the preparation method of the mesoporous polydopamine-gallium composite nanoparticles, the centrifuging rate is 12000 rpm and the centrifuging time is 10 min.

[0058] In a preferred embodiment of the present application, the preparation method of the mesoporous polydopamine nanoparticles comprises the following steps: adding poloxamer 407 into a mixed solvent composed of anhydrous ethanol and water, stirring until the solution becomes clear and transparent, adding dopamine, and stirring uniformly; then adding mesitylene under stirring, stirring uniformly to obtain a mixed solution; and then adding an aqueous ammonia solution into the mixed solution under stirring, stirring at room temperature, and after the reaction is completed, centrifuging the reacted solution at room temperature, and washing to obtain the mesoporous polydopamine nanoparticles.

[0059] Illustratively, in the preparation method of the mesoporous polydopamine nanoparticles, the washing is as follows: first washing once with a mixed solvent of anhydrous ethanol and deionized water (volume ratio of 1:1), then washing once with anhydrous ethanol, and finally washing once with deionized water.

[0060] In a preferred embodiment of the present application, the volume ratio of mesitylene to the aqueous ammonia solution is 2:5.

[0061] The embodiments of the present application also provide the use of the above-mentioned multi-component inulin hydrogel in the preparation of a drug for treating inflammatory bowel disease.

[0062] The embodiments of the present application also provide the use of the above-mentioned multi-component inulin hydrogel in the preparation of a drug for treating non-alcoholic steatohepatitis.

[0063] The mechanism of the present application is as follows:

[0064] 1. The mechanism of the multi-component inulin hydrogel of the present application in treating inflammatory bowel disease

[0065] Acute phase intervention: MPDA-Ga nanoparticles rapidly remove excess reactive oxygen species in the intestine, reducing oxidative stress damage; at the same time, through the antibacterial effect of gallium ions, the excessive proliferation of potential pathogenic bacteria is controlled.

[0066] Repair phase dominance: ALG provides key nutrients for the rapid regeneration of intestinal epithelial cells; short-chain fatty acids such as butyrate produced by inulin fermentation directly strengthen tight junctions and promote the secretion of mucin, the main component of intestinal mucosal mucus, promoting the repair of physical and chemical defense barriers.

[0067] Long-term regulation: Inulin, as a prebiotic, continuously regulates the intestinal flora structure, increases beneficial bacteria, and inhibits pro-inflammatory bacteria, establishing a long-term stable internal environment from the microecological level and preventing the recurrence of inflammatory bowel disease.

[0068] Immune regulation: Short-chain fatty acids produced by inositol in endosomes fermented by probiotics can promote the differentiation of regulatory T cells, maintain normal immune tolerance in the intestinal environment, and prevent excessive immune response in the body.

[0069] In summary, the combined action of multiple components in the present application can promote the repair of the multiple intestinal barriers (including physical barrier, chemical barrier, immune barrier, and microbial barrier) of the intestinal tract of inflammatory bowel disease, and treat inflammatory bowel disease.

[0070] 2. Mechanism of the multi-component inulin hydrogel of the present application for treating non-alcoholic steatohepatitis

[0071] Intestinal barrier repair: mainly through the combined application of ALG, MPDA-Ga nanoparticles, and inulin, the above multiple intestinal barriers are comprehensively played, and the damaged intestinal barrier due to high-fat diet and other factors is effectively repaired, reducing the entry of enterogenous endotoxin into the liver from the source.

[0072] Reducing the burden on the liver: the reduction of endotoxin entering the liver directly reduces the activation level of Kupffer cells in the liver, inhibits the activation of inflammatory pathways such as Toll-like receptor 4, thereby significantly reducing the inflammatory response and fatty degeneration of the liver.

[0073] Systemic improvement: beneficial metabolites (such as SCFAs) produced by inulin-regulated flora enter the systemic circulation, not only improving insulin resistance (an important predisposing factor for NASH), but also directly producing beneficial metabolic regulation effects on the liver.

[0074] Unless otherwise specified, the room temperature in the present application is 25±2℃.

[0075] The raw materials used in the embodiments of the present application are all commercially available. As an example, the inulin is purchased from Shengong Bioengineering (Shanghai) Co., Ltd., model A602227-0100; the serum is fetal bovine serum, purchased from Wuhan Ponusai Life Science and Technology Co., Ltd.; the double antibodies are penicillin and streptomycin, purchased from Gibco; the NCM460 cells are purchased from Wuhan Shengn Biological Technology Co., Ltd.; and the C57BL / 6 mice are purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0076] It should be noted that the details not described in the present application are all conventional operating means in the art and are not the focus of the present application.

[0077] The technical solutions of the present application are further illustrated by the following examples.

[0078] Example 1

[0079] The present embodiment provides a preparation method of mesoporous polydopamine-gallium composite nanoparticles, and the steps are as follows:

[0080] (1) Preparation of mesoporous polydopamine nanoparticles: 5 mL of anhydrous ethanol and 5 mL of deionized water were stirred and mixed uniformly at room temperature to obtain a mixed solvent; 0.1 g of poloxamer 407 was weighed and added to the above mixed solvent, stirred at room temperature for 5 min until completely dissolved (the solution became clear and transparent), 0.05 g of dopamine was added and stirred to dissolve; 200 μL of 1,3,5-trimethylbenzene was added dropwise under stirring, and the above components were stirred and blended at room temperature for 30 min to obtain a mixed solution; then 500 μL of ammonia water solution (25%-28% by mass concentration) was added dropwise to the above mixed solution under stirring, and stirred at room temperature for 2 h. After the reaction was completed, the reacted solution was centrifuged at a speed of 12000 rpm for 10 min at room temperature, first washed once with a mixed solvent of anhydrous ethanol and deionized water (volume ratio 1:1), then washed once with anhydrous ethanol, and finally washed once with deionized water to obtain mesoporous polydopamine nanoparticles, denoted as MPDA, which was resuspended and dispersed with deionized water to obtain a 5 mg / mL MPDA deionized water dispersion, ready for use;

[0081] (2) Preparation of mesoporous polydopamine-gallium composite nanoparticles: 50 mg of gallium nitrate (Ga(NO3)3) was dissolved in 500 μL of deionized water to obtain a Ga(NO3)3 solution, which was added to 4 mL of mesoporous polydopamine nanoparticle water dispersion with a concentration of 5 mg / mL, stirred for 12 h, and the product was collected and centrifuged at a speed of 12000 rpm for 10 min, and washed with deionized water for 3 times to obtain mesoporous polydopamine-gallium composite nanoparticles, denoted as MPDA-Ga, which was resuspended and dispersed with deionized water to obtain a 5 mg / mL MPDA-Ga deionized water dispersion.

[0082] MPDA and MPDA-Ga prepared in this example were morphologically characterized using a transmission electron microscope (TEM), a TEM image of MPDA is shown in Figure 1 a TEM image of MPDA-Ga is shown in Figure 2 It can be seen that the mesoporous polydopamine and the nanoparticles after loading gallium ions are in a mesoporous spherical structure, and the diameters of the two kinds of nanoparticles are both 200-250 nm, and the loading of gallium ions does not change the particle size of the mesoporous polydopamine.

[0083] MPDA-Ga prepared in this example was analyzed by X-ray photoelectron spectroscopy, and the obtained spectrum is shown in Figure 3 It can be seen that the X-ray photoelectron spectrum of the mesoporous polydopamine-gallium composite nanoparticles has a clear characteristic peak of gallium element.

[0084] Example 2

[0085] This example provides a preparation method of a multi-component inulin hydrogel, and the steps are as follows:

[0086] 0.6 g of inulin was weighed and added to 900 μL of deionized water, heated in a 60°C water bath, and stirred at 1000 rpm for 15 min to obtain an inulin solution; 100 mg of L-alanyl-L-glutamine (ALG) was weighed and added to the above inulin solution, and stirred for 5 min to obtain a mixed solution; 100 μL of MPDA-Ga deionized water dispersion prepared in Example 1 with a concentration of 5 mg / mL was added to the above mixed solution (the final concentration of MPDA-Ga was 500 μg / mL), and the stirring and mixing was continued for 5 min, and finally it was left to stand at room temperature for 12 h, thereby obtaining a multi-component inulin hydrogel, which was an inulin hydrogel loaded with ALG and MPDA-Ga, and was denoted as PGaA Gel.

[0087] The inulin hydrogel loaded with ALG and MPDA-Ga prepared in Example 2 was freeze-dried, and the microstructure of the section was observed by scanning electron microscope, and the obtained results are shown in Figure 4 It can be seen that it has a typical porous sponge-like hydrogel structure.

[0088] Figure 5 is the rheogram of the inulin hydrogel loaded with ALG and MPDA-Ga prepared in Example 2 measured by a rheometer, it can be seen that the storage modulus G' measured is greater than the loss modulus G'', which shows an elastic solid behavior, which is consistent with the rheological properties of the gel, and the network structure can be stably maintained.

[0089] Comparative Example 1

[0090] This comparative example provides a preparation method of a hydrogel, and the steps are as follows:

[0091] Take 0.6 g of inulin and add it to 1 mL of deionized water, heat in a 60°C water bath, stir at 1000 rpm for 15 min, and obtain an inulin solution. Take 100 mg of L-alanyl-L-glutamine (ALG) and add it to the above inulin solution, stir for 5 min, and obtain a mixed solution. Finally, stand at room temperature for 12 h, and obtain a single-component inulin hydrogel, which is an ALG-loaded inulin hydrogel, and is recorded as ALG Gel.

[0092] Comparative Example 2

[0093] This comparative example provides a method for preparing a single-component inulin hydrogel, and the steps are as follows:

[0094] Take 0.6 g of inulin and add it to 1 mL of deionized water, heat in a 60°C water bath, stir at 1000 rpm for 15 min, and obtain an inulin solution. Take 100 mg of L-alanyl-L-glutamine (ALG) and add it to the above inulin solution, stir for 5 min, and obtain a mixed solution. Finally, stand at room temperature for 12 h, and obtain a single-component inulin hydrogel, which is an ALG-loaded inulin hydrogel, and is recorded as ALG Gel.

[0095] Comparative Example 3

[0096] This comparative example provides a method for preparing a single-component inulin hydrogel, and the steps are as follows:

[0097] Take 0.6 g of inulin and add it to 900 μL of deionized water, heat in a 60°C water bath, stir at 1000 rpm for 15 min, and obtain an inulin solution. Take 100 μL of MPDA-Ga deionized water dispersion prepared in Example 1 with a concentration of 5 mg / mL and add it to the above inulin solution (the final concentration of MPDA-Ga is 500 μg / mL), continue to stir and mix for 5 min, and finally stand at room temperature for 12 h, and obtain a single-component inulin hydrogel, which is an MPDA-Ga-loaded inulin hydrogel, and is recorded as MPDA-Ga Gel.

[0098] Comparative Example 4

[0099] This comparative example provides a method for preparing a single-component inulin hydrogel, and the steps are as follows:

[0100] Take 0.6 g of inulin and add it to 900 μL of deionized water, heat in a 60°C water bath, stir at 1000 rpm for 15 min, and obtain an inulin solution. Take 100 μL of MPDA-Ga deionized water dispersion prepared in Example 1 with a concentration of 5 mg / mL and add it to the above inulin solution (the final concentration of MPDA-Ga is 500 μg / mL), continue to stir and mix for 5 min, and finally stand at room temperature for 12 h, and obtain a single-component inulin hydrogel, which is an MPDA-Ga-loaded inulin hydrogel, and is recorded as MPDA-Ga Gel.

[0101] Test Example 1 In Vitro Free Radical Scavenging Ability of Mesoporous Polydopamine-Gallium Composite Nanoparticles in Example 1

[0102] Mix 0.2 mL of potassium persulfate solution (2 mmol / L) and 0.2 mL of ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) solution (7 mmol / L) to generate ABTS free radicals in the dark for 12 h. Then, dilute the solution 16 times with PBS buffer (pH = 7.4) to obtain the ABTS free radical solution. Then, mix 0.5 mL of MPDA-Ga aqueous dispersion solution (0, 1.25, 2.5, 5, 10, 20 μg / mL) with 0.5 mL of the ABTS free radical solution obtained above, and react in the dark for 30 min. Centrifuge all the mixed solutions at 12000 rpm for 10 min, and take the supernatant to measure the UV-visible spectrum at 731 nm. Calculate the clearance rate according to the following formula: clearance rate (%) = (A0-A) / A0x100%, wherein A0 is the absorbance measured when the concentration of MPDA-Ga is 0, and A is the absorbance measured at other concentrations.

[0103] Figure 6 The statistical results of the clearance rate of the mesoporous polydopamine-gallium composite nanoparticles in Example 1 for scavenging ABTS free radicals are shown in the graph. As the concentration of MPDA-Ga increases, the clearance rate of scavenging ABTS free radicals also increases, indicating that MPDA-Ga has obvious free radical scavenging ability.

[0104] Test Example 2 Cell toxicity of mesoporous polydopamine-gallium composite nanoparticles in Example 1

[0105] Culture NCM460 cells with 1640 medium (containing 10% (volume percent) serum, 1% (volume percent) double antibody), inoculate 100 μL (density 1x10 5 Absorb the supernatant, prepare 1640 medium with different concentrations of MPDA-Ga (0, 20, 50, 100, 200 μg / mL), and add 100 μL of medium to each well, 6 wells for each concentration, and continue to incubate for 24 h. At the same time, set up a control group without cells, and incubate for 24 h. After incubation, add 11 μL of CCK-8 reagent to each well, incubate in the dark at 37°C for 30 min, and measure the absorbance value at 450 nm with a microplate reader. Subtract the average absorbance value of the control group at the same concentration from the absorbance value of each well of cells to exclude the influence of MPDA-Ga on the absorbance value. The average absorbance value of the group with 0 concentration of MPDA-Ga represents 100% cell viability, and the cell viability of other groups is calculated.

[0106] Figure 7 The cell viability graph after incubation with different MPDA-Ga concentrations shows that MPDA-Ga has little cytotoxicity, and the cell viability remains about 90% at a high concentration of 200 μg / mL.

[0107] Example 3 Effect of inulin hydrogel loaded with L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticles in Example 2 on repairing intestinal barrier of mice

[0108] Healthy 7-week-old female C57BL / 6 mice were randomly divided into 3 groups according to 4 mice per group (n = 4): (1) healthy group, denoted as healthy; (2) disease model group, denoted as DSS; (3) drug (inulin hydrogel loaded with L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticles in Example 2) treatment group, denoted as PGaAGel. After grouping, the mice were adaptively fed for one week, and normal diet and water were provided; the drinking water of the mice in groups (2) and (3) was replaced with 2.5wt% dextran sulfate sodium salt (DSS) aqueous solution for 5 days, and from the 6th day, the normal drinking water was replaced, and the mice in group (3) were orally administered with PGaAGel every day for 5 days.

[0109] After treatment, the intestinal permeability of the mice was detected by gavage of fluorescein isothiocyanate dye-labeled dextran (FITC-dextran), and the specific method was as follows: another healthy mouse without treatment was taken, the whole blood was collected by orbital bleeding with an anticoagulant tube, and the blood was immediately gently inverted and mixed several times after collection to obtain an anticoagulated whole blood sample; the anticoagulated whole blood sample was centrifuged (2000g, 10min) at 4°C, and the upper plasma layer (light yellow liquid) was carefully aspirated with a pipette, and the aspirated plasma was immediately aliquoted into pre-cooled centrifuge tubes. Under light-proof conditions, a known concentration of FITC-dextran stock solution (250 μg / mL) was prepared with PBS, and the FITC-dextran stock solution was diluted with the above-mentioned mouse plasma as standard samples of different concentrations (0, 2, 5, 10, 25, 50 μg / mL), and the volume of each concentration was 100 μL. The microplate reader was set: the excitation wavelength was 485 nm, and the emission wavelength was 530 nm. 100 μL of the standard sample was added to a black 96-well plate, and the fluorescence intensity value was read. The standard curve was plotted with the standard sample concentration as the abscissa and the corresponding fluorescence intensity as the ordinate. Then, a FITC-dextran solution with a concentration of 25 mg / mL was prepared with PBS buffer (pH = 7.4), and 200 μL of the FITC-dextran solution was accurately injected into the stomach of the mouse using a gavage needle. After 4h, the mouse plasma was collected by the same method as above to detect the fluorescence intensity value, and then the FITC-dextran concentration in the plasma of each mouse was calculated using the standard curve.

[0110] The statistical graph of FITC-dextran concentration in the plasma of mice in each group is shown below. Figure 8 As shown, the FITC-dextran content in the plasma of mice in the DSS group was several times higher than that of mice orally administered PGaA Gel, indicating that PGaA Gel effectively reduced intestinal permeability and the degree of intestinal leakage in mice, playing an important role in the repair of the intestinal barrier.

[0111] Test Example 4: The effect of inulin hydrogel loaded with L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticles in Example 2 on the treatment of inflammatory bowel disease in mice.

[0112] Seven-week-old healthy female C57BL / 6 mice were randomly divided into seven groups of six (n=6): (1) healthy group (denoted as healthy); (2) disease model group (denoted as DSS); (3) inulin hydrogel treatment group (denoted as Inulin Gel); (4) inulin hydrogel treatment group (denoted as MPDA Gel) loaded with mesoporous polydopamine nanoparticles; (5) inulin hydrogel treatment group (denoted as MPDA-Ga Gel) loaded with mesoporous polydopamine-gallium composite nanoparticles; (6) inulin hydrogel treatment group (denoted as ALG Gel) loaded with L-alanyl-L-glutamine; and (7) inulin hydrogel treatment group (denoted as PGaA Gel) loaded with L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticles. After being divided into groups, the mice were given an acclimatization diet for one week with normal food and water. At the start of the experiment, except for the healthy group, the drinking water of the other six groups of mice was replaced with a 2.5% sodium dextran sulfate (DSS) aqueous solution on days 1-5. Then, on day 6, the drinking water of all groups of mice was replaced with normal drinking water again, and each group of mice was given 200 μL of the gel drug by gavage daily from day 6 to day 10. On day 11, the mice were euthanized, and colon tissue was harvested by dissection. The weight of each mouse was recorded daily during the experiment from day 0 to day 11.

[0113] Figure 9 The graph shows the changes in body weight of mice after treatment with different groups of hydrogels for inflammatory bowel disease. DSS-induced inflammatory bowel disease in mice causes a significant and sustained decrease in body weight. After oral gavage treatment with various inulin hydrogels, the weight loss symptoms were significantly relieved. The inulin hydrogel (PGaA Gel) loaded with L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticles prepared in Example 2 showed the best relief effect.

[0114] Figure 10 and Figure 11The photographs and length measurements of the colon of mice collected on the 11th day after different groups of hydrogel treatment of mice with inflammatory bowel disease, a major feature of DSS-induced inflammatory bowel disease in intestinal macroscopic pathology is the shortening of the overall length of the colon. It can be seen that the average length of the colon of healthy mice is about 6.78 cm, and the disease group is only 4.83 cm, and after treatment with PGaA Gel, the average length of the colon of mice is restored to 6.52 cm, close to the healthy group.

[0115] Example 5 Effect of inulin hydrogel loaded with L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticles on the treatment of non-alcoholic steatohepatitis in mice in Example 2

[0116] 7-week-old healthy male C57BL / 6 mice were randomly divided into 4 groups according to 6 in each group (n=6): (1) healthy group (recorded as healthy); (2) non-alcoholic steatohepatitis disease model group (recorded as CDAHFD); (3) non-alcoholic steatohepatitis + intestinal barrier damage disease model group (recorded as CDAHFD+DSS); (4) treatment group of inulin hydrogel loaded with L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticles (recorded as CDAHFD+PGaA Gel). The mice were adaptively fed for one week, and normal diet and water were provided. Choline-deficient amino acid high-fat diet (CDAHFD) was used to induce the occurrence of non-alcoholic steatohepatitis in mice, and the mice in groups (2), (3) and (4) were fed with CDAHFD feed from the 0th week to the 10th week, and the mice in group (1) were fed with ordinary ingredient control feed. During this period, the mice in group (3) were given 1% DSS aqueous solution for one week; the mice in group (4) were given PGaA Gel by gavage every two days from the 5th week to the 10th week. After the experiment was completed at the 10th week, the mouse orbital blood was taken into an EP tube, and the blood was allowed to clot at room temperature for 2 h, and the yellowish serum was separated, centrifuged at 4°C, 3000g, 10 min, and the upper serum was carefully collected and detected by alanine substrate method and aspartic acid substrate method to detect the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of mice.

[0117] Figure 12 and Figure 13The figure is the level detection of alanine aminotransferase and aspartate aminotransferase in mouse serum respectively, compared with the healthy group, the ALT and AST levels in the blood of the non-alcoholic fatty liver disease model group of mice are significantly increased; the ALT and AST levels of the non-alcoholic fatty liver disease + intestinal barrier damage disease model group of mice are more increased, indicating that the damage of the intestinal barrier is closely related to the progression of non-alcoholic fatty liver disease of the mice; and after the repair of the intestinal barrier by PGaA Gel, the ALT and AST levels in the blood of the mice are obviously decreased, and the non-alcoholic fatty liver disease of the mice is effectively treated.

[0118] The above merely describes the preferred embodiments of the present application, but the protection scope of the present application is not limited to this. Any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered in the protection scope of the present application.

Claims

1. A multi-component inulin hydrogel, characterized in that, The raw materials of the multi-component inulin hydrogel include L-alanyl-L-glutamine, mesoporous polydopamine-gallium composite nanoparticles and inulin.

2. The multi-component inulin hydrogel according to claim 1, characterized in that, The mass ratio of the inulin, L-alanyl-L-glutamine and mesoporous polydopamine-gallium composite nanoparticles is 6: (0.5-1): (0.002-0.005).

3. A method of preparing a multi-component inulin hydrogel according to any one of claims 1-2, characterized in that, The method comprises the following steps: L-alanyl-L-glutamine is added into the inulin solution and stirred uniformly to obtain a mixed solution; The aqueous dispersion of mesoporous polydopamine-gallium composite nanoparticles is added into the mixed solution and stirred uniformly, and the mixture is left to stand at room temperature to obtain the multi-component inulin hydrogel.

4. The method of preparing a multi-component inulin hydrogel according to claim 3, characterized in that, The preparation method of the inulin solution is as follows: inulin is added into water, heated in a water bath and stirred uniformly to obtain the inulin solution.

5. The method of preparing a multi-component inulin hydrogel according to claim 3, characterized in that, The preparation method of the mesoporous polydopamine-gallium composite nanoparticles comprises the following steps: the aqueous solution of soluble gallium salt is added into the aqueous dispersion of mesoporous polydopamine nanoparticles, stirred to react, the product is collected, centrifuged and washed with water to obtain the mesoporous polydopamine-gallium composite nanoparticles.

6. The method of preparing a multi-component inulin hydrogel according to claim 5, characterized in that, The mass ratio of the soluble gallium salt and the mesoporous polydopamine nanoparticles is 5:

2.

7. The method of preparing a multi-component inulin hydrogel according to claim 6, characterized in that, The preparation method of the mesoporous polydopamine nanoparticles comprises the following steps: poloxamer 407 is added into a mixed solvent composed of anhydrous ethanol and water, stirred until the solution becomes clear and transparent, dopamine is added and stirred uniformly; then 1,3,5-trimethylbenzene is added under stirring and stirred uniformly to obtain a mixed solution; then aqueous ammonia solution is added into the mixed solution under stirring, and the mixture is left to stand and react at room temperature; after the reaction is completed, the reacted solution is centrifuged at room temperature, washed and dried to obtain the mesoporous polydopamine nanoparticles.

8. The method of preparing a multi-component inulin hydrogel according to claim 7, characterized in that, The mass ratio of the poloxamer 407 and the dopamine is 1:0.

5.

9. Use of the multi-component inulin hydrogel according to any one of claims 1-2 in the preparation of a medicament for treating inflammatory bowel disease.

10. Use of the multi-component inulin hydrogel according to any one of claims 1-2 in the preparation of a medicament for treating non-alcoholic steatohepatitis.

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