CN116173071B - Preparation method and application of MnNiPCN@PVP nano enzyme - Google Patents
Preparation method and application of MnNiPCN@PVP nano enzyme Download PDFInfo
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 15
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 239000003814 drug Substances 0.000 claims description 11
- 239000011780 sodium chloride Substances 0.000 claims description 11
- 229910016897 MnNi Inorganic materials 0.000 claims description 9
- 229940079593 drug Drugs 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 5
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims description 5
- 229910021586 Nickel(II) chloride Inorganic materials 0.000 claims description 5
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- 239000011565 manganese chloride Substances 0.000 claims description 5
- 229940099607 manganese chloride Drugs 0.000 claims description 5
- QMMRZOWCJAIUJA-UHFFFAOYSA-L nickel dichloride Chemical compound Cl[Ni]Cl QMMRZOWCJAIUJA-UHFFFAOYSA-L 0.000 claims description 5
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 4
- 238000010304 firing Methods 0.000 claims description 4
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
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- 238000001291 vacuum drying Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- 229960000633 dextran sulfate Drugs 0.000 claims description 2
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- 206010009887 colitis Diseases 0.000 claims 1
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- TVZRAEYQIKYCPH-UHFFFAOYSA-N 3-(trimethylsilyl)propane-1-sulfonic acid Chemical compound C[Si](C)(C)CCCS(O)(=O)=O TVZRAEYQIKYCPH-UHFFFAOYSA-N 0.000 description 4
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- NBGAYCYFNGPNPV-UHFFFAOYSA-N 2-aminooxybenzoic acid Chemical class NOC1=CC=CC=C1C(O)=O NBGAYCYFNGPNPV-UHFFFAOYSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
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- 241001465754 Metazoa Species 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
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- 230000008439 repair process Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/44—Elemental carbon, e.g. charcoal, carbon black
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/32—Manganese; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Pharmacology & Pharmacy (AREA)
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- Animal Behavior & Ethology (AREA)
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- Optics & Photonics (AREA)
- Nanotechnology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Nutrition Science (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a preparation method and application of MnNiPCN@PVP nano enzyme, which can effectively remove ROS in cells/bodies, thereby relieving inflammation of lesion sites, promoting anti-inflammatory immune response and having great significance in treating Inflammatory Bowel Disease (IBD). The potential toxicity of the nano material can be reduced by oral administration, and the PVP can reduce the absorption of the gastrointestinal tract after encapsulation so as to reach the colon part.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method of MnNiPCN@PVP nano enzyme and application of the MnNiPCN@PVP nano enzyme in preparation of a medicament for treating ulcerative colitis.
Background
Inflammatory Bowel Disease (IBD) is a chronic gastrointestinal inflammation that can cause an imbalance in intestinal microorganisms, an impaired intestinal mucosal barrier, and a reduced immune function. At present, antibiotics, aminosalicylates, corticosteroids and other medicaments can reduce inflammation and repair intestinal barriers. However, since the drug cannot form specific targeted therapy on the colon, the drug cannot be effectively delivered to the inflammatory region, so that the curative effect of the drug is reduced, and adverse reactions are generated on other organs. Therefore, there is increasing interest in exploring SOD artificial enzymes suitable for use in vivo.
The nano-enzyme is used as a novel artificial enzyme, which not only can show the unique property of nano-materials, but also can simulate various activities of natural enzymes. More importantly, some studies indicate that new choices of anti-inflammatory therapies can be obtained with nanoenzymes having SOD-like activity through appropriate strategies. However, the application of nano-enzymes with SOD-like activity still faces many limitations such as poor biocompatibility, weak acid tolerance, unsatisfactory catalytic activity, etc.
Disclosure of Invention
In order to solve the technical problems, the invention constructs the MnNiPCN@PVP nano enzyme. Scavenging Reactive Oxygen Species (ROS) is more efficient than non-catalytic antioxidants, while being more stable than free enzymes. Here, mnNiPCN nanoparticles are coated with polyvinylpyrrolidone (PVP) to improve their biocompatibility and acid resistance. Thereby reducing its potential nanotoxicity. We show that MnNiPCN@PVP is stable in the stomach when orally administered, and can clear excessive ROS at colon lesions, thereby alleviating inflammation at the lesions, promoting anti-inflammatory immune response, and finally effectively treating sodium dextran sulfate (DSS) -induced IBD in a mouse model.
In order to achieve the technical purpose, the invention is realized by the following technical scheme:
the preparation method of the MnNiPCN@PVP nano enzyme comprises the following steps:
s1: gradually adding NaCl, manganese chloride, nickel chloride, glucose and urea into deionized water, stirring and dissolving to obtain a clear solution, and then freeze-drying;
s2: heating the mixture to 800 ℃ in a tubular furnace at a heating rate of 5 ℃/min; firing for 2 hours in argon atmosphere;
s3: finally, washing for many times by using distilled water, removing the NaCl template, and vacuum drying to obtain the three-dimensional MnNi/N-doped porous carbon nano-sheet-MnNi/N@PCN;
s4: mnNi/N@PCN is added to ethanol containing PVP; after ultrasonic treatment for 30min, stirring for 2h, and drying at 60 ℃ overnight to finally obtain the product MnNiPCN@PVP.
Preferably, the mass ratio of NaCl, manganese chloride, nickel chloride, glucose and urea is as follows: 7:0.66:0.44:1:1;
preferably, the solid-to-liquid ratio of the MnNi/N@PCN to PVP is 1:1;
preferably, the heating temperature of S2 is up to 800 ℃, and the firing is carried out for 2 hours under the argon atmosphere; s3, drying at 60 ℃ for 24 hours; and S4, carrying out ultrasonic treatment for 30min, stirring for 2h, and drying at 60 ℃ overnight.
The beneficial effects of the invention are as follows:
(1) The nano-enzyme preparation method is simple, does not need complex instruments and equipment, has low cost and is expected to be used for industrial production.
(2) The oral nano-drug can effectively remove the ROS in cells/bodies, thereby relieving the inflammation of lesion parts, promoting anti-inflammatory immune response and having important significance for treating Inflammatory Bowel Disease (IBD).
(3) The oral nano-drug can reduce the potential toxicity of nano-materials by an oral administration mode, and can reduce the absorption of gastrointestinal tracts after PVP encapsulation so as to reach colon parts.
Drawings
FIG. 1 is a graph showing the results of treatment of mice with the prepared nanoenzyme; (a) colonography of different treatments (B) colonography HE staining of different treated mice (C) colonography length results of different treatments (D) weight profile of nano-drug treated mice;
FIG. 2 is a graph of the results of biocompatibility of a nano-drug; (A) blood routine results chart (B) blood biochemical analysis results chart (C) results chart of hemolysis experiment (D) HE staining chart of mice heart liver, spleen, lung and kidney.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The preparation method of the MnNiPCN@PVP nano enzyme specifically comprises the following steps:
preparation of mnnipcn@pvp: briefly, 3.5g NaCl,0.33g manganese chloride, 0.218g nickel chloride, 0.5g glucose, 0.5g urea were gradually added to 12.5mL deionized water, dissolved with stirring to give a clear solution, and then freeze-dried. Subsequently, the mixture was heated to 800 ℃ in a tube furnace at a heating rate of 5 ℃/min. Firing for 2 hours under argon atmosphere. Finally, washing for multiple times by using distilled water, removing the NaCl template, and vacuum drying at 60 ℃ for 24 hours to obtain the three-dimensional MnNi/N-doped porous carbon nano-sheet (MnNi/N@PCN). 200mg of MnNi/N@PCN was added to 60 ml of ethanol containing 200mg PVP. After ultrasonic treatment for 30min, stirring for 2h, and drying at 60 ℃ overnight to finally obtain the product MnNiPCN@PVP.
Example 2
Based on MnNiPCN@PVP prepared in example 1, the MnNiPCN@PVP is used for animal experiments of enteritis mouse models;
(1) Cell viability assay: RAW264.7 (mouse macrophage) was attached to a 96-well plate at a density of 1X 104 for 24h, and then nanoenzymes (MnNiPCN@PVP) at different concentrations (1-100. Mu.g mL-1) were introduced for further incubation for 24h. Cell viability was determined using CCK-8 according to the manufacturer's instructions.
(2) Intracellular ROS scavenging ability: will have a density of 1X 10 6 Individual cells/well of RAW264.7 cells were seeded in 6 well plates for 24h, then suspensions of the materials at different concentrations were incubated for 12h, 200ng/mLLPS was introduced into the RAW264.7 cells, and incubated for 2h. Intracellular ROS were detected with fluorescent probe 5. Mu. MDFH-DA and analyzed by flow cytometry.
(3) Firstly, establishing a mouse colonitis model, namely, 28g-30g male SPFKM mice are randomly divided into three groups, and the mice are fed with 5% (w/v) DSS for 7 days every day. The control group only provided normal water to healthy mice. After the model is established, 2mgkg of the injection is orally injected into the mice -1 Mnnipcn@pvp or PBS. Body weight was assessed daily during the 11 day trial periodIs a variation of (c). On the last day of the experiment, mice were sacrificed and the entire colon and organs were resected. The colon length was measured and gently rinsed with physiological saline.
(4) Biocompatibility: normal male mice were divided into two groups of 3 mice each, and normal saline and mnnipcn@pvp were intravenously injected every other day. After 7d, mice were sacrificed and heart, liver, spleen, lung, kidney and other major organs were HE stained. The blood of the mice is collected at the same time for routine examination and biochemical analysis. Hemolysis analysis one mouse blood was collected and Red Blood Cells (RBCs) were collected by centrifugation at 1500rpm for 15 minutes and washed three times with physiological saline. The 0.3mLRBC saline suspension (5%) was then resuspended in 6mL saline. Erythrocyte suspensions (0.1 mL) were added to 1mL of different concentrations (3.125, 6.25, 12.5, 25, 50 and 100. Mu.gmL -1 ) Is added to the aqueous dispersion of physiological salt. The mixture was incubated at 37℃for 3 hours. Ultrapure water and saline were used as positive and negative controls, respectively. Finally, after centrifugation at 12000rpm for 15 minutes, the absorbance of the supernatant was measured at 540 nm.
The invention is further described below with reference to the accompanying drawings.
(1) Fig. 1, panel a shows a graph of colon effect after experimental treatment, panel C shows a statistical graph of colon length, from which it can be seen that the colon length of mice in the material group is restored to be comparable to that in the control group, and panel D shows a graph of HE staining results of the colon, from which it can be seen that DSS induces irregular, damaged, inflammatory cell infiltration of submucosal epithelium in mice. After treatment, these lesions were all significantly improved, comparable to the control group. At the same time, the E plot shows that the body weight during the treatment period also returns to be comparable to the control group.
(2) In FIG. 2, the results of analysis of mouse blood in panel A show that the white blood cell number and the red blood cell number of DSS group are higher, the difference of biochemical results of blood is not great in panel B and C, the hemolytic experiment result is better, the material concentration is 100 mug/mL, and the hemolysis rate is about 5%. Graph D shows no significant differences between the control and experimental groups, indicating that the nanoparticle has no significant systemic toxicity.
In the description of the present specification, the descriptions of the terms "one embodiment," "example," "specific example," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended only to assist in the explanation of the invention. The preferred embodiments are not exhaustive or to limit the invention to the precise form disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.
Claims (2)
- The preparation method of the MnNiPCN@PVP nano enzyme is characterized by comprising the following steps of:s1: gradually adding NaCl, manganese chloride, nickel chloride, glucose and urea into deionized water, stirring and dissolving to obtain a clear solution, and then freeze-drying;s2: heating the mixture in a tubular furnace at a heating rate of 5 ℃/min; firing under argon atmosphere;s3: finally, washing for many times by using distilled water, removing the NaCl template, and vacuum drying to obtain the three-dimensional MnNi/N-doped porous carbon nano-sheet-MnNi/N@PCN;s4: mnNi/N@PCN is added to ethanol containing PVP; stirring after ultrasonic treatment, and drying overnight to finally obtain a product MnNiPCN@PVP;the mass ratio of NaCl, manganese chloride, nickel chloride, glucose and urea is as follows: 7:0.66:0.44:1:1;the solid-liquid ratio of the MnNi/N@PCN to PVP is 1:1;the S2 is heated to 800 ℃ and is fired for 2 hours in argon atmosphere; s3, drying at 60 ℃ for 24 hours; and S4, carrying out ultrasonic treatment for 30min, stirring for 2h, and drying at 60 ℃ overnight.
- 2. The preparation method of the MnNiPCN@PVP nano enzyme according to claim 1, wherein the prepared MnNiPCN@PVP nano enzyme is applied to preparation of a sodium dextran sulfate induced colitis drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN202310129709.0A CN116173071B (en) | 2023-02-17 | 2023-02-17 | Preparation method and application of MnNiPCN@PVP nano enzyme |
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| CN103303912A (en) * | 2013-07-05 | 2013-09-18 | 黑龙江大学 | Preparation method of high-specific-surface-area porous nitrogen-doped graphitizing carbon nanomaterial |
| CN107633952A (en) * | 2017-09-21 | 2018-01-26 | 北京化工大学 | A kind of nickel manganese composite oxide nano sheet film materials and its preparation method and application |
| CN111785947A (en) * | 2020-07-23 | 2020-10-16 | 合肥国轩高科动力能源有限公司 | Composite negative electrode material and preparation method and application thereof |
| CN111773243A (en) * | 2020-07-17 | 2020-10-16 | 南京大学 | Medicine for inflammatory bowel disease and preparation method and application thereof |
| CN114345349A (en) * | 2022-01-07 | 2022-04-15 | 南开大学 | Preparation method and application of bimetallic nanoenzyme |
| CN115548265A (en) * | 2022-09-01 | 2022-12-30 | 天津大学 | Preparation method of self-supporting three-dimensional porous carbon embedded nanocrystalline active material composite foam |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103303912A (en) * | 2013-07-05 | 2013-09-18 | 黑龙江大学 | Preparation method of high-specific-surface-area porous nitrogen-doped graphitizing carbon nanomaterial |
| CN107633952A (en) * | 2017-09-21 | 2018-01-26 | 北京化工大学 | A kind of nickel manganese composite oxide nano sheet film materials and its preparation method and application |
| CN111773243A (en) * | 2020-07-17 | 2020-10-16 | 南京大学 | Medicine for inflammatory bowel disease and preparation method and application thereof |
| CN111785947A (en) * | 2020-07-23 | 2020-10-16 | 合肥国轩高科动力能源有限公司 | Composite negative electrode material and preparation method and application thereof |
| CN114345349A (en) * | 2022-01-07 | 2022-04-15 | 南开大学 | Preparation method and application of bimetallic nanoenzyme |
| CN115548265A (en) * | 2022-09-01 | 2022-12-30 | 天津大学 | Preparation method of self-supporting three-dimensional porous carbon embedded nanocrystalline active material composite foam |
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