Preparation method of a platinum-carbon-nitrogen artificial biocatalyst, active oxygen scavenging hydrogel, and its products and applications

Abstract

The present invention provides a platinum-carbon-nitrogen artificial biocatalyst, a method for preparing a reactive oxygen species scavenging hydrogel, and its products and applications. The catalyst equips a platinum cluster on a carbon nitride framework. The Pt-N coordinated active center effectively accepts and transfers electrons, has more outstanding catalytic and reactive oxygen species scavenging properties, and can efficiently eliminate reactive oxygen species generated by acute hypoxia after stem cell transplantation and improve the hypoxic environment. At the same time, a combined chemical and physical crosslinking method is adopted. Ultraviolet light excites the double bond chemical crosslinking of acryloylated β-cyclodextrin. The cavity in the acryloylated β-cyclodextrin and the aromatic residue in the gelatin molecule undergo weak host-guest physical crosslinking. The ultraviolet chemical crosslinking provides gel scaffold stability and injectability. The host-guest physical crosslinking is conducive to cell adhesion and migration. The prepared hydrogel can not only improve the acute hypoxic microenvironment and scavenging reactive oxygen species, but also promote cell adhesion, migration and growth and meet the clinical needs of stem cell transplantation.

Description

Technical Field

[0001] The present invention relates to the field of oral medical materials, and in particular to a platinum-carbon-nitrogen artificial biocatalyst, a preparation method of an active oxygen scavenging hydrogel, and products and applications thereof. Background Art

[0002] A person has only two sets of teeth in their lifetime: deciduous teeth and permanent teeth. Deciduous teeth are gradually replaced by permanent teeth by age six. However, once permanent teeth are lost, new teeth will not grow, making it crucial to take good care of them. With increasingly diverse diets and recreational activities, diseases such as pulpitis and apical periodontitis are becoming more common in young permanent teeth, leading to stunted root development, enlarged apical foramina, root resorption, and even tooth loss. Traditional treatments, including pulpotomy and apexoplasty, have limited success rates in older patients. Furthermore, molar surgery is difficult and has a relatively low success rate, ultimately requiring root canal therapy. Root canal therapy involves removing the inflamed pulp, preparing and disinfecting the root canal, and then sealing it tightly. However, this method only preserves tooth tissue but does not promote continued root development or closure of the apical foramina. Furthermore, teeth deprived of pulpal nutrition can suffer from discoloration and fracture. Therefore, achieving biological repair and functional regeneration of damaged pulp to promote continued tooth development is a key goal.

[0003] Regenerative dental pulp is a new concept in root canal treatment, which uses tissue regeneration technology to repair the dental pulp, restore the dental pulp function, and promote tooth development and functional recovery. The main method of dental pulp regeneration is to transplant human dental pulp stem cells isolated from deciduous teeth and permanent teeth into the root canal as seed cells, and use the multidirectional differentiation potential of stem cells to achieve dental pulp regeneration. Currently, many studies have inoculated dental pulp stem cells into scaffold materials and then implanted them into the root canal to promote dental pulp regeneration. However, due to the special anatomical structure of the dental pulp cavity and root canal, acute hypoxia often occurs after implantation, generating a large amount of reactive oxygen such as superoxide anions that damage dental pulp stem cells, resulting in transplant failure.

[0004] Organisms primarily rely on superoxide dismutase and catalase to eliminate reactive oxygen species. However, under abnormal physiological conditions such as acute hypoxia, the body struggles to remove the large amounts of reactive oxygen species produced on its own. Prior research has found that while nanoparticles such as cerium oxide possess superoxide dismutase and catalase activity, they exhibit significant superoxide dismutase and catalase mimicking activity only when the particle size is less than 5 nm. Excessively small particle sizes not only make synthesis difficult but also pose significant biological toxicity.

[0005] Therefore, it is of great significance to develop a material for regenerative dental pulp transplantation that has a simple synthesis method, low biological toxicity, and good active oxygen scavenging effect. Summary of the Invention

[0006] The present invention aims to address the lack of a material for regenerative dental pulp transplantation that is simple to synthesize, has low biotoxicity, and exhibits excellent reactive oxygen species scavenging properties in the prior art. The present invention provides a platinum-carbon-nitrogen artificial biocatalyst, a method for preparing a reactive oxygen species scavenging hydrogel, and its products and applications. This catalyst incorporates platinum clusters on a carbon nitride framework. Platinum has more d electrons, which facilitates chemical bond formation during the catalytic process. Furthermore, more unoccupied d orbitals accept electrons during the catalytic process, demonstrating a higher propensity to form coordination complexes. The Pt-N coordinated active centers effectively accept and transfer electrons, exhibiting superior catalytic and reactive oxygen species scavenging properties. These catalysts can effectively eliminate reactive oxygen species generated by acute hypoxia following stem cell transplantation and improve the hypoxic environment.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A method for preparing a platinum-carbon-nitrogen artificial biocatalyst comprises the following steps:

[0009] Step 1: slowly heating melamine to below 550°C, maintaining the temperature for 2-3 hours, then cooling to room temperature, washing, and drying to obtain carbon nitride;

[0010] Step 2: Add the carbon nitride obtained in step 1 to a sodium hypochlorite aqueous solution, stir for 4-6 days for oxidation and peeling, filter, and wash;

[0011] Step 3: Add the filter cake obtained in step 2 to water and perform continuous ultrasonication for more than 90 minutes, let it stand, take the upper liquid, centrifuge, collect the upper solution, and freeze-dry to obtain relatively uniform small sheets of carbon nitride;

[0012] Step 4: Add the material obtained in step 3 to water and perform ultrasonic treatment; then, add chloroplatinic acid hexahydrate dropwise in an oil bath and stir for 4-6 hours;

[0013] Step 5: Filter, wash, and dry the sample obtained in step 4 to obtain a platinum-carbon-nitrogen artificial biocatalyst; the platinum content in the platinum-carbon-nitrogen artificial biocatalyst is 2.5wt%-5wt%.

[0014] The present invention provides a method for preparing a platinum-carbon-nitrogen artificial biocatalyst. First, melamine is subjected to high-temperature treatment to obtain carbon nitride, which is then oxidatively exfoliated using an aqueous sodium hypochlorite solution. The filter cake is then dissolved in water and subjected to continuous sonication. The supernatant liquid is precipitated and collected, and the supernatant liquid is centrifuged to obtain a solution phase that is freeze-dried. The freeze-dried material is added to water, and hexahydrated chloroplatinic acid is added dropwise in an oil bath. The reaction is stirred to prepare the platinum-carbon-nitrogen artificial biocatalyst. By controlling the preparation conditions and the amount of platinum added, the catalyst is not only low in toxicity but also exhibits an effective catalytic effect. The catalyst incorporates platinum clusters on a carbon nitride framework. Platinum has more d electrons, which facilitates chemical bond formation during the catalytic process, and more unoccupied d orbitals accept electrons during the catalytic process, exhibiting a higher tendency to form coordination complexes. The Pt-N coordinated active centers effectively accept and transfer electrons, exhibiting superior catalytic properties for scavenging reactive oxygen species, and are capable of efficiently eliminating reactive oxygen species generated by acute hypoxia after stem cell transplantation and improving the hypoxic environment.

[0015] Furthermore, in step 1, the melamine is slowly heated to 500° C. to 550° C. Studies have shown that the heating rate should not be too fast and the temperature should not exceed 550° C., otherwise the carbon nitride yield will be reduced and impurities will be introduced.

[0016] Furthermore, in step 1, melamine is heated to 500° C. to 550° C. at a heating rate of 1° C. / min to 5° C. / min.

[0017] Furthermore, in step 2, 1.5-3 mL of sodium hypochlorite is added to every 10 mL of water in the sodium hypochlorite aqueous solution.

[0018] Furthermore, in step 3, the filter cake is added to water and continuously sonicated for 90 to 120 minutes. Continuous sonication for more than 90 minutes, and after precipitation, the upper liquid is centrifuged and the solution phase is collected and dried to ensure that relatively uniform carbon nitride with smaller flakes is obtained.

[0019] Furthermore, in step 4, the temperature of the oil bath is 60°C to 80°C.

[0020] Furthermore, in step 5, the platinum content of the platinum-carbon-nitrogen artificial biocatalyst obtained is 3wt%-4.5wt%. Studies have found that artificial catalysts with a platinum content of 2.5wt%-5wt% have good active oxygen scavenging effect and biosafety. A platinum content of 3wt%-4.5wt% can show even better active oxygen scavenging effect.

[0021] Another object of the present invention is to protect the product prepared by the above method.

[0022] The catalyst product is prepared by the above-mentioned preparation method of the platinum-carbon-nitrogen artificial biocatalyst.

[0023] Using platinum as the active center, the appropriate ratio not only reduces the toxicity of the material but also exerts an effective catalytic effect. Artificial catalysts with a platinum content of 2.5wt%-5wt% have good active oxygen scavenging and biosafety, achieving unexpected technical results.

[0024] A method for preparing an active oxygen scavenging hydrogel comprises the following steps:

[0025] S1. Adding acryloyl β-cyclodextrin to PBS to obtain a first material, wherein the mass concentration of acryloyl β-cyclodextrin in the first material is 10% to 20%;

[0026] S2. dissolving gelatin in the first material to obtain a second material; the mass concentration of gelatin in the second material is 8% to 15%;

[0027] S3, adding LAP to the second material to obtain a third material; the mass concentration of LAP in the third material is 0.1% to 0.3%;

[0028] S4, adding the catalyst product as described above to the third material to obtain a fourth material; the mass concentration of the catalyst product in the fourth material is 0.04% to 0.06%;

[0029] S5. Using ultraviolet light to irradiate, the fourth material undergoes a cross-linking reaction to obtain an active oxygen scavenging hydrogel.

[0030] The active oxygen scavenging hydrogel provided by the present application is prepared by mixing acryloyl β-cyclodextrin, PBS, gelatin, LAP and platinum-carbon nitrogen artificial biocatalyst, and controlling the amount of each raw material added to ensure good stability of the hydrogel; then a cross-linking reaction is carried out by ultraviolet light irradiation to obtain an active oxygen scavenging hydrogel. Specifically, acryloyl β-cyclodextrin can be assembled with gelatin through a host-guest assembly to form a hydrogel (ACD) as a cell scaffold. Due to the host-guest assembly characteristics, cells can adhere and migrate, differentiate and grow in the hydrogel. Loading the synthesized artificial biocatalyst into the host-guest assembly hydrogel (PACD) is not only beneficial to the enzyme simulation activity stability and reduced biological toxicity, but also beneficial to cell migration and growth, opening up a new path for dental pulp regeneration. At the same time, this application uses a combined chemical and physical crosslinking method. Ultraviolet light excites the double bond chemical crosslinking of acryloylated β-cyclodextrin. The cavity in the acryloylated β-cyclodextrin and the aromatic residues in the gelatin molecule undergo weak host-guest physical crosslinking. The ultraviolet chemical crosslinking (larger force) provides gel scaffold stability and injectability, while the host-guest physical crosslinking (smaller force) facilitates cell adhesion and migration. The prepared hydrogel can not only improve the acute hypoxic microenvironment, scavenge reactive oxygen species, and promote cell adhesion, migration and growth, but also meet the clinical needs of stem cell transplantation, facilitating its promotion and application.

[0031] Hydrogels are a class of polymers with three-dimensional network structures. They offer advantages such as high water content, good biocompatibility, and tunable structural and mechanical properties. They are widely used in tissue engineering scaffolds, drug delivery vehicles, and flexible sensors. Gelatin, derived from animal skin or bone, is a type of protein obtained by partial hydrolysis of collagen. Modification of gelatin can produce hydrogels that chemically crosslink under the influence of ultraviolet or visible light, exhibiting properties suitable for cell growth and biodegradability. However, chemically crosslinked hydrogels make it difficult for cells to migrate and grow within them.

[0032] Hydrogels are a common type of tissue engineering scaffold, but most hydrogels, such as GelMA, are formed by chemical crosslinking. It is difficult for cells to adhere and migrate by breaking through chemical bonds, so physical crosslinking is the goal. β-cyclodextrin molecules contain cavities that can undergo weak host-guest physical crosslinking with aromatic residues in gelatin molecules. However, it is difficult to form a stable gel scaffold with weak host-guest crosslinking alone. Therefore, β-cyclodextrin is acrylated and then crosslinked by UV light to form a stable gel scaffold. UV chemical crosslinking not only enables the hydrogel to be injected into the narrow root canals of the mouth, but also improves the stability of the hydrogel scaffold. The host-guest physical crosslinking makes the gel more suitable for the adhesion and migration of dental pulp stem cells, which is more conducive to the regeneration of dental pulp tissue.

[0033] Furthermore, in said S1, PBS is phosphate buffered saline (pH 7.4).

[0034] Furthermore, the type of gelatin may be gelatin (A) type or gelatin (B) type.

[0035] Furthermore, LAP is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0036] Furthermore, the mass concentration of the platinum-carbon nitrogen artificial catalyst is 0.04% to 0.06%. If the concentration is too low, it cannot effectively remove active oxygen, and if the concentration is too high, it affects biological safety.

[0037] Furthermore, in S5, 10 mW / cm 2 Irradiate with UV light for 5-10 minutes.

[0038] Furthermore, in S1, the acryloyl β-cyclodextrin is prepared by the following method:

[0039] I. Add β-cyclodextrin to N,N-dimethylformamide, add triethylamine to the solution, and place in an ice-water bath and stir;

[0040] II. Slowly add acryloyl chloride to the solution obtained in step I, stir, and filter;

[0041] III. Collect the filtrate produced in step II, add excess acetone, filter and wash, and vacuum dry to obtain acryloyl β-cyclodextrin.

[0042] Furthermore, for every 10 g of β-cyclodextrin, 120-200 mL of N,N-dimethylformamide, 3-4 mL of triethylamine and 2.2-3 mL of acryloyl chloride are mixed.

[0043] The active oxygen scavenging hydrogel is prepared by the above-mentioned method for preparing the active oxygen scavenging hydrogel.

[0044] Hydrogels that improve the acute hypoxic microenvironment, eliminate reactive oxygen species, promote cell adhesion, migration and growth, and meet the clinical needs of stem cell transplantation are easy to promote and apply.

[0045] The application of the above-mentioned reactive oxygen species scavenging hydrogel in dental pulp transplantation and regeneration.

[0046] The reactive oxygen species scavenging hydrogel provided in this application can effectively improve the acute hypoxic environment after root canal dental pulp stem cells (DPSCs) transplantation. In particular, its reactive oxygen species scavenging properties can largely avoid the reactive oxygen species hazards caused by hypoxia, greatly improve the survival rate of dental pulp stem cell transplantation, and facilitate promotion and application.

[0047] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0048] 1. The present invention provides a method for preparing a platinum-carbon-nitrogen artificial biocatalyst. First, melamine is subjected to high temperature to obtain carbon nitride, which is then oxidatively exfoliated using an aqueous sodium hypochlorite solution. The filter cake is then dissolved in water and subjected to continuous sonication. The supernatant liquid is precipitated and collected. The supernatant liquid is centrifuged and the solution phase is collected and freeze-dried. The freeze-dried material is added to water, and chloroplatinic acid hexahydrate is added dropwise in an oil bath. The reaction is stirred to prepare the platinum-carbon-nitrogen artificial biocatalyst. By controlling the preparation conditions and the amount of platinum added, the catalyst not only reduces the toxicity of the material but also exhibits an effective catalytic effect. The catalyst incorporates platinum clusters on a carbon nitride framework. Platinum has more d electrons, which facilitates chemical bond formation during the catalytic process. Furthermore, more unoccupied d orbitals accept electrons during the catalytic process, resulting in a higher propensity to form coordination complexes. The Pt-N coordinated active centers effectively accept and transfer electrons, resulting in superior catalytic properties for scavenging reactive oxygen species. This catalyst can effectively eliminate reactive oxygen species generated by acute hypoxia after stem cell transplantation and improve the hypoxic environment.

[0049] 2. The active oxygen scavenging hydrogel provided by the present application is prepared by mixing acryloylated β-cyclodextrin, PBS, gelatin, LAP and platinum-carbon nitrogen artificial biocatalyst, and controlling the amount of each raw material added to ensure good stability of the hydrogel; then, a cross-linking reaction is carried out by ultraviolet light irradiation to obtain an active oxygen scavenging hydrogel. Specifically, acryloylated β-cyclodextrin can be assembled with gelatin through a host-guest assembly to form a hydrogel (ACD) as a cell scaffold. Due to the host-guest assembly characteristics, cells can adhere and migrate, differentiate and grow in the hydrogel. Loading the synthesized artificial biocatalyst into the host-guest assembly hydrogel (PACD) is not only beneficial to the enzyme simulation activity stability and reduced biological toxicity, but also beneficial to cell migration and growth, opening up a new path for dental pulp regeneration. At the same time, this application uses a combination of chemical and physical crosslinking methods. Ultraviolet light excites the double bond chemical crosslinking of acryloylated β-cyclodextrin. The cavity in the acryloylated β-cyclodextrin and the aromatic residues in the gelatin molecule undergo weak host-guest physical crosslinking. The ultraviolet chemical crosslinking (larger force) provides gel scaffold stability and injectability, while the host-guest physical crosslinking (smaller force) facilitates cell adhesion and migration. The prepared hydrogel can not only improve the acute hypoxic microenvironment, scavenge reactive oxygen species, and promote cell adhesion, migration and growth, and meet the clinical requirements of stem cell transplantation, facilitating its promotion and application.

[0050] 3. The reactive oxygen species scavenging hydrogel provided in this application can effectively improve the acute hypoxic environment after root canal dental pulp stem cells (DPSCs) transplantation. In particular, its reactive oxygen species scavenging properties can largely avoid the reactive oxygen species hazards caused by hypoxia, greatly improve the survival rate of dental pulp stem cell transplantation, and facilitate promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the appearance of the active oxygen scavenging hydrogel prepared in Example 1.

[0052] Figure 2 This is a comparison of the photos before and after UV gelation prepared in Example 1.

[0053] Figure 3 This is an electron micrograph of the active oxygen scavenging hydrogel prepared in Example 1.

[0054] Figure 4 This is a graph showing the test results of superoxide dismutase-like activity in Example 1.

[0055] Figure 5 This is a graph showing the test results of the catalase-like activity in Example 1.

[0056] Figure 6 This is a diagram showing the protective effect of dental pulp stem cells in Example 1.

[0057] Figure 7 Figure 2 shows the cell adhesion gel. DETAILED DESCRIPTION

[0058] The present invention will be described in detail below with reference to the accompanying drawings.

[0059] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0060] In the following examples, PBS (1×, pH 7.4, 500 mL) was purchased from Sewell Biotechnology Co., Ltd., and gelatin (type A, 100 g) and LAP (250 mg) were purchased from Aladdin Biochemical Technology Co., Ltd.

[0061] Example 1

[0062] Preparation of platinum-carbon-nitrogen artificial biocatalyst

[0063] Step 1: 10 mg of melamine was heated to 550°C at a heating rate of 5°C / min and maintained for 2 hours. The mixture was then cooled to room temperature, washed, dried, washed three times with deionized water, and dried in a vacuum at 60°C.

[0064] Step 2: Take 0.5 g of the material obtained in step 1 and add it to 10 mL of ultrapure water containing 2 mL of sodium hypochlorite, stir at 40° C. for 4-6 days, filter, and wash;

[0065] Step 3: The filter cake obtained in step 2 was added to 20 mL of water and continuously sonicated for 90 min. After standing for 12 h, the upper liquid was taken, centrifuged at 10,000 rpm for 10 min, the liquid phase was collected, and freeze-dried;

[0066] Step 4: Take 50 mg of the material obtained in step 3 and add it to 25 mL of water, and ultrasonicate for 60 minutes; then, add chloroplatinic acid hexahydrate dropwise in an oil bath at 70°C, and stir at 70°C for 5 hours;

[0067] Step 5: Filter the sample obtained in step 4, wash it with deionized water, and dry it in vacuo at 60° C. to obtain a platinum-carbon-nitrogen artificial biocatalyst; and control the addition ratio of chloroplatinic acid hexahydrate so that the platinum content in the platinum-carbon-nitrogen artificial biocatalyst is 3.5 wt %.

[0068] Preparation of acryloyl β-cyclodextrin

[0069] I. Add 10 g of β-cyclodextrin to 150 mL of N,N-dimethylformamide, add 3.5 mL of triethylamine to the solution, and place in an ice-water bath and stir;

[0070] II. Take 2.5 mL of acryloyl chloride and slowly add it to the solution obtained in step I, stir for 12 hours and then filter;

[0071] III. Collect the filtrate produced in step II, add excess acetone, filter and wash, and vacuum dry to obtain acryloyl β-cyclodextrin.

[0072] Preparation of reactive oxygen species scavenging hydrogel

[0073] S1. Adding acryloyl β-cyclodextrin to PBS to obtain a first material, wherein the mass concentration of acryloyl β-cyclodextrin in the first material is 12%;

[0074] S2. dissolving gelatin in the first material to obtain a second material; the mass concentration of gelatin in the second material is 10%;

[0075] S3, adding LAP to the second material to obtain a third material; the mass concentration of LAP in the third material is 0.1%;

[0076] S4, adding the above-mentioned catalyst product to the third material to obtain a fourth material; the concentration of the catalyst product in the fourth material is 0.05%;

[0077] S5, using 10mW / cm 2 The fourth material was irradiated with an ultraviolet lamp for 8 minutes to undergo a cross-linking reaction to obtain an active oxygen scavenging hydrogel.

[0078] The appearance of the active oxygen scavenging hydrogel prepared in Example 1 is as follows: Figure 1 As shown. The specific operation is to put the mixed active oxygen scavenging gel (PACD) prepared by S4 into an elbow syringe and store it in a dark place at 37°C. After the dental pulp stem cells are transplanted into the root canal, PACD is injected into the root canal and pulp cavity, and ultraviolet light is used to form active oxygen scavenging hydrogel. The comparison of photos before and after UV gelation is shown in the figure below. Figure 2 As shown. Before UV irradiation, PACD has fluidity, which is convenient for injection into small and complex root canals in the oral cavity. After UV irradiation, it cross-links to form a stable gel. The electron micrograph of the prepared active oxygen scavenging hydrogel is shown in Figure 3 Electron microscopy showed that the gel had abundant pores, which facilitated the PtCN to scavenge active oxygen species, and also facilitated cell adhesion, migration, growth, and tissue regeneration.

[0079] A solution was prepared with 1 mg potassium superoxide, 1 mL dimethyl sulfoxide, and 3 mg 18-crown ether-6, and 500 μL of the above solution was added to the EP tube. The control group did not add any materials. The PtCN group added PtCN to a final concentration of 50 μg / mL. The PACD group added 50 μL of UV-crosslinked PACD gel. After stirring for 10 minutes, 5 μL of dimethyl sulfoxide solution containing nitrogen blue tetrazolium (10 mg / mL) was added. The absorbance at 680 nm was measured to obtain the relative residual amount of superoxide anions in each group. Both PtCN and PACD have superoxide anion scavenging effects and have superoxide dismutase mimicking activity. The reason why PACD is not as good as PtCN is that the PtCN in the gel will not be suddenly released to exert its effect. The test results of superoxide dismutase and catalase mimicking activity are shown in Figure 2. Figure 4 shown.

[0080] 40 μL of hydrogen peroxide (1M) was added to 2.96 mL of phosphate buffer, and 1 mL of the above liquid was added to the EP tube. No material was added to the control group. PtCN was added to the PtCN group to a final concentration of 50 μg / mL. 100 μL of ACD was added to the ACD group, and 100 μL of PACD was added to the PACD group. At 0, 5, 10, 20, and 40 minutes of reaction, 100 μL of the above reaction solution was added to 100 μL of titanium sulfate solution and mixed, and the relative remaining amount of hydrogen peroxide was detected by measuring the absorbance at 405 nm. The hydrogen peroxide content in the control group and the ACD group did not change much, while the hydrogen peroxide in the PtCN and PACD groups gradually decreased with the increase of reaction time, showing catalase simulation activity. The specific test results are as follows: Figure 5 shown.

[0081] After dental pulp stem cells were seeded and adhered to the wall, the control group was replaced with fresh culture medium, and the other groups were replaced with fresh culture medium containing 2% (v / v) hydrogen peroxide. PtCN (final concentration 50 μg / mL), ACD [10% (v / v)] and PACD [10% (v / v)] were quickly added and incubated for 1 hour. The culture medium and materials were removed and the cell activity was detected by CCK-8. The results showed that hydrogen peroxide killed dental pulp stem cells, while PtCN and PACD could protect cells from hydrogen peroxide damage. Figure 6 shown.

[0082] After disinfecting ACD and PACD, dental pulp stem cells were inoculated and incubated for 24 hours. Microscopic observation showed that dental pulp stem cells adhered to both ACD and PACD, and the cells showed good cell morphology. Figure 7 shown.

[0083] After testing, the artificial catalyst prepared in Example 1 has good catalytic scavenging effect of active oxygen and biosafety, provides gel scaffold stability and injectability, and the host-guest physical crosslinking (smaller force) is beneficial to cell adhesion and migration.

[0084] Example 2

[0085] Preparation of platinum-carbon-nitrogen artificial biocatalyst

[0086] Step 1: 10 mg of melamine was heated to 500°C at a heating rate of 1°C / min and maintained for 3 hours. The mixture was then cooled to room temperature, washed, dried, washed three times with deionized water, and dried under vacuum at 60°C.

[0087] Step 2: Take 0.5 g of the material obtained in step 1 and add it to 10 mL of ultrapure water containing 1.5 mL of sodium hypochlorite, stir at 40° C. for 4-6 days, filter, and wash;

[0088] Step 3: The filter cake obtained in step 2 was added to 20 mL of water and continuously ultrasonicated for 100 min. After standing for 12 h, the upper liquid was taken, centrifuged at 10,000 rpm for 10 minutes, the solution phase was collected, and freeze-dried;

[0089] Step 4: Take 50 mg of the material obtained in step 3 and add it to 25 mL of water, and ultrasonicate for 60 minutes; then, add chloroplatinic acid hexahydrate dropwise in an oil bath at 60°C, and keep stirring at 60°C for 6 hours;

[0090] Step 5: Filter the sample obtained in step 4, wash it with deionized water, and dry it in vacuo at 60° C. to obtain a platinum-carbon-nitrogen artificial biocatalyst; and control the addition ratio of chloroplatinic acid hexahydrate so that the platinum content in the platinum-carbon-nitrogen artificial biocatalyst is 2.5 wt %.

[0091] Preparation of acryloyl β-cyclodextrin

[0092] I. Add 10 g of β-cyclodextrin to 120 mL of N,N-dimethylformamide, add 3 mL of triethylamine to the solution, and place in an ice-water bath and stir;

[0093] II. Take 2.2 mL of acryloyl chloride and slowly add it to the solution obtained in step I, stir for 10 hours and then filter;

[0094] III. Collect the filtrate produced in step II, add excess acetone, filter and wash, and vacuum dry to obtain acryloyl β-cyclodextrin.

[0095] Preparation of reactive oxygen species scavenging hydrogel

[0096] S1. Adding acryloyl β-cyclodextrin to PBS to obtain a first material, wherein the mass concentration of acryloyl β-cyclodextrin in the first material is 15%;

[0097] S2. dissolving gelatin in the first material to obtain a second material; the mass concentration of gelatin in the second material is 15%;

[0098] S3, adding LAP to the second material to obtain a third material; the mass concentration of LAP in the third material is 0.05%;

[0099] S4, adding the above-mentioned catalyst product to the third material to obtain a fourth material; the concentration of the catalyst product in the fourth material is 0.04%;

[0100] S5, using 10mW / cm 2 The fourth material was irradiated with an ultraviolet lamp for 5 minutes to undergo a cross-linking reaction to obtain an active oxygen scavenging hydrogel.

[0101] Example 3

[0102] Preparation of platinum-carbon-nitrogen artificial biocatalyst

[0103] Step 1: 10 mg of melamine was heated to 520°C at a heating rate of 3°C / min and maintained for 2.5 hours. The mixture was then cooled to room temperature, washed, dried, washed three times with deionized water, and dried in a vacuum at 60°C.

[0104] Step 2: Take 0.5 g of the material obtained in step 1 and add it to 10 mL of ultrapure water containing 3 mL of sodium hypochlorite, stir at 40° C. for 4-6 days, filter, and wash;

[0105] Step 3: Add the filter cake obtained in step 2 to 20 mL of water and perform continuous ultrasonication for 120 min. After standing for 12 h, take the upper liquid, centrifuge at 10,000 rpm for 10 minutes, collect the liquid phase, and freeze-dry;

[0106] Step 4: Take 50 mg of the material obtained in step 3 and add it to 25 mL of water, and ultrasonicate for 60 minutes; then, add chloroplatinic acid hexahydrate dropwise in an oil bath at 80°C, and keep stirring at 80°C for 4 hours;

[0107] Step 5: Filter the sample obtained in step 4, wash it with deionized water, and dry it in vacuo at 60° C. to obtain a platinum-carbon-nitrogen artificial biocatalyst; and control the addition ratio of chloroplatinic acid hexahydrate so that the platinum content in the platinum-carbon-nitrogen artificial biocatalyst is 5 wt%.

[0108] Preparation of acryloyl β-cyclodextrin

[0109] I. Add 10 g of β-cyclodextrin to 200 mL of N,N-dimethylformamide, add 4.0 mL of triethylamine to the solution, and place in an ice-water bath with stirring;

[0110] II. Slowly add 3.0 mL of acryloyl chloride to the solution obtained in step I, stir for 18 h, and then filter;

[0111] III. Collect the filtrate produced in step II, add excess acetone, filter and wash, and vacuum dry to obtain acryloyl β-cyclodextrin.

[0112] Preparation of reactive oxygen species scavenging hydrogel

[0113] S1. Adding acryloyl β-cyclodextrin to PBS to obtain a first material, wherein the mass concentration of acryloyl β-cyclodextrin in the first material is 20%;

[0114] S2. dissolving gelatin in the first material to obtain a second material; the mass concentration of gelatin in the second material is 8%;

[0115] S3, adding LAP to the second material to obtain a third material; the mass concentration of LAP in the third material is 0.15%;

[0116] S4, adding the above-mentioned catalyst product to the third material to obtain a fourth material; the concentration of the catalyst product in the fourth material is 0.06%;

[0117] S5, using 10mW / cm 2 The fourth material was irradiated with an ultraviolet lamp for 10 minutes to undergo a cross-linking reaction to obtain an active oxygen scavenging hydrogel.

[0118] Comparative Example 1

[0119] Comparative Example 1 Compared with Example 1, the platinum content in the platinum-carbon-nitrogen artificial biocatalyst was adjusted to 1.5 wt %. The specific preparation process is as follows:

[0120] Preparation of platinum-carbon-nitrogen artificial biocatalyst

[0121] Step 1: 10 mg of melamine was heated to 550°C at a heating rate of 5°C / min and maintained for 2 hours. The mixture was then cooled to room temperature, washed, dried, washed three times with deionized water, and dried in a vacuum at 60°C.

[0122] Step 2: Take 0.5 g of the material obtained in step 1 and add it to 10 mL of ultrapure water containing 2 mL of sodium hypochlorite, stir at 40° C. for 4-6 days, filter, and wash;

[0123] Step 3: The filter cake obtained in step 2 was added to 20 mL of water and continuously sonicated for 90 min. After standing for 12 h, the upper liquid was taken, centrifuged at 10,000 rpm for 10 min, the liquid phase was collected, and freeze-dried;

[0124] Step 4: Take 50 mg of the material obtained in step 3 and add it to 25 mL of water, and ultrasonicate for 60 minutes; then, add chloroplatinic acid hexahydrate dropwise in an oil bath at 70°C, and stir at 70°C for 5 hours;

[0125] Step 5: Filter the sample obtained in step 4, wash it with deionized water, and dry it in vacuo at 60° C. to obtain a platinum-carbon-nitrogen artificial biocatalyst; and control the addition ratio of chloroplatinic acid hexahydrate so that the platinum content in the platinum-carbon-nitrogen artificial biocatalyst is 1.5 wt %.

[0126] Preparation of acryloyl β-cyclodextrin

[0127] I. Add 10 g of β-cyclodextrin to 150 mL of N,N-dimethylformamide, add 3.5 mL of triethylamine to the solution, and place in an ice-water bath and stir;

[0128] II. Take 2.5 mL of acryloyl chloride and slowly add it to the solution obtained in step I, stir for 12 hours and then filter;

[0129] III. Collect the filtrate produced in step II, add excess acetone, filter and wash, and vacuum dry to obtain acryloyl β-cyclodextrin.

[0130] Preparation of reactive oxygen species scavenging hydrogel

[0131] S1. Adding acryloyl β-cyclodextrin to PBS to obtain a first material, wherein the mass concentration of acryloyl β-cyclodextrin in the first material is 12%;

[0132] S2. dissolving gelatin in the first material to obtain a second material; the mass concentration of gelatin in the second material is 10%;

[0133] S3, adding LAP to the second material to obtain a third material; the mass concentration of LAP in the third material is 0.1%;

[0134] S4, adding the above-mentioned catalyst product to the third material to obtain a fourth material; the concentration of the catalyst product in the fourth material is 0.05%;

[0135] S5, using 10mW / cm 2 The fourth material was irradiated with an ultraviolet lamp for 8 minutes to undergo a cross-linking reaction to obtain an active oxygen scavenging hydrogel.

[0136] Comparative Example 2

[0137] Comparative Example 1 Compared with Example 1, the platinum content in the platinum-carbon-nitrogen artificial biocatalyst was adjusted to 8 wt %. The specific preparation process is as follows:

[0138] Preparation of platinum-carbon-nitrogen artificial biocatalyst

[0139] Step 1: 10 mg of melamine was heated to 550°C at a heating rate of 5°C / min and maintained for 2 hours. The mixture was then cooled to room temperature, washed, dried, washed three times with deionized water, and dried in a vacuum at 60°C.

[0140] Step 2: Take 0.5 g of the material obtained in step 1 and add it to 10 mL of ultrapure water containing 2 mL of sodium hypochlorite, stir at 40° C. for 4-6 days, filter, and wash;

[0141] Step 3: The filter cake obtained in step 2 was added to 20 mL of water and continuously sonicated for 90 min. After standing for 12 h, the upper liquid was taken, centrifuged at 10,000 rpm for 10 min, the liquid phase was collected, and freeze-dried;

[0142] Step 4: Take 50 mg of the material obtained in step 3 and add it to 25 mL of water, and ultrasonicate for 60 minutes; then, add chloroplatinic acid hexahydrate dropwise in an oil bath at 70°C, and stir at 70°C for 5 hours;

[0143] Step 5: Filter the sample obtained in step 4, wash it with deionized water, and dry it in vacuo at 60° C. to obtain a platinum-carbon-nitrogen artificial biocatalyst; and control the addition ratio of chloroplatinic acid hexahydrate so that the platinum content in the platinum-carbon-nitrogen artificial biocatalyst is 8 wt %.

[0144] Preparation of acryloyl β-cyclodextrin

[0145] I. Add 10 g of β-cyclodextrin to 150 mL of N,N-dimethylformamide, add 3.5 mL of triethylamine to the solution, and place in an ice-water bath and stir;

[0146] II. Take 2.5 mL of acryloyl chloride and slowly add it to the solution obtained in step I, stir for 12 hours and then filter;

[0147] III. Collect the filtrate produced in step II, add excess acetone, filter and wash, and vacuum dry to obtain acryloyl β-cyclodextrin.

[0148] Preparation of reactive oxygen species scavenging hydrogel

[0149] S1. Adding acryloyl β-cyclodextrin to PBS to obtain a first material, wherein the mass concentration of acryloyl β-cyclodextrin in the first material is 12%;

[0150] S2. dissolving gelatin in the first material to obtain a second material; the mass concentration of gelatin in the second material is 10%;

[0151] S3, adding LAP to the second material to obtain a third material; the mass concentration of LAP in the third material is 0.1%;

[0152] S4, adding the above-mentioned catalyst product to the third material to obtain a fourth material; the concentration of the catalyst product in the fourth material is 0.05%;

[0153] S5, using 10mW / cm 2 The fourth material was irradiated with an ultraviolet lamp for 8 minutes to undergo a cross-linking reaction to obtain an active oxygen scavenging hydrogel.

[0154] The same test method as in Example 1 was used to test the properties of the catalysts and hydrogels prepared in Examples 2-3 and Comparative Examples 1-2. The specific test results are shown in Table 1.

[0155] Table 1

[0156]

[0157]

[0158] The results of different examples show that the Pt-CN artificial catalyst has a good effect on scavenging reactive oxygen species by changing various parameters and raw material ratios within a given range. The higher the platinum content, the better the catalytic scavenging effect of reactive oxygen species, and can better protect cells from reactive oxygen species damage. However, in the comparative example, when the platinum content is below the given range, the reactive oxygen species scavenging effect is poor, and it is unable to effectively protect cells from reactive oxygen species damage. When the platinum content exceeds the given range, although the reactive oxygen species scavenging effect is good, due to the toxicity of the artificial catalyst with a high platinum content, the relative cell viability is also poor.

[0159] The above are merely preferred embodiments of the present application and only specifically describe the technical principles of the present application. These descriptions are intended only to explain the principles of the present application and should not be construed in any way as limiting the scope of protection of the present application. Based on the explanations herein, any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present application, as well as other specific implementations of the present application that can be conceived by those skilled in the art without inventive effort, shall be included within the scope of protection of the present application.

Claims

1. A method for preparing an active oxygen scavenging hydrogel, characterized in that: The following steps are involved: S1. Adding acryloyl β-cyclodextrin to PBS to obtain a first material, wherein the mass concentration of acryloyl β-cyclodextrin in the first material is 10% to 20%; S2. dissolving gelatin in the first material to obtain a second material; the mass concentration of gelatin in the second material is 8% to 15%; S3, adding LAP to the second material to obtain a third material; the mass concentration of LAP in the third material is 0.1% to 0.3%; S4, adding a platinum-carbon-nitrogen artificial biocatalyst to the third material to obtain a fourth material; the concentration of the catalyst product in the fourth material is 0.04% to 0.06%; S5. Using ultraviolet light to irradiate, the fourth material undergoes a cross-linking reaction to obtain an active oxygen scavenging hydrogel; the platinum-carbon-nitrogen artificial biocatalyst is prepared by the following method: Step 1: slowly heating melamine to below 550°C, maintaining the temperature for 2-3 hours, then cooling to room temperature, washing, and drying to obtain carbon nitride; Step 2: Add the carbon nitride obtained in step 1 to a sodium hypochlorite aqueous solution, stir for 4-6 days for oxidation and peeling, filter, and wash; Step 3: Add the filter cake obtained in step 2 to water and perform continuous ultrasonication for more than 90 minutes, let it stand, take the upper liquid, centrifuge, collect the upper solution, and freeze-dry to obtain relatively uniform small sheets of carbon nitride; Step 4: Add the material obtained in step 3 to water and perform ultrasonic treatment; then, add chloroplatinic acid hexahydrate dropwise in an oil bath and stir for 4-6 hours; Step 5: Filter, wash, and dry the sample obtained in step 4 to obtain a platinum-carbon-nitrogen artificial biocatalyst; the platinum content in the platinum-carbon-nitrogen artificial biocatalyst is 2.5wt%-5wt%.

2. The method for preparing the active oxygen scavenging hydrogel according to claim 1, wherein: In the step 1, the temperature of melamine is slowly raised to 500° C. to 550° C.

3. The method for preparing the active oxygen scavenging hydrogel according to claim 2, wherein: In the step 1, melamine is heated to 500° C. to 550° C. at a heating rate of 1° C. / min to 5° C. / min.

4. The method for preparing the active oxygen scavenging hydrogel according to claim 1, wherein: In step 4, the temperature of the oil bath is 60°C to 80°C.

5. The method for preparing the active oxygen scavenging hydrogel according to any one of claims 1 to 4, characterized in that: In step 5, the platinum content in the obtained platinum-carbon-nitrogen artificial biocatalyst is 3wt%-4.5wt%.

6. The method for preparing the active oxygen scavenging hydrogel according to claim 1, wherein: In S1, acryloyl β-cyclodextrin was prepared by the following method: I. Add β-cyclodextrin to N,N-dimethylformamide, add triethylamine to the solution, and place in an ice-water bath and stir; II. Slowly add acryloyl chloride to the solution obtained in step I, stir, and filter; III. Collect the filtrate produced in step II, add excess acetone, filter and wash, and vacuum dry to obtain acryloyl β-cyclodextrin.

7. The active oxygen scavenging hydrogel prepared by the method for preparing the active oxygen scavenging hydrogel according to any one of claims 1 to 6.

8. Use of the active oxygen scavenging hydrogel according to any one of claims 1 to 6 in preparing a material for transplantation and regeneration of dental pulp.

Images