CN114081897B - Selenium-doped Prussian blue nanoenzyme for regulating intestinal cells to treat colitis and preparation method and application thereof - Google Patents
Selenium-doped Prussian blue nanoenzyme for regulating intestinal cells to treat colitis and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a selenium-doped Prussian blue nano enzyme for regulating intestinal cells to treat colitis and a preparation method and application thereof. The selenium-doped Prussian blue nanoenzyme has good active oxygen scavenging capacity and the functions of inhibiting lipid peroxidation of intestinal epithelial cells and T cell differentiation, and can be used for relieving intestinal inflammatory diseases.
Description
Technical Field
The invention belongs to the technical field of nano materials, and particularly relates to a preparation method of selenium-doped Prussian blue nano enzyme for regulating intestinal cells to treat colitis.
Background
Ulcerative Colitis (UC) is a chronic inflammatory bowel disease of unknown etiology affecting the colon and rectum. UC occurs due to multiple factors, including genetic background, environment and mucus immunoregulation imbalance, which are all important causes of intestinal inflammation. Chronic inflammation in patients with colitis can lead not only to abdominal pain and diarrhea, but also to disruption of the gastrointestinal epithelial barrier, dysregulation of cell types and intestinal microflora in the colon, the appearance of which can significantly reduce the quality of life for patients with inflammatory bowel disease. UC has now developed as a global burden, with a great impact on developed countries and a greatly increased impact on developing countries. Current clinical interventions for inflammatory bowel disease mainly involve oral or intravenous injection of small molecule drugs (such as salicylates), corticosteroids, and immunosuppressants. Although these treatments may cure a subset of patients, they are not well suited for treatment of patients who develop resistance to colorectal hyperplasia or cancer associated with colitis. In order to solve this troublesome medical problem, development of a novel therapeutic method for safely and reliably treating inflammatory bowel disease has received high attention from a large number of researchers.
Reactive Oxygen Species (ROS) are a class of oxygen-containing one-electron by-products generated by oxygen molecules during cellular respiration and organism metabolism, including superoxide radicals, hydroxyl radicals, hydrogen peroxide, and the like. Although the etiology and pathogenesis of inflammatory bowel disease is not well defined, numerous studies have demonstrated excessive accumulation of ROS at inflammatory sites in the colon. Many animal and clinical studies have demonstrated that high levels of ROS result in oxidative damage to proteins, lipids, and DNA, further resulting in increased mucosal damage, ulceration of the mucosa, and exacerbation of intestinal inflammation. Therefore, strategies that are effective in scavenging ROS have received a great deal of attention. Some nano-drugs loaded with ROS-scavenging enzymes have been successfully developed and applied for intracolonic administration for treatment of colitis. Despite achieving a certain therapeutic effect, the inherent disadvantages of natural enzymes have prevented their further development and use. Recently, nanoenzymes with ROS scavenging ability have gained new applications in the field of medical materials, which have diverse mimic enzyme activities, helping to protect tissues from oxidative damage of endogenous activities and promoting tissue regeneration. In addition, the nano enzyme also has the advantages of simple preparation, high stability, good catalytic activity and the like, and the nano enzyme can gradually become a substitute of the traditional enzyme and the natural enzyme. Prussian Blue (PB) is a nano enzyme with more application prospects in recent years, and due to good enzyme simulation properties of Catalase (CAT) and superoxide dismutase (SOD), the Prussian Blue (PB) can effectively remove ROS, and has good application in various inflammatory diseases. However, PB alone scavenges reactive oxygen species, which are difficult to deal with in complex immune cell imbalances at the site of colitis.
Disclosure of Invention
In order to solve the problems of inflammatory reaction of various cells at the inflammatory part of the colon, high preparation cost, low catalytic activity and the like of the traditional nano enzyme, the invention constructs the selenium-doped Prussian blue nano enzyme for regulating intestinal cells to treat enteritis.
In order to solve the technical problem, the invention adopts the following technical scheme:
the invention firstly discloses a selenium-doped Prussian blue nano enzyme for regulating intestinal cells to treat enteritis, which is characterized in that: the selenium-doped Prussian blue nano enzyme takes hollow mesoporous Prussian blue as a core, and selenium element is doped on the surface of the hollow mesoporous Prussian blue. The particle size of the selenium-doped Prussian blue nano enzyme is 120-140 nm. On the basis of the existing Prussian blue, the invention further improves the synthesis method to enable the Prussian blue to be in a hollow mesoporous structure, and a layer of selenium is covered on the outer layer through a hard template method, so that the Prussian blue contains the activity of glutathione peroxidase (GPx), and the enzyme can play the potential of reversing the lipid peroxidation process of intestinal epithelial cells like GPX4 enzyme of the cells. Meanwhile, selenium is an essential trace mineral, and is incorporated into proteins to form selenoproteins, which can inhibit the differentiation of T cells into CD4 + T, relieving intestinal inflammation.
Further, the valence state of the selenium element doped on the surface is 0 valence state.
The preparation method of the selenium-doped Prussian blue nano enzyme comprises the following steps:
(1) Dissolving 2.5-3.5g PVP in 30mL of HCL solution with the concentration of 0.01-0.05M, adding 100-150mg of potassium ferricyanide powder, and stirring until the mixture is uniformly mixed; placing the obtained mixed solution in an oven to react for 24 hours at the temperature of 80 ℃; after the reaction is finished, centrifuging, washing with water, and freeze-drying to obtain Prussian blue powder;
(2) Dissolving 20-30mg Prussian blue powder in 30mL of HCL solution with the concentration of 0.8-1M, adding 200-300mg PVP, stirring at room temperature for 3-3.5h, and placing in an oven for reaction at 140 ℃ for 3h; after the reaction is finished, centrifuging, washing and freeze-drying to obtain hollow mesoporous Prussian blue powder;
(3) Adding 4-6mL of 2mg/mL sodium selenite solution into 2mL hollow mesoporous Prussian blue aqueous dispersion, stirring at normal temperature for reaction for 20-30min, slowly adding 8-12mL of 2mg/mL ascorbic acid solution, and reacting at normal temperature for 3h; after the reaction is finished, centrifuging, dialyzing for 24h in a dialysis bag with Mw =3500Da, and freeze-drying to obtain selenium-doped Prussian blue nano-enzyme powder; wherein the mass ratio of the ascorbic acid to the sodium selenite is 2.
The selenium-doped Prussian blue nano enzyme has the following characteristics: not only can remove hydroxyl free radicals, but also has the function of glutathione peroxidase; no obvious hemolytic behavior and cytotoxicity; has the capability of eliminating various active oxygen; the selenium element can inhibit lipid peroxidation of epithelial cells of the intestinal tract and restore the barrier of the intestinal tract; the selenium element can inhibit T cell differentiation and reduce T cell mediated inflammatory reaction.
The selenium-doped Prussian blue nanoenzyme has the function of regulating intestinal cells, has no obvious toxicity to mammalian cells, has good biological safety, and is used for preparing the nanoenzyme for regulating the intestinal cells and treating colitis.
The mechanism for regulating intestinal tract cells to treat colitis by using the selenium-doped Prussian blue nanoenzyme provided by the invention is as follows: prussian blue has good mimic enzyme properties of Catalase (CAT), superoxide dismutase (SOD) and the like, can effectively remove ROS, and has good application in various inflammatory diseases. Selenium (Se) is an essential nutrient for human body and has good biocompatibility. In addition, selenium, which is an important component of glutathione peroxidase (GPx), has high GPx enzyme activity, and can exert the potential of reversing lipid peroxide process like GPX4 enzyme of cells. Meanwhile, selenium is an essential trace mineral, and is incorporated into proteins to form selenoproteins, which can inhibit the differentiation of T cells into CD4 + T, relieving intestinal inflammation.
The invention has the following beneficial effects:
1. according to the invention, the selenite is reduced on the surface of the hollow mesoporous Prussian blue to form the monovalent selenium element, the method is simple, and the obtained selenium-doped Prussian blue nanoenzyme has good stability and dispersibility.
2. Compared with the single prussian blue, the selenium-doped prussian blue nano enzyme further improves the capacity and variety of eliminating active oxygen, and can be used for treating various inflammatory diseases.
3. The selenium-doped Prussian blue nanoenzyme has the potential of inhibiting lipid peroxidation of intestinal epithelial cells and T cell differentiation, and can be used for relieving intestinal inflammatory diseases.
4. The selenium-doped Prussian blue nanoenzyme is used as a novel nanoenzyme capable of regulating intestinal cells to relieve enteritis, and remarkably relieves the enteritis in a DSS (dextran sulfate) induced prevention and delay colitis model and a TNBS (trinitrobenzenesulfonic acid) induced rat Crohn's disease model.
5. The nano enzyme has good biological safety and is beneficial to clinical use.
Drawings
FIG. 1 is a schematic synthesis of the present invention.
Fig. 2 is a transmission electron micrograph of the selenium-doped prussian blue nanoenzyme prepared in example 1.
FIG. 3 is an X-ray photoelectron spectrum of the selenium-doped Prussian blue nanoenzyme prepared in example 1.
Fig. 4 is a diagram of in vitro hydroxyl radical scavenging of the selenium-doped prussian blue nanoenzyme prepared in example 1.
Fig. 5 is a diagram of the selenium-doped prussian blue proteasome external mimic glutathione peroxidase prepared in example 1.
Fig. 6 is a graph of cellular reactive oxygen species staining of the selenium-doped prussian blue nanoenzyme prepared in example 1.
FIG. 7 is a graph of cell death and staining of selenium-doped Prussian blue nanoenzymes prepared in example 1.
Fig. 8 is a cell inhibitory lipid peroxide map of the selenium-doped prussian blue nanoenzyme prepared in example 1.
FIG. 9 is a hemolysis experiment diagram of the selenium-doped Prussian blue nanoenzyme prepared in example 1.
Fig. 10 is a diagram of MTT experiment of the selenium-doped prussian blue nanoenzyme prepared in example 1.
Fig. 11 is a statistical graph of colon length after prevention and treatment of colitis in a mouse by selenium-doped prussian blue nanoenzymes prepared in example 1.
Fig. 12 is a statistical plot of colon length after delayed treatment of colitis in mice with selenium-doped prussian blue nanoenzymes prepared in example 1.
Fig. 13 is a flow chart of the selenium-doped prussian blue nanoenzyme prepared in example 1 for treatment of crohn's disease in rats.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, embodiments accompanying examples are described in detail below. The following is merely exemplary and illustrative of the inventive concept and those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.
Example 1
In this example, selenium-doped prussian blue nanoenzyme was prepared as follows:
(1) Dissolving 3.0g of PVP in 30mL of HCL solution with the concentration of 0.01M, adding 132mg of potassium ferricyanide powder, and stirring until the mixture is uniformly mixed; placing the obtained mixed solution in an oven to react for 24 hours at the temperature of 80 ℃; after the reaction, the mixture was centrifuged at 14000rpm for 10min, washed with water 3 times, and lyophilized to obtain Prussian blue powder (PB NCs).
(2) Dissolving 30mg of Prussian blue powder in 30mL of HCL solution with the concentration of 1M, adding 300mg of PVP, stirring at room temperature for 3h, and placing in an oven for reaction at 140 ℃ for 3h; after the reaction is finished, 14000rpm is carried out, centrifugation is carried out for 10min, water washing is carried out for 3 times, and freeze-drying is carried out, so as to obtain hollow mesoporous Prussian blue powder (HMPB NCs).
(3) Adding 6mg/mL sodium selenite solution with the same volume as the hollow mesoporous Prussian blue aqueous dispersion liquid of 2mg/mL, stirring at normal temperature for reaction for 30 minutes, slowly adding 12mg/mL ascorbic acid solution with the same volume as the hollow mesoporous Prussian blue aqueous dispersion liquid, and reacting at normal temperature for 3 hours; after the reaction, centrifugation, dialysis (dialysis in a dialysis bag with Mw =3500Da for 24 h) and lyophilization were performed to obtain selenium-doped prussian blue nano-enzyme powder (Se-HMPB NCs).
FIG. 2 is a transmission electron microscope image of the selenium-doped Prussian blue nanoenzyme obtained in the present embodiment, and it can be seen from the image that the nanoenzyme has a nanosheet structure, and the diameter is 120-140 nm.
Fig. 3 is an X-ray photoelectron spectrum of the selenium-doped prussian blue nanoenzyme obtained in this example, and it can be seen from the diagram that the valence of selenium element contained in the nanoenzyme is 0.
Fig. 4 is a hydroxyl radical scavenging diagram of the selenium-doped prussian blue nanoenzyme obtained in this example, which is characterized by the following steps: salicylic Acid (SA) is used as a chromogenic substrate, and the ability of the selenium-doped Prussian blue nanoenzyme to remove hydroxyl radicals is detected in an acetic acid/sodium acetate (HAc/NaAc) buffer solution. First 100. Mu.L of 20mM H 2 O 2 With 100. Mu.L of 10mM FeSO 4 ·7H 2 O is added into 400. Mu.L HAc/NaAc buffer solution and mixed for reaction for 10min. Then adding 300 mu L of different materials (PB, HMPB or Se-HMPB) into the reaction system (the final concentration of the materials in the system is 25, 50 or 100 mu g/mL) for reaction for 1h, and setting FeSO 4 +H 2 O 2 Is a positive control. Finally, 100. Mu.L of 18mM SA was added and the reaction was developed in the dark for 15min, and then the UV absorption at 510nm was measured. As can be seen from the figure, the capacity of eliminating hydroxyl radicals of the selenium-doped Prussian blue nanoenzyme with the concentration of 100 mu g/mL is about 65%.
Fig. 5 is a diagram of in vitro simulated glutathione peroxidase of the selenium-doped prussian blue nanoenzyme obtained in the present example, wherein the characterization method comprises the following steps: and (3) detecting the GPx enzyme-like activity of the selenium-doped Prussian blue nano-enzyme in a PBS (phosphate buffer solution) by taking dinitrobenzoic acid (DTNB) as a substrate. First, a sample (PB, HMPB or Se-HMPB) having a final concentration of 100. Mu.g/mL was mixed with GSH having a final concentration of 1mM and H having a final concentration of 2mM 2 O 2 The mixture was reacted for 1h in the dark. Then, 100. Mu.L of the reaction mixture was added to 800. Mu.L of PBSAfter mixing, 100. Mu.L of 1mM DTNB was added, and the mixture was developed in the dark for 10min. The UV absorption at 412nm was determined after centrifugation at 3000rpm for 5 min. As can be seen from the figure, the ability of 100. Mu.g/mL selenium-doped Prussian blue nanoenzymes to consume GSH is about 70%.
Fig. 6 is a cell reactive oxygen species staining chart of the selenium-doped prussian blue nanoenzyme obtained in this example, which is characterized by the following steps: to verify the clear effect of selenium-doped Prussian blue nanoenzymes on intracellular reactive oxygen species, 100. Mu.L (1X 10) per well was seeded in 96-well plates 5 ) And (3) incubating for 24h. The supernatant was aspirated and 3mM H was prepared in DMEM 2 O 2 100 mu L of the prepared solution is added into each well for incubation for 4h. The experiments were divided into the following groups: control (fresh Medium), 3mM H 2 O 2 、100μg/mL PB、100μg/mL Se-HMPB、3mM H 2 O 2 +100μg/mL PB、3mM H 2 O 2 + 100. Mu.g/mL Se-HMPB. After incubation, the supernatant was aspirated, washed three times with PBS, 10. Mu.M DCFH-DA dye solution prepared with DMEM was added to each well, incubated at 37 ℃ for 30min in the dark, washed three times with serum-free medium, and photographed with a fluorescence microscope. As can be seen from the figure, H 2 O 2 The +100 mu g/mL Se-HMPB group has no obvious green fluorescence, which indicates that the Se-HMPB group has better capacity of eliminating active oxygen in cells.
Fig. 7 is a cell death and viability staining chart of the selenium-doped prussian blue nanoenzyme obtained in this example, which is characterized by the following steps: to demonstrate that selenium-doped prussian blue nanoenzymes can protect NCM460 cells by scavenging ROS. After 24H incubation in the above experiment, the supernatant was aspirated and 3mM H in DMEM 2 O 2 100 mu L of the prepared solution is added into each well for incubation for 4h. The experiments were divided into the following groups: control (fresh Medium), 3mM H 2 O 2 、50μg/mL PB、100μg/mL PB、50μg/mL Se-HMPB、100μg/mL Se-HMPB、3mM H 2 O 2 +50μg/mL PB、3mM H 2 O 2 +100μg/mL PB、3mM H 2 O 2 +50μg/mL Se-HMPB、3mM H 2 O 2 + 100. Mu.g/mL Se-HMPB. After the incubation is finished, preparing Calcein-AM/PI dye by PBS, adding 100 mu L of dye into each hole after sucking supernatant, incubating for 30min at 37 ℃ under dark condition, washing for 2 times by PBS, adding 100 mu L of PBS to resuspend cellsFluorescence photography was performed. As can be seen from the figure, 3mM H 2 O 2 The +100 mu g/mL Se-HMPB group has no obvious red fluorescence, which shows that the Se-HMPB group has better protection effect on intestinal epithelial cells.
Fig. 8 is a diagram of cytostatic lipid peroxides of the selenium-doped prussian blue nanoenzyme prepared in this example, which is characterized by the following steps: to demonstrate that selenium-doped prussian blue nanoenzymes can inhibit lipid peroxidation in cells. 1mL (5X 10) of the seed was inoculated into each well of a 24-well plate 5 ) And (3) incubating for 24h. After the incubation, the supernatant was aspirated, 1. Mu.g/mL of LPS was prepared in DMEM, and 1mL of the prepared solution was added to each well for incubation for 12 hours. The experiments were divided into the following groups: control (fresh medium), 1. Mu.g/mL LPS, 100. Mu.g/mL PB, 100. Mu.g/mL Se-HMPB, 1. Mu.g/mL LPS + 100. Mu.g/mL PB, 1. Mu.g/mL LPS + 100. Mu.g/mL Se-HMPB. After incubation, 100. Mu.L of IP cell lysate was added to each well and lysed for 30min on ice. After complete lysis of the cells, the supernatant was collected by centrifugation (12000 g, 10min, 4 ℃). And detecting the MDA level of the cell lysis supernatant by adopting a lipid oxide MDA detection kit. As can be seen from the figure, se-HMPB NCs are effective in reducing intracellular lipid peroxide levels by only 9.2nmol/10 5 cell。
Fig. 9 is a hemolysis experiment chart of the selenium-doped prussian blue nanoenzyme obtained in this example, which is characterized by the following steps: diluting Se-HMPB NCs aqueous dispersion to 25 mug/mL, 50 mug/mL, 100 mug/mL, 150 mug/mL and 200 mug/mL, adding 0.2mL of the solutions with different concentrations into 0.2mL of treated blood (500 mug L of fresh blood and 4.5mL of physiological saline for centrifugal washing for 5-8 times, wherein the centrifugal rotation speed is 3000rpm, the centrifugal time is 10min, discarding supernatant after supernatant is clear and transparent, using physiological saline for constant volume to 5 mL), mixing the solutions with 0.6mL of physiological saline, incubating for 4h at 37 ℃, centrifuging for 10min at 3000rpm, sucking supernatant to measure the absorbance at OD 541nm, and calculating the hemolysis rate. From the figure, the hemolysis rate of the selenium-doped prussian blue nano-enzyme is lower than 5%, which indicates that the biocompatibility of the material is good.
Fig. 10 is an MTT experimental graph of the selenium-doped prussian blue nanoenzyme obtained in this example, which is characterized by: each well was inoculated with 100. Mu.L (1X 10) 4 ) The NCM460 cells of (1), incubated for 24h.The supernatant was aspirated, and Se-HMPB (0, 25, 50, 100, 150, 200, 300. Mu.g/mL) was dispensed in DMEM at various concentrations, 100. Mu.L/well, and incubated for 24h. After the incubation was complete, 20. Mu.L of MTT (5 mg/mL) was added to each well and the incubator was incubated for 4h. Finally, the supernatant was aspirated and the culture was stopped by adding 150 μ L DMSO per well. Incubating at 37 ℃ in dark for 10min, and measuring the absorbance at 490 nm. The graph shows that the cell survival rates of the selenium-doped Prussian blue nano-enzyme are higher than 85%, which indicates that the biological safety of the material is good.
To verify the ability of the selenium-doped prussian blue nanoenzyme obtained in this example to alleviate colitis, the following tests were performed: in the prophylactic treatment colitis model, normal mice were randomized into 6 groups (n =7 per group): (1) PBS + Normal Drinking Water (2) PBS +3% DSS (3) PB (10 mg/kg) +3% DSS (4) Se-HMPB (10 mg/kg) +3% DSS (5) PB (20 mg/kg) +3% DSS (6) Se-HMPB (20 mg/kg) +3% DSS. Experimental group mice were fed with 3% DSS for 7 consecutive days, and the administration group was administered through the tail vein on days 1, 3, and 5, and mice were sacrificed on day nine, and the colon length of each group of mice was recorded. As can be seen from FIG. 11, the colon lengths of the Se-HMPB (20 mg/kg) +3% DSS group mice were all close to 7cm, comparable to the colon lengths of the normal group mice.
To further verify the ability of the selenium-doped prussian blue nanoenzyme obtained in this example to alleviate colitis, the following tests were performed: in the delayed treatment colitis model, normal mice were randomized into 6 groups (n =7 per group): (1) PBS + Normal Drinking Water (2) PBS +3% DSS (3) PB (10 mg/kg) +3% DSS (4) Se-HMPB (10 mg/kg) +3% DSS (5) PB (20 mg/kg) +3% DSS (6) Se-HMPB (20 mg/kg) +3% DSS. Experimental group mice were fed with 3-DSS for 7 consecutive days, administration group was administered through the tail vein on days 9, 11, and 13, mice were sacrificed on day 16, and colon length of each group of mice was recorded. As can be seen from FIG. 12, the colon lengths of the Se-HMPB (20 mg/kg) +3% DSS group mice were all close to 7.3cm, comparable to the colon lengths of the normal group mice.
To further verify the effect of the selenium-doped prussian blue nanoenzyme obtained in this example in inhibiting T cell differentiation to treat crohn's disease, the following tests were performed: in the rat crohn's disease model, normal mice were randomized into 3 groups (n =7 per group): (1) PBS + TNBS (2) PB (10 mg/kg) + TNBS (3) Se-HMPB (10 mg/kg) + TNBS. Large experimental groupMice were enema-treated with TNBS every other day for 14 consecutive days, and the administration groups were administered through tail vein on days 3, 6, 9, 12, and 14, and rats were sacrificed on day 16. Cells were dissected out of the colon ends and extracted and detected on the machine after labeling the cells with Anti-CD3, anti-CD45 and Anti-CD 4. As can be seen from FIG. 13, the Se-HMPB (10 mg/kg) + TNBS group of rats has CD4 + The number of T positive cells accounts for about 11.3% of the total immune cells, which is significantly lower than that of the control group.
The above description is intended to be illustrative of the preferred embodiment of the present invention and should not be taken as limiting the invention, but rather, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
Claims (9)
1. A selenium-doped Prussian blue nano-enzyme for regulating intestinal cells to treat colitis is characterized in that: the selenium-doped Prussian blue nano enzyme takes hollow mesoporous Prussian blue as a core, and selenium element is doped on the surface of the hollow mesoporous Prussian blue; the preparation method of the selenium-doped Prussian blue nano enzyme comprises the following steps:
(1) Dissolving 2.5-3.5g PVP in 30mL of HCL solution with the concentration of 0.01-0.05M, adding 100-150mg of potassium ferricyanide powder, and stirring until the mixture is uniformly mixed; placing the obtained mixed solution in an oven to react for 24 hours at the temperature of 80 ℃; after the reaction is finished, centrifuging, washing with water, and freeze-drying to obtain Prussian blue powder;
(2) Dissolving 20-30mg Prussian blue powder in 30mL of HCL solution with the concentration of 0.8-1M, adding 200-300mg PVP, stirring at room temperature for 3-3.5h, and placing in an oven for reaction at 140 ℃ for 3h; after the reaction is finished, centrifuging, washing and freeze-drying to obtain hollow mesoporous Prussian blue powder;
(3) Adding 4-6mL of 2mg/mL sodium selenite solution into 2mL hollow mesoporous Prussian blue aqueous dispersion, stirring at normal temperature for reaction for 20-30min, slowly adding 8-12mL of 2mg/mL ascorbic acid solution, and reacting at normal temperature for 3h; after the reaction is finished, centrifuging, dialyzing and freeze-drying to obtain selenium-doped Prussian blue nano-enzyme powder; wherein the mass ratio of the ascorbic acid to the sodium selenite is 2.
2. The selenium-doped prussian blue nanoenzyme of claim 1, wherein: the particle size of the selenium-doped Prussian blue nano-enzyme is 120-140 nm, and the valence state of the selenium element doped on the surface is 0 valence.
3. The selenium-doped prussian blue nanoenzyme of claim 1, wherein: the dialysis was performed in a dialysis bag with Mw =3500Da for 24h.
4. The use of the selenium-doped prussian blue nanoenzyme of claim 1 or 2, wherein: used for preparing the nano enzyme for regulating intestinal cells and treating the colitis.
5. Use according to claim 4, characterized in that: the selenium-doped Prussian blue nanoenzyme can not only remove hydroxyl free radicals, but also has the function of glutathione peroxidase.
6. Use according to claim 4, characterized in that: the selenium-doped Prussian blue nanoenzyme has no hemolytic behavior and cytotoxicity.
7. Use according to claim 4, characterized in that: the selenium-doped Prussian blue nanoenzyme has the capability of eliminating active oxygen.
8. Use according to claim 4, characterized in that: the selenium element contained in the selenium-doped Prussian blue nanoenzyme can inhibit lipid peroxidation of epithelial cells of the intestinal tract and restore the barrier of the intestinal tract.
9. Use according to claim 4, characterized in that: the selenium element contained in the selenium-doped Prussian blue nanoenzyme can inhibit T cell differentiation and reduce T cell-mediated inflammatory reaction.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111700909A (en) * | 2020-06-30 | 2020-09-25 | 上海市第六人民医院 | Application of prussian blue in preparation of medicine for treating acute pancreatitis |
| CN112186182A (en) * | 2020-09-10 | 2021-01-05 | 青岛科技大学 | One-dimensional hollow carbon-coated iron selenide nanotube composite electrode material and preparation method thereof |
| CN113116922A (en) * | 2021-03-11 | 2021-07-16 | 浙江大学 | Application of selenium preparation in preparation of medicine for treating Crohn's disease |
| CN113456816A (en) * | 2021-06-10 | 2021-10-01 | 华侨大学 | Self-oxygen-supplying hollow Prussian blue nanoparticles and preparation method and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111700909A (en) * | 2020-06-30 | 2020-09-25 | 上海市第六人民医院 | Application of prussian blue in preparation of medicine for treating acute pancreatitis |
| CN112186182A (en) * | 2020-09-10 | 2021-01-05 | 青岛科技大学 | One-dimensional hollow carbon-coated iron selenide nanotube composite electrode material and preparation method thereof |
| CN113116922A (en) * | 2021-03-11 | 2021-07-16 | 浙江大学 | Application of selenium preparation in preparation of medicine for treating Crohn's disease |
| CN113456816A (en) * | 2021-06-10 | 2021-10-01 | 华侨大学 | Self-oxygen-supplying hollow Prussian blue nanoparticles and preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Nanoenzymes in disease diagnosis and therapy;Zhengze Yu等;《Chem. Commun》;20201231;第15513-15524页 * |
| Nanoparticle-based therapeutics of infammatory bowel diseases: a narrative review of the current state and prospects;Mei Yang等;《Journal of Bio-X Research》;20201215;第157-173页 * |
| Nanozyme-mediated catalytic nanotherapy for inflammatory bowel disease;Jiulong Zhao等;《Theranostics》;20190413;第9卷(第10期);第2843-2855页 * |
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