HMPB / Mn3O4 nano-enzyme with multi-enzyme activity as well as preparation method and application of HMPB / Mn3O4 nano-enzyme

Abstract

The invention discloses an HMPB / Mn3O4 nano-enzyme with multi-enzyme activity as well as a preparation method and application of the HMPB / Mn3O4 nano-enzyme. According to the nano-enzyme, HMPB with a hollow mesoporous structure is used as a carrier, and nano Mn3O4 is loaded on the inner surface and the outer surface of the HMPB through a self-assembly method; the HMPB / Mn3O4 nano-enzyme provided by the invention has good multi-enzyme activity, has no obvious cytotoxicity to NCM460 and RAW264.7 cells, can improve related symptoms of mice with DSS-induced ulcerative colitis, and is expected to become a novel potential medicine for treating ulcerative colitis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a novel HMPB / Mn3O4 nanozyme with multi-enzyme activity. The present invention also relates to a preparation method of the novel HMPB / Mn3O4 nanozyme with multi-enzyme activity and its application in the treatment of ulcerative colitis. Background Art

[0002] Inflammatory bowel disease (IBD) is a type of idiopathic intestinal disease that poses a serious threat to millions of patients worldwide every year. Compared with the healthy intestine, the intestinal microenvironment of IBD shows increased levels of reactive oxygen species (ROS), immune dysfunction, and damage to the intestinal mucosa and epithelial cells. Among them, the long-term accumulation of ROS in the body may cause many chronic diseases in the host. At present, the purpose of relieving enteritis is mainly achieved by using anti-inflammatory drugs, immunomodulators, monoclonal antibodies, antibiotics or bioengineered probiotics to eliminate inflammation and inhibit intestinal inflammatory reactions. However, the above methods can only relieve inflammation, reduce the frequency of relapses, and prevent complications, but cannot completely cure IBD, and some drugs have certain side effects. Therefore, it is necessary to develop new anti-inflammatory biomaterials for the treatment of IBD.

[0003] Studies have shown that removing excess ROS is crucial to inhibiting the development of enteritis. Among the ROS removal methods, nanozymes with high activity, simple preparation and good stability are an effective method. At present, nanozymes used in the treatment of IBD have been developed one after another, such as cerium dioxide nanozymes, iron-based nanozymes, manganese-based nanozymes and other non-metal-based nanozymes. However, most of the nanozymes reported for the treatment of IBD usually only have single or dual enzyme catalytic activity. Therefore, the design and development of new nanozymes with multi-enzyme activity is expected to further improve the treatment effect of IBD.

[0004] So far, there has been no research report on hollow mesoporous Prussian blue (HMPB) loaded with Mn3O4 nanozymes with multi-enzyme activity. Summary of the invention

[0005] In view of this, the first object of the present invention is to provide a HMPB / Mn3O4 nanozyme with multi-enzyme activity, which effectively solves the problems of few types of traditional nanozyme mimics and low catalytic activity.

[0006] The second object of the present invention is to provide a method for preparing a novel HMPB / Mn3O4 nanozyme with multi-enzyme activity with low preparation cost.

[0007] The third object of the present invention is to provide a novel HMPB / Mn3O4 nanozyme with multi-enzyme activity for use in the treatment of ulcerative colitis, so as to solve various inflammatory reactions caused by ulcerative colitis.

[0008] To this end, the first technical solution provided by the present invention is as follows:

[0009] A HMPB / Mn3O4 nanozyme with multi-enzyme activity, wherein the nanozyme is a hollow mesoporous Prussian blue (HMPB) with nano-Mn3O4 loaded on the inner and outer surfaces.

[0010] Furthermore, in the above-mentioned HMPB / Mn3O4 nanozyme with multi-enzyme activity, the mass ratio of Mn3O4 and HMPB is: 1-5:3-15.

[0011] Furthermore, the above-mentioned HMPB / Mn3O4 nanozyme with multi-enzyme activity comprises four enzymes including catalase-like enzyme, superoxide dismutase, peroxidase and glutathione peroxidase.

[0012] The second technical solution provided by the present invention is a method for preparing the HMPB / Mn3O4 nanozyme with multi-enzyme activity described in the first technical solution, wherein nano-Mn3O4 and HMPB are dispersed in anhydrous ethanol and stirred evenly, and the resulting solution is centrifuged, washed and dried to obtain the HMPB / Mn3O4 nanozyme.

[0013] Furthermore, in the preparation method of the above-mentioned HMPB / Mn3O4 nanozyme with multi-enzyme activity, the mass ratio of the nano-Mn3O4 to HMPB is: 1-5:3-15, and the mass volume ratio of the sample and anhydrous ethanol in the reaction system is 3mg:2ml.

[0014] Furthermore, in the method for preparing the HMPB / Mn3O4 nanozyme with multi-enzyme activity, the stirring temperature is 25°C and the time is 24-36h.

[0015] Furthermore, in the method for preparing the HMPB / Mn3O4 nanozyme with multi-enzyme activity, the centrifugation time is 15 min and the rotation speed is 11000 r / min.

[0016] Furthermore, in the method for preparing the above-mentioned HMPB / Mn3O4 nanozyme with multi-enzyme activity, the drying temperature is 60-100°C and the drying time is 5-24h.

[0017] Furthermore, the preparation method of the above-mentioned HMPB / Mn3O4 nanozyme with multi-enzyme activity, the HMPB preparation method is: dissolving mesoporous Prussian blue and PVP in HCl solution, performing hydrothermal reaction, and obtaining HMPB.

[0018] The third technical solution provided by the present invention is the use of the above-mentioned HMPB / Mn3O4 nanozyme with multi-enzyme activity as a drug for preparing a drug for treating ulcerative colitis.

[0019] Compared with the prior art, the technical solution provided by the present invention has the following advantages and beneficial effects:

[0020] (1) The method for preparing HMPB / Mn3O4 nanozyme of the present invention is simple, has high synthesis efficiency, low cost, good repeatability, and controllable content of each component. Nano-Mn3O4 is evenly loaded on the inner and outer surfaces of HMPB and is not easy to fall off.

[0021] (2) The HMPB / Mn3O4 nanozyme prepared by the present invention has excellent multi-enzyme-like activity and no obvious cytotoxicity due to the synergistic effect of the components.

[0022] (3) The HMPB / Mn3O4 nanozyme prepared by the present invention can significantly improve the relevant symptoms of DSS-induced ulcerative colitis in mice (including bloody stools, weight loss and shortened colon), and can be used as a new potential drug for the treatment of ulcerative colitis, thereby solving the problems of various inflammatory reactions caused by ulcerative colitis and the high cost of traditional nanozyme preparation, the single type of simulated enzyme, and the low catalytic activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a transmission electron microscopy (TEM) image of the HMPB / Mn3O4 nanozyme prepared in Example 1 of the present invention;

[0024] Figure 2 IR spectra of HMPB, Mn3O4 and HMPB / Mn3O4 nanozymes in Example 1 of the present invention;

[0025] Figure 3 This is a comparison chart of the four enzyme-like activities of HMPB and HMPB / Mn3O4 nanozymes in Example 1 of the present invention, wherein: a is peroxidase-like activity, b is superoxide dismutase-like activity, c is glutathione peroxidase-like activity, and d is catalase-like activity.

[0026] Figure 4 Schematic diagram of the cytotoxic activity of HMPB / Mn3O4 nanozyme on NCM460 and RAW264.7 cells in Example 1 of the present invention;

[0027] Figure 5 This is a diagram of bloody stools in different groups of mice in the prevention model.

[0028] Figure 6 Data of (a) weight change and (b) colon length of mice in different groups for prevention model

[0029] Figure 7 Data graphs of (a) body weight changes and (b) colon length of mice in different groups of the treatment model; DETAILED DESCRIPTION

[0030] The technical solution of the present application will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0031] Example 1

[0032] This embodiment provides a HMPB / Mn3O4 nanozyme, which is prepared by the following method:

[0033] 1) Preparation of mesoporous Prussian blue PB

[0034] 4.5 g PVP and 198 mg K3[Fe(CN)6] were dissolved in 60 mL 0.01 M HCl solution and stirred magnetically until fully dissolved. The resulting mixed solution was transferred to an oven and reacted at 80 °C for 24 h. After the reaction, the mixture was cooled to room temperature and the mesoporous PB nanoparticles were collected by centrifugation, washed with water and ethanol, and dried at 60 °C for 5 h to obtain mesoporous Prussian blue PB.

[0035] 2) Preparation of HMPB nanoparticles

[0036] 35 mg of the mesoporous Prussian blue PB prepared in step 1) and 175 mg of PVP were dissolved in 35 mL of 1.0 M HCl solution, stirred at room temperature for 4 h, and the resulting mixed solution was transferred to a hydrothermal reactor and reacted at 140° C. for 3 h; after the reaction, the mixture was cooled to room temperature, and HMPB nanoparticles were collected by centrifugation, washed with water and ethanol, and dried at 60° C. for 5 h to obtain hollow mesoporous Prussian blue powder HMPB;

[0037] 3) Preparation of Mn3O4

[0038] 598 mg of MnCl2·4H2O was dissolved in 30 mL of ethanolamine and stirred at room temperature for 5 h to turn into a dark brown suspension. Mn3O4 nanoparticles were collected by centrifugation, washed with water and ethanol, and dried at 60 °C for 5 h to obtain Mn3O4;

[0039] 4) Preparation of HMPB / Mn3O4 nanozyme

[0040] 6 mg HMPB and 3 mg Mn3O4 were added to 6 mL anhydrous ethanol in sequence, stirred at 11000 r / min for 24 h at room temperature in the dark, washed with water and ethanol in sequence, then centrifuged at 11000 r / min for 15 min, and dried at 60°C for 5 h to obtain HMPB / Mn3O4 nanozyme.

[0041] Figure 1 TEM image of HMPB / Mn3O4 nanozyme prepared in this example; Figure 1 It can be seen that the Mn3O4 nanoparticles are uniformly dispersed on the inner and outer surfaces of HMPB; Figure 2 This is the infrared spectrum of the sample prepared in this example. The HMPB / Mn3O4 nanozyme has typical infrared peaks of HMPB and Mn3O4, proving the successful preparation of the composite material.

[0042] Example 2

[0043] This embodiment provides a HMPB / Mn3O4 nanozyme, whose preparation steps and process parameters are the same as those of Example 1, and the difference from Example 1 is: 3 mg of HMPB and 1 mg of Mn3O4 described in step 4).

[0044] Example 3

[0045] This embodiment provides a HMPB / Mn3O4 nanozyme, whose preparation steps and process parameters are the same as those in Example 1, and the difference from Example 1 is: 5 mg of HMPB and 1 mg of Mn3O4 described in step 4).

[0046] Example 4

[0047] This embodiment provides a HMPB / Mn3O4 nanozyme, whose preparation steps and process parameters are the same as those in Example 1, and the difference from Example 1 is: 6 mg of HMPB and 1 mg of Mn3O4 described in step 4).

[0048] Example 5

[0049] This embodiment provides a HMPB / Mn3O4 nanozyme, which is different from Example 1 in that: 4 mg of HMPB and 1 mg of Mn3O4 are described in step 4).

[0050] Example 6

[0051] This embodiment provides a HMPB / Mn3O4 nanozyme, which is different from Example 1 in that: Step 4) is: 6 mg HMPB and 3 mg Mn3O4 are added to 6 mL anhydrous ethanol in sequence, stirred at 11000 r / min for 36 hours at room temperature in the dark, washed with water and ethanol in sequence, then centrifuged at 11000 r / min for 15 minutes, and dried at 70°C for 24 hours to obtain HMPB / Mn3O4 nanozyme.

[0052] Example 7

[0053] This embodiment provides a HMPB / Mn3O4 nanozyme, which is different from Example 1 in that: Step 4) is to add 6 mg HMPB and 3 mg Mn3O4 to 6 mL anhydrous ethanol in sequence, stir at 11000 r / min for 30 h at room temperature in the dark, wash with water and ethanol in sequence, then centrifuge at 11000 r / min for 15 min, and dry at 65°C for 15 h to obtain HMPB / Mn3O4 nanozyme.

[0054] In order to demonstrate the advantages of the technical solution provided by this application, a test example of the technical solution provided by this application is given below:

[0055] Test Example 1

[0056] Example 1 of the present invention provides a method for testing the activity of multiple enzymes of HMPB / Mn3O4 nanozymes, comprising the following steps:

[0057] The HMPB / Mn3O4 nanozymes of different concentrations provided in Example 1 (20 μL, 100-500 μg / mL) were placed in acetic acid-sodium acetate buffer (940 μL, pH=5), and then the reaction substrates TMB (20 μL, 2 mM in DMSO) and H2O2 (20 μL, 10%) were added, and the reaction was carried out for 2 min. The absorbance at 300-800 nm was measured. The POD mimetic enzyme catalyzed the oxidation reaction of TMB and accelerated the formation of colored products, which simulated the role of the POD enzyme in removing peroxides in vivo;

[0058] The different concentrations of HMPB / Mn3O4 nanozymes (400μL, 100-500μg / mL) provided in Example 1 were placed in a detection solution (800μL), wherein riboflavin (100μL, 1.2mM), NBT (150μL, 2mM) and EDTA·Na2 (400μL, 0.1M) were sequentially added to PBS (5.8mL, pH=7.8, 10mM), and the mixed solution was irradiated under a xenon lamp for 3min, and the absorption peak of the purple compound in the blank group at 400-800nm ​​was measured, and the absorbance at the wavelength corresponding to the absorption peak of the sample before and after illumination was measured. This simulates the capture of electrons from excited riboflavin by the SOD enzyme, thereby reducing the possibility of electron transfer to NBT, thereby reducing the reduction of NBT to a purple compound;

[0059] The HMPB / Mn3O4 nanozyme (50 μL, 40 μg / mL) provided in Example 1 was added to the detection working solution (40 μL) in the glutathione peroxidase detection kit (NADPH method), wherein the detection working solution (40 μL) includes glutathione peroxidase detection buffer (35 μL), NADPH (62.5 mM, 2 μL), GSH (75 mM, 2 μL) and glutathione reductase (1 μL), and then peroxide reagent solution (30 mM, 10 μL) was added, and the absorbance at a wavelength of 340 nm was detected by an ELISA instrument, and recorded every 1 min for 10 min. This simulates that glutathione peroxidase helps to reduce H2O2 to H2O and convert GSH to GSSG at the same time, and GSH is generated with the help of coenzymes NADPH and GR, so that the absorbance of NADPH decreases over time;

[0060] The HMPB / Mn3O4 nanozyme (500 μL, 1 mg / mL) provided in Example 1 was placed in deionized water (3410 μL) and H2O2 (590 μL, 30%), and the generation of bubbles was observed and recorded at 0-5 min, which simulated the reaction of CAT enzyme with H2O2 to generate water and oxygen;

[0061] The results of various enzyme activity tests are as follows Figure 3 As shown, from Figure 3 a It can be seen that the HMPB / Mn3O4 nanozyme provided by the present invention has excellent multi-enzyme activity.

[0062] Test Example 2

[0063] The cytotoxic activity of the HMPB / Mn3O4 nanozyme prepared in Example 1 on NCM460 and RAW264.7 cells was tested. The cytotoxicity test process included: after incubating the cells (3000 cells / well) in a 96-well plate for 24 hours, adding different concentrations of Mn3O4 / HMPB nanozyme and incubating for another 24 hours, and detecting cell viability with CCK8 reagent.

[0064] The results of cell experiment test of new nanozymes with multiple enzyme activities are as follows Figure 4 As shown, from Figure 4 It can be seen that the HMPB / Mn3O4 nanozyme provided by the present invention has no obvious cytotoxicity to NCM460 and RAW264.7 cells.

[0065] Test Example 3

[0066] In Example 4 of the present invention, the HMPB / Mn3O4 nanozyme prepared in Example 1 was subjected to a mouse ulcerative colitis prevention experiment test. The tested mice were 6-8 week old C57BL / 6 male mice. The test indicators included changes in mouse weight, colon length, and bloody stools.

[0067] The process of testing the weight change, colon length, and blood in stool of mice included randomly dividing the mice into 4 groups: Control, PBS + 2.5% DSS, 5-ASA (30 mg / kg) + 2.5% DSS, and HMPB / Mn3O4 (10 mg / kg) + 2.5% DSS. The mice in the experimental group were fed 2.5% DSS for 7 consecutive days (0-6 days), and the drug-treated group was orally gavaged on days 0, 2, 4, and 6, and the mice were killed on day 7. The weight of each mouse was recorded every day starting from day 0, and the mice were dissected on day 7 to record the colon length and blood in stool of each group of mice.

[0068] The results of the test of the new multi-enzyme nanozyme to prevent ulcerative colitis in mice are as follows Figure 5-6 As shown in the figure, it can be seen that the HMPB / Mn3O4 nanozyme provided by the present invention can improve the relevant symptoms of DSS-induced ulcerative colitis in mice (including bloody stools, weight loss and shortened colon in mice), and is expected to become a new potential drug for preventing ulcerative colitis.

[0069] Test Example 4

[0070] In Example 4 of the present invention, the HMPB / Mn3O4 nanozyme prepared in Example 1 was subjected to a pre-treatment experimental test on ulcerative colitis in mice. The tested mice were 6-8 week old C57BL / 6 male mice, and the test indicators included changes in mouse weight and colon length.

[0071] The process of testing the weight change and colon length of mice included randomly dividing the mice into 4 groups: Control, PBS + 2.5% DSS, 5-ASA (30 mg / kg) + 2.5% DSS, HMPB / Mn3O4 (10 mg / kg) + 2.5% DSS. The mice in the experimental group were fed with 2.5% DSS for 7 consecutive days (0-6 days), and the mice in the drug-treated group were orally gavaged on days 7, 9, and 11, and the mice were killed on day 12. The weight of each mouse was recorded every day starting from day 0, and the colon length of each group of mice was recorded by dissecting the mice on day 12.

[0072] Results of testing of novel nanozymes with multiple enzyme activities to treat ulcerative colitis in mice Figure 7 As shown in the figure, it can be seen that the HMPB / Mn3O4 nanozyme provided by the present invention can improve the relevant symptoms of DSS-induced ulcerative colitis mice (including weight loss and colon shortening), and is expected to become a new potential drug for the treatment of ulcerative colitis.

[0073] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A HMPB / Mn3O4 nanozyme with multi-enzyme activity, characterized in that: The nanozyme is HMPB with nano-Mn3O4 loaded on the inner and outer surfaces.

2. The HMPB / Mn3O4 nanozyme with multi-enzyme activity according to claim 1, characterized in that: The mass ratio of Mn3O4 to HMPB is 1-5:3-15.

3. The HMPB / Mn3O4 nanozyme with multi-enzyme activity according to claim 1, characterized in that: The multi-enzyme comprises four enzymes: catalase-like enzyme, superoxide dismutase, peroxidase and glutathione peroxidase.

4. The method for preparing the HMPB / Mn3O4 nanozyme with multi-enzyme activity according to claim 1, characterized in that: Nano-Mn3O4 and HMPB are dispersed in anhydrous ethanol and stirred evenly. The resulting solution is washed by centrifugation and dried to obtain HMPB / Mn3O4 nanozyme.

5. The method for preparing the HMPB / Mn3O4 nanozyme with multi-enzyme activity according to claim 4, characterized in that: The stirring temperature is 25°C and the stirring time is 24-36h.

6. The method for preparing the HMPB / Mn3O4 nanozyme with multi-enzyme activity according to claim 4, characterized in that: The centrifugal time is 15 minutes and the rotation speed is 11000 r / min.

7. The method for preparing the HMPB / Mn3O4 nanozyme with multi-enzyme activity according to claim 4, characterized in that: The drying temperature is 60-70°C and the drying time is 5-24h.

8. Use of the HMPB / Mn3O4 nanozyme with multi-enzyme activity as claimed in claim 1 as a drug for the treatment of ulcerative colitis.

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