CN114099704B - Simple preparation method and application of ultra-small nano selenium metal frame composite material - Google Patents
Simple preparation method and application of ultra-small nano selenium metal frame composite material Download PDFInfo
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Abstract
The application provides an ultra-small nano selenium metal organic frame composite material and a preparation method and application thereof, wherein PEG400 and selenium powder are added into a beaker, and the mixture is stirred after ultrasonic treatment to uniformly disperse the selenium powder in the PEG 400; transferring the beaker to a magnetic stirring heater, stirring while heating, naturally cooling after reacting for a certain time, transferring to a centrifuge tube, centrifuging, taking an upper layer solution after centrifuging, and finally obtaining the PEG-SepPs; the nano-selenium metal organic frame composite material is prepared by using the raw materials comprising the PEG-Sepps and the metal salt solution, and is resuspended in a buffer solution. The ultra-small nano selenium metal organic frame composite material provided by the application has the effect of effectively removing free radicals, has good antioxidation capability, can effectively reverse the damage caused by oxidative stress, and improves the brain damage caused by cerebral ischemia reperfusion.
Description
Technical Field
The application belongs to the field of metal-organic frame materials, and particularly relates to an ultra-small nano selenium metal-organic frame composite material, a preparation method and application thereof.
Background
The central nervous system is the central control system of the human body, and slight lesions or injuries can have immeasurable consequences. Cerebral stroke is one of the main causes of adult disability in recent years. Due to the high oxygen consumption and lipid-rich nature of the brain, many diseases associated with oxidative stress damage can occur after injury, which produces deleterious reactive nitrogen and oxygen species, such as peroxides and superoxides, that trigger a range of biochemical reactions and neuroinflammation.
Currently, the massive use of early recanalization technology in the ischemic stroke treatment stage has prompted more focus on reperfusion injury caused by blood flow reperfusion. Ischemia reperfusion injury refers to the situation in which the degree of tissue injury increases rapidly after a certain period of time has elapsed after the recovery of blood flow from a tissue suffering from ischemia. Ischemia reperfusion injury is mainly due to the generation of a large amount of active oxygen free radicals after reperfusion, and excessive generation of the active oxygen free radicals can mediate inflammation and immune response by stimulating the expression of cytokines and adhesion molecules, so that the inflammation is further damaged, the neural network is secondarily damaged, and the ischemia reperfusion injury is finally caused. In the case of non-early recanalization patients, the mechanism of ischemia reperfusion injury described above is also present due to collateral circulation compensation, spontaneous recanalization, and the like. Therefore, reducing damage to brain tissue by reactive oxygen species cerebral vascular systems and neural networks following cerebral ischemic injury, particularly during ischemic stroke reperfusion, may be a therapeutic target for ischemic stroke. The search for better treatment methods has important application prospect.
Selenium is a trace element essential to the human body. Selenium is a constituent of a variety of amino acids in the human body, such as selenocysteine, selenomethionine and selenocysteine. Thus, elemental selenium has a wide range of physiological and pharmacological properties in humans, including anti-tumor, antioxidant, and the like. In the human body, selenium element enters the human body and can be converted into selenium-containing amino acid, and finally the selenium-containing protein is formed to play a role. Selenoprotein is produced by altering the intracellular oxidation level.
Metal organic framework materials are widely studied and developed for use in various catalytic reactions due to their excellent catalytic properties. Research shows that different metal organic framework materials have different capabilities of simulating antioxidant natural enzymes and have the potential of being applied to scavenging a large number of free radicals generated by reperfusion of ischemic cerebral apoplexy.
While there is much evidence in animal studies that a number of vascular nerve unit protectants against the ischemic mechanism have not been demonstrated in clinical studies, and are not generalized by clinical guidelines. However, edaravone, which only takes free radical scavenging as a main action mechanism, is approved and promoted in Asian countries such as Japan and China based on the evidence of the edaravone in a multi-center clinical study. However, since the phase III clinical study of NXY-059, which also has radical scavenging as the main mechanism, ends up failing, the radical scavenger has not been approved and promoted by European and American countries.
Disclosure of Invention
In view of the above-described drawbacks of the prior art, an object of the present application is to provide a method for preparing an ultra-small nano-selenium metal organic framework composite material.
Another object of the application is to provide an ultra-small nano-selenium metal organic framework composite.
The application further aims at providing an application of the ultra-small nano-selenium metal organic framework composite material in preparing a medicament for treating ischemic stroke.
The patent proposal provides a preparation method of an ultra-small nano selenium metal organic framework composite material, which comprises the following steps
(1) Preparation of ultra-small nano-selenium (PEG-Sepps): adding PEG400 and selenium powder into a beaker, and stirring after ultrasonic treatment to uniformly disperse the selenium powder in the PEG 400; transferring the beaker to a magnetic stirring heater, stirring while heating, naturally cooling after reacting for a certain time, transferring to a centrifuge tube for centrifugal treatment, centrifuging, taking an upper solution, removing unreacted selenium powder, and finally obtaining the PEG-Sepps;
(2) Preparing an ultra-small nano selenium metal organic frame composite material: the nano-selenium metal organic frame composite material is prepared by using the raw materials comprising the PEG-Sepps and the metal salt solution, and the ultra-small nano-selenium metal organic frame composite material is resuspended in a buffer solution.
As a first embodiment, the ultra-small nano-selenium metal organic framework composite material in the step (2) is Se@ZIF-8, the metal salt solution is a solution of zinc nitrate hexahydrate, and the buffer solution is methanol.
The step (2) comprises: mixing the PEG-Sepps with a solution in which the dimethylimidazole is dissolved, stirring until the PEG-Sepps are dispersed, adding polyvinylpyrrolidone, stirring until the PEG-Sepps are dissolved, finally adding an aqueous solution of zinc nitrate hexahydrate, uniformly stirring, standing, centrifuging to obtain Se@ZIF-8, and then washing with methanol and re-suspending in the methanol.
As a second embodiment, the ultra-small nano-selenium metal organic framework composite material in the step (2) is Se@MIL-101, the metal salt solution is DMF solution in which ferric chloride hexahydrate and terephthalic acid are dissolved, and the buffer solution is DMF.
The step (2) comprises: mixing the PEG-Sepps with DMF solution in which ferric chloride hexahydrate and terephthalic acid are dissolved, stirring uniformly, transferring into a Teflon reaction kettle, reacting for a certain time at 100-140 ℃ to obtain Se@MIL-101, and then re-suspending in DMF after washing for a plurality of times with DMF solution.
As a third embodiment, the ultra-small nano-selenium metal organic frame composite material in the step (2) is Se@HKUST-1, the metal salt solution is a DMF solution of copper chloride, and the buffer solution is DMF.
The step (2) comprises: mixing PEG-Sepps with DMF solution of cupric chloride uniformly by ultrasonic, adding DMF solution in which trimesic acid is dissolved into the mixed solution, adding acetic acid, rapidly heating to 130-170 ℃ after sealing a mouth by a rubber plug, maintaining for a period of time, naturally cooling, centrifuging to obtain Se@HKUST-1, washing by using DMF solution, resuspension and preserving at low temperature.
As a fourth embodiment, the ultra-small nano-selenium metal organic framework composite material in the step (2) is Se@Ru-Mn-MOF, and the metal salt solution is MnCl 2 DMF and Ru (POPCOOH) 3 DMF of (2) and buffer solution of DMF;
the step (2) comprises: taking the PEG-SeNPs and MnCl 2 Is mixed with DMF solution of (2) and ultrasonic homogenized, ru (POPCOOH) is added into the mixed solution 3 Heating to 140-180 ℃ after sealing a mouth by a rubber plug, maintaining for a period of time, naturally cooling to below 100 ℃, quenching to room temperature by ice water bath, centrifugally collecting solids, and obtaining the Se@Ru-Mn-MOF, and then washing and resuspension by using DMF and preserving at low temperature.
And (3) transferring the waste water into a centrifugal tube for centrifugal treatment in the step (1), wherein the centrifugal speed is 1500-2500rpm, and the centrifugal time is 9-11min.
The application also provides the ultra-small nano-selenium metal organic frame composite material prepared by the preparation method, which is characterized in that the ultra-small nano-selenium metal organic frame composite material is a material loaded by taking a metal organic frame as a carrier.
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.
The application also provides an application of the ultra-small nano-selenium metal organic frame composite material prepared by the preparation method in preparation of a medicament for treating ischemic stroke, and the application of the material with the ultra-small nano-selenium loaded by taking the metal organic frame as a carrier in treatment of ischemic stroke.
The medicine can improve brain injury caused by cerebral ischemia reperfusion by scavenging free radicals and resisting oxidation, reverses injury caused by oxidative stress, and is used for treating ischemic stroke.
The improvement of this patent brings the following advantage:
(1) The preparation method of the ultra-small nano selenium metal organic frame composite material provided by the embodiment of the application has relatively mild reaction conditions, and does not need harsh reaction conditions, such as high temperature and high pressure; the reaction steps are simple and clear, and the method is easy for experiments and mass production.
(2) According to experimental examples of the application, the four ultra-small nano selenium metal organic frame composite materials have the effect of effectively removing free radicals, have good oxidation resistance, and have good capability of repeatedly removing free radicals and hydroxy free radicals. Can effectively reverse the damage caused by oxidative stress, improve the brain damage caused by cerebral ischemia reperfusion, and achieve the effect of efficiently protecting brain tissues.
(3) The application combines the metal organic frame nano material with catalytic capability with the ultra-small nano selenium, and fully exerts the advantages of large specific surface area, high catalytic efficiency and the like of the metal organic frame material. Meanwhile, the metal organic frame material can be used as a carrier of the ultra-small nano-selenium in vivo, and the absorption and the application of the ultra-small nano-selenium in vivo are promoted.
(4) Experimental examples show that the metal organic frame material loaded with the ultra-small nano-selenium at the same concentration has more excellent capability of scavenging free radicals than the single ultra-small nano-selenium and the single metal organic frame material. Provides more evidence for the neuroprotection mechanism of the nano material and the clinical application of the nano material in ischemia reperfusion therapy in future.
(5) As a further improvement, the application adopts four typical metal organic frame materials to load the ultra-small nano-selenium, thereby creatively preparing the composite nano-material of the ultra-small nano-selenium and the metal organic frame materials and providing reference for the ultra-small nano-selenium loaded by other metal organic frame materials.
(6) As a further improvement, the application is found by comparing sodium selenite, sodium selenate, selenocysteine, selenomethionine, chitosan modified nano-selenium, lentinan modified nano-selenium, ultra-small nano-selenium (PEG-Sepps) and four metal organic frame materials: the four inorganic selenium of sodium selenite, sodium selenate, selenocysteine and selenomethionine have no antioxidation capability, the chitosan modified nano selenium, the lentinan modified nano selenium and the ultra-small nano selenium (PEG-Sepps) have weak antioxidation capability, the four metal-organic frame materials independently have lower antioxidation capability, and the ultra-small nano selenium metal-organic frame material can effectively improve the antioxidation capability by about 1.3-2.6 times compared with the ultra-small nano selenium, so that references are provided for the improvement of the antioxidation capability of other selenium-containing nano particles.
(7) By carrying out an ischemic reperfusion mouse experiment at a cell level and an animal level, we find that four ultra-small nano selenium metal organic framework composite materials are damaged due to oxidative stress, thereby achieving the function of efficiently protecting brain tissues.
Drawings
FIG. 1 is a transmission electron microscope image and an elemental analysis image of four ultra-small nano-selenium metal organic framework composite materials; wherein, four kinds of ultra-small nano selenium metal organic frame composite materials are respectively: (1) Se@ZIF-8, (2) Se@MIL-101, (3) Se@Ru-Mn-MOF, (4) Se@HKUST-1;
FIG. 2 shows that four ultra-small nano-selenium metal organic framework composite materials reverse the damage of free radical damage models to cells;
FIG. 3 is a graph showing apoptosis of human neuroblastoma cells after treatment with a flow cytometer to detect a free radical damage model;
FIG. 4 shows the detection of active oxygen levels in human neuroblastoma cells of four ultra-small nano-selenium metal organic framework composites following free radical damage using a fluorogenic microplate reader;
fig. 5 is a graph of brain injury area of mice treated with four ultra-small nano-selenium metal organic framework composites under an ischemia reperfusion model.
Detailed Description
Other advantages and effects of the present application will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present application with reference to specific examples. The application may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present application.
At present, the nano technology and the discovery of nano materials with enzyme-like activity promote us to take new ideas for treating cerebral apoplexy and related diseases. Compared with natural enzymes, nanoenzymes exhibit high stability, durable catalytic activity and multifunctional enzyme properties. After the enzyme activity evaluation of the nano selenium and the metal organic framework nano particles, the nano enzyme is used for treating ischemic cerebral apoplexy. The nano-enzyme with biological safety has good prospect in the aspect of treating cerebral apoplexy and related diseases.
Example 1
(1) Preparation of PEG-SeNPs (ultra-small nano-selenium): adding polyethylene glycol (PEG) 400 and selenium powder into a beaker, and stirring after ultrasonic treatment to uniformly disperse the selenium powder in the PEG 400; transferring the beaker to a magnetic stirring heater, stirring while heating, naturally cooling after reacting for one period, transferring to a centrifuge tube, centrifuging, taking an upper solution after centrifuging, removing unreacted selenium powder, and finally obtaining the PEG-Sepps;
(2) Preparing an ultra-small nano selenium metal organic frame composite material: the nano-selenium metal organic frame composite material is prepared by using the raw materials comprising the PEG-Sepps and the metal salt solution, and is resuspended in a buffer solution.
Example 2
(1) Preparation of PEG-SeNPs: 10mL of PEG400 is added into a beaker, then 20mg of selenium powder is weighed, and stirring is carried out for 5 minutes after ultrasonic treatment, so that the selenium powder is completely and uniformly dispersed in the PEG 400; the transferring beaker is placed on a magnetic stirring heater, heated to the temperature of about 205 ℃, stirred while being heated, naturally cooled after reacting for 1 hour, transferred to a centrifuge tube, centrifuged at 2000rpm for 10 minutes, and then taken out of the upper layer solution, unreacted selenium powder is removed, and finally PEG-Sepps is obtained, wherein the concentration of the obtained PEG-Sepps is 16-17mM.
(2) Preparing an ultra-small nano selenium and zinc-containing organic framework composite material Se@ZIF-8: mixing 2 mLPEG-Sepps with 10mL solution with 20.94mg of dimethyl imidazole dissolved therein, stirring until PEG-Sepps are completely dispersed, then adding 400mg of polyvinylpyrrolidone (PVP), stirring until the mixture is completely dissolved, finally adding 10mL of solution with 75.12mg of zinc nitrate hexahydrate dissolved therein, uniformly stirring, standing the mixture to room temperature overnight (12 h), centrifuging the mixture at 12000rpm, and obtaining Se@ZIF-8, washing the mixture with a large amount of methanol, and re-suspending the mixture in the methanol.
Or preparing the composite material Se@MIL-101 of the ultra-small nano selenium and the iron-containing organic frame: 1ml of PEG-Sepps was stirred uniformly with 20ml of DMF solution in which 13.42mg of ferric chloride hexahydrate and 12.02mg of terephthalic acid were dissolved, then transferred to a Teflon reaction kettle, reacted at 120℃for 12 hours to prepare Se@MIL-101, washed 3 times with DMF and resuspended in DMF.
Or preparing the ultra-small nano selenium and copper-containing organic framework composite material Se@HKUST-1: 1ml of PEG-modified PEG-Sepps and 5ml of DMF dissolved with 0.15mmol of copper chloride are mixed uniformly by ultrasonic, 5ml of DMF solution dissolved with 0.1mmol of trimesic acid is added into the mixed solution, 2-3 drops of acetic acid are added, the temperature is quickly heated to 150 ℃ after the mouth is sealed by a rubber plug, the temperature is kept for 10min, centrifugation is carried out at 8000rmp and 5min after natural cooling, se@HKUST-1 is prepared, and the mixed solution is washed and resuspended by equal volume of DMF and then is placed at 4 ℃ for standby.
Or preparing the ultra-small nano selenium and manganese-containing ruthenium organic framework composite material Se@Ru-Mn-MOF: 1ml of PEG-modified PEG-Sepps was taken with 0.05mmol of MnCl dissolved therein 2 5ml of DMF was mixed and sonicated uniformly, and 5ml of Ru (POPCOOH) dissolved in 0.05mmol was added to the mixture 3 Heating to 160 ℃ after sealing a mouth by a rubber plug, keeping for 10min, naturally cooling to below 100 ℃, quenching to room temperature by ice water bath, centrifuging 8000rmp for 5min, collecting solid, preparing Se@Ru-Mn-MOF, washing with equal volume DMF, and suspending at 4 ℃ for later use.
Example 3
(1) Preparation of PEG-SeNPs: 8mL of PEG400 is added into a beaker, 17mg of selenium powder is weighed, and stirring is carried out for 4 minutes after ultrasonic treatment, so that the selenium powder is completely and uniformly dispersed in the PEG 400; the transferring beaker is placed on a magnetic stirring heater, heated to the temperature of about 200 ℃, stirred while being heated, naturally cooled after reacting for 1 hour, transferred to a centrifuge tube, centrifuged at 1800rpm for 11min, and then taken out of the upper layer solution, unreacted selenium powder is removed, and finally PEG-Sepps is obtained, wherein the concentration of the obtained PEG-Sepps is 16-20mM.
(2) Preparing an organic framework composite material Se@ZIF-8 of ultra-small nano selenium and zinc: 1.8mL of PEG-Sepps was mixed with 8.8mL of a solution containing 19mg of dimethylimidazole, stirred until the PEG-Sepps were completely dispersed, then 380mg of polyvinylpyrrolidone was added, stirred until complete dissolution, finally 9mL of an aqueous solution containing 68mg of zinc nitrate hexahydrate was added, stirred uniformly, then allowed to stand overnight (11 h) at 11000rpm, centrifuged to give Se@ZIF8, washed with a large amount of methanol, and resuspended in methanol.
Or preparing an organic framework composite material Se@MIL-101 of the ultra-small nano selenium and iron: 0.9ml of PEG-Sepps was taken and stirred well with 18.5ml of DMF solution in which 12.15mg of ferric chloride hexahydrate and 10.9mg of terephthalic acid were dissolved, then transferred to a Teflon reactor and reacted at 115℃for 13 hours to prepare Se@MIL-101, which was washed 4 times with DMF and resuspended in DMF.
Or preparing an organic framework composite material Se@HKUST-1 of the ultra-small nano selenium and copper: mixing 0.9ml of PEG-modified PEG-Sepps with 4.8ml of DMF dissolved with 0.14mmol of copper chloride uniformly by ultrasonic, adding 4.8ml of DMF dissolved with 0.09mmol of trimesic acid into the mixed solution, adding 2-3 drops of acetic acid, quickly heating to 145 ℃ after sealing a mouth by a rubber plug, keeping for 11min, naturally cooling, centrifuging for 7500rmp and 6min to prepare Se@HKUST-1, washing and resuspension by equal volume of DMF, and then placing at 4 ℃ for standby.
Or preparing an organic framework composite material Se@Ru-Mn-MOF of the ultra-small nano selenium and the manganese ruthenium: 0.9ml of PEG-modified PEG-SeNPs was taken with 0.04mmol of MnCl dissolved therein 2 4.5ml of DMF was mixed and sonicated uniformly, and 4.5ml of Ru (POPCOOH) dissolved in 0.04mmol was added to the mixture 3 Heating to 155 ℃ after sealing the mouth by a rubber plug, keeping for 11min, naturally cooling to 95 ℃ and quenching to room temperature by an ice water bath, centrifugally collecting solids at 7500rmp for 6min to prepare Se@Ru-Mn-MOF, washing with equal volume of DMFAnd after re-suspending, the mixture is placed at 4 ℃ for standby.
The four ultra-small nano-selenium metal organic framework composite materials prepared by the method of the embodiment 2 are subjected to the following experiment.
Experimental example 1
Four ultra-small nano-selenium metal organic frame composite materials were tested using transmission electron microscopy and elemental analysis, and the results are shown in figure 1. The four ultra-small nano selenium metal organic frame composite materials are respectively (the following is the same): se@ZIF-8, se@MIL-101, se@Ru-Mn-MOF, se@HKUST-1. The transmission electron microscope image and the element analysis image of the four ultra-small nano-selenium metal organic frame composite materials shown in the figure 1 show that the preparation of the four ultra-small nano-selenium loaded metal organic frame materials is successful. 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.
Experimental example 2
And detecting the antioxidant capacity of sodium selenite, sodium selenate, selenocysteine, selenomethionine, chitosan modified nano-selenium, lentinan modified nano-selenium, ultra-small nano-selenium (PEG-Sepps) and four nano-selenium metal organic frame materials by using an ABTS total antioxidant capacity detection method. Sodium selenite, sodium selenate, selenocysteine and selenomethionine have no obvious antioxidant capacity. The chitosan modified nano-selenium, the lentinan modified nano-selenium and the ultra-small nano-selenium (PEG-Sepps) have a small amount of antioxidant capacity; the oxidation resistance of the four ultra-small nano-selenium metal organic frame materials formed by compounding the metal organic frame materials is enhanced compared with that of the single ultra-small nano-selenium (PEG-Sepps), and the oxidation resistance is enhanced by about 1.3-2.6 times compared with that of the single ultra-small nano-selenium, so that the addition of the metal organic frame has remarkable effect of improving the oxidation resistance of the nano-selenium.
Experimental example 3
In the experimental example, a human brain neuroblastoma cell (SH-SY 5Y) cell line is selected as an object of a cell model, free radicals generated by cells are simulated by adding 20 mu M of tertiary butyl hydroperoxide, and finally the survival rate of the SH-SY5Y cells is detected by an MTT method, so that the protection effect of four ultra-small nano selenium metal organic frame composite materials on the cells in the free radical injury SH-SY5Y cell model is evaluated, and the result is shown in figure 2. As can be seen from the cell viability in fig. 2, the cell viability after the injury of t-butyl hydroperoxide is about 70%, the cell viability after the addition of nano-selenium is improved to some extent, and the cell viability after the treatment of the four ultra-small nano-selenium metal organic frame composite materials is obviously improved relative to the injury group and the single ultra-small nano-selenium group (Se group in the figure), which indicates that the four nano-selenium metal organic frame composite materials can effectively protect cells from being injured by free radicals.
Experimental example 4
In the experimental example, a human brain neuroblastoma cell (SH-SY 5Y) cell line is selected as an object of a cell model, free radicals generated by cells are simulated by adding 20 mu M of tertiary butyl hydroperoxide, and finally the apoptosis proportion of the SH-SY5Y cells is detected by a flow cytometry to evaluate the protection effect of the four ultra-small nano selenium metal organic frame composite materials on the SH-SY5Y cells in the cell damage model by the free radical damage model, and the result is shown in figure 3. From fig. 3, it can be seen from the cell survival rate that the apoptosis ratio of the cells after the t-butyl hydroperoxide is damaged is about 40%, the apoptosis ratio of the cells after the ultra-small nano selenium is added is reduced, and the apoptosis ratio of the four ultra-small nano selenium metal organic frame composite materials after being treated is obviously reduced compared with the damage group and the independent nano selenium group, which indicates that the four ultra-small nano selenium metal organic frame composite materials can effectively protect the cells from being damaged by free radicals.
Experimental example 5
In this experimental example, a human brain neuroblastoma cell (SH-SY 5Y) cell line is selected as an object of a cell model, free radicals generated by cells are simulated by adding 20 mu M of tertiary butyl hydroperoxide, and finally, the level of active oxygen in SH-SY5Y cells is detected by detecting a fluorescent dye through the active oxygen in DCFH-DA cells. The protection effect of the four ultra-small nano selenium metal organic framework composite materials on SH-SY5Y cells in a cell damage model by a free radical damage model is evaluated, and the result is shown in figure 4. From FIG. 4, it can be observed that the intracellular active oxygen level is obviously increased after the tert-butyl hydroperoxide is added, and the intracellular active oxygen level is obviously reduced after the four ultra-small nano-selenium metal organic frame composite materials are added. The results show that the four ultra-small nano selenium metal organic framework composite materials can effectively reduce the active oxygen level in cells caused by free radicals.
Experimental example 6
In the experimental example, a C57 mouse is selected as an experimental object, and a cerebral apoplexy ischemia reperfusion model is prepared by using Middle Cerebral Artery Occlusion (MCAO) operation. Reperfusion was accomplished by removing the rubber stopper 40 minutes after blocking blood flow in the middle cerebral artery of the mouse using a silicone stopper. Then four nano-selenium metal organic framework composite materials are continuously given to the mice for 3 days, the brain tissues of the mice are taken out on the fourth day, the brain tissues are uniformly divided into 5 parts by a blade, each thickness is about 2mm, then the brain tissues are dyed by using 2,3, 5-triphenyltetrazolium chloride (TTC), and finally the situation after the TTC dyeing of the brain tissues is shot by using a camera. As shown in FIG. 5, it can be seen from TTC staining that the Sham group is a control group, and the brain tissue of the mice is not damaged, so that the brain tissue is red after TTC staining. The white area of the mouse brain tissue of the Saline group is obviously larger than that of the Sham group, and then the white area of the mouse brain tissue treated by the four nano-selenium metal organic frame composite materials is obviously smaller than that of the Saline group, so that the four ultra-small nano-selenium metal organic frame composite materials can effectively protect the mouse brain tissue from damage caused by cerebral apoplexy ischemia reperfusion.
The application researches the treatment effect of four metal organic frame materials of ultra-small nano-selenium in ischemic cerebral apoplexy reperfusion injury through experimental examples 1-6. Experiments show that compared with the single metal organic frame material or nano-selenium, the metal organic frame material loaded with the ultra-small nano-selenium has better effect of treating ischemic cerebral apoplexy reperfusion injury.
Through a transmission electron microscope and elemental analysis, four metal organic frame materials loaded with ultra-small nano selenium can be successfully prepared. Meanwhile, the application uses the human neuroblastoma cells after free radical injury to verify that the human neuroblastoma cells reverse the injury caused by free radicals, and can reduce the level of ROS in cells. Finally, the performance of the four metal organic frame materials loaded with the ultra-small nano-selenium is verified in a mouse model of ischemic cerebral apoplexy reperfusion injury, and the four metal organic frame materials loaded with the ultra-small nano-selenium are found to be capable of reversing cerebral tissue injury caused by ischemic cerebral apoplexy reperfusion injury and effectively reducing the area of cerebral tissue necrosis. The application also discloses application of the ultra-small nano selenium metal organic frame composite material in preparing a medicament for treating ischemic stroke.
In conclusion, the application not only shows the preparation method of nano-selenium loaded by the metal organic framework material, but also provides more evidence for the nerve protection mechanism of the nano-material and the clinical application of the nano-material in ischemia reperfusion therapy in future.
The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.
Claims (4)
1. The preparation method of the ultra-small nano selenium metal organic framework composite material is characterized by comprising the following steps of:
(1) Preparation of ultra-small nano-selenium (PEG-Sepps): adding PEG400 and selenium powder into a beaker, and stirring after ultrasonic treatment to uniformly disperse the selenium powder in the PEG 400; transferring the beaker to a magnetic stirring heater, heating while stirring, naturally cooling after reacting for a certain time, transferring to a centrifuge tube for centrifugal treatment at 1500-2500rpm for 9-11min, taking an upper solution after centrifugation, removing unreacted selenium powder, and finally obtaining the PEG-Sepps;
(2) Preparing an ultra-small nano selenium metal organic framework composite material Se@ZIF-8: mixing the PEG-Sepps with a solution in which dimethyl imidazole is dissolved, stirring until the PEG-Sepps are dispersed, then adding polyvinylpyrrolidone, stirring until the PEG-Sepps are dissolved, finally adding a solution of zinc nitrate hexahydrate, uniformly stirring, standing, centrifuging to obtain Se@ZIF-8, and then washing with methanol and re-suspending in the methanol;
or preparing the ultra-small nano selenium metal organic framework composite material Se@MIL-101: mixing the PEG-Sepps with DMF solution in which ferric chloride hexahydrate and terephthalic acid are dissolved, stirring uniformly, transferring into a Teflon reaction kettle, reacting for a certain time at 100-140 ℃ to obtain Se@MIL-101, and then re-suspending in DMF after washing for many times with DMF solution;
or preparing the ultra-small nano selenium metal organic framework composite material Se@HKUST-1: mixing the PEG-Sepps with DMF solution of copper chloride uniformly by ultrasonic, adding DMF solution in which trimesic acid is dissolved into the mixed solution, adding acetic acid, quickly heating to 130-170 ℃ after sealing a mouth by a rubber plug, keeping for a period of time, naturally cooling, centrifuging to obtain Se@HKUST-1, washing by using DMF solution, resuspension, and preserving at low temperature;
or preparing the ultra-small nano selenium metal organic framework composite material Se@Ru-Mn-MOF: taking the PEG-SeNPs and MnCl 2 Is mixed with DMF solution of (2) and ultrasonic homogenized, ru (POPCOOH) is added into the mixed solution 3 Heating to 140-180 ℃ after sealing a mouth by a rubber plug, maintaining for a period of time, naturally cooling to below 100 ℃, quenching to room temperature by ice water bath, centrifugally collecting solids, and obtaining the Se@Ru-Mn-MOF, and then washing and resuspension by using DMF and preserving at low temperature.
2. The ultra-small nano-selenium metal organic frame composite material prepared by the preparation method of claim 1 is characterized in that the material is a material loaded with nano-selenium by taking a metal organic frame as a carrier, and comprises any one of Se@ZIF-8, se@MIL-101, se@HKUST-1 and Se@Ru-Mn-MOF.
3. The application of the ultra-small nano-selenium metal organic frame composite material prepared by the preparation method of claim 1 in preparing a medicament for treating ischemic stroke, which is characterized in that the application of the material taking the metal organic frame as a carrier to load the ultra-small nano-selenium in treating ischemic stroke.
4. The use of the ultra-small nano-selenium metal organic framework composite material according to claim 3 for preparing a medicament for treating ischemic stroke, wherein the medicament is used for treating ischemic stroke by scavenging free radicals and resisting oxidation, reversing injury caused by oxidative stress, and improving brain injury caused by cerebral ischemia reperfusion.
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