CN114129508A - Combined material based on metal nano-enzyme material and temperature sensitive gel and construction method thereof - Google Patents
Combined material based on metal nano-enzyme material and temperature sensitive gel and construction method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of composite materials, and particularly relates to a composite material based on a metal nano-enzyme material and temperature-sensitive gel and a construction method thereof, wherein the composite material comprises the metal nano-enzyme and the temperature-sensitive gel, the mixing ratio of the metal nano-enzyme to the temperature-sensitive gel is 1-10 mug: 1mL, the composite material provided by the invention has stable heat resistance and cold resistance, a freeze-dried product has good water solubility, the temperature-sensitive gel belongs to high-molecular hydrogel, the temperature-sensitive gel is liquid at low temperature and is condensed at high temperature, the change of the state is reversible along with the temperature, meanwhile, the composite material has various characteristics of a polymeric material, such as a network structure and high water content, the metal nano-enzyme material and the temperature-sensitive gel are combined, the enzyme can stay in a target action part for a longer time to play a greater role, and the composite material is divided into a pre-encapsulation water needle or a freeze-dried preparation before use, the prefilled water needle can be directly added with a needle head for clysis, gastric lavage and the like; the freeze-dried preparation is directly used after being dissolved according to the proportion before freeze-drying, and is convenient and quick.
Description
Technical Field
The invention relates to the technical field of composite materials, in particular to a composite material based on a metal nano enzyme material and temperature sensitive gel and a construction method thereof.
Background
At present, metal nano-enzyme (such as iron-based nano-enzyme, manganese-based nano-enzyme and the like) has gradually become a interdisciplinary material and is more and more emphasized, for example, the manganese-based nano-enzyme is a nano-material which takes manganese as an active center and has natural enzyme activity. In addition to playing an important role in photosynthesis, divalent Mn ions are also important in hydrolysis and phosphotransferase. In addition, the higher valencies of manganese are the redox centers of ribonucleotide reductase, catalase, peroxidase and superoxide dismutase (SOD) in mitochondria. In 2016, researchers found that octahedral manganese oxide nanomaterials have superoxide dismutase-like properties, and they combined the properties with magnetic resonance imaging techniques to achieve the goal of improving nuclear magnetic resonance contrast with manganese oxide. This is because the content of SOD in the tumor tissue is reduced and the content of superoxide anion is increased, and the contrast of magnetic resonance imaging is related to the concentration of superoxide anion, and the lower the concentration of superoxide anion, the better the contrast effect. The relaxation time of manganese oxide is greatly increased when it is contacted with superoxide radical. Recent research results show that the dendritic trimanganese tetroxide nanoparticles have larger pore diameters, have activities of both superoxide dismutase-like and catalase-like, and the activities depend on size and shape, so that the elimination rate of superoxide anions can exceed fifty percent, and the Parkinson disease is effectively protected from a cell level.
However, the research on the application of mimic enzymes to living organisms in vivo has been slow, and it has been found that, although mimic enzymes have good oxidase activity and catalase activity, they also have biocompatibility and administration route problems. So far, the preparation of mature mimic enzyme preparations is not reported, more administration modes of the existing mimic enzyme are not standard, and the mimic enzyme is directly delivered after being dispersed by purified water and the like, so that the mimic enzyme has short retention time at a target action part and cannot effectively play a role.
Disclosure of Invention
The invention aims to provide a composite material based on a metal nano enzyme material and temperature-sensitive gel and a construction method thereof so as to solve the technical problems mentioned in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a composite material based on a metal nano-enzyme material and temperature-sensitive gel comprises the components of the metal nano-enzyme and the temperature-sensitive gel, wherein the mixing ratio of the metal nano-enzyme to the temperature-sensitive gel is 1-10 mug: 1 mL.
A construction method of a combined material based on a metal nano enzyme material and temperature sensitive gel comprises the following steps:
step 1: preparation of metal nano enzyme material
Dissolving a metal compound in absolute ethyl alcohol, magnetically stirring until the metal compound is completely dissolved, transferring the mixture into a polytetrafluoroethylene reaction kettle, reacting for 24 hours at the temperature of 100-120 ℃, cooling to room temperature, and adding water for washing to obtain a final product;
step 2: preparation of polylactic acid temperature sensitive gel
Setting the preheating temperature of a reaction kettle to be 80-120 ℃, adding PEG under the protection of inert gas, continuously synthesizing for 6-12 h when the PEG is heated and melted and the temperature is reduced to normal temperature under the protection of positive pressure of the inert gas, adding a crosslinking catalyst, then adding D, L-lactide, setting the temperature of the reaction kettle to be 150-180 ℃ under the protection of the inert gas, introducing cold circulating water into a jacket of the reaction kettle after the synthesis is finished, reducing the temperature of a liquid material to normal temperature, adding a proper amount of purified water into a washing tank, pouring the liquid material into the washing tank, strongly stirring for dissolving, standing, removing a supernatant, taking out a solid polylactic acid polymer into a stainless steel container, configuring a PBS buffer solution, controlling the temperature of the solution to be lower than normal temperature, adding the polylactic acid polymer into the PBS buffer solution, stirring for dissolving, and freeze-drying for later use;
and step 3: building up composite materials
And (3) mixing the products prepared in the steps (1) and (2), swirling, wherein the mixing ratio is 1-10 mu g:1mL, and freeze-drying or filling the mixed product by using a pre-filling and sealing syringe.
Preferably, in step 1, the ratio of manganese acetate to absolute ethyl alcohol is: 1.2-1.5 g: 60-100 mL.
Preferably, in the step 2, the molecular weight of PEG is 1500-2500, and the ratio of PEG to D, L-lactide to the crosslinking catalyst is 1:2: 0.009.
Preferably, the use mode of the combined material based on the metal nano-enzyme material and the temperature-sensitive gel is that purified water is adopted for preparation and use.
The invention has the beneficial effects that:
(1) the composite material provided by the invention has stable heat resistance and cold resistance, the freeze-dried product has good water solubility, the temperature-sensitive gel belongs to high-molecular hydrogel, the gel is liquid at low temperature and is condensed at high temperature, the change of the state is reversible along with the temperature, and meanwhile, the composite material has various characteristics of a polymeric material, such as a network structure and high water content.
(2) The composite material provided by the invention can be divided into a pre-filled and sealed water needle or a freeze-dried preparation before use, and the pre-filled and sealed water needle can be directly added with a needle head for clysis, gastric lavage and the like; the freeze-dried preparation is directly used after being dissolved according to the proportion before freeze-drying, and is convenient and quick.
(3) The composite material provided by the invention has the advantages that the antioxidant enzyme activity of the metal-based mimic enzyme and the catalase activity are not reduced compared with the activity of the pure mimic enzyme.
(4) The phase transition temperature of the composite material provided by the invention can be controlled by controlling the molecular weight of the block, and after the composite material is compounded, compared with the pure temperature-sensitive gel, the phase transition temperature has no obvious change, so that the phase transition characteristic of the temperature-sensitive gel can be realized, and the enzyme activity of the metal-based mimic enzyme can be considered.
Drawings
FIG. 1 is a scanning electron microscope photograph of manganese-based nanoenzyme in an embodiment of the present invention;
FIG. 2 is a graph comparing mimetic enzyme and composite superoxide enzyme activities in accordance with an embodiment of the present invention;
FIG. 3 is a graph comparing the activity of a mimic enzyme and the catalase in a composite material in an example of the present invention;
FIG. 4 is a blank control intestinal histopathological section of an embodiment of the invention;
FIG. 5 is a pathological section of intestinal tissue after single manganese-based nanoenzyme intervention in an embodiment of the present invention;
FIG. 6 is a pathological section of a simple temperature sensitive gel dried intestine tissue according to an embodiment of the present invention;
FIG. 7 is a pathological section of intestinal tissue after composite material drying in an embodiment of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example (b):
the metal-based mimic enzyme is manganese-based mimic enzyme, and comprises the following steps:
step 1: accurately weighing 1.225gMn (CH)3COO)2·4H2Dissolving O in 60mL of absolute ethanol, magnetically stirring until completely dissolving, and mixingTransferring the mixture into a polytetrafluoroethylene reaction kettle, reacting for 24 hours at 120 ℃, cooling the product to room temperature, and adding water to wash for three times to obtain a final product;
step 2: taking a reaction kettle, setting the preheating temperature to 120 ℃, adding PEG under the protection of inert gas, when the PEG is heated and melted, reducing the temperature to normal temperature, adding a crosslinking catalyst under the protection of positive pressure of the inert gas, then adding D, L-lactide, setting the temperature of the reaction kettle to 150 ℃ under the protection of the inert gas, continuously synthesizing for 12 hours, after the synthesis is finished, introducing cooling water into a jacket of the reaction kettle for circulation, reducing the temperature of liquid materials to normal temperature, adding a proper amount of purified water into a washing tank, placing the liquid materials into the washing tank, stirring and dissolving the liquid materials by strong force, standing, removing supernatant, taking out solids into a stainless steel container, preparing a PBS buffer solution, controlling the temperature of the solution to be lower than normal temperature, adding a polylactic acid polymer into the PBS buffer solution, stirring and dissolving the polylactic acid polymer, and freeze-drying the polylactic acid polymer for later use, wherein the molecular weight of the PEG is 1500-2500: d, L-lactide: the mass ratio of the crosslinking catalyst is 1:2: 0.009;
PBS buffer: disodium hydrogen phosphate 5g, sodium dihydrogen phosphate 0.3g, polylactic acid polymer 100g and purified water 600 mL.
And step 3: mixing the products prepared in the step 1 and the step 2, swirling, mixing the mixture in a ratio of 10 mug to 1mL, and freeze-drying the mixed product.
The manganese-based nanoenzyme composite material prepared by the method has stable heat resistance, cold resistance and water solubility, and the microstructure of the manganese-based nanoenzyme composite material can be seen through a high-resolution scanning electron microscope, and is polyhedral as shown in two figures in figure 1.
The manganese-based nanoenzyme and nanoenzyme composite synthesized in example 1 were subjected to a superoxide radical scavenging ability test and a hydrogen peroxide scavenging ability test, respectively:
(1) superoxide radical scavenging ability test: adding the reagents according to the following table, quickly mixing the reagents uniformly, reacting the mixture in a water bath at 35 ℃, taking a blank as a reference, placing the blank in a quartz cuvette at the wavelength of 320nm at intervals of 0.5min, measuring the A value once, and continuously recording the A value until the reaction reaches the balance.
| Reagent | Blank space | Self-oxidation | Mn3O4 | PLG | PLG-Mn3O4 |
| 50mM Tris-HCl buffer (mL) pH8.2 | 4.5 | 4.5 | 4.5 | 4.5 | 4.5 |
| 45mM pyrogallol (. mu.L) | 10 | 10 | 10 | 10 | |
| Distilled water (mu L) | 20 | 10 | |||
| 1mg/ml Mn3O4(μL) | 10 |
(2) Scavenging ability of hydrogen peroxide
Adding the reagents according to the following table, quickly mixing the reagents uniformly, reacting the mixture in a water bath at 37 ℃, placing the mixture in a quartz cuvette at the wavelength of 240nm at intervals of 0.5min to measure the A value once, and continuously recording the A value until the reaction reaches the balance. (the amount of the sample in the reaction system should be confirmed again during the operation)
Superoxide radical scavenging ability is shown in FIG. 2, and hydrogen peroxide scavenging ability is shown in FIG. 3. Through comparison of the two materials before and after compounding, the speed of the composite material is higher when the scavenging capacity of the superoxide radical is tested by using a simple manganese-based mimic enzyme, and the reaction speed of the composite material is lower when the scavenging capacity of the superoxide radical is tested, which shows that the temperature-sensitive gel can slow down the reaction speed of the mimic enzyme and a substrate, and the reaction degree tends to be consistent with the extension of the reaction time. In the hydrogen peroxide removal capability test, the reaction trend and the reaction speed of the pure manganese-based mimic enzyme and the mimic enzyme composite material tend to be consistent, and the reaction degree tends to be consistent. This also indicates that the enzymatic activity of the mimetic enzyme itself is not reduced by this complexing. The enzyme activities of the pure mimic enzyme and the mimic enzyme composite material tend to be consistent.
In order to show the application of the manganese-based mimic enzyme and the mimic enzyme composite material, a mouse enteritis model is designed, and the simple mimic enzyme and the composite material are respectively used for enema treatment.
Experimental grouping information
Grouping information is shown in the table, 12 mice in each group are cultured for three days in an adaptive manner, the mice are fed with the feed normally, drinking water is cooled after being boiled, then the 1 st group continuously and freely drinks the boiled drinking water, 2-4 groups are replaced with drinking water with the concentration of 4% Dextran Sodium Sulfate (DSS), the mice are fed normally, daily observation is carried out, and the bloody stool and other conditions of the mice can be observed in about 5-7 days. On day 7, two mice were sacrificed at random in each group (including the first group) to confirm the modelling of the enteritis model. Each group was administered as an enema according to the protocol in Table 1 three times consecutively with one day intervals. After administration, each group of sacrificed mice was dissected the day after the administration and the tissues were fixed in 4% paraformaldehyde for HE staining microscopy. After another 7 days of continuous culture, each group of the remaining mice was sacrificed, dissected, photographed, and collected. In order to avoid the death of the mice caused by individual difference, another 5 mice are independently taken and one cage is used for DSS water feeding modeling synchronously, and if the death exists in other groups, the supplementation is carried out.
Finally, the rectal tissue of the mouse is taken, fixed in 4% paraformaldehyde, and subjected to pathological diagnosis and cumulative scoring according to HE staining. The scoring results are shown in the table below, with higher scores leading to more severe disease.
Pathological diagnosis scoring data
| Group of | Group name | Inflammation pathology scoring | Overall score for pathological diagnosis |
| 1 | Negative of | 0 | 0 |
| 2 | Positive for | 17 | 41 |
| 3 | Manganese oxide mimic enzyme | 9 | 18 |
| 4 | Temperature-sensitive gel material | 12 | 22 |
| 5 | Thermo-sensitive manganese oxide mimic enzyme composite material | 8 | 14 |
The pathological sections are shown in FIGS. 4-7. Pathological data show that the temperature-sensitive manganese oxide mimic enzyme composite material has the minimum value in the intervention of the acute enteritis of the mice, namely inflammation pathological score or pathological diagnosis total score. The mimic enzyme can better play a role in the body of an animal and scavenge Reactive Oxygen Species (ROS) after the composite material is used. Ensuring recovery of the target inflammatory site. In pathological sections, it can be observed that the intestinal injury of a positive group is large, the intestinal injury of a negative group is basically zero, a pure manganese oxide group and a pure temperature-sensitive gel group play a certain role in the recovery of enteritis, and the pathological sections of the composite material show that the intervention effect on the enteritis of the intestinal tracts is better than that of the pure two materials.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. A composite material based on a metal nano enzyme material and temperature sensitive gel is characterized in that: the temperature-sensitive gel comprises metal nano-enzyme and temperature-sensitive gel, wherein the mixing ratio of the metal nano-enzyme to the temperature-sensitive gel is 1-10 mu g:1 mL.
2. A method for constructing a composite material based on a metal nano enzyme material and temperature sensitive gel is characterized by comprising the following steps: the construction method comprises the following steps:
step 1: preparation of metal nano enzyme material
Dissolving a metal compound in absolute ethyl alcohol, magnetically stirring until the metal compound is completely dissolved, transferring the mixture into a polytetrafluoroethylene reaction kettle, reacting for 24 hours at the temperature of 100-120 ℃, cooling to room temperature, and adding water for washing to obtain a final product;
step 2: preparation of polylactic acid temperature sensitive gel
Setting the preheating temperature of the reaction kettle to be 80-120 ℃, adding polyethylene glycol under the protection of inert gas, reducing the temperature to normal temperature when the polyethylene glycol is heated and melted, adding a crosslinking catalyst under the protection of positive pressure of inert gas, then adding D, L-lactide, under the protection of inert gas, setting the temperature of the reaction kettle to be 150-180 ℃, continuously reacting and synthesizing for 6-12 h, after the synthesis is finished, introducing cold circulating water into the jacket of the reaction kettle, reducing the temperature of the liquid material to normal temperature, adding a proper amount of purified water into a washing tank, pouring the liquid material into the washing tank, stirring strongly for dissolving, standing, removing supernatant, taking out solid polylactic acid polymer, putting into a stainless steel container, preparing a phosphate buffer salt solution, controlling the temperature of the solution to be lower than the normal temperature, putting the polylactic acid polymer into a PBS buffer solution, stirring and dissolving, and freeze-drying for later use;
and step 3: building up composite materials
And (3) mixing the products prepared in the steps (1) and (2), swirling, wherein the mixing ratio is 1-10 mu g:1mL, and freeze-drying or filling the mixed product by using a pre-filling and sealing syringe.
3. The method for constructing the combined material based on the metal nano-enzyme material and the temperature-sensitive gel as claimed in claim 2, wherein the method comprises the following steps: in the step 1, the proportion of manganese acetate and absolute ethyl alcohol is as follows: 1.2-1.5 g: 60-100 mL.
4. The method for constructing the combined material based on the metal nano-enzyme material and the temperature-sensitive gel as claimed in claim 2, wherein the method comprises the following steps: in the step 2, the molecular weight of PEG is 1500-2500, and the mass ratio of PEG, D, L-lactide to the cross-linking catalyst is 1:2: 0.009.
5. The method for constructing the combined material based on the metal nano-enzyme material and the temperature-sensitive gel as claimed in claim 2, wherein the method comprises the following steps: the use mode is that purified water is adopted for configuration and use.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114797889A (en) * | 2022-04-12 | 2022-07-29 | 上海工程技术大学 | Fe 3 O 4 @MnO 2 -CeO 2 Nano material and preparation method and application thereof |
| CN114797889B (en) * | 2022-04-12 | 2023-10-17 | 上海工程技术大学 | Fe (Fe) 3 O 4 @MnO 2 -CeO 2 Nanometer material and preparation method and application thereof |
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