WO2023082216A1 - Simple preparation method for and application of ultra-small selenium nanoparticle-loaded metal framework composite - Google Patents
Simple preparation method for and application of ultra-small selenium nanoparticle-loaded metal framework composite Download PDFInfo
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- WO2023082216A1 WO2023082216A1 PCT/CN2021/130489 CN2021130489W WO2023082216A1 WO 2023082216 A1 WO2023082216 A1 WO 2023082216A1 CN 2021130489 W CN2021130489 W CN 2021130489W WO 2023082216 A1 WO2023082216 A1 WO 2023082216A1
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- selenium
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- organic framework
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- 239000011669 selenium Substances 0.000 title claims abstract description 167
- 229910052711 selenium Inorganic materials 0.000 title claims abstract description 115
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 31
- 229910052751 metal Inorganic materials 0.000 title claims abstract description 13
- 239000002184 metal Substances 0.000 title claims abstract description 13
- 239000002131 composite material Substances 0.000 title claims description 13
- 239000002105 nanoparticle Substances 0.000 title abstract description 7
- 239000012924 metal-organic framework composite Substances 0.000 claims abstract description 61
- 238000003756 stirring Methods 0.000 claims abstract description 26
- 230000006378 damage Effects 0.000 claims abstract description 25
- 229920002565 Polyethylene Glycol 400 Polymers 0.000 claims abstract description 12
- 239000012266 salt solution Substances 0.000 claims abstract description 9
- 238000010438 heat treatment Methods 0.000 claims abstract description 8
- 238000001816 cooling Methods 0.000 claims abstract description 6
- 230000036542 oxidative stress Effects 0.000 claims abstract description 6
- 230000002000 scavenging effect Effects 0.000 claims abstract description 6
- 208000029028 brain injury Diseases 0.000 claims abstract description 5
- 201000006474 Brain Ischemia Diseases 0.000 claims abstract description 4
- 206010008120 Cerebral ischaemia Diseases 0.000 claims abstract description 4
- 206010008118 cerebral infarction Diseases 0.000 claims abstract description 4
- 239000002994 raw material Substances 0.000 claims abstract description 4
- 239000000463 material Substances 0.000 claims description 58
- 239000000243 solution Substances 0.000 claims description 46
- 239000012621 metal-organic framework Substances 0.000 claims description 34
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 30
- 208000032382 Ischaemic stroke Diseases 0.000 claims description 17
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- KKEYFWRCBNTPAC-UHFFFAOYSA-N Terephthalic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)C=C1 KKEYFWRCBNTPAC-UHFFFAOYSA-N 0.000 claims description 12
- 238000005119 centrifugation Methods 0.000 claims description 12
- 239000013239 manganese-based metal-organic framework Substances 0.000 claims description 12
- 239000000872 buffer Substances 0.000 claims description 9
- QMKYBPDZANOJGF-UHFFFAOYSA-N benzene-1,3,5-tricarboxylic acid Chemical compound OC(=O)C1=CC(C(O)=O)=CC(C(O)=O)=C1 QMKYBPDZANOJGF-UHFFFAOYSA-N 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 7
- XIOUDVJTOYVRTB-UHFFFAOYSA-N 1-(1-adamantyl)-3-aminothiourea Chemical compound C1C(C2)CC3CC2CC1(NC(=S)NN)C3 XIOUDVJTOYVRTB-UHFFFAOYSA-N 0.000 claims description 6
- 229940044631 ferric chloride hexahydrate Drugs 0.000 claims description 6
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 claims description 6
- 238000003760 magnetic stirring Methods 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 5
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 5
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 5
- YSWBFLWKAIRHEI-UHFFFAOYSA-N 4,5-dimethyl-1h-imidazole Chemical compound CC=1N=CNC=1C YSWBFLWKAIRHEI-UHFFFAOYSA-N 0.000 claims description 4
- 239000004809 Teflon Substances 0.000 claims description 4
- 229920006362 Teflon® Polymers 0.000 claims description 4
- 230000003064 anti-oxidating effect Effects 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 4
- 239000005457 ice water Substances 0.000 claims description 4
- 238000007789 sealing Methods 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 238000002525 ultrasonication Methods 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims 3
- 230000000694 effects Effects 0.000 abstract description 8
- 238000000034 method Methods 0.000 abstract description 8
- 230000010410 reperfusion Effects 0.000 abstract description 7
- 230000003647 oxidation Effects 0.000 abstract description 4
- 238000007254 oxidation reaction Methods 0.000 abstract description 4
- 230000006931 brain damage Effects 0.000 abstract description 3
- 231100000874 brain damage Toxicity 0.000 abstract description 3
- 239000007853 buffer solution Substances 0.000 abstract description 3
- 238000000527 sonication Methods 0.000 abstract 1
- 229940091258 selenium supplement Drugs 0.000 description 92
- 150000003254 radicals Chemical class 0.000 description 22
- 210000005013 brain tissue Anatomy 0.000 description 14
- CIHOLLKRGTVIJN-UHFFFAOYSA-N tert‐butyl hydroperoxide Chemical compound CC(C)(C)OO CIHOLLKRGTVIJN-UHFFFAOYSA-N 0.000 description 12
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- 239000003963 antioxidant agent Substances 0.000 description 10
- 206010063837 Reperfusion injury Diseases 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
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- 239000013384 organic framework Substances 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 7
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- 206010029260 Neuroblastoma Diseases 0.000 description 6
- 208000006011 Stroke Diseases 0.000 description 6
- ZKZBPNGNEQAJSX-UHFFFAOYSA-N selenocysteine Natural products [SeH]CC(N)C(O)=O ZKZBPNGNEQAJSX-UHFFFAOYSA-N 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 6
- FDKWRPBBCBCIGA-REOHCLBHSA-N (2r)-2-azaniumyl-3-$l^{1}-selanylpropanoate Chemical compound [Se]C[C@H](N)C(O)=O FDKWRPBBCBCIGA-REOHCLBHSA-N 0.000 description 5
- PMYDPQQPEAYXKD-UHFFFAOYSA-N 3-hydroxy-n-naphthalen-2-ylnaphthalene-2-carboxamide Chemical compound C1=CC=CC2=CC(NC(=O)C3=CC4=CC=CC=C4C=C3O)=CC=C21 PMYDPQQPEAYXKD-UHFFFAOYSA-N 0.000 description 5
- FDKWRPBBCBCIGA-UWTATZPHSA-N D-Selenocysteine Natural products [Se]C[C@@H](N)C(O)=O FDKWRPBBCBCIGA-UWTATZPHSA-N 0.000 description 5
- RJFAYQIBOAGBLC-BYPYZUCNSA-N Selenium-L-methionine Chemical compound C[Se]CC[C@H](N)C(O)=O RJFAYQIBOAGBLC-BYPYZUCNSA-N 0.000 description 5
- RJFAYQIBOAGBLC-UHFFFAOYSA-N Selenomethionine Natural products C[Se]CCC(N)C(O)=O RJFAYQIBOAGBLC-UHFFFAOYSA-N 0.000 description 5
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- 229960001881 sodium selenate Drugs 0.000 description 5
- 235000018716 sodium selenate Nutrition 0.000 description 5
- 239000011655 sodium selenate Substances 0.000 description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
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- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
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- 229960001471 sodium selenite Drugs 0.000 description 3
- 235000015921 sodium selenite Nutrition 0.000 description 3
- 239000011781 sodium selenite Substances 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 206010008089 Cerebral artery occlusion Diseases 0.000 description 2
- 229920001661 Chitosan Polymers 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000013528 artificial neural network Methods 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- 239000010949 copper Substances 0.000 description 2
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- 230000004054 inflammatory process Effects 0.000 description 2
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- 201000007309 middle cerebral artery infarction Diseases 0.000 description 2
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- JULROCUWKLNBSN-UHFFFAOYSA-N selenocystine Chemical compound OC(=O)C(N)C[Se][Se]CC(N)C(O)=O JULROCUWKLNBSN-UHFFFAOYSA-N 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
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- 238000004627 transmission electron microscopy Methods 0.000 description 2
- 239000011701 zinc Substances 0.000 description 2
- PKDBCJSWQUOKDO-UHFFFAOYSA-M 2,3,5-triphenyltetrazolium chloride Chemical compound [Cl-].C1=CC=CC=C1C(N=[N+]1C=2C=CC=CC=2)=NN1C1=CC=CC=C1 PKDBCJSWQUOKDO-UHFFFAOYSA-M 0.000 description 1
- AHDSRXYHVZECER-UHFFFAOYSA-N 2,4,6-tris[(dimethylamino)methyl]phenol Chemical compound CN(C)CC1=CC(CN(C)C)=C(O)C(CN(C)C)=C1 AHDSRXYHVZECER-UHFFFAOYSA-N 0.000 description 1
- 206010069729 Collateral circulation Diseases 0.000 description 1
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- 240000000599 Lentinula edodes Species 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 208000036110 Neuroinflammatory disease Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 description 1
- 102000008114 Selenoproteins Human genes 0.000 description 1
- 108010074686 Selenoproteins Proteins 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- KRSZDIGCQWBYNU-UHFFFAOYSA-N [Mn].[Ru] Chemical compound [Mn].[Ru] KRSZDIGCQWBYNU-UHFFFAOYSA-N 0.000 description 1
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- XLZOVRYBVCMCGL-BPNVQINPSA-L disodium;4-[(z)-[tert-butyl(oxido)azaniumylidene]methyl]benzene-1,3-disulfonate Chemical compound [Na+].[Na+].CC(C)(C)[N+](\[O-])=C\C1=CC=C(S([O-])(=O)=O)C=C1S([O-])(=O)=O XLZOVRYBVCMCGL-BPNVQINPSA-L 0.000 description 1
- QELUYTUMUWHWMC-UHFFFAOYSA-N edaravone Chemical compound O=C1CC(C)=NN1C1=CC=CC=C1 QELUYTUMUWHWMC-UHFFFAOYSA-N 0.000 description 1
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Images
Classifications
-
- 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
- A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
Definitions
- the invention belongs to the field of metal-organic framework materials, and in particular relates to an ultra-small nano-selenium metal-organic framework composite material and a preparation method and application thereof.
- the central nervous system is the centralized control system of the human body, and a slight lesion or injury will cause immeasurable consequences. Stroke is one of the main causes of adult disability in recent years. Due to factors such as high oxygen consumption and lipid-rich brain, many diseases related to oxidative stress damage will occur after injury, which produces harmful reactive nitrogen and oxygen species, such as superoxide and superoxide, triggering A series of biochemical reactions and neuroinflammation.
- Ischemia-reperfusion injury refers to the situation in which the degree of tissue damage increases rapidly after the blood flow is restored to the tissue subjected to ischemia for a certain period of time. Ischemia-reperfusion injury is mainly due to the generation of a large number of reactive oxygen species free radicals after reperfusion. Excessive production of reactive oxygen species can mediate inflammation and immune responses by stimulating the expression of cytokines and adhesion molecules, and eventually lead to further damage from inflammation. , leading to secondary injury of the neural network, eventually leading to ischemia-reperfusion injury.
- Selenium is an essential trace element for the human body.
- selenium is a constituent of several amino acids, such as selenocysteine, selenomethionine and selenocystine. Therefore, selenium has a wide range of physiological and pharmacological properties in the human body, including anti-tumor, anti-oxidation and other aspects.
- selenium enters the human body, it will be converted into selenium-containing amino acids, and finally form selenium-containing proteins to function.
- Selenoproteins work by altering intracellular oxidation levels.
- Metal-organic frameworks have been widely researched and developed for various catalytic reactions due to their excellent catalytic properties. Studies have shown that different metal-organic framework materials have different abilities to mimic antioxidant natural enzymes, and have the potential to be applied to scavenge a large number of free radicals produced by ischemic stroke and reperfusion.
- the purpose of the present invention is to provide a method for preparing ultra-small nano-selenium metal-organic framework composites.
- Another object of the present invention is to provide an ultra-small nanometer selenium metal organic framework composite material.
- Another object of the present invention is to provide the application of ultra-small nanometer selenium metal organic framework composite material in the preparation of medicine for treating ischemic stroke.
- This patent solution provides a method for preparing ultra-small nano-selenium metal-organic framework composite materials, including the following steps
- PEG-SeNPs ultra-small nano-selenium
- the ultra-small nano-selenium metal-organic framework composite material described in step (2) is Se@ZIF-8
- the metal salt solution is a solution of zinc nitrate hexahydrate
- the buffer solution is methanol.
- Step (2) includes: mixing the PEG-SeNPs with a solution in which dimethylimidazole is dissolved, stirring until the PEG-SeNPs are dispersed, then adding polyvinylpyrrolidone, stirring until dissolved, and finally adding zinc nitrate hexahydrate Aqueous solution, stirring evenly, standing still and centrifuging to obtain the Se@ZIF-8, then washing with methanol and resuspending in methanol.
- the ultra-small nano-selenium metal-organic framework composite material described in step (2) is Se@MIL-101, and the metal salt solution is a DMF solution in which ferric chloride hexahydrate and terephthalic acid have been dissolved , the buffer is DMF.
- Step (2) includes: mixing the PEG-SeNPs with a DMF solution in which ferric chloride hexahydrate and terephthalic acid have been dissolved, stirring evenly, then transferring to a Teflon reactor, and reacting at 100-140°C After a certain period of time, the Se@MIL-101 was prepared, washed with DMF solution several times and then resuspended in DMF.
- the ultra-small nano-selenium metal-organic framework composite material described in step (2) is Se@HKUST-1
- the metal salt solution is a DMF solution of copper chloride
- the buffer solution is DMF.
- Step (2) includes: taking the DMF solution of PEG-SeNPs and cupric chloride, mixing and ultrasonically uniform, adding the DMF solution dissolved in trimesic acid to the mixed solution, then adding acetic acid, sealing the mouth with the rubber stopper and heating rapidly Keep it at 130-170°C for a period of time, and then centrifuge after natural cooling to obtain the Se@HKUST-1, then wash and resuspend with DMF solution, and store at low temperature.
- the ultra-small nano-selenium metal-organic framework composite material described in step (2) is Se@Ru- Mn -MOF, and the metal salt solution is MnCl DMF solution and Ru(POPCOOH) DMF Solution, the buffer is DMF;
- Step (2) includes: taking the PEG-SeNPs and the DMF solution of MnCl 2 and mixing them uniformly by ultrasonication, adding the DMF solution of Ru(POPCOOH) 3 into the mixed solution, sealing the mouth with a rubber stopper and heating to 140-180°C, keeping After a period of time, the temperature was naturally lowered to below 100°C and then quenched to room temperature in an ice-water bath, and the solid was collected by centrifugation to prepare the Se@Ru-Mn-MOF, which was then washed and resuspended with DMF and stored at low temperature.
- the centrifugation speed is 1500-2500rpm, and the centrifugation time is 9-11min.
- the present invention also provides the ultra-small nano-selenium metal-organic framework composite material prepared by the above-mentioned preparation method, which is characterized in that the ultra-small nano-selenium is loaded with the metal-organic framework as a carrier.
- nano-selenium metal-organic framework composite material is any one of Se@ZIF-8, Se@MIL-101, Se@HKUST-1, and Se@Ru-Mn-MOF.
- the present invention also provides the application of the ultra-small nano-selenium metal-organic framework composite material prepared by the above-mentioned preparation method in the preparation of drugs for the treatment of ischemic stroke. Stroke application.
- the medicine reverses the damage caused by oxidative stress by scavenging free radicals and anti-oxidation, improves the brain damage caused by cerebral ischemia-reperfusion, and is used for the treatment of ischemic stroke.
- the preparation method of the ultra-small nano-selenium metal-organic framework composite material provided in the examples of the present application has relatively mild reaction conditions and does not require harsh reaction conditions, such as high temperature and high pressure; the reaction steps are simple and clear, and are easy to experiment and large-scale production.
- the four ultra-small nano-selenium metal-organic framework composites in the embodiments of the application have the effect of effectively scavenging free radicals, have good oxidation resistance, and have good repeated scavenging free radicals and scavenging capacity for hydroxyl radicals. It can effectively reverse the damage caused by oxidative stress and improve the brain damage caused by cerebral ischemia-reperfusion, so as to achieve the effect of protecting brain tissue efficiently.
- This application combines metal organic framework nanomaterials with catalytic ability with ultra-small nano-selenium to give full play to the advantages of large specific surface area and high catalytic efficiency of metal organic framework materials.
- the metal-organic framework material can be used as a carrier of ultra-small nano-selenium in the body to promote the absorption and application of ultra-small nano-selenium in the body.
- the metal organic framework material loaded with ultra-small nano-selenium has a better ability to scavenge free radicals than the single ultra-small nano-selenium and the single metal organic framework material at the same concentration. It provides more evidence for the neuroprotective mechanism of nanomaterials and its clinical application in ischemia-reperfusion therapy in the future.
- this application selects four typical metal-organic framework materials to load ultra-small nano-selenium, and innovatively prepares a composite nanomaterial of ultra-small nano-selenium and metal-organic framework materials, which can be used for other metal-organic framework materials.
- Ultra-small nano-selenium provides a reference.
- the application is modified by sodium selenate, sodium selenate, selenocysteine, selenomethionine, chitosan modified nano-selenium, lentinan modified nano-selenium, ultra-small nano-selenium ( PEG-SeNPs) and four metal-organic framework materials were found: sodium selenite, sodium selenate, selenocysteine, selenomethionine, these four inorganic selenium have no antioxidant capacity, chitosan modified nano-selenium, shiitake mushroom Polysaccharide-modified nano-selenium and ultra-small nano-selenium (PEG-SeNPs) have weak antioxidant capacity, and the four separate metal-organic framework materials have low antioxidant capacity, and ultra-small nano-selenium metal-organic framework materials are relatively small Nano-selenium can effectively increase its antioxidant capacity by about 1.3-2.6 times, which provides a reference for other
- Figure 1 shows the transmission electron microscope images and elemental analysis diagrams of four ultra-small nano-selenium metal-organic framework composites; among them, the four ultra-small nano-selenium metal-organic framework composites are: 1Se@ZIF-8, 2Se@MIL-101 , 3Se@Ru-Mn-MOF, 4Se@HKUST-1;
- Figure 2 shows that four kinds of ultra-small nano-selenium metal-organic framework composites reverse free radical damage model to cell damage;
- Fig. 3 is the cell apoptosis diagram of the human neuroblastoma cells treated by the free radical damage model detected by flow cytometry;
- Figure 4 shows the use of a fluorescent microplate reader to detect the level of reactive oxygen species in human neuroblastoma cells after free radical damage by four ultra-small nano-selenium metal-organic framework composites;
- Fig. 5 is a map of brain injury area of mice treated with four kinds of ultra-small nano-selenium metal-organic framework composite materials under the ischemia-reperfusion model.
- nanozymes exhibit high stability, long-lasting catalytic activity, and multifunctional enzymatic properties.
- the treatment of ischemic stroke diseases was performed using nanozymes. This class of biosafe nanozymes has shown promising prospects in the treatment of stroke and related diseases.
- PEG-SeNPs (ultra-small nano-selenium): Add polyethylene glycol (PEG) 400 and selenium powder in a beaker, stir after ultrasonic to make the selenium powder evenly dispersed in PEG 400; transfer the beaker to magnetic stirring On the heater, stir while heating, cool naturally after a period of reaction, transfer to a centrifuge tube for centrifugation, take the upper layer solution after centrifugation, remove unreacted selenium powder, and finally obtain the PEG-SeNPs;
- PEG polyethylene glycol
- PEG-SeNPs Preparation of PEG-SeNPs: Add 10mL PEG 400 to a beaker, then weigh 20mg of selenium powder, and stir for 5 minutes after ultrasonication to completely and evenly disperse the selenium powder in PEG 400; transfer the beaker to a magnetic stirring heater, and heat to The temperature is about 205°C, stir while heating, and cool naturally after reacting for 1 hour. After transferring to a centrifuge tube and centrifuging at a speed of 2000rpm for 10 minutes, take the upper layer solution, remove unreacted selenium powder, and finally obtain PEG-SeNPs. The obtained PEG-SeNPs The concentration is 16-17mM.
- Ultra-small nano-selenium and iron-containing organic framework composite material Se@MIL-101 take 1ml PEG-SeNPs and dissolve 13.42mg ferric chloride hexahydrate and 12.02mg terephthalic acid in 20ml DMF solution, stir evenly, and then Transfer to a Teflon reactor and react at 120°C for 12 hours to prepare Se@MIL-101, wash with DMF 3 times and resuspend in DMF.
- PEG-SeNPs Preparation of PEG-SeNPs: Add 8mL PEG 400 to a beaker, then weigh 17mg of selenium powder, stir for 4 minutes after ultrasonication to completely and evenly disperse the selenium powder in PEG 400; transfer the beaker to a magnetic stirring heater, and heat to The temperature is about 200°C, stirring while heating, reacting for 1 hour, cooling naturally, transferring to a centrifuge tube, centrifuging at a speed of 1800rpm for 11min, taking the upper layer solution, removing unreacted selenium powder, and finally obtaining PEG-SeNPs, the obtained PEG-SeNPs The concentration is 16-20mM.
- the organic framework composite material Se@Ru-Mn-MOF of ultra-small nano-selenium and manganese ruthenium take 0.9ml of PEG-modified PEG-SeNPs and 4.5ml of DMF dissolved in 0.04mmol MnCl 2 and mix it evenly with ultrasonic, and mix it with Add 4.5ml of DMF solution with 0.04mmol Ru(POPCOOH) 3 dissolved in the solution, seal the mouth with a rubber stopper, heat to 155°C, keep it for 11min, cool down naturally to below 95°C, and then cool to room temperature in an ice-water bath, centrifuge at 7500rmp for 6min The solid was collected to prepare Se@Ru-Mn-MOF, which was washed and resuspended with an equal volume of DMF and placed at 4°C for use.
- the four ultra-small nano-selenium metal-organic framework composites prepared by the method in Example 2 were subjected to the following experiments.
- the four ultra-small nano-selenium metal-organic framework composites are (the same below): Se@ZIF-8, Se@MIL-101, Se@Ru-Mn-MOF, Se@HKUST-1.
- the transmission electron microscope images and elemental analysis images of the four ultra-small nano-selenium metal-organic framework composite materials shown in Figure 1 show that the four metal-organic framework materials loaded with ultra-small nano-selenium were successfully prepared.
- 1 corresponds to Se@ZIF-8
- 2 corresponds to Se@MIL-101
- 3 corresponds to Se@Ru-Mn-MOF
- 4 corresponds to Se@HKUST-1.
- Sodium selenite, sodium selenate, selenocysteine, selenomethionine, chitosan-modified nano-selenium, lentinan-modified nano-selenium, ultra-small nano-selenium (PEG-SeNPs) ) and the antioxidant capacity of four nano-selenium metal-organic framework materials were tested.
- Sodium selenite, sodium selenate, selenocysteine and selenomethionine have no significant antioxidant capacity.
- Chitosan-modified nano-selenium, lentinan-modified nano-selenium, and ultra-small nano-selenium have a small amount of antioxidant capacity; the anti-oxidative properties of four ultra-small nano-selenium metal-organic frameworks formed by compounding with metal-organic framework materials Compared with the single ultra-small nano-selenium (PEG-SeNPs), the oxidation ability is enhanced, and the anti-oxidation ability is about 1.3-2.6 times stronger than that of the single ultra-small nano-selenium, indicating that the addition of metal-organic frameworks can improve the antioxidant capacity of nano-selenium have a significant effect.
- the human brain neuroblastoma cell line (SH-SY5Y) was selected as the object of the cell model, and the free radicals produced by the cells were simulated by adding 20 ⁇ M tert-butyl hydroperoxide, and finally the SH-SY5Y cells were detected by the MTT method
- the survival rate of four ultra-small nano-selenium metal-organic framework composites was used to evaluate the protective effect on cells in the free radical damage SH-SY5Y cell model, and the results are shown in Figure 2.
- the cell survival rate after tert-butyl hydroperoxide damage is about 70%, and the survival rate has increased after adding nano-selenium, while four ultra-small nano-selenium metal organic Compared with the injury group and the single ultra-small nano-selenium group (Se group in the figure), the cell survival rate after the frame composite treatment was significantly increased, indicating that the four nano-selenium metal-organic framework composites can effectively protect cells from cell damage. suffer from free radical damage.
- the human brain neuroblastoma cell line (SH-SY5Y) was selected as the object of the cell model, and the free radicals produced by the cells were simulated by adding 20 ⁇ M tert-butyl hydroperoxide, and finally the SH-SY5Y was detected by flow cytometry.
- the apoptosis ratio of SY5Y cells was used to evaluate the protective effect of four ultra-small nano-selenium metal-organic framework composites on SH-SY5Y cells in the free radical damage model versus cell damage model, and the results are shown in Figure 3.
- the cell survival rate in Figure 3 the cell apoptosis ratio after tert-butyl hydroperoxide damage was about 40%, and the apoptosis ratio decreased after adding ultra-small nano-selenium, while the four Compared with the injured group and the single nano-selenium group, the percentage of cell apoptosis after treatment with ultra-small nano-Se metal-organic framework composites was significantly lower, indicating that the four ultra-small nano-Se metal-organic framework composites can effectively protect cells from suffer from free radical damage.
- the human brain neuroblastoma cell line (SH-SY5Y) was selected as the object of the cell model, and the free radicals produced by the cells were simulated by adding 20 ⁇ M tert-butyl hydroperoxide, and finally the reactive oxygen species in the DCFH-DA cells Detection of fluorescent dyes to detect the level of reactive oxygen species in SH-SY5Y cells.
- the results are shown in Figure 4.
- C57 mice were selected as the experimental subjects, and the middle cerebral artery occlusion (MCAO) operation was used to prepare the stroke ischemia-reperfusion model.
- Reperfusion was accomplished by removing the rubber stopper after occluding the blood flow in the middle cerebral artery of the mouse for 40 min using a silicone stopper.
- TTC 2,3,5-triphenyltetrazolium chloride
- the white area of the brain tissue of the mice in the Saline group was significantly larger than that of the Sham group, and then the white area of the brain tissue of the mice treated with four kinds of nano-selenium metal-organic framework composite materials was significantly smaller than that of the Saline group, indicating that the four ultra-small Nano-selenium metal-organic framework composites can effectively protect mouse brain tissue from damage caused by stroke ischemia-reperfusion.
- the present application explores the therapeutic effect of four ultra-small nano-selenium metal organic framework materials on ischemic stroke reperfusion injury. It has been found through experiments that, compared with metal-organic framework materials or nano-selenium alone, the metal-organic framework material loaded with ultra-small nano-selenium has a better effect on the treatment of ischemic stroke reperfusion injury.
- this application not only demonstrates the preparation method of metal-organic framework materials loaded with nano-selenium, but also provides more evidence for the neuroprotective mechanism of nanomaterials and their future clinical application in ischemia-reperfusion therapy.
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Abstract
An ultra-small selenium nanoparticle-loaded metal-organic framework composite, and a preparation method therefor and an application thereof. The method comprises: adding PEG 400 and selenium powder into a beaker, and stirring after sonication to uniformly disperse the selenium powder in the PEG 400; transferring the beaker to a magnetic stirrer, stirring while heating, naturally cooling after reacting for a certain period of time, transferring into a centrifugal tube for centrifuging, obtaining a supernate after centrifuging, and finally obtaining PEG-SeNPs; and preparing a selenium nanoparticle-loaded metal-organic framework composite by using raw materials comprising the PEG-SeNPs and a metal salt solution, and suspending the selenium nanoparticle-loaded metal-organic framework composite in a buffer solution. The ultra-small selenium nanoparticle-loaded metal-organic framework composite has an effect of effectively scavenging free radicals, and good oxidation resistance, and can effectively reverse damage caused by oxidative stress, and relieve brain damage caused after cerebral ischemia reperfusion.
Description
本发明属于金属有机框架材料领域，尤其涉及超小纳米硒金属有机框架复合材料及其制备方法和应用。The invention belongs to the field of metal-organic framework materials, and in particular relates to an ultra-small nano-selenium metal-organic framework composite material and a preparation method and application thereof.
中枢神经***是人体的集中控制***，轻微的病变或损伤都会造成不可估量的后果。脑卒中是近年来引起成年人残疾的主要原因之一。由于大脑的高耗氧量和富含脂质等因素，在损伤后会产生许多与氧化应激损伤相关的疾病，它产生有害的活性氮和氧物种，如过氧化物和超氧化物，引发一系列的生化反应和神经炎症。The central nervous system is the centralized control system of the human body, and a slight lesion or injury will cause immeasurable consequences. Stroke is one of the main causes of adult disability in recent years. Due to factors such as high oxygen consumption and lipid-rich brain, many diseases related to oxidative stress damage will occur after injury, which produces harmful reactive nitrogen and oxygen species, such as superoxide and superoxide, triggering A series of biochemical reactions and neuroinflammation.
目前，在缺血性脑卒中治疗阶段，早期再通技术的大量使用促使更多目光聚焦于血流再灌注引起的再灌注损伤。缺血再灌注损伤，是指遭受一定时间缺血的组织恢复血流后，组织损伤程度迅速增剧的情况。缺血再灌注损伤主要是因为再灌注后有生成大量活性氧自由基，活性氧自由基的过量产生可通过刺激细胞因子和粘附分子的表达而介导炎症和免疫反应，最终导致炎症进一步损伤，导神经网络的继发性损伤，最终导致缺血再灌注损伤。而即使在非早期再通患者中，由于侧枝循环代偿、自发再通等原因，上述缺血再灌注损伤的机制同样存在。因此，在脑缺血损伤后，特别是缺血性卒中再灌注过程中，降低活性氧自由基脑血管***和神经网络对脑组织的损伤可能是缺血性卒中的治疗靶点。寻找更佳的治疗方法有重要应用前景。At present, in the treatment stage of ischemic stroke, the extensive use of early recanalization technology has prompted more attention to focus on the reperfusion injury caused by blood reperfusion. Ischemia-reperfusion injury refers to the situation in which the degree of tissue damage increases rapidly after the blood flow is restored to the tissue subjected to ischemia for a certain period of time. Ischemia-reperfusion injury is mainly due to the generation of a large number of reactive oxygen species free radicals after reperfusion. Excessive production of reactive oxygen species can mediate inflammation and immune responses by stimulating the expression of cytokines and adhesion molecules, and eventually lead to further damage from inflammation. , leading to secondary injury of the neural network, eventually leading to ischemia-reperfusion injury. Even in patients with non-early recanalization, due to collateral circulation compensation, spontaneous recanalization and other reasons, the above-mentioned mechanism of ischemia-reperfusion injury also exists. Therefore, after cerebral ischemic injury, especially during the reperfusion process of ischemic stroke, reducing the damage of reactive oxygen species free radicals to brain tissue and neural network may be the therapeutic target of ischemic stroke. Finding a better treatment method has important application prospects.
硒是人体必须的微量元素。在人体内，硒是多种氨基酸的组成成分，例如硒代半胱氨酸、硒代蛋氨酸和硒代胱氨酸。因此，硒元素在人体中具有广泛的生理和药理特性，这其中包括抗肿瘤、抗氧化等方面。在人体内，硒元素进入人 体内会转化为含硒的氨基酸，最终形成含硒的蛋白质发挥功能。硒蛋白通过改变细胞内的氧化水平。Selenium is an essential trace element for the human body. In humans, selenium is a constituent of several amino acids, such as selenocysteine, selenomethionine and selenocystine. Therefore, selenium has a wide range of physiological and pharmacological properties in the human body, including anti-tumor, anti-oxidation and other aspects. In the human body, when selenium enters the human body, it will be converted into selenium-containing amino acids, and finally form selenium-containing proteins to function. Selenoproteins work by altering intracellular oxidation levels.
金属有机框架材料由于其优异的催化性能被广泛研究开发用于各种催化反应中。有研究表明，不同的金属有机框架材料具有不同的模拟抗氧化天然酶的能力，具有应用于清除缺血性脑卒中再灌注产生的大量自由基的潜能。Metal-organic frameworks have been widely researched and developed for various catalytic reactions due to their excellent catalytic properties. Studies have shown that different metal-organic framework materials have different abilities to mimic antioxidant natural enzymes, and have the potential to be applied to scavenge a large number of free radicals produced by ischemic stroke and reperfusion.
针对缺血机制的众多血管神经单元保护剂虽然在动物研究中获得很多证据，但均未获得临床研究证实，而不被临床指南推广。但唯有以清除自由基为主要作用机制的依达拉奉基于其在一项多中心临床研究中的证据，获得了日本和中国等亚洲国家的批准和指南推广使用。但由于同样以自由基清除为主要机制的NXY-059III期临床研究以失败而告终，自由基清除剂未获得欧美国家的批准和指南推广。Although many vascular and neural unit protective agents targeting ischemic mechanisms have obtained a lot of evidence in animal studies, none of them have been confirmed by clinical studies and have not been promoted by clinical guidelines. However, only Edaravone, whose main mechanism of action is to scavenge free radicals, has been approved and promoted by guidelines in Asian countries such as Japan and China based on its evidence in a multi-center clinical study. However, due to the failure of the phase III clinical study of NXY-059, which also uses free radical scavenging as the main mechanism, free radical scavenger has not been approved and promoted by European and American countries.
发明内容Contents of the invention
鉴于以上所述现有技术的缺点，本发明的目的在于提供超小纳米硒金属有机框架复合材料的制备方法。In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide a method for preparing ultra-small nano-selenium metal-organic framework composites.
本发明的另一目的在于提供超小纳米硒金属有机框架复合材料。Another object of the present invention is to provide an ultra-small nanometer selenium metal organic framework composite material.
本发明的再一目的在于提供超小纳米硒金属有机框架复合材料在制备治疗缺血性卒中药物的应用。Another object of the present invention is to provide the application of ultra-small nanometer selenium metal organic framework composite material in the preparation of medicine for treating ischemic stroke.
本专利方案提供超小纳米硒金属有机框架复合材料的制备方法，包括以下步骤This patent solution provides a method for preparing ultra-small nano-selenium metal-organic framework composite materials, including the following steps
(1)制备超小纳米硒(PEG-SeNPs)：在烧杯中加入PEG 400和硒粉，超声后搅拌使硒粉均匀分散在PEG 400中；转移所述烧杯至磁力搅拌加热器上，边加热边搅拌，反应一定时间后自然冷却，转移到离心管进行离心处理，离心后取上层溶液，去除未反应的硒粉，最后获得所述PEG-SeNPs；(1) Preparation of ultra-small nano-selenium (PEG-SeNPs): add PEG 400 and selenium powder in a beaker, stir after ultrasonic so that the selenium powder is evenly dispersed in PEG 400; transfer the beaker to a magnetic stirring heater, and heat it While stirring, react for a certain period of time, cool naturally, transfer to a centrifuge tube for centrifugation, take the upper layer solution after centrifugation, remove unreacted selenium powder, and finally obtain the PEG-SeNPs;
(2)制备超小纳米硒金属有机框架复合材料：使用包括有所述PEG-SeNPs和金属盐溶液的原料制得所述纳米硒金属有机框架复合材料，并将所述超小纳 米硒金属有机框架复合材料重悬于缓冲液中。(2) Preparation of ultra-small nano-selenium metal-organic framework composites: use the raw materials comprising the PEG-SeNPs and metal salt solution to prepare the nano-selenium metal-organic framework composites, and prepare the ultra-small nano-selenium metal-organic frameworks Framework composites were resuspended in buffer.
作为第一种实施方式，步骤(2)中所述的超小纳米硒金属有机框架复合材料为Se@ZIF-8，金属盐溶液为六水合硝酸锌的溶液，缓冲液为甲醇。As a first embodiment, the ultra-small nano-selenium metal-organic framework composite material described in step (2) is Se@ZIF-8, the metal salt solution is a solution of zinc nitrate hexahydrate, and the buffer solution is methanol.
步骤(2)包括：取所述PEG-SeNPs与有溶解了二甲基咪唑的溶液混合，搅拌至所述PEG-SeNPs分散，然后加入聚乙烯吡咯烷酮，搅拌至溶解，最后加入六水合硝酸锌的水溶液，搅拌均匀后静置后离心处理，制得所述Se@ZIF-8，然后用甲醇清洗并重悬至甲醇中。Step (2) includes: mixing the PEG-SeNPs with a solution in which dimethylimidazole is dissolved, stirring until the PEG-SeNPs are dispersed, then adding polyvinylpyrrolidone, stirring until dissolved, and finally adding zinc nitrate hexahydrate Aqueous solution, stirring evenly, standing still and centrifuging to obtain the Se@ZIF-8, then washing with methanol and resuspending in methanol.
作为第二种实施方式，步骤(2)中所述的超小纳米硒金属有机框架复合材料为Se@MIL-101，金属盐溶液为溶解了六水合氯化铁和对苯二甲酸的DMF溶液，缓冲液为DMF。As a second embodiment, the ultra-small nano-selenium metal-organic framework composite material described in step (2) is Se@MIL-101, and the metal salt solution is a DMF solution in which ferric chloride hexahydrate and terephthalic acid have been dissolved , the buffer is DMF.
步骤(2)包括：取所述PEG-SeNPs与溶解了六水合氯化铁和对苯二甲酸的DMF溶液混合，搅拌均匀，然后转移至特氟龙反应釜中，在100-140℃中反应一定时间，制得所述Se@MIL-101，然后用DMF溶液多次清洗后重悬于DMF中。Step (2) includes: mixing the PEG-SeNPs with a DMF solution in which ferric chloride hexahydrate and terephthalic acid have been dissolved, stirring evenly, then transferring to a Teflon reactor, and reacting at 100-140°C After a certain period of time, the Se@MIL-101 was prepared, washed with DMF solution several times and then resuspended in DMF.
作为第三种实施方式，步骤(2)中所述的超小纳米硒金属有机框架复合材料为Se@HKUST-1，金属盐溶液为氯化铜的DMF溶液，缓冲液为DMF。As a third embodiment, the ultra-small nano-selenium metal-organic framework composite material described in step (2) is Se@HKUST-1, the metal salt solution is a DMF solution of copper chloride, and the buffer solution is DMF.
步骤(2)包括：取所述PEG-SeNPs与氯化铜的DMF溶液混合超声均匀，向混合液中加入溶解了均苯三甲酸的DMF溶液，再加入乙酸，橡皮塞封好口后迅速加热到130-170℃，保持一段时间，自然降温后离心，制得所述Se@HKUST-1，然后使用DMF溶液洗涤重悬后低温保存。Step (2) includes: taking the DMF solution of PEG-SeNPs and cupric chloride, mixing and ultrasonically uniform, adding the DMF solution dissolved in trimesic acid to the mixed solution, then adding acetic acid, sealing the mouth with the rubber stopper and heating rapidly Keep it at 130-170°C for a period of time, and then centrifuge after natural cooling to obtain the Se@HKUST-1, then wash and resuspend with DMF solution, and store at low temperature.
作为第四种实施方式，步骤(2)中所述的超小纳米硒金属有机框架复合材料为Se@Ru-Mn-MOF，金属盐溶液为MnCl
2的DMF溶液和Ru(POPCOOH)
3的DMF溶液，缓冲液为DMF；
As a fourth embodiment, the ultra-small nano-selenium metal-organic framework composite material described in step (2) is Se@Ru- Mn -MOF, and the metal salt solution is MnCl DMF solution and Ru(POPCOOH) DMF Solution, the buffer is DMF;
步骤(2)包括：取所述PEG-SeNPs与MnCl
2的DMF溶液混合超声均匀，向混合液中加入Ru(POPCOOH)
3的DMF溶液，橡皮塞封好口后加热到140-180℃，保持一段时间，自然降温至低于100℃后冰水浴骤冷至室温，离心收集固体，制 得所述Se@Ru-Mn-MOF，然后使用DMF洗涤重悬后低温保存。
Step (2) includes: taking the PEG-SeNPs and the DMF solution of MnCl 2 and mixing them uniformly by ultrasonication, adding the DMF solution of Ru(POPCOOH) 3 into the mixed solution, sealing the mouth with a rubber stopper and heating to 140-180°C, keeping After a period of time, the temperature was naturally lowered to below 100°C and then quenched to room temperature in an ice-water bath, and the solid was collected by centrifugation to prepare the Se@Ru-Mn-MOF, which was then washed and resuspended with DMF and stored at low temperature.
进一步，步骤(1)中所述的转移到离心管进行离心处理，离心转速1500-2500rpm，离心时间9-11min。Further, transfer to a centrifuge tube as described in step (1) for centrifugation, the centrifugation speed is 1500-2500rpm, and the centrifugation time is 9-11min.
本发明还提供上述的制备方法制得的超小纳米硒金属有机框架复合材料，其特征在于，是以金属有机框架为载体负载了超小纳米硒的材料。The present invention also provides the ultra-small nano-selenium metal-organic framework composite material prepared by the above-mentioned preparation method, which is characterized in that the ultra-small nano-selenium is loaded with the metal-organic framework as a carrier.
进一步，所述纳米硒金属有机框架复合材料是Se@ZIF-8、Se@MIL-101、Se@HKUST-1、Se@Ru-Mn-MOF中的任意一种材料。Further, the nano-selenium metal-organic framework composite material is any one of Se@ZIF-8, Se@MIL-101, Se@HKUST-1, and Se@Ru-Mn-MOF.
本发明还提供上述的制备方法制得的超小纳米硒金属有机框架复合材料在制备治疗缺血性卒中药物的应用，是以金属有机框架为载体负载超小纳米硒的材料在治疗缺血性卒中的应用。The present invention also provides the application of the ultra-small nano-selenium metal-organic framework composite material prepared by the above-mentioned preparation method in the preparation of drugs for the treatment of ischemic stroke. Stroke application.
所述药物通过清除自由基和抗氧化，逆转氧化应激引起的损伤，改善脑缺血性再灌注后引起的脑部损伤，用于缺血性卒中的治疗。The medicine reverses the damage caused by oxidative stress by scavenging free radicals and anti-oxidation, improves the brain damage caused by cerebral ischemia-reperfusion, and is used for the treatment of ischemic stroke.
本专利的改进带来如下优点：The improvement of this patent brings following advantages:
(1)本申请实施例提供的超小纳米硒金属有机框架复合材料的制备方法，反应条件相对温和，不需要苛刻的反应条件，例如高温高压；反应步骤简单明确，易于实验和大规模生产。(1) The preparation method of the ultra-small nano-selenium metal-organic framework composite material provided in the examples of the present application has relatively mild reaction conditions and does not require harsh reaction conditions, such as high temperature and high pressure; the reaction steps are simple and clear, and are easy to experiment and large-scale production.
(2)由本申请实验例可知，本申请实施例四种超小纳米硒金属有机框架复合材料具有有效清除自由基的效果，抗氧化能力好，且其具有良好的重复清除自由基的能力和清除羟基自由基的能力。能够有效逆转氧化应激引起的损伤，改善脑缺血性再灌注后引起的脑部损伤，从而达到高效保护脑组织的效果。(2) It can be seen from the experimental examples of the application that the four ultra-small nano-selenium metal-organic framework composites in the embodiments of the application have the effect of effectively scavenging free radicals, have good oxidation resistance, and have good repeated scavenging free radicals and scavenging capacity for hydroxyl radicals. It can effectively reverse the damage caused by oxidative stress and improve the brain damage caused by cerebral ischemia-reperfusion, so as to achieve the effect of protecting brain tissue efficiently.
(3)本申请将具有催化能力的金属有机框架纳米材料与超小纳米硒相结合，充分发挥金属有机框架材料大比表面积、催化效率高等优点。同时金属有机框架材料能够作为超小纳米硒在体内的运载载体，促进超小纳米硒在体内的吸收与应用。(3) This application combines metal organic framework nanomaterials with catalytic ability with ultra-small nano-selenium to give full play to the advantages of large specific surface area and high catalytic efficiency of metal organic framework materials. At the same time, the metal-organic framework material can be used as a carrier of ultra-small nano-selenium in the body to promote the absorption and application of ultra-small nano-selenium in the body.
(4)由实验例可知，在相同浓度下负载了超小纳米硒的金属有机框架材料比单独的超小纳米硒与单独的金属有机框架材料具有更加优异的清除自由基的能 力。为纳米材料的神经保护机制及今后在缺血再灌注治疗中的临床应用提供了更多的证据。(4) It can be seen from the experimental examples that the metal organic framework material loaded with ultra-small nano-selenium has a better ability to scavenge free radicals than the single ultra-small nano-selenium and the single metal organic framework material at the same concentration. It provides more evidence for the neuroprotective mechanism of nanomaterials and its clinical application in ischemia-reperfusion therapy in the future.
(5)作为进一步的改进，本申请通过选取四种典型金属有机框架材料负载超小纳米硒，创新的制备出超小纳米硒与金属有机框架材料的复合纳米材料，为其他金属有机框架材料负载超小纳米硒提供了参考。(5) As a further improvement, this application selects four typical metal-organic framework materials to load ultra-small nano-selenium, and innovatively prepares a composite nanomaterial of ultra-small nano-selenium and metal-organic framework materials, which can be used for other metal-organic framework materials. Ultra-small nano-selenium provides a reference.
(6)作为进一步的改造，本申请通过对比***钠、硒酸钠、硒代半胱氨酸、硒代蛋氨酸、壳聚糖修饰纳米硒、香菇多糖修饰纳米硒、超小纳米硒(PEG-SeNPs)和四种金属有机框架材料发现：***钠、硒酸钠、硒代半胱氨酸、硒代蛋氨酸这四种无机硒没有抗氧化能力，壳聚糖修饰纳米硒、香菇多糖修饰纳米硒、超小纳米硒(PEG-SeNPs)具有微弱的抗氧化能力，单独的四种金属有机框架材料具有较低的抗氧化能力，而超小纳米硒金属有机框架材料相对于超小纳米硒能够有效提升其抗氧化能力约1.3-2.6倍，为其他含硒纳米粒子进行抗氧化能力改造提供了参考。(6) As a further improvement, the application is modified by sodium selenate, sodium selenate, selenocysteine, selenomethionine, chitosan modified nano-selenium, lentinan modified nano-selenium, ultra-small nano-selenium ( PEG-SeNPs) and four metal-organic framework materials were found: sodium selenite, sodium selenate, selenocysteine, selenomethionine, these four inorganic selenium have no antioxidant capacity, chitosan modified nano-selenium, shiitake mushroom Polysaccharide-modified nano-selenium and ultra-small nano-selenium (PEG-SeNPs) have weak antioxidant capacity, and the four separate metal-organic framework materials have low antioxidant capacity, and ultra-small nano-selenium metal-organic framework materials are relatively small Nano-selenium can effectively increase its antioxidant capacity by about 1.3-2.6 times, which provides a reference for other selenium-containing nanoparticles to transform their antioxidant capacity.
(7)通过在细胞水平与动物水平进行缺血性再灌注小鼠实验，我们发现四种超小纳米硒金属有机框架复合材料氧化应激引起的损伤，从而达到高效保护脑组织的功能。(7) Through ischemia-reperfusion mouse experiments at the cellular level and animal level, we found that four ultra-small nano-selenium metal-organic framework composites can effectively protect brain tissue from damage caused by oxidative stress.
图1为四种超小纳米硒金属有机框架复合材料的透射电镜图与元素分析图；其中，四种超小纳米硒金属有机框架复合材料分别为：①Se@ZIF-8、②Se@MIL-101、③Se@Ru-Mn-MOF、④Se@HKUST-1；Figure 1 shows the transmission electron microscope images and elemental analysis diagrams of four ultra-small nano-selenium metal-organic framework composites; among them, the four ultra-small nano-selenium metal-organic framework composites are: ①Se@ZIF-8, ②Se@MIL-101 , ③Se@Ru-Mn-MOF, ④Se@HKUST-1;
图2显示四种超小纳米硒金属有机框架复合材料逆转自由基损伤模型对细胞损伤；Figure 2 shows that four kinds of ultra-small nano-selenium metal-organic framework composites reverse free radical damage model to cell damage;
图3为使用流式细胞仪检测自由基损伤模型处理后的人神经母细胞瘤细胞的细胞凋亡图；Fig. 3 is the cell apoptosis diagram of the human neuroblastoma cells treated by the free radical damage model detected by flow cytometry;
图4显示使用荧光酶标仪检测在自由基损伤后四种超小纳米硒金属有机框架 复合材料对人神经母细胞瘤细胞细胞内活性氧水平；Figure 4 shows the use of a fluorescent microplate reader to detect the level of reactive oxygen species in human neuroblastoma cells after free radical damage by four ultra-small nano-selenium metal-organic framework composites;
图5为在缺血再灌注模型下的小鼠使用四种超小纳米硒金属有机框架复合材料治疗后小鼠脑部损伤面积图。Fig. 5 is a map of brain injury area of mice treated with four kinds of ultra-small nano-selenium metal-organic framework composite materials under the ischemia-reperfusion model.
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention.
目前，纳米技术与对具有类酶活性纳米材料的发现，促使我们对脑卒中及其相关疾病的治疗采取新的思路。与天然酶相比，纳米酶表现出高稳定性、持久的催化活性和多功能的酶类特性。通过使用不同的纳米硒与金属有机框架纳米粒子的类酶活性评价后，运用纳米酶对缺血性脑卒中疾病的治疗。这类具有生物安全性的纳米酶在治疗脑卒中及其相关疾病方面显示出良好的前景。At present, nanotechnology and the discovery of nanomaterials with enzyme-like activity have prompted us to adopt new ideas for the treatment of stroke and related diseases. Compared with natural enzymes, nanozymes exhibit high stability, long-lasting catalytic activity, and multifunctional enzymatic properties. After evaluating the enzyme-like activity of different nano-selenium and metal-organic framework nanoparticles, the treatment of ischemic stroke diseases was performed using nanozymes. This class of biosafe nanozymes has shown promising prospects in the treatment of stroke and related diseases.
实施例1Example 1
(1)制备PEG-SeNPs(超小纳米硒)：在烧杯中加入聚乙二醇(PEG)400和硒粉，超声后搅拌使硒粉均匀分散在PEG 400中；转移所述烧杯至磁力搅拌加热器上，边加热边搅拌，反应一段后自然冷却，转移到离心管中离心，离心后取上层溶液，去除未反应的硒粉，最后获得所述PEG-SeNPs；(1) Preparation of PEG-SeNPs (ultra-small nano-selenium): Add polyethylene glycol (PEG) 400 and selenium powder in a beaker, stir after ultrasonic to make the selenium powder evenly dispersed in PEG 400; transfer the beaker to magnetic stirring On the heater, stir while heating, cool naturally after a period of reaction, transfer to a centrifuge tube for centrifugation, take the upper layer solution after centrifugation, remove unreacted selenium powder, and finally obtain the PEG-SeNPs;
(2)制备超小纳米硒金属有机框架复合材料：使用包括有所述PEG-SeNPs和金属盐溶液的原料制得所述纳米硒金属有机框架复合材料，并将所述纳米硒金属有机框架复合材料重悬于缓冲液中。(2) Preparation of ultra-small nano-selenium metal-organic framework composites: use the raw materials including the PEG-SeNPs and metal salt solution to prepare the nano-selenium metal-organic framework composites, and compound the nano-selenium metal-organic frameworks Material was resuspended in buffer.
实施例2Example 2
(1)制备PEG-SeNPs：在烧杯中加入10mL PEG 400，然后称取20mg硒粉，超声后搅拌5分钟使硒粉完全均匀分散在PEG 400中；转移烧杯置磁力搅拌加热器上，加热至温度约205℃，边加热边搅拌，反应1小时后自然冷却，转移到 离心管后在转速2000rpm离心10min后取上层溶液，去除未反应的硒粉，最后获得PEG-SeNPs，所得PEG-SeNPs的浓度为16-17mM。(1) Preparation of PEG-SeNPs: Add 10mL PEG 400 to a beaker, then weigh 20mg of selenium powder, and stir for 5 minutes after ultrasonication to completely and evenly disperse the selenium powder in PEG 400; transfer the beaker to a magnetic stirring heater, and heat to The temperature is about 205°C, stir while heating, and cool naturally after reacting for 1 hour. After transferring to a centrifuge tube and centrifuging at a speed of 2000rpm for 10 minutes, take the upper layer solution, remove unreacted selenium powder, and finally obtain PEG-SeNPs. The obtained PEG-SeNPs The concentration is 16-17mM.
(2)制备超小纳米硒与含锌有机框架复合材料Se@ZIF-8：取2mL PEG-SeNPs与有溶解了20.94mg二甲基咪唑的10mL溶液混合，搅拌至PEG-SeNPs完全分散，然后加入400mg聚乙烯吡咯烷酮(PVP)，搅拌至完全溶解，最后加入溶解了75.12mg六水合硝酸锌的10ml溶液，搅拌均匀后静置至室温过夜(12h)，12000rpm离心，制得Se@ZIF-8，用大量甲醇清洗，重悬至甲醇中。(2) Preparation of ultra-small nano-selenium and Zn-containing organic framework composite material Se@ZIF-8: Take 2 mL of PEG-SeNPs and mix with 10 mL of solution dissolved in 20.94 mg of dimethylimidazole, stir until the PEG-SeNPs are completely dispersed, and then Add 400mg of polyvinylpyrrolidone (PVP), stir until it is completely dissolved, and finally add 10ml of a solution in which 75.12mg of zinc nitrate hexahydrate has been dissolved, stir evenly, let stand at room temperature overnight (12h), and centrifuge at 12000rpm to obtain Se@ZIF-8 , washed with copious amounts of methanol, and resuspended in methanol.
或者制备超小纳米硒与含铁有机框架复合材料Se@MIL-101：取1ml PEG-SeNPs与溶解了13.42mg六水合氯化铁和12.02mg对苯二甲酸的20mlDMF溶液中，搅拌均匀，然后转移至特氟龙反应釜中，在120℃反应12h，制得Se@MIL-101，用DMF洗3次后重悬与DMF中。Or prepare ultra-small nano-selenium and iron-containing organic framework composite material Se@MIL-101: take 1ml PEG-SeNPs and dissolve 13.42mg ferric chloride hexahydrate and 12.02mg terephthalic acid in 20ml DMF solution, stir evenly, and then Transfer to a Teflon reactor and react at 120°C for 12 hours to prepare Se@MIL-101, wash with DMF 3 times and resuspend in DMF.
或者制备超小纳米硒与含铜有机框架复合材料Se@HKUST-1：取1ml的PEG修饰的PEG-SeNPs与溶解有0.15mmol氯化铜的5mlDMF混合超声均匀，向混合液中加入5ml溶解有0.1mmol均苯三甲酸的DMF溶液，再加入2-3滴乙酸，橡皮塞封好口后迅速加热到150℃，保持10min，自然降温后8000rmp、5min离心，制得Se@HKUST-1，等体积DMF洗涤重悬后置于4℃备用。Or prepare ultra-small nano-selenium and copper-containing organic framework composite material Se@HKUST-1: Take 1ml of PEG-modified PEG-SeNPs and 5ml DMF dissolved in 0.15mmol copper chloride, mix and sonicate evenly, add 5ml dissolved in the mixed solution Add 2-3 drops of acetic acid to a DMF solution of 0.1mmol trimesic acid, heat up to 150°C quickly after the rubber stopper is sealed, keep it for 10min, and then centrifuge at 8000rmp for 5min after natural cooling to prepare Se@HKUST-1, etc. Wash and resuspend with volume DMF and store at 4°C for use.
或者制备超小纳米硒与含锰钌有机框架复合材料Se@Ru-Mn-MOF：取1ml的PEG修饰的PEG-SeNPs与溶解有0.05mmol MnCl
2的5mlDMF混合超声均匀，向混合液中加入5ml溶解有0.05mmol Ru(POPCOOH)
3的DMF溶液，橡皮塞封好口后加热到160℃，保持10min，自然降温低于100℃后冰水浴骤冷至室温，8000rmp、5min离心收集固体，制得Se@Ru-Mn-MOF，等体积DMF洗涤重悬后置于4℃备用。
Or prepare ultra-small nano-selenium and manganese-containing ruthenium organic framework composite material Se@Ru-Mn-MOF: take 1ml of PEG-modified PEG-SeNPs and 5ml DMF dissolved in 0.05mmol MnCl 2 , mix and ultrasonically evenly, add 5ml to the mixture DMF solution with 0.05mmol Ru(POPCOOH) 3 dissolved, sealed with a rubber stopper, heated to 160°C, kept for 10min, cooled naturally to below 100°C in an ice-water bath to room temperature, centrifuged at 8000rmp, 5min to collect the solid, obtained Se@Ru-Mn-MOF was washed and resuspended with an equal volume of DMF and placed at 4°C for use.
实施例3Example 3
(1)制备PEG-SeNPs：在烧杯中加入8mL PEG 400，然后称取17mg硒粉，超声后搅拌4分钟使硒粉完全均匀分散在PEG 400中；转移烧杯置磁力搅拌加热器上，加热至温度约200℃，边加热边搅拌，反应1小时后自然冷却，转移到 离心管后在转速1800rpm离心11min后取上层溶液，去除未反应的硒粉，最后获得PEG-SeNPs，所得PEG-SeNPs的浓度为16-20mM。(1) Preparation of PEG-SeNPs: Add 8mL PEG 400 to a beaker, then weigh 17mg of selenium powder, stir for 4 minutes after ultrasonication to completely and evenly disperse the selenium powder in PEG 400; transfer the beaker to a magnetic stirring heater, and heat to The temperature is about 200°C, stirring while heating, reacting for 1 hour, cooling naturally, transferring to a centrifuge tube, centrifuging at a speed of 1800rpm for 11min, taking the upper layer solution, removing unreacted selenium powder, and finally obtaining PEG-SeNPs, the obtained PEG-SeNPs The concentration is 16-20mM.
(2)制备超小纳米硒与锌的有机框架复合材料Se@ZIF-8：取1.8mL PEG-SeNPs与有溶解了19mg二甲基咪唑的8.8mL溶液混合，搅拌至PEG-SeNPs完全分散，然后加入380mg聚乙烯吡咯烷酮，搅拌至完全溶解，最后加入溶解了68mg六水合硝酸锌的9ml水溶液，搅拌均匀后静置至室温过夜(11h)，11000rpm离心，制得Se@ZIF-8，用大量甲醇清洗，重悬至甲醇中。(2) Preparation of ultra-small nano-selenium and zinc organic framework composite Se@ZIF-8: Take 1.8mL PEG-SeNPs and mix with 8.8mL solution dissolved in 19mg dimethylimidazole, stir until PEG-SeNPs are completely dispersed, Then add 380mg of polyvinylpyrrolidone, stir until it is completely dissolved, and finally add 9ml of aqueous solution in which 68mg of zinc nitrate hexahydrate has been dissolved, stir evenly, let it stand at room temperature overnight (11h), and centrifuge at 11000rpm to obtain Se@ZIF-8. Wash with methanol and resuspend in methanol.
或者制备超小纳米硒与铁的有机框架复合材料Se@MIL-101：取0.9ml的PEG-SeNPs与溶解了12.15mg六水合氯化铁和10.9mg对苯二甲酸的18.5ml的DMF溶液中，搅拌均匀，然后转移至特氟龙反应釜中，在115℃反应13h，制得Se@MIL-101，用DMF洗4次后重悬与DMF中。Or prepare ultra-small nano-selenium and iron organic framework composite Se@MIL-101: Take 0.9ml of PEG-SeNPs and dissolve 12.15mg of ferric chloride hexahydrate and 10.9mg of terephthalic acid in 18.5ml of DMF solution , stirred evenly, then transferred to a Teflon reactor, and reacted at 115°C for 13h to prepare Se@MIL-101, which was washed 4 times with DMF and then resuspended in DMF.
或者制备超小纳米硒与铜的有机框架复合材料Se@HKUST-1：取0.9ml的PEG修饰的PEG-SeNPs与溶解有0.14mmol氯化铜的4.8mlDMF混合超声均匀，向混合液中加入4.8ml溶解有0.09mmol均苯三甲酸的DMF溶液，再加入2-3滴乙酸，橡皮塞封好口后迅速加热到145℃，保持11min，自然降温后7500rmp、6min离心，制得Se@HKUST-1，等体积DMF洗涤重悬后置于4℃备用。Or prepare the organic framework composite material Se@HKUST-1 of ultra-small nano-selenium and copper: take 0.9ml of PEG-modified PEG-SeNPs and 4.8ml DMF dissolved in 0.14mmol copper chloride, mix and sonicate evenly, add 4.8 ml of DMF solution with 0.09 mmol of trimesic acid dissolved in it, then add 2-3 drops of acetic acid, heat up to 145°C quickly after the rubber stopper is sealed, keep it for 11 min, and centrifuge at 7500rmp for 6 min after natural cooling to prepare Se@HKUST- 1. Wash and resuspend with an equal volume of DMF and store at 4°C for use.
或者制备超小纳米硒与锰钌的有机框架复合材料Se@Ru-Mn-MOF：取0.9ml的PEG修饰的PEG-SeNPs与溶解有0.04mmol MnCl
2的4.5ml是DMF混合超声均匀，向混合液中加入4.5ml溶解有0.04mmol Ru(POPCOOH)
3的DMF溶液，橡皮塞封好口后加热到155℃，保持11min，自然降温低于95℃后冰水浴骤冷至室温，7500rmp、6min离心收集固体，制得Se@Ru-Mn-MOF，等体积DMF洗涤重悬后置于4℃备用。
Or prepare the organic framework composite material Se@Ru-Mn-MOF of ultra-small nano-selenium and manganese ruthenium: take 0.9ml of PEG-modified PEG-SeNPs and 4.5ml of DMF dissolved in 0.04mmol MnCl 2 and mix it evenly with ultrasonic, and mix it with Add 4.5ml of DMF solution with 0.04mmol Ru(POPCOOH) 3 dissolved in the solution, seal the mouth with a rubber stopper, heat to 155°C, keep it for 11min, cool down naturally to below 95°C, and then cool to room temperature in an ice-water bath, centrifuge at 7500rmp for 6min The solid was collected to prepare Se@Ru-Mn-MOF, which was washed and resuspended with an equal volume of DMF and placed at 4°C for use.
将实施例2方法制备得到的四种超小纳米硒金属有机框架复合材料进行如下实验。The four ultra-small nano-selenium metal-organic framework composites prepared by the method in Example 2 were subjected to the following experiments.
实验例1Experimental example 1
使用透射电镜和元素分析对四种超小纳米硒金属有机框架复合材料进行检 测，结果如图1所示。四种超小纳米硒金属有机框架复合材料分别为(下同)：Se@ZIF-8、Se@MIL-101、Se@Ru-Mn-MOF、Se@HKUST-1。如图1所示的四种超小纳米硒金属有机框架复合材料的透射电镜图与元素分析图，显示负载超小纳米硒的四种金属有机框架材料制备成功。图中，①对应Se@ZIF-8、②对应Se@MIL-101、③对应Se@Ru-Mn-MOF、④对应Se@HKUST-1。Using transmission electron microscopy and elemental analysis, four ultra-small nano-selenium metal-organic framework composites were detected, and the results are shown in Figure 1. The four ultra-small nano-selenium metal-organic framework composites are (the same below): Se@ZIF-8, Se@MIL-101, Se@Ru-Mn-MOF, Se@HKUST-1. The transmission electron microscope images and elemental analysis images of the four ultra-small nano-selenium metal-organic framework composite materials shown in Figure 1 show that the four metal-organic framework materials loaded with ultra-small nano-selenium were successfully prepared. In the figure, ① corresponds to Se@ZIF-8, ② corresponds to Se@MIL-101, ③ corresponds to Se@Ru-Mn-MOF, and ④ corresponds to Se@HKUST-1.
实验例2Experimental example 2
使用ABTS总抗氧化能力检测法对***钠、硒酸钠、硒代半胱氨酸、硒代蛋氨酸、壳聚糖修饰纳米硒、香菇多糖修饰纳米硒、超小纳米硒(PEG-SeNPs)和四种纳米硒金属有机框架材料的抗氧化能力进行检测。***钠、硒酸钠、硒代半胱氨酸和硒代蛋氨酸并无明显的抗氧化能力。壳聚糖修饰纳米硒、香菇多糖修饰纳米硒和超小纳米硒(PEG-SeNPs)具有少量抗氧化能力；通过与金属有机框架材料复合后形成的四种超小纳米硒金属有机框架材料的抗氧化能力比单独的超小纳米硒(PEG-SeNPs)有所增强，相对于单独超小纳米硒，抗氧化能力增强约1.3-2.6倍，说明金属有机框架的加入对于提升纳米硒的抗氧化能力具有显著性的效果。Sodium selenite, sodium selenate, selenocysteine, selenomethionine, chitosan-modified nano-selenium, lentinan-modified nano-selenium, ultra-small nano-selenium (PEG-SeNPs) ) and the antioxidant capacity of four nano-selenium metal-organic framework materials were tested. Sodium selenite, sodium selenate, selenocysteine and selenomethionine have no significant antioxidant capacity. Chitosan-modified nano-selenium, lentinan-modified nano-selenium, and ultra-small nano-selenium (PEG-SeNPs) have a small amount of antioxidant capacity; the anti-oxidative properties of four ultra-small nano-selenium metal-organic frameworks formed by compounding with metal-organic framework materials Compared with the single ultra-small nano-selenium (PEG-SeNPs), the oxidation ability is enhanced, and the anti-oxidation ability is about 1.3-2.6 times stronger than that of the single ultra-small nano-selenium, indicating that the addition of metal-organic frameworks can improve the antioxidant capacity of nano-selenium have a significant effect.
实验例3Experimental example 3
本实验例选取了人脑神经母细胞瘤细胞(SH-SY5Y)细胞系作为细胞模型的对象，通过加入20μM叔丁基过氧化氢模拟细胞产生的自由基，最后通过MTT法检测SH-SY5Y细胞的存活率，以评价四种超小纳米硒金属有机框架复合材料在自由基损伤SH-SY5Y细胞模型中对细胞的保护作用，结果如图2所示。由图2可以从细胞存活率中看出，在叔丁基过氧化氢损伤后的细胞存活率约为70％，加入了纳米硒后存活率有所上升，而四种超小纳米硒金属有机框架复合材料处理后的细胞存活率明显相对于损伤组和单独超小纳米硒组(图中的Se组)均上升明显，说明四种纳米硒金属有机框架复合材料能够有效保护细胞，免受细胞遭受自由基的损伤。In this experiment example, the human brain neuroblastoma cell line (SH-SY5Y) was selected as the object of the cell model, and the free radicals produced by the cells were simulated by adding 20 μM tert-butyl hydroperoxide, and finally the SH-SY5Y cells were detected by the MTT method The survival rate of four ultra-small nano-selenium metal-organic framework composites was used to evaluate the protective effect on cells in the free radical damage SH-SY5Y cell model, and the results are shown in Figure 2. As can be seen from the cell survival rate in Figure 2, the cell survival rate after tert-butyl hydroperoxide damage is about 70%, and the survival rate has increased after adding nano-selenium, while four ultra-small nano-selenium metal organic Compared with the injury group and the single ultra-small nano-selenium group (Se group in the figure), the cell survival rate after the frame composite treatment was significantly increased, indicating that the four nano-selenium metal-organic framework composites can effectively protect cells from cell damage. suffer from free radical damage.
实验例4Experimental example 4
本实验例选取了人脑神经母细胞瘤细胞(SH-SY5Y)细胞系作为细胞模型的对象，通过加入20μM叔丁基过氧化氢模拟细胞产生的自由基，最后通过流式细胞仪检测SH-SY5Y细胞的细胞凋亡比例来评价四种超小纳米硒金属有机框架复合材料在自由基损伤模型对细胞损伤模型中对SH-SY5Y细胞的保护作用，结果如图3所示。由图3可以从细胞存活率中看出，在叔丁基过氧化氢损伤后的细胞细胞凋亡比例约为40％，加入了超小纳米硒后细胞凋亡比例有所下降，而四种超小纳米硒金属有机框架复合材料处理后的细胞凋亡比例明显相对于损伤组和单独纳米硒组均下降明显，说明四种超小纳米硒金属有机框架复合材料能够有效保护细胞，免受细胞遭受自由基的损伤。In this experiment example, the human brain neuroblastoma cell line (SH-SY5Y) was selected as the object of the cell model, and the free radicals produced by the cells were simulated by adding 20 μM tert-butyl hydroperoxide, and finally the SH-SY5Y was detected by flow cytometry. The apoptosis ratio of SY5Y cells was used to evaluate the protective effect of four ultra-small nano-selenium metal-organic framework composites on SH-SY5Y cells in the free radical damage model versus cell damage model, and the results are shown in Figure 3. As can be seen from the cell survival rate in Figure 3, the cell apoptosis ratio after tert-butyl hydroperoxide damage was about 40%, and the apoptosis ratio decreased after adding ultra-small nano-selenium, while the four Compared with the injured group and the single nano-selenium group, the percentage of cell apoptosis after treatment with ultra-small nano-Se metal-organic framework composites was significantly lower, indicating that the four ultra-small nano-Se metal-organic framework composites can effectively protect cells from suffer from free radical damage.
实验例5Experimental example 5
本实验例选取了人脑神经母细胞瘤细胞(SH-SY5Y)细胞系作为细胞模型的对象，通过加入20μM叔丁基过氧化氢模拟细胞产生的自由基，最后通过DCFH-DA细胞内活性氧检测荧光染料来检测SH-SY5Y细胞内活性氧水平。以评价四种超小纳米硒金属有机框架复合材料在自由基损伤模型对细胞损伤模型中对SH-SY5Y细胞的保护作用，结果如图4所示。由图4我们可以观察到细胞内活性氧水平在加入了叔丁基过氧化氢后有明显的上升，而加入四种超小纳米硒金属有机框架复合材料后，细胞内活性氧水平上升幅度明显下降。该结果说明四种超小纳米硒金属有机框架复合材料能够有效降低细胞内由于自由基引起的活性氧水平。In this experiment example, the human brain neuroblastoma cell line (SH-SY5Y) was selected as the object of the cell model, and the free radicals produced by the cells were simulated by adding 20 μM tert-butyl hydroperoxide, and finally the reactive oxygen species in the DCFH-DA cells Detection of fluorescent dyes to detect the level of reactive oxygen species in SH-SY5Y cells. To evaluate the protective effect of four ultra-small nano-selenium metal-organic framework composites on SH-SY5Y cells in the free radical damage model to the cell damage model, the results are shown in Figure 4. From Figure 4, we can observe that the level of active oxygen in the cells increased significantly after adding tert-butyl hydroperoxide, and after adding four kinds of ultra-small nano-selenium metal-organic framework composites, the level of active oxygen in cells increased significantly decline. The results indicated that the four ultra-small nano-Se metal-organic framework composites could effectively reduce the level of reactive oxygen species caused by free radicals in cells.
实验例6Experimental example 6
本实验例选取C57小鼠作为实验对象，使用大脑中动脉阻塞(MCAO)手术进行脑卒中缺血再灌注模型的制备。通过使用硅胶塞在小鼠大脑中动脉阻塞血流40分钟后，取出橡胶塞完成再灌注。然后连续3天尾静脉给予小鼠四种纳米硒金属有机框架复合材料，在第四天取出小鼠的脑组织，用刀片将脑组织均匀分切成5份，每篇厚度约为2mm，然后使用2，3，5－三苯基氯化四氮唑(TTC)对脑组织进行染色，最后使用相机拍摄脑组织TTC染色后的情况。如图5所示， 通过TTC染色我们可以看出，Sham组为对照组，小鼠脑组织无损伤，故TTC染色后脑组织均为红色。Saline组的小鼠脑组织白色面积明显比Sham组的面积大，然后通过给予四种纳米硒金属有机框架复合材料治疗后的小鼠脑组织白色面积明显比Saline组减小，说明四种超小纳米硒金属有机框架复合材料能够有效保护小鼠脑组织免受脑卒中缺血再灌注引起的损伤。In this experiment example, C57 mice were selected as the experimental subjects, and the middle cerebral artery occlusion (MCAO) operation was used to prepare the stroke ischemia-reperfusion model. Reperfusion was accomplished by removing the rubber stopper after occluding the blood flow in the middle cerebral artery of the mouse for 40 min using a silicone stopper. Then four kinds of nano-selenium metal-organic framework composite materials were given to the mice through the tail vein for 3 consecutive days, and the brain tissue of the mice was taken out on the fourth day, and the brain tissue was evenly divided into 5 parts with a blade, each with a thickness of about 2 mm, and then Use 2,3,5-triphenyltetrazolium chloride (TTC) to stain the brain tissue, and finally use a camera to take pictures of the brain tissue after TTC staining. As shown in Figure 5, we can see from TTC staining that the Sham group was the control group, and the brain tissue of the mice was not damaged, so the brain tissue was all red after TTC staining. The white area of the brain tissue of the mice in the Saline group was significantly larger than that of the Sham group, and then the white area of the brain tissue of the mice treated with four kinds of nano-selenium metal-organic framework composite materials was significantly smaller than that of the Saline group, indicating that the four ultra-small Nano-selenium metal-organic framework composites can effectively protect mouse brain tissue from damage caused by stroke ischemia-reperfusion.
通过实验例1-6本申请探究了四种超小纳米硒的金属有机框架材料在缺血性脑卒中再灌注损伤的治疗效果。经实验发现，相比于单独的金属有机框架材料或纳米硒，负载了超小纳米硒的金属有机框架材料具有更好的治疗缺血性脑卒中再灌注损伤的效果。Through experimental examples 1-6, the present application explores the therapeutic effect of four ultra-small nano-selenium metal organic framework materials on ischemic stroke reperfusion injury. It has been found through experiments that, compared with metal-organic framework materials or nano-selenium alone, the metal-organic framework material loaded with ultra-small nano-selenium has a better effect on the treatment of ischemic stroke reperfusion injury.
经过透射电镜和元素分析可以显示成功制备了负载超小纳米硒的四种金属有机框架材料。同时本申请使用自由基损伤后的人神经母细胞瘤细胞验证其逆转了自由基引起的损伤，同时能够降低细胞内ROS水平。最终，本申请在缺血性脑卒中再灌注损伤的小鼠模型中验证这四种负载超小纳米硒的金属有机框架材料的性能，发现这四种负载了超小纳米硒的金属有机框架材料可以逆转缺血性脑卒中再灌注引起的脑组织损伤，能够有效减少脑组织坏死的面积。本发明还公开了采用超小纳米硒金属有机框架复合材料在制备治疗缺血性卒中药物的应用。Transmission electron microscopy and elemental analysis show that four metal organic framework materials loaded with ultra-small nano-selenium have been successfully prepared. At the same time, this application uses human neuroblastoma cells after free radical damage to verify that it reverses the damage caused by free radicals and can reduce the level of intracellular ROS. Finally, this application verified the performance of these four metal organic framework materials loaded with ultra-small nano-selenium in the mouse model of ischemic stroke reperfusion injury, and found that these four metal-organic framework materials loaded with ultra-small nano-selenium It can reverse the brain tissue damage caused by ischemic stroke reperfusion, and can effectively reduce the area of brain tissue necrosis. The invention also discloses the application of the ultra-small nanometer selenium metal organic framework composite material in the preparation of medicine for treating ischemic stroke.
综上所述，本申请不仅展示了金属有机框架材料负载纳米硒的制备方法，而且为纳米材料的神经保护机制及今后在缺血再灌注治疗中的临床应用提供了更多的证据。In summary, this application not only demonstrates the preparation method of metal-organic framework materials loaded with nano-selenium, but also provides more evidence for the neuroprotective mechanism of nanomaterials and their future clinical application in ischemia-reperfusion therapy.
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应该以权利要求的保护范围为准。The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed in the present invention can easily think of changes or Replacement should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.
Claims (10)
- 超小纳米硒金属有机框架复合材料的制备方法，其特征在于，包括以下步骤：The preparation method of the ultra-small nanometer selenium metal organic framework composite material is characterized in that it comprises the following steps:(1)制备超小纳米硒(PEG-SeNPs)：在烧杯中加入PEG 400和硒粉，超声后搅拌使硒粉均匀分散在PEG 400中；转移所述烧杯至磁力搅拌加热器上，边加热边搅拌，反应一定时间后自然冷却，转移到离心管进行离心处理，离心后取上层溶液，去除未反应的硒粉，最后获得所述PEG-SeNPs；(1) Preparation of ultra-small nano-selenium (PEG-SeNPs): add PEG 400 and selenium powder in a beaker, stir after ultrasonic so that the selenium powder is evenly dispersed in PEG 400; transfer the beaker to a magnetic stirring heater, and heat it While stirring, react for a certain period of time, cool naturally, transfer to a centrifuge tube for centrifugation, take the upper layer solution after centrifugation, remove unreacted selenium powder, and finally obtain the PEG-SeNPs;(2)制备超小纳米硒金属有机框架复合材料：使用包括有所述PEG-SeNPs和金属盐溶液的原料制得所述纳米硒金属有机框架复合材料，并将所述超小纳米硒金属有机框架复合材料重悬于缓冲液中。(2) Preparation of ultra-small nano-selenium metal-organic framework composites: use the raw materials comprising the PEG-SeNPs and metal salt solution to prepare the nano-selenium metal-organic framework composites, and prepare the ultra-small nano-selenium metal-organic frameworks Framework composites were resuspended in buffer.
- 根据权利要求1所述的超小纳米硒金属有机框架复合材料的制备方法，其特征在于，步骤(2)中所述的超小纳米硒金属有机框架复合材料为Se@ZIF-8，金属盐溶液为六水合硝酸锌的溶液，缓冲液为甲醇；The preparation method of the ultra-small nano-selenium metal-organic framework composite material according to claim 1, wherein the ultra-small nano-selenium metal-organic framework composite material described in step (2) is Se@ZIF-8, a metal salt The solution is a solution of zinc nitrate hexahydrate, and the buffer is methanol;步骤(2)包括：取所述PEG-SeNPs与有溶解了二甲基咪唑的溶液混合，搅拌至所述PEG-SeNPs分散，然后加入聚乙烯吡咯烷酮，搅拌至溶解，最后加入六水合硝酸锌的溶液，搅拌均匀后静置后离心处理，制得所述Se@ZIF-8，然后用甲醇清洗并重悬至甲醇中。Step (2) includes: mixing the PEG-SeNPs with a solution in which dimethylimidazole is dissolved, stirring until the PEG-SeNPs are dispersed, then adding polyvinylpyrrolidone, stirring until dissolved, and finally adding zinc nitrate hexahydrate solution, stirred evenly, left to stand and then centrifuged to obtain the Se@ZIF-8, then washed with methanol and resuspended in methanol.
- 根据权利要求1所述的超小纳米硒金属有机框架复合材料的制备方法，其特征在于，步骤(2)中所述的超小纳米硒金属有机框架复合材料为Se@MIL-101，金属盐溶液为溶解了六水合氯化铁和对苯二甲酸的DMF溶液，缓冲液为DMF；The preparation method of the ultra-small nano-selenium metal-organic framework composite material according to claim 1, wherein the ultra-small nano-selenium metal-organic framework composite material described in step (2) is Se@MIL-101, a metal salt The solution is a DMF solution that has dissolved ferric chloride hexahydrate and terephthalic acid, and the buffer is DMF;步骤(2)包括：取所述PEG-SeNPs与溶解了六水合氯化铁和对苯二甲酸的DMF溶液混合，搅拌均匀，然后转移至特氟龙反应釜中，在100-140℃中反应一定时间，制得所述Se@MIL-101，然后用DMF溶液多次清洗后重悬于DMF 中。Step (2) includes: mixing the PEG-SeNPs with the DMF solution in which ferric chloride hexahydrate and terephthalic acid are dissolved, stirring evenly, then transferring to a Teflon reactor, and reacting at 100-140°C After a certain period of time, the Se@MIL-101 was prepared, washed with DMF solution several times and then resuspended in DMF.
- 根据权利要求1所述的超小纳米硒金属有机框架复合材料的制备方法，其特征在于，步骤(2)中所述的超小纳米硒金属有机框架复合材料为Se@HKUST-1，金属盐溶液为氯化铜的DMF溶液，缓冲液为DMF；The preparation method of the ultra-small nano-selenium metal-organic framework composite material according to claim 1, wherein the ultra-small nano-selenium metal-organic framework composite material described in step (2) is Se@HKUST-1, a metal salt The solution is a DMF solution of copper chloride, and the buffer is DMF;步骤(2)包括：取所述PEG-SeNPs与氯化铜的DMF溶液混合超声均匀，向混合液中加入溶解了均苯三甲酸的DMF溶液，再加入乙酸，橡皮塞封好口后迅速加热到130-170℃，保持一段时间，自然降温后离心，制得所述Se@HKUST-1，然后使用DMF溶液洗涤重悬后低温保存。Step (2) includes: taking the PEG-SeNPs and the DMF solution of copper chloride, mixing and ultrasonically uniform, adding the DMF solution dissolved in trimesic acid to the mixed solution, then adding acetic acid, sealing the mouth with the rubber stopper and heating rapidly Keep it at 130-170°C for a period of time, and then centrifuge after natural cooling to obtain the Se@HKUST-1, then wash and resuspend with DMF solution, and store at low temperature.
- 根据权利要求1所述的超小纳米硒金属有机框架复合材料的制备方法，其特征在于，步骤(2)中所述的超小纳米硒金属有机框架复合材料为Se@Ru-Mn-MOF，金属盐溶液为MnCl 2的DMF溶液和Ru(POPCOOH) 3的DMF溶液，缓冲液为DMF； The preparation method of the ultra-small nano-selenium metal-organic framework composite material according to claim 1, wherein the ultra-small nano-selenium metal-organic framework composite material described in step (2) is Se@Ru-Mn-MOF, The metal salt solution is MnCl DMF solution and Ru(POPCOOH) DMF solution , buffer is DMF;步骤(2)包括：取所述PEG-SeNPs与MnCl 2的DMF溶液混合超声均匀，向混合液中加入Ru(POPCOOH) 3的DMF溶液，橡皮塞封好口后加热到140-180℃，保持一段时间，自然降温至低于100℃后冰水浴骤冷至室温，离心收集固体，制得所述Se@Ru-Mn-MOF，然后使用DMF洗涤重悬后低温保存。 Step (2) includes: taking the PEG-SeNPs and the DMF solution of MnCl 2 and mixing them uniformly by ultrasonication, adding the DMF solution of Ru(POPCOOH) 3 into the mixed solution, sealing the mouth with a rubber stopper and heating to 140-180°C, keeping After a period of time, the temperature was naturally lowered to below 100°C and then quenched to room temperature in an ice-water bath, and the solid was collected by centrifugation to prepare the Se@Ru-Mn-MOF, which was then washed and resuspended with DMF and stored at low temperature.
- 根据权利要求1所述的超小纳米硒金属有机框架复合材料的制备方法，其特征在于，步骤(1)中所述的转移到离心管进行离心处理，离心转速1500-2500rpm，离心时间9-11min。The preparation method of ultra-small nano-selenium metal organic framework composite material according to claim 1, is characterized in that, described in step (1) is transferred to centrifuge tube and carries out centrifugal treatment, centrifugal speed 1500-2500rpm, centrifugation time 9- 11min.
- 根据权利要求1-6任一项所述的制备方法制得的超小纳米硒金属有机框架复合材料，其特征在于，是以金属有机框架为载体负载了纳米硒的材料。The ultra-small nano-selenium metal-organic framework composite material prepared according to the preparation method described in any one of claims 1-6 is characterized in that it is a material loaded with nano-selenium on a metal-organic framework as a carrier.
- 根据权利要求1-6任一项所述的制备方法制得的超小纳米硒金属有机框 架复合材料，其特征在于，所述超小纳米硒金属有机框架复合材料是Se@ZIF-8、Se@MIL-101、Se@HKUST-1、Se@Ru-Mn-MOF中的任意一种材料。According to the ultra-small nano-selenium metal-organic framework composite material prepared by the preparation method described in any one of claims 1-6, it is characterized in that the ultra-small nano-selenium metal-organic framework composite material is Se@ZIF-8, Se Any material in @MIL-101, Se@HKUST-1, Se@Ru-Mn-MOF.
- 根据权利要求1-6任一项所述的制备方法制得的超小纳米硒金属有机框架复合材料在制备治疗缺血性卒中药物的应用，其特征在于，是以金属有机框架为载体负载超小纳米硒的材料在治疗缺血性卒中的应用。The application of the ultra-small nanometer selenium metal-organic framework composite material prepared according to the preparation method described in any one of claims 1-6 in the preparation of a drug for treating ischemic stroke is characterized in that the metal-organic framework is used as a carrier to load super Application of small nano-selenium materials in the treatment of ischemic stroke.
- 根据权利要求9所述的超小纳米硒金属有机框架复合材料在制备治疗缺血性卒中药物的应用，其特征在于，所述药物通过清除自由基和抗氧化，逆转氧化应激引起的损伤，改善脑缺血性再灌注后引起的脑部损伤，用于缺血性卒中的治疗。The application of the ultra-small nanometer selenium metal organic framework composite material according to claim 9 in the preparation of a drug for the treatment of ischemic stroke, characterized in that the drug reverses the damage caused by oxidative stress by scavenging free radicals and anti-oxidation, Improve the brain injury caused by cerebral ischemia-reperfusion, and it is used for the treatment of ischemic stroke.
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