Injectable metal nanoparticle hybrid hydrogel material as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to an injectable metal nanoparticle hybrid hydrogel material as well as a preparation method and application thereof. According to the injectable metal nanoparticle hybrid hydrogel material provided by the invention, oxidized dextran, aminated gelatin, adipic dihydrazide and other components are mixed, so that in-situ forming of hydrogel on a complex injured wound surface can be realized; meanwhile, cerium oxide nanoparticles doped in the hydrogel can be uniformly dispersed in the hydrogel and efficiently remove reactive oxygen species (ROS), in addition, an imine bond and a hydrazone bond for constructing a hydrogel skeleton serve as reversible bonds and can respond to microenvironment changes, and removal of the ROS with structural stability during tissue use is regulated, controlled and optimized; therefore, the invention provides the high-performance injectable metal nanoparticle hybrid hydrogel material, and the technical problem that injectable hydrogel in the prior art is relatively low in performance is solved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical materials, and particularly relates to an injectable metal nanoparticle hybrid hydrogel material and a preparation method and application thereof. BACKGROUND

[0002] As a functional material with unique physical and chemical and mechanical properties, the structure of a hydrogel is a soft and hydrophilic three-dimensional network system constructed by cross-linked polymers. Such a function enables the hydrogel to mimic the cell microenvironment and adapt to the tissue structure well. Due to excellent rheological properties and strong structural stability, the hydrogel has excellent biodegradability and biocompatibility, and has been widely used in biomedical materials.

[0003] Currently, the mainstream hydrogel biomedical material is prepared into a shaped hydrogel in advance and then implanted into a wound surface. However, the site and shape of the damaged wound surface are complex and diverse, and the pre-shaped hydrogel is difficult to adapt to the damaged wound surface. By developing an injectable hydrogel, the hydrogel can be shaped in situ at the damaged wound surface, which can better adapt to the damaged wound surface. Oxidative free radicals ROS are generated at the damaged wound surface to kill bacteria, resist infection and promote healing. However, for special groups such as diabetics, high blood sugar state mediates oxidative stress, generating a large amount of oxidative free radicals ROS, so that the damaged wound surface is in a state of oxidative damage greater than healing, which makes it difficult for the damaged wound surface to heal. For the damaged wound surface of special groups such as diabetics, an antioxidant active ingredient needs to be used to promote the healing of the damaged wound surface. In addition to diabetics, special groups with high oxidative stress-related diseases such as chronic dermatitis and neurodegenerative diseases (Alzheimer's disease) also need to use antioxidant active ingredients to treat high oxidative stress.

[0004] Generally, antioxidant active ingredients such as ascorbic acid are consumed after removing oxidative free radicals ROS, and need to be continuously supplemented, which is not suitable for the treatment of chronic diseases such as diabetes. Cerium is one of the most abundant rare earth metals, which exists in two cationic states (Ce 3+ and Ce 4+ ), has a crystal cubic fluorite lattice structure and a highly reactive surface, and the two valence states (Ce 3+ / Ce 4+ ) can be converted to each other while removing free radicals, so that the cerium antioxidant active ingredient can be reused without frequent replenishment, which can remove oxidative free radicals ROS for a long time and is suitable for the treatment of chronic high oxidative stress diseases. Due to its high specific surface area, the surface of cerium oxide nanoparticles (CeO2 NP) exposes a large amount of Ce 3+ / Ce 4+The active sites can more efficiently contact and react with oxidative free radicals ROS, greatly improving the free radical scavenging efficiency, so that the cerium oxide nanoparticles (CeO2 NP) loaded into the hydrogel has a broad application prospect in treating various high oxidative stress related diseases.

[0005] However, the cerium oxide nanoparticles (CeO2 NP) have poor dispersibility, and it is difficult to uniformly add them to various systems, such as injectable hydrogels and other aqueous media. Due to the high surface energy characteristics, the particles are prone to uncontrollable agglomeration, resulting in a decrease in specific surface area and insufficient exposure of active sites, thereby reducing their antioxidant and catalytic properties. Therefore, the performance of the current injectable hydrogel loaded with antioxidant active ingredients is low, and it is difficult to meet the treatment needs of various high oxidative stress related diseases. SUMMARY

[0006] Therefore, the present application provides an injectable metal nanoparticle hybrid hydrogel material, a preparation method and an application, to solve the technical problem of low performance of the prior art injectable hydrogel.

[0007] The first aspect of the present application provides an injectable metal nanoparticle hybrid hydrogel material, the raw material composition of which comprises: polyethylene glycol and derivative modified cerium oxide nanoparticles, oxidized dextran, aminated gelatin and adipic acid dihydrazide.

[0008] Preferably, the injectable metal nanoparticle hybrid hydrogel material comprises 1 part by mass of polyethylene glycol and derivative modified cerium oxide nanoparticles, 50 to 200 parts by mass of oxidized dextran, 50 to 400 parts by mass of aminated gelatin and 1 to 10 parts by mass of adipic acid dihydrazide.

[0009] Among them, 1 part by mass of polyethylene glycol and derivative modified cerium oxide nanoparticles, for 50 to 200 parts by mass of oxidized dextran, can be "an integer between 50 and 200", for example, 50, 51, 52... or 100 parts by mass of oxidized dextran; and 50 to 400 parts by mass of aminated gelatin, can be "an integer between 50 and 400", for example, 50, 51, 52... or 400 parts by mass of aminated gelatin; and 1 to 10 parts by mass of adipic acid dihydrazide, can be "an integer between 1 and 10", for example, 1, 2, 3... or 10 parts by mass of adipic acid dihydrazide.

[0010] Preferably, the polyethylene glycol and derivative modified cerium oxide nanoparticles comprise 1 part by mass of cerium oxide nanoparticles and 10 to 100 parts by mass of polyethylene glycol and derivatives.

[0011] Among them, for 10 to 100 parts by mass of polyethylene glycol and derivatives, the amount is "an integer between 10 and 100".

[0012] Preferably, the polyethylene glycol and its derivatives are selected from at least one of PEG2000, PEG4000, DSPE-PEG2000 (distearoylphosphatidylethanolamine grafted polyethylene glycol), DSPE-PEG4000, DSPE-MPEG2000 (distearoylphosphatidylethanolamine grafted methoxypolyethylene glycol), DSPE-MPEG4000.

[0013] Preferably, the injectable metal nanoparticle hybrid hydrogel material further comprises a solvent selected from at least one of water, normal saline, PBS, and culture medium.

[0014] Preferably, the mass ratio of the polyethylene glycol and its derivatives modified cerium oxide nanoparticles, oxidized dextran, aminated gelatin, and adipic acid dihydrazide, and the solvent is 0.2-1:10-20:10-40:0.2-2:200-600.

[0015] Wherein, the first digit after the decimal point in 0.2-1 can be an integer between 1 and 9, the amount in 10-20 can be an integer between 10 and 20, the first digit after the decimal point in 0.2-2 can be an integer between 1 and 9, and the amount in 200-600 can be an integer between 200 and 600.

[0016] Preferably, the mass ratio of the polyethylene glycol and its derivatives modified cerium oxide nanoparticles, oxidized dextran, aminated gelatin, and adipic acid dihydrazide, and the solvent is 0.4:20:20:0.5:300-500.

[0017] The second aspect of the present application provides a preparation method of an injectable metal nanoparticle hybrid hydrogel material, which can prepare the injectable metal nanoparticle hybrid hydrogel material of the first aspect; the preparation method comprises the following steps:

[0018] The preparation step of cerium oxide nanoparticles: cerium source, dispersant, and surfactant are used to prepare cerium oxide nanoparticles by an oil thermal method;

[0019] The preparation step of the surface modified cerium oxide nanoparticle dispersion: 1 part by mass of cerium oxide nanoparticles and 10-100 parts by mass of polyethylene glycol and its derivatives are mixed and ultrasonicated to obtain polyethylene glycol and its derivatives modified cerium oxide nanoparticles, which are then configured into a polyethylene glycol and its derivatives modified cerium oxide nanoparticle dispersion;

[0020] The preparation step of the oxidized dextran solution: after oxidizing the hydroxyl groups in the dextran molecules to obtain oxidized dextran, an oxidized dextran aqueous solution is configured;

[0021] The preparation step of the amino gelatin solution: after activating the carboxyl in the gelatin molecule by an activating agent and an amino donor and introducing a primary amino group to obtain an amino gelatin, the amino gelatin is configured into an amino gelatin aqueous solution;

[0022] The mixing step: mixing the configured adipic acid dihydrazide aqueous solution, the amino gelatin aqueous solution, the oxidized dextran aqueous solution, and the polyethylene glycol and derivative modified cerium oxide nanoparticle dispersion to obtain an injectable metal nanoparticle hybrid hydrogel material.

[0023] Preferably, in the preparation step of the cerium oxide nanoparticles, the process of the oil thermal method comprises: reacting the cerium source, the dispersant, and the surfactant at a temperature of 200-320°C at a stirring speed of 500-2000 rpm for 30-120 min.

[0024] In the above, 200-320°C can be "an integer Celsius degree between 200 and 320", and 500-2000 rpm can be "an integer rotating speed between 50 and 2000", and 30-120 min can be "an integer time between 30 and 120".

[0025] Preferably, in the preparation step of the cerium oxide nanoparticles, the cerium source is selected from at least one of cerium nitrate hexahydrate, anhydrous cerium chloride, or cerium acetylacetone, the dispersant is selected from at least one of oleylamine, dodecylamine, or oleic acid, and the surfactant is selected from at least one of octadecene, trioctylamine, or dibenzyl ether.

[0026] Preferably, in the preparation step of the cerium oxide nanoparticles, the molar ratio of the cerium source, the dispersant, and the surfactant is 1 mmol: 10-20 ml: 10-20 ml.

[0027] In the above, 10-20 ml can be "an integer milliliter between 10 and 20".

[0028] Preferably, in the preparation step of the surface-modified cerium oxide nanoparticle dispersion, the ultrasonic time is 30-120 min.

[0029] In the above, 30-120 min can be "an integer time between 30 and 120".

[0030] Preferably, in the preparation step of the surface-modified cerium oxide nanoparticle dispersion, the mass percentage of the configured polyethylene glycol and derivative modified cerium oxide nanoparticle dispersion is 0.1-1%.

[0031] In the above, the first digit after the decimal point in 0.1-1% can be "an integer percentage between 1 and 9".

[0032] Preferably, in the preparation step of the oxidized dextran solution, the reaction temperature for selective oxidation of hydroxyl groups is 10~45℃ and the time is 1~8h;

[0033] Among them, 10~45℃ can be "an integer degree Celsius between 10 and 45", and 1~8h can be "an integer time between 1 and 8".

[0034] Preferably, in the step of preparing the oxidized dextran solution, the oxidant is selected from at least one of sodium periodate and potassium periodate.

[0035] Preferably, in the preparation step of the oxidized dextran solution, the mass-to-volume ratio of dextran to oxidant is 1~10g:10~100ml, and the mass fraction of the oxidant is 5~20%.

[0036] Among them, 1~10g can be "an integer number of grams between 1 and 10", 10~100ml can be "an integer number of milliliters between 10 and 100", and 5~20% can be "an integer percentage between 5 and 20".

[0037] Preferably, in the preparation step of the oxidized dextran solution, the mass percentage of the prepared oxidized dextran aqueous solution is 10-20%.

[0038] Among them, 10~20% can be "an integer percentage between 10 and 20".

[0039] Preferably, in the preparation step of the aminated gelatin solution, the pH is 4-6, the reaction temperature is 30-40℃, and the time is 4-10h during the process of activating the carboxyl groups in the gelatin molecules and attaching primary amino groups.

[0040] Among them, pH is 4~6, which can be 4, 5, or 6, and "the first decimal place can be an integer between 1 and 9, or 0", while 30~40℃ can be "an integer between 30 and 40 degrees Celsius", and 4~10h can be "an integer between 4 and 10 hours".

[0041] Preferably, in the preparation step of the aminated gelatin solution, the activator is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and dicyclohexylcarbodiimide (DCC), and the amino donor is selected from at least one of ethylenediamine and hexamethylenediamine.

[0042] Preferably, in the preparation step of the aminated gelatin solution, the mass-volume ratio of gelatin, activator and amino donor is: 1~10g: 1~4g: 5~30ml;

[0043] Among them, 1~10g can be "an integer number of grams between 1 and 10", 1~4g can be "an integer number of grams between 1 and 4", and 5~30min can be "an integer number of milliliters between 5 and 30".

[0044] Preferably, in the preparation step of the aminated gelatin solution, the mass percentage of the prepared aminated gelatin aqueous solution is 10-20%.

[0045] Among them, 10~20% can be "an integer percentage between 10 and 20".

[0046] Preferably, the mass percentage of the prepared adipic acid dihydrazide aqueous solution is 5-10%;

[0047] Among them, 5~10% can be "an integer percentage between 5 and 10".

[0048] The third aspect of this application provides the application of an injectable metal nanoparticle hybrid hydrogel material in the preparation of anti-inflammatory and antioxidant therapeutic biomedical materials, sustained-release biomedical materials, or tissue engineering materials.

[0049] The fourth aspect of this application provides an in-situ molded metal nanoparticle hybrid hydrogel, which is synthesized in-situ from an injectable metal nanoparticle hybrid hydrogel material as described in the first aspect, including an in-situ molded hydrogel and cerium oxide nanoparticles modified with polyethylene glycol and its derivatives.

[0050] The cerium oxide nanoparticles modified with polyethylene glycol and its derivatives are dispersed in an in-situ formed hydrogel.

[0051] The hydrophilic three-dimensional cross-linked network framework of the in-situ formed hydrogel is dynamically constructed based on the imine bonds and hydrazone bonds formed by oxidized dextran with aminoated gelatin and adipic acid dihydrazide, respectively.

[0052] Preferably, the in-situ formed metal nanoparticle hybrid hydrogel comprises, by weight, 1 part by weight of cerium oxide nanoparticles modified with polyethylene glycol and its derivatives, 50-200 parts by weight of oxidized dextran, 50-400 parts by weight of aminated gelatin, and 1-10 parts by weight of adipic acid dihydrazide.

[0053] Among them, 1 part by mass of cerium oxide nanoparticles modified with polyethylene glycol and its derivatives can be an integer between 50 and 200 parts by mass of oxidized dextran, such as 50, 51, 52, ... or 100 parts by mass of oxidized dextran; and 50 to 400 parts by mass of aminated gelatin can be an integer between 50 and 400, such as 50, 51, 52, ... or 400 parts by mass of aminated gelatin; as for 1 to 10 parts by mass of adipic acid dihydrazide, it can be an integer between 1 and 10, such as 1, 2, 3, ... or 10 parts by mass of adipic acid dihydrazide.

[0054] Preferably, the cerium oxide nanoparticles modified with polyethylene glycol and its derivatives in the in-situ formed metal nanoparticle hybrid hydrogel include 1 part by mass of cerium oxide nanoparticles and 10 to 100 parts by mass of polyethylene glycol and its derivatives.

[0055] For 10 to 100 parts by mass of polyethylene glycol and its derivatives, the amount is "an integer between 10 and 100".

[0056] Compared with the prior art, the injectable metal nanoparticle hybrid hydrogel material provided in this application has at least the following beneficial effects:

[0057] 1. This application provides an injectable metal nanoparticle hybrid hydrogel material, comprising cerium oxide nanoparticles modified with polyethylene glycol and its derivatives, oxidized dextran, aminoated gelatin, and adipate dihydrazide, etc., which is a high-performance hydrogel material that is injectable and can be molded in situ, can efficiently remove ROS, and dynamically responds to the microenvironment of damaged wounds.

[0058] 2. The injectable metal nanoparticle hybrid hydrogel material provided in this application can improve the efficiency of ROS removal by controlling the mass ratio of polyethylene glycol and its derivatives-modified cerium oxide nanoparticles to 1:10~50.

[0059] 3. The injectable metal nanoparticle hybrid hydrogel material provided in this application has a wide range of applications. It can be used as an anti-inflammatory and antioxidant biomedical material for oxidative stress treatment, or as a sustained-release biomedical material based on the dynamic response of the microenvironment of the damaged wound. It can also be used as a tissue engineering material. Attached Figure Description

[0060] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0061] Figure 1 The particle size test results are for the cerium oxide nanoparticles and the cerium oxide nanoparticles modified with polyethylene glycol and its derivatives provided in Example 1 of this application.

[0062] Figure 2 The dispersion stability test results of cerium oxide nanoparticles and cerium oxide nanoparticles modified with polyethylene glycol and its derivatives provided in Example 1 of this application were analyzed by transmission electron microscopy.

[0063] Figure 3 The dispersion stability test results of dispersions of 0.1% by mass of the cerium oxide nanoparticles modified with polyethylene glycol and its derivatives provided in Examples 1-3 of this application are presented.

[0064] Figure 4 The injectability test results are for the injectable metal nanoparticle hybrid hydrogel material provided in Example 1 of this application;

[0065] Figure 5 Images of the injectable metal nanoparticle hybrid hydrogel material provided in Example 1 of this application, before and after gelation.

[0066] Figure 6 The images show the injectable metal nanoparticle hybrid hydrogel materials provided in Examples 1-3 of this application after gelation.

[0067] Figure 7 The experimental test results of scavenging oxidative free radicals (ROS) using the injectable metal nanoparticle hybrid hydrogel material provided in the embodiments of this application are shown in the figure.

[0068] Figure 8 The figures show the experimental test results of scavenging oxidative free radicals (ROS) in the injectable metal nanoparticle hybrid hydrogel materials provided in Examples 4-5 of this application. Detailed Implementation

[0069] This application provides an injectable metal nanoparticle hybrid hydrogel material, its preparation method, and its application, which addresses the technical problem of low performance of injectable hydrogels in the prior art.

[0070] The technical solutions of this application will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0071] Given the current limitations of injectable hydrogels loaded with antioxidant active ingredients, which have low performance and cannot meet the treatment needs of various diseases related to high oxidative stress, this application provides an injectable metal nanoparticle hybrid hydrogel material. The composition of the provided injectable metal nanoparticle hybrid hydrogel material includes: cerium oxide nanoparticles modified with polyethylene glycol and its derivatives, oxidized dextran, aminoated gelatin, and adipate dihydrazide, etc.

[0072] In the injectable metal nanoparticle hybrid hydrogel material provided in this application, oxidized dextran, before and immediately after mixing with components such as aminated gelatin and adipate dihydrazide, is a flowing injectable liquid. After standing for a period of time, oxidized dextran forms imine bonds (-C=N-) and hydrazone bonds (-C=NNH-) with aminated gelatin and adipate dihydrazide, respectively, constructing a hydrophilic three-dimensional cross-linked network framework for the hydrogel. This enables in-situ molding, and the hydrophilic three-dimensional cross-linked network framework of the hydrogel is used to fill solvents such as water, thereby enabling the in-situ molding of biocompatible / degradable hydrogels on complex wound surfaces. Furthermore, the cerium oxide nanoparticles modified with polyethylene glycol and its derivatives in the hydrogel material, due to the surface modification with polyethylene glycol and its derivatives, enhance the hydrophilicity of the cerium oxide nanoparticles, allowing them to be uniformly dispersed in the hydrogel material, preventing agglomeration, and resulting in a large surface area with a large amount of Ce exposed on the surface. 3+ / Ce 4+The active sites can efficiently react with oxidative free radicals (ROS), significantly improving the free radical scavenging efficiency and thus significantly enhancing the hydrogel performance. This allows the hydrogel to scavenge ROS levels in patients with oxidative stress down to near normal physiological levels. Furthermore, since imine bonds (-C=N-) and hydrazone bonds (-C=NNH-) are reversible dynamic crosslinking bonds, the hydrophilic three-dimensional crosslinked network framework of the hydrogel constructed based on imine bonds (-C=N-) / hydrazone bonds (-C=NNH-) in this application can respond to changes in the microenvironment. It can regulate and optimize the structural stability during tissue use according to changes in the microenvironment of the damaged wound, control the release amount of cerium oxide nanoparticles modified with doped polyethylene glycol and its derivatives, and regulate and optimize the scavenging of oxidative free radicals (ROS). Therefore, the injectable metal nanoparticle hybrid hydrogel material provided in this application is a high-performance hydrogel material that is injectable and in-situ moldable, can efficiently scavenge ROS, and dynamically responds to the microenvironment of the damaged wound, overcoming the shortcomings of current injectable hydrogels with relatively low performance.

[0073] Meanwhile, for cerium oxide nanoparticles modified with polyethylene glycol and its derivatives, when the amount of polyethylene glycol and its derivatives is small, the cerium oxide nanoparticles cannot be successfully modified. For example, when the mass ratio of polyethylene glycol and its derivatives to cerium oxide nanoparticles is 1:1, the cerium oxide nanoparticles cannot be modified, which reduces the performance of the injectable metal nanoparticle hybrid hydrogel material. Therefore, in this application, the mass ratio of polyethylene glycol and its derivatives to cerium oxide nanoparticles is further controlled to be 1:10~50; where 10~50 can be "an integer between 10 and 50".

[0074] The following section will provide a detailed explanation of injectable metal nanoparticle hybrid hydrogel materials, combining the preparation method and experimental testing.

[0075] Example 1

[0076] This embodiment provides a method for preparing an injectable metal nanoparticle hybrid hydrogel material. The preparation method includes: a step of preparing cerium oxide nanoparticles, a step of preparing a surface-modified cerium oxide nanoparticle dispersion, a step of preparing an oxidized dextran solution, a step of preparing an aminated gelatin solution, and a mixing step.

[0077] The preparation steps of cerium oxide nanoparticles include: weighing 15 ml of dispersant oleylamine, 15 ml of surfactant octadecene, and 1 mmol of cerium source acetylacetone solid; dissolving the weighed cerium source acetylacetone solid in oleylamine and then adding the oleylamine and octadecene mixture, reacting at 280°C with a stirring speed of 1000 rpm for 60 minutes, washing three times with anhydrous ethanol to remove oleylamine and oleic acid, obtaining cerium oxide nanoparticles (CeO2 NP), which are then dissolved in cyclohexane solution for later use.

[0078] The preparation steps of the surface-modified cerium oxide nanoparticle dispersion include: weighing cerium oxide nanoparticles and distearylphosphatidylethanolamine-grafted polyethylene glycol (DSPE-PEG2000) at a mass ratio of 1:20; dissolving the cerium oxide nanoparticles and distearylphosphatidylethanolamine-grafted polyethylene glycol in an appropriate amount of dichloromethane; modifying the surface of the cerium oxide nanoparticles with distearylphosphatidylethanolamine-grafted polyethylene glycol by ultrasonic treatment for 60 minutes; washing three times with water to obtain cerium oxide nanoparticles modified with polyethylene glycol and its derivatives, named CeO2 NP-PEG; and then preparing a dispersion with a mass percentage of 0.4% using water as a solvent for later use, i.e., the concentration of cerium oxide nanoparticles modified with polyethylene glycol and its derivatives is about 0.4 g / 100 mL (4000 μg / 1 mL).

[0079] The preparation steps of the oxidized dextran solution include: weighing 6g of dextran with an average molecular weight of 10000, 100ml of water, and 40ml of sodium periodate solution with a mass percentage of 10%; after dissolving the weighed dextran in water, slowly adding the sodium periodate solution as an oxidant dropwise to the dextran solution, and then reacting at 25℃ in the dark for 4h to selectively oxidize the hydroxyl groups in the dextran molecules to aldehyde groups; next, the product is placed in a dialysis bag (MWCO=3500), dialyzed for 3 days for purification, and the final product is obtained by lyophilization; it is then prepared as a 10% oxidized dextran aqueous solution for later use.

[0080] The preparation steps of the aminated gelatin solution include: weighing 5g gelatin, 100ml PBS solution, 2.675g activator 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 15mL amino donor ethylenediamine; completely dissolving the weighed gelatin in the PBS solution, adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and dissolving it completely, then slowly adding ethylenediamine, adjusting the pH to 5.0 with an appropriate amount of 5M HCl, and then making up the volume to 400mL with PBS at pH = 5, stirring in a constant temperature water bath at 37℃ for 6h; finally, placing the product into a dialysis bag (MWCO=3500), dialyzing for 3 days for purification, obtaining the final product by lyophilization, and preparing a 20% (w / w) aminated gelatin aqueous solution for later use.

[0081] The mixing steps include: weighing adipic acid dihydrazide and deionized water in a mass ratio of 1:19, and dissolving the adipic acid dihydrazide in water to prepare a 5% (w / w) aqueous solution of adipic acid dihydrazide; then mixing the surface-modified cerium oxide nanoparticle dispersion, oxidized dextran aqueous solution, aminated gelatin aqueous solution, and adipic acid dihydrazide aqueous solution uniformly in a ratio of 1:2:1:0.1 to obtain an injectable metal nanoparticle hybrid hydrogel material.

[0082] Example 2

[0083] This embodiment provides a method for preparing an injectable metal nanoparticle hybrid hydrogel material. The preparation method includes: a step of preparing cerium oxide nanoparticles, a step of preparing a surface-modified cerium oxide nanoparticle dispersion, a step of preparing an oxidized dextran solution, a step of preparing an aminated gelatin solution, and a mixing step.

[0084] The preparation steps of cerium oxide nanoparticles include: weighing 10 ml of dispersant oleylamine, 10 ml of surfactant octadecene, and 1 mmol of cerium source acetylacetone solid; dissolving the weighed cerium source acetylacetone solid in oleylamine and then adding the oleylamine and octadecene mixture, reacting at 300°C with a stirring speed of 1500 rpm for 50 minutes, washing three times with anhydrous ethanol to remove oleylamine and oleic acid, obtaining cerium oxide nanoparticles (CeO2 NP), which are then dissolved in cyclohexane solution for later use.

[0085] The preparation steps of the surface-modified cerium oxide nanoparticle dispersion include: weighing cerium oxide nanoparticles and distearylphosphatidylethanolamine-grafted polyethylene glycol (DSPE-PEG2000) at a mass ratio of 1:30; dissolving the cerium oxide nanoparticles and distearylphosphatidylethanolamine-grafted polyethylene glycol in an appropriate amount of dichloromethane; modifying the surface of the cerium oxide nanoparticles with distearylphosphatidylethanolamine-grafted polyethylene glycol by ultrasonic treatment for 60 minutes; washing three times with water to obtain cerium oxide nanoparticles modified with polyethylene glycol and its derivatives, named CeO2 NP-PEG; and then preparing a dispersion with a mass percentage of 0.4% using water as a solvent for later use.

[0086] The preparation steps of the oxidized dextran solution include: weighing 6g of dextran with an average molecular weight of 10000, 100ml of water, and 40ml of sodium periodate solution with a mass percentage of 10%; after dissolving the weighed dextran in water, slowly adding the sodium periodate solution as an oxidant dropwise to the dextran solution, and then reacting at 25℃ in the dark for 2h to selectively oxidize the hydroxyl groups in the dextran molecules to aldehyde groups; next, the product is placed in a dialysis bag (MWCO=3500), dialyzed for 3 days for purification, and the final product is obtained by lyophilization; and then it is prepared as a 10% oxidized dextran aqueous solution for later use.

[0087] The preparation steps of the aminated gelatin solution include: weighing 3g of gelatin, 50ml of PBS solution, 1.3375g of activator 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 9mL of amino donor ethylenediamine; completely dissolving the weighed gelatin in the PBS solution, adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide until completely dissolved, and slowly adding ethylenediamine, then adjusting the pH to 5.0 with an appropriate amount of 5M HCl, and then making up the volume to 200mL with PBS at pH = 5, stirring in a constant temperature water bath at 37℃ for 6h; finally, the product is placed in a dialysis bag (MWCO=3500), dialyzed for 3 days for purification, and the final product is obtained by lyophilization, and a 20% (w / w) aminated gelatin aqueous solution is prepared for later use.

[0088] The mixing steps include: weighing adipic acid dihydrazide and deionized water in a mass ratio of 1:19, and dissolving the adipic acid dihydrazide in water to prepare a 5% (w / w) aqueous solution of adipic acid dihydrazide; then mixing the surface-modified cerium oxide nanoparticle dispersion, oxidized dextran aqueous solution, aminated gelatin aqueous solution, and adipic acid dihydrazide aqueous solution uniformly in a ratio of 1:2:1:0.1 to obtain an injectable metal nanoparticle hybrid hydrogel material.

[0089] Example 3

[0090] This embodiment provides a method for preparing an injectable metal nanoparticle hybrid hydrogel material. The preparation method includes: a step of preparing cerium oxide nanoparticles, a step of preparing a surface-modified cerium oxide nanoparticle dispersion, a step of preparing an oxidized dextran solution, a step of preparing an aminated gelatin solution, and a mixing step.

[0091] The preparation steps of cerium oxide nanoparticles include: weighing 20 ml of dispersant oleylamine, 15 ml of surfactant octadecene, and 1 mmol of cerium source acetylacetone solid; dissolving the weighed cerium source acetylacetone solid in oleylamine and adding it to the oleylamine and octadecene mixture, reacting at 280°C with a stirring speed of 1500 rpm for 70 minutes, washing three times with anhydrous ethanol to remove oleylamine and oleic acid, obtaining cerium oxide nanoparticles (CeO2 NP), which are then dissolved in cyclohexane solution for later use.

[0092] The preparation steps of the surface-modified cerium oxide nanoparticle dispersion include: weighing cerium oxide nanoparticles and distearylphosphatidylethanolamine-grafted polyethylene glycol (DSPE-PEG2000) at a mass ratio of 1:25; dissolving the cerium oxide nanoparticles and distearylphosphatidylethanolamine-grafted polyethylene glycol in an appropriate amount of dichloromethane; modifying the surface of the cerium oxide nanoparticles with distearylphosphatidylethanolamine-grafted polyethylene glycol by ultrasonic treatment for 60 minutes; washing three times with water to obtain cerium oxide nanoparticles modified with polyethylene glycol and its derivatives, named CeO2 NP-PEG; and then preparing a dispersion with a mass percentage of 0.4% using water as a solvent for later use.

[0093] The preparation steps of the oxidized dextran solution include: weighing 5g of dextran with an average molecular weight of 10000, 100ml of water, and 30ml of sodium periodate solution with a mass percentage of 10%; after dissolving the weighed dextran in water, slowly adding the sodium periodate solution as an oxidant dropwise to the dextran solution, and then reacting at 25℃ in the dark for 4h to selectively oxidize the hydroxyl groups in the dextran molecules to aldehyde groups; next, the product is placed in a dialysis bag (MWCO=3500), dialyzed for 3 days for purification, and the final product is obtained by lyophilization; and then it is prepared as a 10% oxidized dextran aqueous solution for later use.

[0094] The preparation steps of the aminated gelatin solution include: weighing 5g of gelatin, 200ml of PBS solution, 2.675g of activator 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 15mL of amino donor ethylenediamine; completely dissolving the weighed gelatin in the PBS solution, adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and dissolving it completely, then slowly adding ethylenediamine, adjusting the pH to 5.0 with an appropriate amount of 5M HCl, and then bringing the volume to 400mL with PBS at pH = 5, stirring in a constant temperature water bath at 37℃ for 6h; finally, placing the product into a dialysis bag (MWCO = 3500), dialyzing for 3 days for purification, obtaining the final product by lyophilization, and preparing a 20% (w / w) aminated gelatin aqueous solution for later use.

[0095] The mixing steps include: weighing adipic acid dihydrazide and deionized water in a mass ratio of 1:19, and dissolving the adipic acid dihydrazide in water to prepare a 5% (w / w) aqueous solution of adipic acid dihydrazide; then mixing the surface-modified cerium oxide nanoparticle dispersion, oxidized dextran aqueous solution, aminated gelatin aqueous solution, and adipic acid dihydrazide aqueous solution uniformly in a ratio of 1:2:1:0.1 to obtain an injectable metal nanoparticle hybrid hydrogel material.

[0096] Example 4

[0097] This embodiment provides a method for preparing an injectable metal nanoparticle hybrid hydrogel material. The preparation method includes: a step of preparing cerium oxide nanoparticles, a step of preparing a surface-modified cerium oxide nanoparticle dispersion, a step of preparing an oxidized dextran solution, a step of preparing an aminated gelatin solution, and a mixing step.

[0098] The preparation steps of cerium oxide nanoparticles include: weighing 20 ml of dispersant oleylamine, 15 ml of surfactant octadecene, and 1 mmol of cerium source acetylacetone solid; dissolving the weighed cerium source acetylacetone solid in oleylamine and then adding the oleylamine and octadecene mixture, reacting at 280°C with a stirring speed of 1500 rpm for 70 minutes, washing three times with anhydrous ethanol to remove oleylamine and oleic acid, obtaining cerium oxide nanoparticles (CeO2 NP), which are then dissolved in cyclohexane solution for later use.

[0099] The preparation steps of the surface-modified cerium oxide nanoparticle dispersion include: weighing cerium oxide nanoparticles and distearylphosphatidylethanolamine-grafted polyethylene glycol (DSPE-PEG2000) at a mass ratio of 1:1; dissolving the cerium oxide nanoparticles and distearylphosphatidylethanolamine-grafted polyethylene glycol in an appropriate amount of dichloromethane; modifying the surface of the cerium oxide nanoparticles with distearylphosphatidylethanolamine-grafted polyethylene glycol by ultrasonic treatment for 60 minutes; washing three times with water to obtain cerium oxide nanoparticles modified with polyethylene glycol and its derivatives, named CeO2 NP-PEG; and then preparing a dispersion with a mass percentage of 0.4% using water as a solvent for later use, i.e., the concentration of cerium oxide nanoparticles modified with polyethylene glycol and its derivatives is about 0.4 g / 100 mL (4000 μg / 1 mL).

[0100] The preparation steps of the oxidized dextran solution include: weighing 5g of dextran with an average molecular weight of 10000, 100ml of water, and 30ml of sodium periodate solution with a mass percentage of 10%; after dissolving the weighed dextran in water, slowly adding the sodium periodate solution as an oxidant dropwise to the dextran solution, and then reacting at 25℃ in the dark for 4h to selectively oxidize the hydroxyl groups in the dextran molecules to aldehyde groups; next, the product is placed in a dialysis bag (MWCO=3500), dialyzed for 3 days for purification, and the final product is obtained by lyophilization; and then it is prepared as a 10% oxidized dextran aqueous solution for later use.

[0101] The preparation steps of the aminated gelatin solution include: weighing 5g of gelatin, 200ml of PBS solution, 2.675g of activator 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 15mL of amino donor ethylenediamine; completely dissolving the weighed gelatin in the PBS solution, adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and dissolving it completely, then slowly adding ethylenediamine, adjusting the pH to 5.0 with an appropriate amount of 5M HCl, and then bringing the volume to 400mL with PBS at pH = 5, stirring in a constant temperature water bath at 37℃ for 6h; finally, placing the product into a dialysis bag (MWCO = 3500), dialyzing for 3 days for purification, obtaining the final product by lyophilization, and preparing a 20% (w / w) aminated gelatin aqueous solution for later use.

[0102] The mixing steps include: weighing adipic acid dihydrazide and deionized water in a mass ratio of 1:19, and dissolving the adipic acid dihydrazide in water to prepare a 5% (w / w) aqueous solution of adipic acid dihydrazide; then mixing the surface-modified cerium oxide nanoparticle dispersion, oxidized dextran aqueous solution, aminated gelatin aqueous solution, and adipic acid dihydrazide aqueous solution uniformly in a ratio of 1:2:1:0.1 to obtain an injectable metal nanoparticle hybrid hydrogel material.

[0103] Comparative Example 1

[0104] This comparative example provides a method for preparing an injectable metal nanoparticle hybrid hydrogel material, the method including: a preparation step of cerium oxide nanoparticle dispersion, a preparation step of oxidized dextran solution, a preparation step of aminated gelatin solution, and a mixing step.

[0105] The preparation steps of the cerium oxide nanoparticle dispersion include: weighing 20 ml of dispersant oleylamine, 15 ml of surfactant octadecene, and 1 mmol of cerium-derived cerium acetylacetone solid; dissolving the weighed cerium-derived cerium acetylacetone solid in oleylamine, then adding the oleylamine and octadecene mixture, reacting at 280°C with a stirring speed of 1500 rpm for 70 minutes, washing three times with anhydrous ethanol to remove oleylamine and oleic acid, obtaining cerium oxide nanoparticles (CeO2 NP), which are then dissolved in cyclohexane solution for later use; subsequently, using water as a solvent, a dispersion with a mass percentage of 0.4% is prepared for later use, i.e., the concentration of cerium oxide nanoparticles is approximately 0.4 g / 100 mL (4000 μg / 1 mL).

[0106] The preparation steps of the oxidized dextran solution include: weighing 5g of dextran with an average molecular weight of 10000, 100ml of water, and 30ml of sodium periodate solution with a mass percentage of 10%; after dissolving the weighed dextran in water, slowly adding the sodium periodate solution as an oxidant dropwise to the dextran solution, and then reacting at 25℃ in the dark for 4h to selectively oxidize the hydroxyl groups in the dextran molecules to aldehyde groups; next, the product is placed in a dialysis bag (MWCO=3500), dialyzed for 3 days for purification, and the final product is obtained by lyophilization; and then it is prepared as a 10% oxidized dextran aqueous solution for later use.

[0107] The preparation steps of the aminated gelatin solution include: weighing 5g of gelatin, 200ml of PBS solution, 2.675g of activator 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 15mL of amino donor ethylenediamine; completely dissolving the weighed gelatin in the PBS solution, adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and dissolving it completely, then slowly adding ethylenediamine, adjusting the pH to 5.0 with an appropriate amount of 5M HCl, and then bringing the volume to 400mL with PBS at pH = 5, stirring in a constant temperature water bath at 37℃ for 6h; finally, placing the product into a dialysis bag (MWCO = 3500), dialyzing for 3 days for purification, obtaining the final product by lyophilization, and preparing a 20% (w / w) aminated gelatin aqueous solution for later use.

[0108] The mixing steps include: weighing adipic acid dihydrazide and deionized water at a mass ratio of 1:19, and dissolving the adipic acid dihydrazide in water to prepare a 5% (w / w) aqueous solution of adipic acid dihydrazide; then mixing the cerium oxide nanoparticle dispersion, the oxidized dextran aqueous solution, the aminated gelatin aqueous solution, and the adipic acid dihydrazide aqueous solution uniformly at a ratio of 1:2:1:0.1 to obtain an injectable metal nanoparticle hybrid hydrogel material.

[0109] Experimental Example 1

[0110] This experiment tests the dispersion stability and particle size of cerium oxide nanoparticles, polyethylene glycol and its derivatives modified cerium oxide nanoparticles provided in Examples 1-2.

[0111] The particle size testing process was as follows: the cerium oxide nanoparticles dissolved in cyclohexane solution provided in Example 1 were named sample CeO2, and the cerium oxide nanoparticles modified with polyethylene glycol and its derivatives dissolved in aqueous solution provided in Example 1 were named sample CeO2-PEG; subsequently, particle size was measured using a dynamic light scattering (DLS) instrument, and the test results are as follows. Figure 1 As shown; from Figure 1 It can be seen that compared with the CeO2 sample with a particle size distribution concentrated at around 18 nm, the CeO2-PEG sample has a larger particle size, with a particle size distribution concentrated at 43 nm. This indicates that polyethylene glycol and its derivatives have been successfully modified on the surface of cerium oxide nanoparticles, thereby increasing their particle size.

[0112] The process of dispersion stability testing includes:

[0113] The cerium oxide nanoparticles provided in Example 1 were dissolved in water and dropped onto a copper grid to construct the transmission electron microscopy (TEM) sample CeO2 NP. The cerium oxide nanoparticles modified with polyethylene glycol and its derivatives provided in Example 1 were dissolved in water and dropped onto a copper grid to construct the TEM sample CeO2 NP-PEG. The dispersion stability was then tested using TEM, and the results are as follows: Figure 2 As shown; from Figure 2 As shown in the electron microscope images at different magnifications, compared with the CeO2 NP sample, which has poor dispersion stability, particle size of hundreds of nanometers and agglomeration, the CeO2 NP-PEG sample has significantly better dispersion stability, with almost no agglomeration and particle size concentrated in the tens of nanometers.

[0114] Meanwhile, the cerium oxide nanoparticles modified with polyethylene glycol and its derivatives provided in Examples 1 and 2 were respectively prepared into dispersions with a mass percentage of 0.1% using water as a solvent. After standing for 24 hours, the dispersion was observed, and the results are as follows. Figure 3 As shown; from Figure 3 It can be seen that the cerium oxide nanoparticles modified with polyethylene glycol and its derivatives provided in Examples 1 and 2 have good dispersion stability and the dispersions are clear.

[0115] Based on the dispersion stability and particle size tests, it can be seen that after the ultrasonic surface modification of cerium oxide nanoparticles provided in this application embodiment, the cerium oxide nanoparticles will be slightly enlarged, but the dispersion stability in water will be significantly improved, showing potential for application in aqueous media such as injectable hydrogels.

[0116] Experimental Example 2

[0117] This experimental example tests the injectability and in-situ molding of the injectable metal nanoparticle hybrid hydrogel materials provided in Examples 1-3.

[0118] The injectionability and in-situ molding testing process includes:

[0119] The injectable metal nanoparticle hybrid hydrogel material, as provided in Example 1, was transferred into a syringe, placed at room temperature for 5 minutes, and then injected. The results are as follows: Figure 4 As shown; from Figure 4 As can be seen, the injectable metal nanoparticle hybrid hydrogel material provided in Example 1 is "injectable" and the hydrogel can be directly squeezed out from the needle. At the same time, it can be seen that the squeezed hydrogel can present a stable filament rather than a droplet, indicating that the hydrogel gradually crosslinks under this ratio and has the property of being injectable and can maintain its shape. It can be squeezed into various shapes according to different needs.

[0120] The injectable metal nanoparticle hybrid hydrogel material, as provided in Example 1, was placed in a sample vial and allowed to stand at 25°C for 15 minutes to gel. Images before and after gelation are shown below. Figures 5-6 As shown; from Figure 5 It can be seen that during the standing process, the injectable metal nanoparticle hybrid hydrogel material undergoes a transformation from a liquid to a gel state. This is because the injectable metal nanoparticle hybrid hydrogel material contains components such as oxidized dextran, aminated gelatin, and adipic acid diazid. Among these components, the aldehyde group of oxidized dextran undergoes a highly efficient nucleophilic addition reaction with the primary amino group of aminated gelatin, forming a reversible imine bond (-C=N-), i.e., a Schiff base bond; at the same time, adipic acid diazid, as a small molecule crosslinking agent containing a dihydrazide group, has its hydrazide group... (-CONHNH2) can also specifically react with the aldehyde group of oxidized dextran to form hydrazone bonds (-C=NNH-); these two dynamic covalent bonds work synergistically to construct the hydrophilic three-dimensional cross-linked network framework of the hydrogel. After the hydrophilic three-dimensional cross-linked network framework is filled with water solvent, the cerium oxide nanoparticles modified with polyethylene glycol and its derivatives have good dispersion stability in water solvent and can be stably dispersed in it, making it less prone to aggregation. Finally, a hydrogel with uniformly distributed cerium oxide nanoparticles modified with polyethylene glycol and its derivatives is obtained. Figure 6 ).

[0121] Meanwhile, the injectable metal nanoparticle hybrid hydrogel material provided in Example 2 was placed in a sample vial and allowed to stand at 25°C for 15 minutes to form a gel, and the injectable metal nanoparticle hybrid hydrogel material provided in Example 3 was placed in a sample vial and allowed to stand at 25°C for 20 minutes to form a gel. Images of the gelled materials are shown below. Figure 6 As shown; fromFigure 6 It can be seen that the injectable metal nanoparticle hybrid hydrogel materials provided in Examples 2-3 can also provide hydrogels with uniformly distributed cerium oxide nanoparticles modified with polyethylene glycol and its derivatives inside.

[0122] Injectability and in-situ molding tests show that the injectable metal nanoparticle hybrid hydrogel materials provided in Examples 1-3 of this application can be injected and extruded using a syringe, and can gel at room temperature for a short time (15-20 min). This indicates that the injectable metal nanoparticle hybrid hydrogel materials provided in Examples 1-3 of this application can be stored at 4°C for a long time before use, using a surface-modified cerium oxide nanoparticle dispersion, oxidized dextran solution, aminated gelatin solution, and adipic acid dihydrazide solution. When using, the oxidized dextran solution can be mixed with the surface-modified cerium oxide nanoparticle dispersion, oxidized dextran solution, aminated gelatin solution, and adipic acid dihydrazide solution. A dispersion of surface-modified cerium oxide nanoparticles, an aminoated gelatin solution, and an adipic acid dihydrazide solution are placed at the left and right ends of a double-port syringe, respectively. The injectable metal nanoparticle hybrid hydrogel material is then directly injected into the wound surface, forming an in-situ shaped metal nanoparticle hybrid hydrogel. Furthermore, because the in-situ shaped metal nanoparticle hybrid hydrogel provided in this application has uniformly distributed cerium oxide nanoparticles modified with polyethylene glycol and its derivatives, it possesses the potential to effectively scavenge oxidative free radicals (ROS) and is suitable for the treatment of various chronic hyperoxidative stress diseases.

[0123] Experimental Example 3

[0124] To investigate the performance of injectable metal nanoparticle hybrid hydrogel materials in scavenging oxidative free radicals (ROS), this experimental example tested the injectable metal nanoparticle hybrid hydrogel materials provided in Examples 1, 4, and Comparative Example 1 after molding to scavenge ROS.

[0125] The experimental testing procedure for scavenging oxidative free radicals (ROS) using the injectable metal nanoparticle hybrid hydrogel material provided in Example 1 includes:

[0126] Following the steps of Experimental Example 2, the injectable metal nanoparticle hybrid hydrogel material provided in Example 1 was gelled by allowing it to stand at room temperature, resulting in a gelled metal nanoparticle hybrid hydrogel.

[0127] Three groups were set up: a blank group, an oxidative stress group, and an experimental group. Log-phase mouse embryonic fibroblasts (NIH 3T3) cultured for 24 hours to a stable growth stage served as the Blank group, and the level of oxidative free radicals (ROS) under normal physiological conditions was tested. Log-phase mouse embryonic fibroblasts (NIH 3T3) cultured for 24 hours to a stable growth stage, with 400 μM hydrogen peroxide added to induce ROS production, served as the Control group. After adding an appropriate amount of 400 μM hydrogen peroxide to the log-phase mouse embryonic fibroblasts (NIH 3T3) cultured for 24 hours to induce ROS production, the injectable metal nanoparticle hybrid hydrogel provided in Example 1 was added and co-incubated with the mouse embryonic fibroblasts (NIH 3T3) for 24 hours, and the amount of scavenged ROS was calculated. The experimental group was defined as the horizontal group, which consisted of cerium oxide nanoparticles modified with polyethylene glycol and its derivatives at a concentration of 50 μg / ml or 100 μg / ml, and was named Ce-N 50 μg / ml or Ce-N 100 μg / ml.

[0128] The levels of oxidative free radicals (ROS) in the blank group, oxidative stress group, and experimental group were quantified using the ROS fluorescent probe DCFH-DA. After DCFH-DA enters the cells, the resulting DCFH is oxidized by ROS into strongly fluorescent DCF. The level of ROS was quantified based on the fluorescence intensity. Mouse embryonic fibroblasts (NIH 3T3) were stained with 10 μM of the ROS fluorescent probe DCFH-DA for 30 min. Cells were collected for flow cytometry analysis. The results are shown below. Figure 7 As shown; from Figure 7 It can be seen that the hydrogen peroxide-induced oxidative stress group produces an excessive level of oxidative free radicals (ROS), while the experimental group, which is incubated with the injectable metal nanoparticle hybrid hydrogel provided in Example 1, can effectively remove oxidative free radicals (ROS) and reduce the level of oxidative free radicals (ROS) to a level close to that of normal physiological conditions.

[0129] The experimental testing procedures for scavenging oxidative free radicals (ROS) using the injectable metal nanoparticle hybrid hydrogel materials provided in Example 4 and Comparative Example 1 included:

[0130] Following the steps of Experimental Example 2, the injectable metal nanoparticle hybrid hydrogel materials provided in Example 4 and Comparative Example 1 were allowed to stand at room temperature to obtain gelled metal nanoparticle hybrid hydrogels.

[0131] The study set up a blank group, an oxidative stress group, and an experimental group. Log-phase mouse embryonic fibroblasts (NIH 3T3) cultured for 24 hours to a stable growth stage were used as the Blank group to test the level of oxidative free radicals (ROS) under normal physiological conditions. Log-phase mouse embryonic fibroblasts (NIH 3T3) cultured for 24 hours to a stable growth stage, with the addition of 400 μM hydrogen peroxide to induce ROS production, served as the Control group. After adding an appropriate amount of 400 μM hydrogen peroxide to the log-phase mouse embryonic fibroblasts (NIH 3T3) cultured for 24 hours to induce ROS production, the injectable metal nanoparticle hybrid hydrogels provided in Example 4 and Comparative Example 1 were added to the mouse embryonic fibroblasts (NIH 3T3) and co-incubated for 24 hours to calculate the level of scavenged ROS. The concentration of cerium oxide nanoparticles modified with polyethylene glycol and its derivatives in the experimental group was 50 μg / ml.

[0132] The levels of oxidative free radicals (ROS) in the blank group, oxidative stress group, and experimental group were quantified using the ROS fluorescent probe DCFH-DA. After DCFH-DA enters the cells, the resulting DCFH is oxidized by ROS into strongly fluorescent DCF. The level of ROS was quantified based on the fluorescence intensity. Mouse embryonic fibroblasts (NIH 3T3) were stained with 10 μM of the ROS fluorescent probe DCFH-DA for 30 min. Cells were collected for flow cytometry analysis. The results are shown below. Figure 8 As shown; from Figure 8 It can be seen that the hydrogen peroxide-induced oxidative stress group produces an excessive level of oxidative free radicals (ROS), while the experimental group, i.e., after incubation with the injectable metal nanoparticle hybrid hydrogels provided in Example 4 and Comparative Example 1, can effectively remove oxidative free radicals (ROS) and reduce the level of oxidative free radicals (ROS). This indicates that the injectable metal nanoparticle hybrid hydrogels provided in Example 4 and Comparative Example 1 can remove oxidative free radicals (ROS) due to the dispersion of cerium oxide nanoparticles.

[0133] The experimental results of the injectable metal nanoparticle hybrid hydrogel materials provided in Examples 1, 4 and Comparative Example 1 on scavenging oxidative free radicals (ROS) show that although the metal nanoparticle hybrid hydrogel materials provided in Examples 4 and Comparative Example 1 have a certain effect on scavenging ROS, the scavenging effect is not significant. However, the injectable metal nanoparticle hybrid hydrogel material provided in Example 1 can reduce the level of oxidative free radicals (ROS) to a level close to that of normal physiological conditions, and the effect of scavenging ROS is more significant. This is because in the injectable metal nanoparticle hybrid hydrogel material provided in Example 1, the surface of the cerium oxide nanoparticles is modified with polyethylene glycol and its derivatives, which allows the cerium oxide nanoparticles to be uniformly dispersed in the hydrogel, making them less prone to aggregation, and providing a larger surface area, thus providing more active sites for scavenging oxidative free radicals (ROS).

[0134] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. An injectable metal nanoparticle hybrid hydrogel material, characterized in that, include: Cerium oxide nanoparticles modified with polyethylene glycol and its derivatives, oxidized dextran, aminated gelatin, and adipate dihydrazide.

2. The injectable metal nanoparticle hybrid hydrogel material according to claim 1, characterized in that, The product comprises, by weight, 1 part by weight of cerium oxide nanoparticles modified with polyethylene glycol and its derivatives, 50-200 parts by weight of oxidized dextran, 50-400 parts by weight of aminated gelatin, and 1-10 parts by weight of adipic acid dihydrazide.

3. The injectable metal nanoparticle hybrid hydrogel material according to claim 1, characterized in that, The cerium oxide nanoparticles modified with polyethylene glycol and its derivatives include 1 part by mass of cerium oxide nanoparticles and 10 to 100 parts by mass of polyethylene glycol and its derivatives.

4. A method for preparing an injectable metal nanoparticle hybrid hydrogel material, characterized in that, A method for preparing an injectable metal nanoparticle hybrid hydrogel material according to any one of claims 1-3 includes the following steps: Preparation steps of cerium oxide nanoparticles: Cerium source, dispersant and surfactant are used to prepare cerium oxide nanoparticles by oil thermal method; Preparation steps of surface-modified cerium oxide nanoparticle dispersion: 1 part by mass of cerium oxide nanoparticles and 10-100 parts by mass of polyethylene glycol and its derivatives are mixed and ultrasonicated to obtain cerium oxide nanoparticles with polyethylene glycol and its derivatives on the surface, and then prepared as a dispersion of cerium oxide nanoparticles modified with polyethylene glycol and its derivatives. Preparation steps of oxidized dextran solution: After selectively oxidizing the hydroxyl groups in the dextran molecule with an oxidizing agent to obtain oxidized dextran, it is prepared into an aqueous solution of oxidized dextran; Preparation steps of aminated gelatin solution: After activating the carboxyl group in the gelatin molecule with an activator and an amino donor and attaching a primary amino group to obtain aminated gelatin, it is prepared into an aqueous solution of aminated gelatin. The mixing steps are as follows: The prepared aqueous solutions of adipic acid dihydrazide, aminoated gelatin, oxidized dextran, and cerium oxide nanoparticle dispersions modified with polyethylene glycol and its derivatives are mixed to obtain an injectable metal nanoparticle hybrid hydrogel material.

5. The method for preparing an injectable metal nanoparticle hybrid hydrogel material according to claim 4, characterized in that, In the preparation steps of cerium oxide nanoparticles, the oil-thermal method includes: reacting cerium source, dispersant and surfactant at a temperature of 200-320℃ with a stirring speed of 500-2000 rpm for 30-120 min.

6. The method for preparing an injectable metal nanoparticle hybrid hydrogel material according to claim 4, characterized in that, In the preparation of the surface-modified cerium oxide nanoparticle dispersion, the sonication time is 30~120 minutes.

7. The method for preparing an injectable metal nanoparticle hybrid hydrogel material according to claim 4, characterized in that, In the preparation of the oxidized dextran solution, the reaction temperature for selective oxidation of hydroxyl groups is 10~45℃, and the reaction time is 1~8h.

8. The method for preparing an injectable metal nanoparticle hybrid hydrogel material according to claim 4, characterized in that, In the preparation of amination gelatin, the process of activating the carboxyl group in the gelatin molecule and attaching it to the primary amino group involves a pH of 4-6, a reaction temperature of 30-40℃, and a reaction time of 4-10 hours.

9. The use of the injectable metal nanoparticle hybrid hydrogel material according to any one of claims 1-8 in the preparation of anti-inflammatory and antioxidant therapeutic biomedical materials, sustained-release biomedical materials, or tissue engineering materials.

10. An in-situ formed metal nanoparticle hybrid hydrogel, characterized in that, Including in-situ formed hydrogels, cerium oxide nanoparticles modified with polyethylene glycol and its derivatives; The cerium oxide nanoparticles modified with polyethylene glycol and its derivatives are dispersed in an in-situ formed hydrogel. The three-dimensional cross-linked network framework of the in-situ formed hydrogel is dynamically constructed from imine bonds and hydrazone bonds formed by oxidized dextran with aminoated gelatin and adipate dihydrazide, respectively.

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