Preparation method of manganese-copper bimetallic complex nanozyme and its application in alleviating animal inflammatory bowel disease

Abstract

The invention discloses a preparation method of a manganese-copper bimetallic complex nanozyme and an application thereof in alleviating animal inflammatory bowel disease. The invention uses myricetin to complex manganese metal and copper metal, prepares the manganese-copper bimetallic complex nanozyme through a simple precipitation reaction, and then uses TPP to encapsulate the nanozyme to obtain a manganese-copper bimetallic complex nanozyme with a targeting function. The manganese-copper bimetallic complex nanozyme disclosed by the invention has multiple mimetic enzyme activities of superoxide dismutase, peroxidase and catalase. The nanozyme can effectively remove ROS in cells or in vivo, thereby reducing inflammation in the lesion site, and at the same time promotes anti-inflammatory immune response, which is of great significance for treating inflammatory bowel disease IBD. In particular, the manganese-copper bimetallic complex nanozyme can be used for targeted treatment of ulcerative colitis in mice, and has excellent application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for preparing a manganese-copper bimetallic complex nanozyme and an application thereof in alleviating animal inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a major gastrointestinal disease, including Crohn's disease (CD) and ulcerative colitis (UC). It is characterized by chronic inflammation of gastrointestinal tissues, which can cause intestinal microbial imbalance, damage to the intestinal mucosal barrier, and decreased immune function, posing a great threat to human health. Inflammation is essentially a defensive response of the body, which is mainly caused by living tissues to damaging factors and is a self-defense of the body. Many studies have found that the factors that cause inflammation are very wide, such as pathogen infection, long-term stimulation of allergens, ionizing radiation, etc.

[0003] Generally speaking, exogenous sources such as ionizing radiation or chemicals can cause the appearance or accumulation of ROS in cells. Reactive oxygen species (ROS) generally refers to single-electron products containing oxygen atoms such as superoxide radicals, hydroxyl radicals, hydrogen peroxide and ozone that appear in the metabolic process of organisms when the cells of organisms perform respiration. Excess ROS can directly attack RNA, proteins, etc., causing a large number of intestinal tissue cells to die, thereby promoting the progression and severity of IBD. At present, drugs such as antibiotics, aminosalicylate, and corticosteroids can reduce inflammation and repair the intestinal barrier. However, since the drugs cannot form specific targeted treatments in the colon, the drugs cannot be effectively delivered to the inflammatory area, thereby reducing the efficacy of the drugs and causing adverse reactions to other organs. Therefore, there is an increasing interest in exploring SOD artificial enzymes suitable for the body. As a new type of artificial enzyme, nanozymes can not only show the unique properties of nanomaterials, but also simulate the multiple activities of natural enzymes. More importantly, some studies have shown that nanozymes with SOD-like activity can be obtained through appropriate strategies, which is a new option for anti-inflammatory therapy. However, the application of nanozymes with SOD-like activity still faces many limitations, such as poor biocompatibility, weak acid tolerance, and unsatisfactory catalytic activity. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for preparing a manganese-copper bimetallic complex nanozyme and its application in alleviating animal inflammatory bowel disease. The present invention complexes manganese metal and copper metal through myricetin, prepares a manganese-copper bimetallic complex nanozyme through a simple precipitation reaction, and then uses TPP to encapsulate the nanozyme to obtain a manganese-copper bimetallic complex nanozyme with a targeting function; the manganese-copper bimetallic complex nanozyme disclosed in the present invention has multiple mimetic enzyme activities of superoxide dismutase, peroxidase and catalase; the nanozyme can effectively remove ROS in cells or in vivo, thereby reducing inflammation in the lesion site, and at the same time promotes anti-inflammatory immune response, which is of great significance for the treatment of inflammatory bowel disease IBD, especially, the manganese-copper bimetallic complex nanozyme can be used for the targeted treatment of ulcerative colitis in mice, and has excellent application prospects.

[0005] The manganese-copper bimetallic complex nanozyme of the present invention is more effective in removing reactive oxygen species (ROS) than non-catalytic antioxidants, and is more stable than free enzymes; triphenylphosphine TPP is used to coat MnCu-Myr nanoparticles to improve their targeting, biocompatibility and acid resistance, thereby reducing their potential nanotoxicity; when orally administered, MnCu-Myr@TPP is stable in the stomach and can remove excess ROS at the colon lesion, thereby reducing inflammation at the lesion site, while promoting anti-inflammatory immune response, and ultimately effectively treating dextran sodium sulfate (DSS)-induced IBD in a mouse model. In order to address the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a preparation method based on a bimetallic complex nanozyme, which is simple to operate, low in cost, and is expected to be used in industrial production.

[0006] In order to achieve the above technical effects, the following technical solutions are adopted:

[0007] A method for preparing a manganese-copper bimetallic complex nanozyme comprises the following steps:

[0008] Step S1: dissolving manganese acetate tetrahydrate and copper acetate in an ethanol aqueous solution and stirring at room temperature; then adding PVP to enhance its water solubility to obtain a mixed solution A;

[0009] Step S2: adding myricetin to an ethanol aqueous solution, adjusting the pH, and stirring at room temperature to obtain a mixed solution B;

[0010] Step S3: pouring mixed solution A and mixed solution B into a reaction container in sequence, heating in a water bath for reaction; after the reaction is completed, cooling to room temperature; centrifuging the obtained solution, washing with water for multiple times, and centrifuging to obtain a brown-yellow precipitate; drying the brown-yellow precipitate in a vacuum freeze dryer to obtain a MnCu-Myr complex nanozyme;

[0011] Step S4: dispersing TPP in a PBS buffer solution, and then adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC and N-hydroxysulfosuccinimide NHS to activate the carboxyl group of TPP; reacting the mixture of TPP, EDC and NHS at room temperature under stirring; purifying the product with a dialysis bag, and then freeze-drying to obtain pure TPP;

[0012] Step S5: dissolving the MnCu-Myr complex nanozyme obtained in step S3 in an aqueous solution, adding the TPP obtained in step S4, stirring at room temperature, centrifuging, washing with methanol, and vacuum drying to obtain a manganese-copper bimetallic complex nanozyme with targeting function.

[0013] Furthermore, the mass ratio of manganese acetate tetrahydrate, copper acetate, PVP and myricetin is 40-45:6-8:40-45:9-10.

[0014] Furthermore, in the step S1, stirring is performed at room temperature for 1-2 hours; in the step S2, the pH is adjusted to 7.0-7.4; and stirring is performed at room temperature for 1-2 hours.

[0015] Furthermore, in step S3, the water bath is heated to 65°C-75°C, and the reaction is continued for 7h-8h; the resulting solution is centrifuged at 4000-4200rpm for 10-15min, washed with water for multiple times, and centrifuged to obtain a brown-yellow precipitate, which is dried in a vacuum freeze dryer for 24-48h.

[0016] Furthermore, in step S4, the mass ratio of TPP, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC and N-hydroxysulfosuccinimide NHS is 4-4.2:2-2.2:1-1.2.

[0017] Furthermore, in step S4, the components of the PBS buffer solution are Na2HPO4, KH2PO4, NaCl and KCl; and the pH of the PBS buffer solution is 7-7.4.

[0018] Furthermore, in step S4, the mixture of TPP, EDC and NHS is reacted at room temperature for 16-18 hours under stirring; and the product is purified using a 500-1000Da dialysis bag for 24-48 hours.

[0019] Furthermore, in step S4, the mass ratio of the MnCu-Myr complex nanozyme to TPP is 5-5.5:2-2.5; stirring at room temperature for 20-24 hours, centrifuging at 4000-4200 rpm for 10-15 minutes, and washing with methanol for 3-5 times.

[0020] A manganese-copper bimetallic complex nanozyme is prepared by any one of the above preparation methods.

[0021] The manganese-copper bimetallic complex nanozyme is used for targeted therapeutic applications in alleviating inflammatory bowel disease in animals.

[0022] The beneficial effects of the present invention are:

[0023] (1) The nanozyme preparation method of the present invention is simple, does not require complex instruments and equipment, is low in cost, and is expected to be used in industrial production;

[0024] (2) The oral targeted nanomedicine of the present invention can synergistically enhance the effect of myricetin and bimetallic compounds through complexation, effectively remove ROS in cells or in the body, thereby reducing inflammation in the lesion site and promoting anti-inflammatory immune response, which is of great significance for the treatment of inflammatory bowel disease (IBD).

[0025] (3) The oral nanomedicine of the present invention can reduce the potential toxicity of nanomaterials by oral administration. After TPP encapsulation, the absorption in the gastrointestinal tract can be reduced so that the nanomedicine can reach the colon. The nanomedicine has low toxicity to normal cells and good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a SEM image of the Cu-Myr@TPP nanozyme of an embodiment of the present invention;

[0027] Figure 2 This is a SEM image of the Mn-Myr@TPP nanozyme in an embodiment of the present invention;

[0028] Figure 3 This is a SEM image of the MnCu-Myr@TPP nanozyme in an embodiment of the present invention;

[0029] Figure 4 This is a SEM image of the MnCu-Quercetin@TPP nanozyme in an embodiment of the present invention;

[0030] Figure 5 This is a SEM image of the MnCu-Baicalein@TPP nanozyme in an embodiment of the present invention;

[0031] Figure 6 This is the EDS image of the MnCu-Myr@TPP nanozyme in the embodiment of the present invention;

[0032] Figure 7 The ESR spectra of five nanozymes in the embodiments of the present invention;

[0033] Figure 8 This is a comparison of the cytotoxicity of five nanozymes to RAW264.7 cells in the examples of the present invention;

[0034] Fig. 9 This is a diagram showing the effect of intracellular ROS removal in the blank control group in the embodiment of the present invention;

[0035] Fig.10 This is a diagram showing the effect of removing intracellular ROS in the LPS group in an embodiment of the present invention;

[0036] Fig.11 This is a diagram showing the effect of intracellular ROS removal by the Mn-Myr@TPP nanozyme group in an embodiment of the present invention;

[0037] Fig.12 This is a diagram showing the effect of intracellular ROS removal by the MnCu-Baicalein@TPP nanozyme group in an embodiment of the present invention;

[0038] Fig.13 This is a diagram showing the effect of intracellular ROS removal by the MnCu-Quercetin@TPP nanozyme group in an embodiment of the present invention;

[0039] Fig.14 This is a diagram showing the intracellular ROS removal effect of the Cu-Myr@TPP nanozyme group in an embodiment of the present invention;

[0040] Fig.15 This is a diagram showing the effect of intracellular ROS removal by the MnCu-Myr@TPP nanozyme group in an embodiment of the present invention;

[0041] Fig.16 This is a comparison of the ROS scavenging abilities of five nanozymes induced by LPS in the examples of the present invention;

[0042] Fig.17 This is a comparison chart of the antioxidant capacity of five nanozymes in the embodiments of the present invention;

[0043] Fig.18 The effect diagram of the mouse colon after treatment with five nanozymes in the examples of the present invention;

[0044] Fig.19 This is a graph showing the weight changes of mice in each group after being treated with five nanozymes in the examples of the present invention;

[0045] Fig. 20 This is a hemolysis analysis diagram of the MnCu-Myr@TPP nanozyme in an embodiment of the present invention. DETAILED DESCRIPTION

[0046] The present invention is further described below in conjunction with the accompanying drawings, and the protection scope of the present invention is not limited to the following:

[0047] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0048] Example 1: Preparation of manganese-copper bimetallic complex nanozyme

[0049] 0.4 g (1.63 mmol) of manganese acetate tetrahydrate and 0.06 g (0.30 mmol) of copper acetate were dissolved in 20 mL of ethanol aqueous solution and stirred at room temperature for 1 h. 400 mg of PVP was then added to enhance its water solubility.

[0050] Then, 90 mg of myricetin was added to 60 mL of ethanol aqueous solution, the pH was adjusted to 7.8, and stirred at room temperature for 1 h.

[0051] The two solutions were poured into a 250 mL three-necked flask, heated to 80 ° C in a water bath, and the reaction lasted for 8 hours. After the reaction was completed, it was cooled to room temperature. Then, the resulting solution was centrifuged at 4200 rpm for 10 minutes, washed with water several times, and centrifuged to obtain a brown-yellow precipitate. Finally, it was dried in a vacuum freeze dryer for 24 hours to obtain the MnCu-Myr complex nanozyme.

[0052] TPP (400 mg) was dispersed in 50 mL PBS buffer solution (pH 7.4), and then 200 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 100 mg N-hydroxysulfosuccinimide (NHS) were added to activate the carboxyl group of TPP. The mixture of TPP, EDC and NHS was reacted at room temperature for 16 hours under stirring. The TPP product was purified by 500 Da dialysis bag for 48 hours and then freeze-dried to obtain pure TPP.

[0053] 0.2 g of MnCu-Myr complex nanozyme was dissolved in 40 mL of aqueous solution, 80 mg of TPP was added, stirred at room temperature for 24 h, centrifuged at 4200 rpm for 10 min, washed with methanol three times, and vacuum dried to finally obtain the product MnCu-Myr@TPP.

[0054] Example 2: Preparation of manganese monometallic complex nanozyme

[0055] 0.47 g (1.93 mmol) of manganese acetate tetrahydrate was dissolved in 20 mL of ethanol aqueous solution and stirred at room temperature for 1 h. 400 mg of PVP was then added to enhance its water solubility.

[0056] Then, 90 mg of myricetin was added to 60 mL of ethanol aqueous solution, the pH was adjusted to 7.8, and stirred at room temperature for 1 h.

[0057] The two solutions were poured into a 250 mL three-necked flask, heated to 70 ° C in a water bath, and the reaction lasted for 8 hours. Then, the resulting solution was centrifuged at 4200 rpm for 10 minutes, washed with water several times, and centrifuged to obtain a light yellow precipitate. Finally, it was dried in a vacuum freeze dryer for 24 hours to obtain the Mn-Myr complex nanozyme.

[0058] TPP (400 mg) was dispersed in 50 mL PBS buffer solution (pH 7.4), and then 200 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 100 mg N-hydroxysulfosuccinimide (NHS) were added to activate the carboxyl group of TPP. The mixture of TPP, EDC and NHS was reacted at room temperature for 16 hours under stirring. The TPP product was purified by 500 Da dialysis bag for 48 hours and then freeze-dried to obtain pure TPP.

[0059] 0.2 g of Mn-Myr complex nanozyme was dissolved in 40 mL of aqueous solution, 80 mg of TPP was added, stirred at room temperature for 24 h, centrifuged at 4200 rpm for 10 min, washed with methanol three times, and vacuum dried to finally obtain the product Mn-Myr@TPP.

[0060] Example 3: Preparation of copper monometallic complex nanozyme

[0061] 0.38 g (0.193 mmol) of copper acetate was dissolved in 20 mL of ethanol aqueous solution and stirred at room temperature for 1 h. 400 mg of PVP was then added to enhance its water solubility.

[0062] Then, 90 mg of myricetin was added to 60 mL of ethanol aqueous solution, the pH was adjusted to 7.8, and stirred at room temperature for 1 h.

[0063] The two solutions were poured into a 250 mL three-necked flask, heated to 70 ° C in a water bath, and the reaction lasted for 8 hours. Then, the resulting solution was centrifuged at 4200 rpm for 10 minutes, washed with water several times, and centrifuged to obtain a light yellow precipitate. Finally, it was dried in a vacuum freeze dryer for 24 hours to obtain the Cu-Myr complex nanozyme.

[0064] TPP (400 mg) was dispersed in 50 mL PBS buffer solution (pH 7.4), and then 200 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 100 mg N-hydroxysulfosuccinimide (NHS) were added to activate the carboxyl group of TPP. The mixture of TPP, EDC and NHS was reacted at room temperature for 16 hours under stirring. The TPP product was purified by 500 Da dialysis bag for 48 hours and then freeze-dried to obtain pure TPP.

[0065] 0.2 g of Cu-Myr complex nanozyme was dissolved in 40 mL of aqueous solution, 80 mg of TPP was added, stirred at room temperature for 24 h, centrifuged at 4200 rpm for 10 min, washed with methanol three times, and vacuum dried to finally obtain the product Cu-Myr@TPP.

[0066] Example 4: Preparation of Quercetin Chelated MnCu, i.e. MnCu-Quercetin (Myricetin Replaced with Quercetin)

[0067] 0.4 g of manganese acetate tetrahydrate and 0.06 g of copper acetate were dissolved in 20 mL of ethanol aqueous solution and stirred at room temperature for 1 h. 400 mg of PVP was then added to enhance its water solubility.

[0068] Then, 85.5 mg of quercetin was added to 60 mL of ethanol aqueous solution, the pH was adjusted to 7.8, and stirred at room temperature for 1 h.

[0069] The two solutions were poured into a 250 mL three-necked flask, heated to 80 ° C in a water bath, and the reaction lasted for 8 hours. After the reaction was completed, it was cooled to room temperature. Then, the resulting solution was centrifuged at 4200 rpm for 10 minutes, washed with water several times, and centrifuged to obtain a yellow precipitate. Finally, it was dried in a vacuum freeze dryer for 24 hours to obtain the MnCu-Quercetin complex nanozyme.

[0070] TPP (400 mg) was dispersed in 50 mL PBS buffer solution (pH 7.4), and then 200 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 100 mg N-hydroxysulfosuccinimide (NHS) were added to activate the carboxyl group of TPP. The mixture of TPP, EDC and NHS was reacted at room temperature for 16 hours under stirring. The TPP product was purified by 500 Da dialysis bag for 48 hours and then freeze-dried to obtain pure TPP.

[0071] 0.2 g of MnCu-Quercetin complex nanozyme was dissolved in 40 mL of aqueous solution, 80 mg of TPP was added, stirred at room temperature for 24 h, centrifuged at 4200 rpm for 10 min, washed with methanol three times, and vacuum dried to finally obtain the product MnCu-Quercetin@TPP.

[0072] Example 5: Preparation of Baicalein Chelated MnCu, i.e. MnCu-Baicalein (Myricetin Replaced with Baicalein)

[0073] 0.4 g of manganese acetate tetrahydrate and 0.06 g of copper acetate were dissolved in 20 mL of ethanol aqueous solution and stirred at room temperature for 1 h. 400 mg of PVP was then added to enhance its water solubility.

[0074] Then, 76.4 mg of baicalin was added to 60 mL of ethanol aqueous solution, the pH was adjusted to 7.8, and stirred at room temperature for 1 h.

[0075] The two solutions were poured into a 250 mL three-necked flask, heated to 80 ° C in a water bath, and the reaction lasted for 8 hours. After the reaction was completed, it was cooled to room temperature. Then, the resulting solution was centrifuged at 4200 rpm for 10 minutes, washed with water several times, and centrifuged to obtain a precipitate. Finally, it was dried in a vacuum freeze dryer for 24 hours to obtain the MnCu-Baicalein complex nanozyme.

[0076] TPP (400 mg) was dispersed in 50 mL PBS buffer solution (pH 7.4), and then 200 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 100 mg N-hydroxysulfosuccinimide (NHS) were added to activate the carboxyl group of TPP. The mixture of TPP, EDC and NHS was reacted at room temperature for 16 hours under stirring. The TPP product was purified by 500 Da dialysis bag for 48 hours and then freeze-dried to obtain pure TPP.

[0077] 0.2 g of MnCu-Baicalein complex nanozyme was dissolved in 40 mL of aqueous solution, 80 mg of TPP was added, stirred at room temperature for 24 h, centrifuged at 4200 rpm for 10 min, washed with methanol three times, and vacuum dried to finally obtain the product MnCu-Baicalein@TPP.

[0078] Based on the MnCu-Myr@TPP, Mn-Myr@TPP, Cu-Myr@TPP, MnCu-Quercetin@TPP, and MnCu-Baicalein@TPP prepared in Examples 1-5, their basic properties such as structure were evaluated and characterized, and used in animal experiments on intestinal inflammation mouse models:

[0079] (1) Structural characterization: The morphology of the nanozymes was observed by scanning electron microscopy, and the SEM images of each nanozyme were obtained. Energy dispersive X-ray spectroscopy (EDS) was used to perform elemental analysis on the MnCu-Myr@TPP nanozyme.

[0080] (2) ESR energy spectrum of superoxide anion scavenging: 1.8 ml PBS was added to 6 1.5 ml centrifuge tubes respectively, followed by 200 μl methionine (Met), 200 μl riboflavin (VB2), 100 μl ethylenediaminetetraacetic acid (EDTA), and 300 μl nitroblue tetrazolium (NBT). Finally, 200 μl of PBS (control group), MnCu-Myr@TPP, Mn-Myr@TPP, Cu-Myr@TPP, MnCu-Quercetin@TPP, and MnCu-Baicalein@TPP were added to 6 small centrifuge tubes respectively, mixed, illuminated for 5 min, and finally the superoxide anion free radical (·OH) was measured using an ESR spectrometer.

[0081] (3) Cell viability assay: RAW264.7 (mouse macrophages) were cultured at a density of 1×10 4 The 96-well plates were adhered for 24 h, and then different concentrations (1-100 μg mL -1 ) were cultured for another 24 h. Cell viability was determined using CCK-8 according to the manufacturer's instructions.

[0082] (4) Intracellular ROS scavenging ability: The density of 1×10 6 RAW264.7 cells were seeded in 6-well plates for 24 hours, and then 10 μg / mL of MnCu-Myr@TPP, Mn-Myr@TPP, Cu-Myr@TPP, MnCu-Quercetin@TPP, and MnCu-Baicalein@TPP were added to the experimental groups, and the nanozymes were replaced with an equal amount of PBS in the LPS group and the blank control group (control group); after incubation at 37°C for 12 hours, 200 ng / mL LPS was introduced into RAW 264.7 cells, and the blank control group (control group) replaced LPS with an equal amount of PBS and incubated for 2 hours. Intracellular ROS was detected using a fluorescent probe 5 μM DCFH-DA, and analyzed by flow cytometry.

[0083] (5) Antioxidant capacity determination: 7mM ABTS solution was incubated with 2.45mM potassium persulfate overnight to activate ABTS free radicals. Five nanozymes (MnCu-Myr@TPP, Mn-Myr@TPP, Cu-Myr@TPP, MnCu-Quercetin@TPP, MnCu-Baicalein@TPP) were mixed with ABTS free radical solution and incubated for 10 minutes. The absorption of ABTS free radicals at 734nm was measured. The scavenging efficiency of ABTS free radicals was calculated according to the following formula.

[0084] ABTS removal efficiency (%) = ((A ABTS -A Sample ) / A ABTS) *100%

[0085] A ABTS represents the absorbance of ABTS without additional nanozyme treatment (control group), A Sample Represents the absorbance of ABTS after adding nanozyme.

[0086] (6) Experiment on enteritis mouse model: First, establish a mouse colitis model, DSS-induced colitis model: 30 male C57 mice weighing 15g-18g were randomly divided into 7 groups. After 7 days of adaptive feeding, 6 groups of mice were fed 6% (w / v) DSS every day for 3 days. One blank group (control group) only provided normal water to healthy mice, and no colitis was induced. The other 6 groups used DSS to establish a colitis model. After the model was successfully established on the 4th day, except for one DSS group and one blank group, the remaining 5 groups were fed MnCu-Myr@TPP, Mn-Myr@TPP, Cu-Myr@TPP, MnCu-Quercetin@TPP, and MnCu-Baicalein@TPP orally every other day, with a feeding amount of 2 mg / kg -1 Mouse weight; other conditions were the same as the blank group. The blank group and DSS group were fed with the same amount of water when the experimental group was fed with nanozymes, and other conditions were the same. During the experiment, the changes in body weight were evaluated every day. On the last day of the experiment, the mice were killed and the entire colon and organs were removed. The length of the colon was measured and gently flushed with saline.

[0087] (8) Hemolytic analysis: Blood from one mouse was collected by centrifugation at 1500 rpm for 15 minutes, and red blood cells (RBC) were collected and washed three times with saline. Then, 0.3 mL of RBC saline suspension (5%) was resuspended in 6 mL of saline. The RBC suspension (0.1 mL) was added to 1 mL of different concentrations (3.125, 6.25, 12.5, 25, 50, and 100 μg mL -1) in a MnCu-Myr@TPP saline dispersion. The mixture was incubated at 37 °C for 3 h. Ultrapure water and saline were used as positive and negative controls, respectively. Finally, the absorbance of the supernatant was measured at 540 nm after centrifugation at 12000 rpm for 15 min.

[0088] Experimental results and analysis:

[0089] like Figure 1 , Figure 2 , Figure 3 , Figure 4 and Figure 5 As shown, the SEM images of Cu-Myr@TPP, Mn-Myr@TPP, MnCu-Myr@TPP, MnCu-Quercetin@TPP, and MnCu-Baicalein@TPP are shown. It can be seen that the morphology changes after MnCu-Myr is coated on TPP.

[0090] like Figure 6 As shown in the EDS elemental analysis diagram of MnCu-Myr@TPP, it can be seen that the elemental composition of MnCu-Myr@TPP is Mn, Cu, C, N, O, and P, while TPP is composed of P, C, and O. Therefore, it is concluded that TPP successfully coats MnCu-Myr, thereby successfully synthesizing MnCu-Myr@TPP.

[0091] like Figure 7 As shown, the ESR energy spectra of MnCu-Myr@TPP, Mn-Myr@TPP, Cu-Myr@TPP, MnCu-Quercetin@TPP, and MnCu-Baicalein@TPP for scavenging superoxide anions are compared. It can be seen that MnCu-Myr@TPP has the best scavenging effect, and the scavenging effects of other nanozymes are greatly reduced.

[0092] like Figure 8 As shown, the cytotoxicity of MnCu-Myr@TPP, Mn-Myr@TPP, Cu-Myr@TPP, MnCu-Quercetin@TPP, and MnCu-Baicalein@TPP on RAW264.7 cells is compared. It can be seen that when the concentration of MnCu-Myr@TPP reaches 100μg / mL, the cell viability of MnCu-Myr@TPP is still 100%, while that of Mn-Myr@TPP, Cu-Myr@TPP, MnCu-Quercetin@TPP, and MnCu-Baicalein@TPP is only about 60%, which shows that MnCu-Myr@TPP has the least cytotoxicity.

[0093] like Fig. 9 , Fig.10, Fig.11 , Fig.12 , Fig.13 , Fig.14 and Fig.15 As shown, flow cytometry analysis showed that Fig.16 As shown, the ROS content of the blank control group was only 25.4, while after treatment with LPS (200 ng / ml, 3 hours), the ROS in the LPS group increased greatly, reaching 49.5. On this basis, five different nanozymes (10 ug / ml) were used to alleviate the increase in ROS content. The results showed that the ROS content of the MnCu-Myr@TPP group was 26.2, close to the blank control group, indicating that compared with the other four nanozymes, MnCu-Myr@TPP had the best ROS scavenging effect.

[0094] like Fig.17 As shown, compared with other nanozymes, MnCu-Myr@TPP is more effective in removing ABTS, indicating that it has strong antioxidant capacity.

[0095] like Fig.18 As shown in the figure, the colon effect pictures after five nanozyme experiments were treated. It can be seen from the figure that the colon length of mice treated with MnCu-Myr@TPP was restored to the same level as that of the blank group; compared with other nanozymes, MnCu-Myr@TPP had a good inhibitory effect on colitis after treatment. Fig.19 As shown, it can be seen that the body weight during MnCu-Myr@TPP treatment also recovered to the same level as the blank group.

[0096] like Fig. 20 As shown, it can be seen that the hemolytic test results of MnCu-Myr after coating with TPP are good. The material concentration is 100 μg / mL and the hemolysis rate is about 5%, indicating that the hemolysis of the nanoparticles is normal.

[0097] Therefore, in summary, the oral targeted nanomedicine of the present invention can effectively remove ROS in cells or in vivo by complexing myricetin and bimetallic to reduce inflammation in the lesion site, and promote anti-inflammatory immune response, which is of great significance for the treatment of inflammatory bowel disease (IBD). The potential toxicity of nanomaterials can be reduced by oral administration, and TPP encapsulation can reduce gastrointestinal absorption to reach the colon. The nanomedicine has low toxicity to normal cells and good biocompatibility.

[0098] In summary, the present invention discloses a method for preparing a manganese-copper bimetallic complex nanozyme and its application in alleviating animal inflammatory bowel disease. The present invention complexes manganese metal and copper metal through myricetin, prepares a manganese-copper bimetallic complex nanozyme through a simple precipitation reaction, and then uses TPP to encapsulate the nanozyme to obtain a manganese-copper bimetallic complex nanozyme with targeting function; the manganese-copper bimetallic complex nanozyme disclosed by the present invention has multiple mimetic enzyme activities of superoxide dismutase, peroxidase and catalase; the nanozyme can effectively remove ROS in cells or in vivo, thereby reducing inflammation in the lesion site, and at the same time promotes anti-inflammatory immune response, which is of great significance for the treatment of inflammatory bowel disease IBD, especially, the manganese-copper bimetallic complex nanozyme can be used for targeted treatment of ulcerative colitis in mice, and has excellent application prospects.

[0099] At this point, those skilled in the art recognize that, although the embodiments of the present invention have been shown and described in detail herein, many other variations or modifications that conform to the principles of the present invention can still be directly determined or derived based on the contents disclosed in the present invention without departing from the spirit and scope of the present invention. Therefore, the scope of the present invention should be understood and recognized as covering all such other variations or modifications.

Claims

1. A method for preparing a manganese-copper bimetallic complex nanozyme, characterized in that: The preparation method comprises the following steps: Step S1: dissolving manganese acetate tetrahydrate and copper acetate in an ethanol aqueous solution and stirring at room temperature; then adding PVP to enhance its water solubility to obtain a mixed solution A; Step S2: adding myricetin to an ethanol aqueous solution, adjusting the pH, and stirring at room temperature to obtain a mixed solution B; Step S3: pouring mixed solution A and mixed solution B into a reaction container in sequence, heating in a water bath for reaction; after the reaction is completed, cooling to room temperature; centrifuging the obtained solution, washing with water for multiple times, and centrifuging to obtain a brown-yellow precipitate; drying the brown-yellow precipitate in a vacuum freeze dryer to obtain a MnCu-Myr complex nanozyme; Step S4: dispersing TPP in a PBS buffer solution, and then adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide EDC and N-hydroxysulfosuccinimide NHS to activate the carboxyl group of TPP; reacting the mixture of TPP, EDC and NHS at room temperature under stirring; purifying the product with a dialysis bag, and then freeze-drying to obtain pure TPP; Step S5: dissolving the MnCu-Myr complex nanozyme obtained in step S3 in an aqueous solution, adding the TPP obtained in step S4, stirring at room temperature, centrifuging, washing with methanol, and vacuum drying to obtain a manganese-copper bimetallic complex nanozyme with a targeting function; The mass ratio of manganese acetate tetrahydrate, copper acetate, PVP and myricetin is 40-45:6-8:40-45:9-10.

2. A method for preparing a manganese-copper bimetallic complex nanozyme as claimed in claim 1, characterized in that: In the step S1, stirring is performed at room temperature for 1-2 hours; in the step S2, the pH is adjusted to 7.8-8.0; and stirring is performed at room temperature for 1-2 hours.

3. The method for preparing a manganese-copper bimetallic complex nanozyme as claimed in claim 1, characterized in that: In step S3, the water bath is heated to 65°C-75°C, and the reaction is continued for 7 h-8 h; the resulting solution is centrifuged at 4000-4200 rpm for 10-15 min, washed with water for multiple times, and centrifuged to obtain a brown-yellow precipitate, which is dried in a vacuum freeze dryer for 24-36 h.

4. The method for preparing a manganese-copper bimetallic complex nanozyme as claimed in claim 1, characterized in that: In the step S4, the mass ratio of TPP, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC and N-hydroxysulfosuccinimide NHS is 4-5:2-3:1-2.

5. The method for preparing a manganese-copper bimetallic complex nanozyme as claimed in claim 1, characterized in that: In step S4, the components of the PBS buffer solution are Na2HPO4, KH2PO4, NaCl and KCl; and the pH of the PBS buffer solution is 7-7.

4.

6. A method for preparing a manganese-copper bimetallic complex nanozyme as claimed in claim 1, characterized in that: In step S4, the mixture of TPP, EDC and NHS is reacted at room temperature for 16-18 hours under stirring; the product is purified using a 500-1000Da dialysis bag for 24-48 hours.

7. The method for preparing a manganese-copper bimetallic complex nanozyme as claimed in claim 1, characterized in that: In the step S4, the mass ratio of the MnCu-Myr complex nanozyme to TPP is 5-6:2-3; stirring at room temperature for 20-24 h, centrifuging at 4000-4200 rpm for 10-15 min, and washing with methanol for 3-5 times.

8. A manganese-copper bimetallic complex nanozyme, characterized in that: The compound is prepared by any one of the preparation methods in claims 1 to 7.

9. The manganese-copper bimetallic complex nanozyme according to claim 8, characterized in that: The manganese-copper bimetallic complex nanozyme is used for targeted therapeutic applications in alleviating inflammatory bowel disease in animals.

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