Protein hydrogel capable of slowly releasing chlorine dioxide as well as preparation method and application of protein hydrogel

Abstract

The invention discloses a protein hydrogel capable of slowly releasing chlorine dioxide, the protein hydrogel comprises chlorine dioxide and an embedding material for embedding chlorine dioxide, and the embedding material is hydrogel obtained after bovine serum albumin and gelatin are subjected to protein thermal polymerization; the invention further discloses a preparation method and application of the protein hydrogel capable of slowly releasing chlorine dioxide. By adjusting the ratio of bovine serum albumin, gelatin and chlorine dioxide and the adding sequence of all the raw materials, physical and chemical double-crosslinking is carried out through protein thermal polymerization and the oxidation effect of ClO2 on protein, no cross-linking agent is needed, and the preparation is safe, simple, convenient, low in cost, easy to implement and capable of transforming clinics; the prepared protein hydrogel capable of slowly releasing chlorine dioxide responsively releases ClO2 to ambient temperature and protease, is completely degraded under the action of body temperature and enzyme, has an excellent antibacterial effect on immune-escape intracellular porphyromonas gingivalis and a biological membrane thereof, and has anti-inflammatory and alveolar bone regeneration promoting effects in the verification of a constructed periodontitis animal model.

Description

Technical Field

[0001] The invention relates to the technical field of drug sustained release, and in particular to a protein hydrogel capable of sustained-releasing chlorine dioxide, and a preparation method and application thereof. Background Art

[0002] Chronic periodontitis is a multifactorial chronic destructive inflammatory disease with periodontal microorganisms as initiating factors and host immune defense responses involved. It is the main cause of tooth loss in adults and a major public health issue worldwide. Periodontal disease is also considered to be one of the risk factors for several chronic non-communicable diseases, including cardiovascular disease, cancer, chronic respiratory diseases and diabetes. Porphyromonas gingivalis is considered to be the main pathogen of chronic periodontitis. It can invade gingival epithelial cells and osteoblasts through its pili and various components, evade the host's immune defense mechanism, and ultimately lead to microbial community disorder, destructive periodontal inflammation and alveolar bone loss. Since the deep periodontal pockets and complex root bifurcation anatomical structure of chronic periodontitis are difficult to perform perfect mechanical cleaning and scaling, traditional periodontitis treatment recommends the use of antibiotics or antibacterial agents to remove residual plaque and biofilm while mechanical cleaning. However, invasive mechanical scaling is time-consuming and labor-intensive, has high technical sensitivity, and is not effective in regenerating and repairing tissue defects. It may also cause side effects such as tooth sensitivity and local tooth damage. Current local antibiotic delivery systems are prone to drug resistance, have short half-lives, and have unsatisfactory periodontal regeneration effects. Commonly used oral antibacterial agents such as chlorhexidine and hydrogen peroxide have strong cytotoxicity and are not conducive to periodontal regeneration. Therefore, better periodontal medication strategies are urgently needed.

[0003] Chlorine dioxide (CD, molecular formula ClO2) has a strong oxidizing property second only to ozone. It performs disinfection through oxidation reaction rather than chlorination reaction, and produces almost no toxic byproducts, so it is considered to be an efficient and safe disinfectant. ClO2 has a good bactericidal effect even at a relatively low concentration, and has strong diffusivity and permeability, making it more potent than other antibacterial disinfectants in removing anaerobic bacteria and stubborn biofilms in deep periodontal pockets. However, liquid chlorine dioxide preparations have poor stability, and the active ingredients of low-concentration chlorine dioxide solutions will evaporate rapidly and become ineffective after opening, which may be the reason why chlorine dioxide mouthwashes on the market are less effective than other disinfectants; when using high-concentration chlorine dioxide, it is difficult to control the release rate of chlorine dioxide gas, and it is easy to release explosively, and it is irritating to the respiratory system and cytotoxic, so it cannot be directly applied to the body.

[0004] Both natural polymers and synthetic polymers can be used to prepare hydrogels. Extracellular matrix components from natural sources are more conducive to the regeneration and reconstruction of periodontal tissues. Natural polymers mainly include proteins and polysaccharides extracted from plants, such as gelatin, chitosan, alginate, etc. Since natural polymers generally have poor mechanical properties and stability, they often need to be cross-linked or modified to enhance their mechanical properties and stability. Common methods include simple, non-toxic chemical cross-linkers and enzymes for cross-linking. The current chlorine dioxide sustained-release gel component is a synthetic polymer gel, which is difficult to degrade, is not conducive to periodontal regeneration, and cannot be directly used in the treatment of oral infectious bone defect diseases. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a protein hydrogel capable of sustained release of chlorine dioxide, and a preparation method and application thereof. The protein hydrogel capable of sustained release of chlorine dioxide is constructed by utilizing the characteristics of bovine serum albumin and gelatin, two natural protein polymers, such as good biocompatibility, easy gelation, and temperature sensitivity, without the need to add additional cross-linking agents.

[0006] To achieve the above purpose, the technical solution provided by the present invention is as follows:

[0007] A protein hydrogel capable of sustained-release chlorine dioxide comprises chlorine dioxide and an embedding material for embedding chlorine dioxide, wherein the embedding material is a hydrogel obtained by thermal polymerization of bovine serum albumin (BSA) and gelatin; wherein the mass fraction of bovine serum albumin, the mass fraction of gelatin, and the mass fraction of chlorine dioxide in the protein hydrogel capable of sustained-release chlorine dioxide are 2.4%, 15%, and 0.020-0.030% (the final concentration of chlorine dioxide in the obtained product is 200-300 ppm).

[0008] The method for preparing the protein hydrogel capable of sustained-release of chlorine dioxide as described above comprises the following steps:

[0009] (1) adding chlorine dioxide (ClO2) solution (CD) to bovine serum albumin stock solution (BSA) and mixing, letting it stand for 4-6 minutes until its color no longer changes, then adding chlorine dioxide (ClO2) solution and mixing, letting it stand for 4-6 minutes, and adding gelatin stock solution (GEL) and mixing evenly;

[0010] (2) The solution obtained after being mixed evenly in step (1) is subjected to protein thermal polymerization until the solution becomes completely opaque, and then frozen overnight to form a CD / BSA / GEL hydrogel, which is a protein hydrogel capable of sustained release of chlorine dioxide.

[0011] Preferably, the mass concentration of the bovine serum albumin stock solution in step (1) is 24%, which is obtained by dissolving bovine serum albumin powder in deionized water; the gelatin stock solution is obtained by dissolving gelatin in deionized water, and the mass concentration of the gelatin stock solution is 30%.

[0012] Preferably, in step (1), the total volume ratio of chlorine dioxide (ClO2) solution (CD), bovine serum albumin solution (BSA) and gelatin (GEL) is chlorine dioxide (ClO2) solution (CD): bovine serum albumin solution (BSA): gelatin (Gelatin) = 4:1:5; wherein, the chlorine dioxide (ClO2) solution (CD) is added in two times, firstly added at a volume ratio of chlorine dioxide (ClO2): bovine serum albumin solution (BSA) = 10%:10%, and the second addition of chlorine dioxide is 30% of the total volume, where the volume fraction is based on the total liquid volume.

[0013] Preferably, the concentration of the chlorine dioxide (ClO2) solution in step (1) is 1800-2500 ppm.

[0014] Preferably, the protein thermal polymerization in step (2) is polymerization at 95° C. for 2-3 minutes.

[0015] Preferably, the freezing overnight in step (2) is placed in a refrigerator at 4° C. overnight.

[0016] The protein hydrogel capable of sustained release of chlorine dioxide as described above is used in drugs for treating periodontitis.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention adjusts the ratio of bovine serum albumin, gelatin and chlorine dioxide, as well as the order of adding the raw materials, to prepare a physical and chemical double cross-linking by utilizing protein thermal polymerization and the oxidation of protein by ClO2, without the need to add any additional cross-linking agent, and is safe, simple, low-cost, easy to implement and clinically transformed; the prepared protein hydrogel capable of sustained-release of chlorine dioxide can release ClO2 in response to ambient temperature and protease, and can be completely degraded under body temperature and enzyme action, and has an excellent antibacterial effect on immune-escaping intracellular Porphyromonas gingivalis and its biofilm, and has been verified to have anti-inflammatory and alveolar bone regeneration-promoting effects in the constructed animal model of periodontitis, and is expected to be applied to the local drug treatment of periodontitis and peri-implantitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the synthesis of the CD / BSA / GEL hydrogel of the present invention.

[0020] Figure 2This is a scanning electron microscope image of the CD / BSA / GEL hydrogel of the present invention; the left, middle and right images are at different magnification ratios.

[0021] Figure 3 The molecular weight of BSA / GEL hydrogel was analyzed by SDS-PAGE.

[0022] Figure 4 Schematic diagram of the injectability of the CD / BSA / GEL hydrogel of the present invention.

[0023] Figure 5 This is the sustained release curve of the CD / BSA / GEL hydrogel of the present invention.

[0024] Figure 6 This is a graph showing the antibacterial performance of the CD / BSA / GEL hydrogel of the present invention against intracellular bacteria.

[0025] Figure 7 It is the anti-biofilm activity of the CD / BSA / GEL hydrogel of the present invention.

[0026] Figure 8 This is a cytotoxicity test of the CD / BSA / GEL hydrogel of the present invention on MC3T3-E1 cells.

[0027] Fig. 9 These are Micro-CT 3D images (cheek and palate sides) and 2D images of the jaws of rats in each group reconstructed 4 weeks after treatment with the present invention.

[0028] Fig.10 The enzyme response diagram of the CD / BSA / GEL hydrogel prepared by the present invention.

[0029] Fig.11 Schematic diagram of the different evaporation rates of CD / BSA / GEL hydrogel and CD solution containing the same initial ClO2 concentration stored uncovered at room temperature (A) and in a 37°C incubator (B).

[0030] Fig.12 Schematic diagram of the process of exploring the gelation of different ratios of gelatin and protein. DETAILED DESCRIPTION

[0031] The specific implementation methods are described in detail below in conjunction with the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited by the specific implementation methods. Unless otherwise specified, the raw materials and reagents used in the examples are all commercially available. Bovine serum albumin (BSA) was purchased from Solebao, gelatin (Gelatin, GEL) was purchased from McLean (China), and chlorine dioxide was purchased from Lightning Biological Co., Ltd. (analytical grade, 2500ppm). All three are analytical grade. Porphyromonas gingivalis was purchased from Guangdong Provincial Microbiological Culture Collection Center.

[0032] Example 1

[0033] A method for preparing a protein hydrogel capable of sustained-releasing chlorine dioxide, the operating steps are as follows:

[0034] (1) dissolving bovine serum albumin powder in deionized water to obtain a bovine serum albumin stock solution with a mass concentration of 24%, which is set aside; placing gelatin in deionized water and heating it to 30-40° C. to dissolve the gelatin in deionized water to obtain a gelatin stock solution with a mass concentration of 30%, which is set aside; adding 1 mL of chlorine dioxide (ClO2) solution (CD) with a concentration of 2500 ppm to 1 mL of the above-mentioned bovine serum albumin stock solution (BSA) to mix evenly, letting it stand for 4-6 minutes until its color no longer changes, then adding 3 mL of chlorine dioxide (ClO2) solution to mix evenly, letting it stand for 4-6 minutes, and adding 5 mL of the above-mentioned gelatin stock solution to mix evenly after its color no longer changes;

[0035] (2) The solution obtained after mixing in step (1) is subjected to protein thermal polymerization at 95°C for 2-3 minutes until the solution becomes completely opaque, and placed in a refrigerator at 4°C overnight to form a CD / BSA / GEL hydrogel, which is a protein hydrogel capable of sustained release of chlorine dioxide. The final bovine serum protein mass fraction in the obtained protein hydrogel capable of sustained release of chlorine dioxide is 2.4%, the gelatin mass fraction is 15%, and the chlorine dioxide mass fraction is 0.02-0.03% (the final concentration of chlorine dioxide in the obtained product is 200-300ppm).

[0036] The CD / BSA / GEL hydrogel obtained above was scanned by electron microscope. Figure 2 As shown. Figure 2 It can be seen that the CD / BSA / GEL hydrogels prepared by the present invention all have a porous structure with uniform pore size (50-100 μm). The CD / BSA / GEL hydrogel skeleton has a pore size similar to that of BSA, but the skeleton is thicker and no dispersed granular gelatin small molecules are seen (each component is completely cross-linked).

[0037] Example 2

[0038] A method for preparing a protein hydrogel capable of sustained-releasing chlorine dioxide, the operating steps are as follows:

[0039] (1) dissolving bovine serum albumin powder in deionized water to obtain a bovine serum albumin stock solution with a mass concentration of 24%, which is set aside; placing gelatin in deionized water and heating it to 30-40° C. to dissolve the gelatin in the deionized water to obtain a gelatin stock solution with a mass concentration of 30%, which is set aside; adding 1 mL of chlorine dioxide (ClO2) solution (CD) with a concentration of 1800 ppm to 1 mL of the above-mentioned bovine serum albumin stock solution (BSA) and mixing, letting it stand for 4-6 minutes until its color no longer changes, then adding 3 mL of chlorine dioxide (ClO2) solution and mixing, letting it stand for 4-6 minutes, and adding 5 mL of the above-mentioned gelatin stock solution after its color no longer changes and mixing evenly;

[0040] (2) The solution obtained after being mixed evenly in step (1) is subjected to protein thermal polymerization at 95° C. for 2-3 minutes until the solution becomes completely opaque, and then placed in a refrigerator at 4° C. overnight to form a CD / BSA / GEL hydrogel, i.e., a protein hydrogel capable of sustained release of chlorine dioxide.

[0041] Example 3

[0042] A method for preparing a protein hydrogel capable of sustained-releasing chlorine dioxide, the operating steps are as follows:

[0043] (1) dissolving bovine serum albumin powder in deionized water to obtain a bovine serum albumin stock solution with a mass concentration of 24%, which is set aside; placing gelatin in deionized water and heating it to 30-40° C. to dissolve the gelatin in the deionized water to obtain a gelatin stock solution with a mass concentration of 30%, which is set aside; adding 1 mL of chlorine dioxide (ClO2) solution (CD) with a concentration of 2000 ppm to 1 mL of the above-mentioned bovine serum albumin stock solution (BSA) and mixing, letting it stand for 4-6 minutes until its color no longer changes, then adding 3 mL of chlorine dioxide (ClO2) solution and mixing, letting it stand for 4-6 minutes, and adding 5 mL of the above-mentioned gelatin stock solution after its color no longer changes and mixing evenly;

[0044] (2) The solution obtained after being mixed evenly in step (1) is subjected to protein thermal polymerization at 95° C. for 2-3 minutes until the solution becomes completely opaque, and then placed in a refrigerator at 4° C. overnight to form a CD / BSA / GEL hydrogel, i.e., a protein hydrogel capable of sustained release of chlorine dioxide.

[0045] SDS-PAGE electrophoresis analysis:

[0046] BSA+GEL mixture without protein thermal polymerization and BSA / GEL gel denatured by protein thermal polymerization were prepared with the same ratio (BSA: gelatin = 2.4%:15%), and were subjected to SDS-PAGE electrophoresis analysis together with bovine serum albumin (BSA) and gelatin (Gelatin) alone.

[0047] The BSA, Gelatin, BSA+GEL and BSA / GEL obtained above were subjected to SDS-PAGE electrophoresis analysis. The molecular weights of the four substances were as follows: Figure 3 It can be seen that the molecular weight of BSA+GEL is larger than that of BSA, and the BSA / GEL gel is further enlarged (Gelatin is a small molecule and has gone out of the marker range); this indicates that the molecular weights of BSA / GEL, the product of protein thermal polymerization, and BSA+GEL, the protein gel product obtained by simply mixing GEL and BSA without heating, as well as pure BSA, are different, indicating that the degree of cross-linking of the three gel molecules is different.

[0048] The CD / BSA / GEL hydrogel prepared in Example 1 was placed in a 10 ml syringe to test its injectability. The results are as follows: Figure 4 As shown in the figure, it can be seen that the CD / BSA / GEL hydrogel is a pink gel-like gel and is injectable.

[0049] Detection:

[0050] 1. The sustained release of the CD / BSA / GEL hydrogel prepared in Example 1 stored without a cover at room temperature was measured by iodine titration for 14 days. The results are as follows: Figure 5 The CD / BSA / GEL hydrogel curve representing the residual content of ClO2 has a different downward slope from the CD (ClO2) solution curve, indicating that ClO2 can be successfully released.

[0051] The iodine titration method was used to determine the content of chlorine dioxide in CD / BSA / GEL hydrogel. CD / BSA / GEL hydrogel was stored without a cover. 1 ml of CD / BSA / GEL hydrogel was taken at different times after preparation to measure the content of ClO2, and the ClO2 solution with the same initial concentration was used as a control. The sample was placed in an iodine volume bottle containing 7 ml of 3 mol / L H2SO4 solution, 5 ml of 200 g / L KI solution and 100 ml of deionized water, and the reaction was placed in the dark for 5 minutes. 2 ml of 5 g / L starch indicator solution was added until the solution turned blue, and then titrated with 0.01 mol / L Na2S2O3 standard titration solution until the blue just disappeared, and the volume V of the Na2S2O3 standard titration solution used was recorded. Three parallel measurements were performed for each experiment and the average was taken. It should be noted that potassium iodide and chlorine dioxide sustained-release hydrogel were fully mixed and protected from light during the whole process to react, and the change of chlorine dioxide concentration over time was recorded by calculation. The titration is based on the measurement of iodine produced in the redox reaction between chlorine dioxide and excess potassium iodide in an acidic medium (pH ≤ 2) according to the following reaction equation:

[0052] 2ClO2+10Iˉ+8H + =5I2ˉ+Clˉ+4H2O

[0053] Calculation of chlorine dioxide concentration:

[0054] C(ClO2)=V(Na2S2O3)×C(Na2S2O3) / V 0(test solution)

[0055] Note: In the formula, V(Na2S2O3) is the dosage of sodium thiosulfate standard solution (ml), C(Na2S2O3) is the concentration of sodium thiosulfate standard solution (mol / L), and V0 is the sampling volume of chlorine dioxide (ml).

[0056] 2. The antibacterial performance of the CD / BSA / GEL hydrogel prepared in Example 1 against intracellular bacteria is shown in the following results: Figure 6 As shown, the antibiofilm activity of the hydrogels Figure 7 It has excellent anti-anaerobic effect against free anaerobic bacteria, intracellular parasites and biofilm.

[0057] The antibacterial activity of CD / BSA / GEL hydrogel against P. gingivalis intracellular parasites was evaluated by plate colony counting method combined with antibiotic protection method. The groups were as follows: positive control group (PBS), BSA / GEL group (BSA / GEL refers to BSA / GEL gel, which is prepared in the same way as in "SDS-PAGE electrophoresis analysis"), CD group (800 ppm ClO2 and TSB culture medium are prepared at a ratio of 1:9), CD / BSA / GEL group (the "CD / BSA / GEL hydrogel prepared in Example 1). Mouse preosteoblast MC3T3-E1 cells were added to the diluted P.gingivalis suspension for co-culture, washed with PBS, and metronidazole and gentamicin were added for a combined action of 60 minutes to kill bacteria that did not invade the cells. According to the grouping, the corresponding drug-containing culture medium (the drug in the drug-containing culture medium refers to the above-mentioned "PBS, BSA / GEL, CD / BSA / GEL, CD", the culture medium is TSB culture medium, and the preparation ratio is drug: culture medium = 1:9), and incubated in a 37°C incubator for 15 minutes. Wash with PBS, add deionized water to lyse the cells, take 100 μl from each group and spread it on Columbia blood agar plates, and observe the growth results of bacteria on the blood agar plates of each group in an anaerobically cultured in a 37°C incubator ( Figure 6 , Figure 6 In the two figures on the far right, "MIC" refers to the minimum inhibitory concentration of the drug used, which was determined in the preliminary experiment).

[0058] The inhibitory effect of CD / BSA / GEL hydrogel on P.gingivalis biofilm on hydroxyapatite bioceramic sheets (HA sheets) was tested. First, the HA sheets were soaked in artificial saliva and coated, then the artificial saliva was discarded, and 1 ml of Porphyromonas gingivalis suspension (1×10 8CFU / ml), and cultured at 37℃ for 3 days under anaerobic conditions. After the formation of P.gingivalis biofilm on the HA sheet, CD / BSA / GEL hydrogel was added to treat the HA sheet: the control group was treated with saline. The unattached suspended bacteria were washed, and the live and dead bacteria double staining reagent (SYTO 9 / PI) prepared according to the instructions was added, incubated in the dark for 15 minutes, and then placed under a laser confocal microscope for photography ( Figure 7 , left picture is control).

[0059] 3. In the presence of higher concentrations of ClO2, CD / BSA / GEL hydrogel has better biosafety than CD solution and has a good therapeutic effect in animal models of periodontitis.

[0060] In order to understand the in vitro cytotoxic effect of the CD / BSA / GEL hydrogel prepared in Example 1 on mouse preosteoblasts (MC3T3-E1), a cell counting kit-8 (CCK-8) was used for detection and a CD (ClO2) solution of the same concentration was used as a control. 4 MC3T3-E1 cells / mL were inoculated into the lower chamber of a 24-well plate with a chamber, with 500 μL (i.e., 2×10 4 / well). The groups were: drug-free medium control group, three concentration groups of CD solution (1 / 4MIC, 1 / 2MIC, MIC), three concentration groups of CD / BSA / GEL hydrogel (1 / 4MIC, 1 / 2MIC, MIC) and BSA / GEL hydrogel group. After the cells were completely attached, the culture medium of each well was replaced with drug-containing medium according to the group (250μl culture medium was added to the lower chamber, 200μl culture medium and 50μl experimental drugs of corresponding concentration were added to the upper chamber). After 4 hours of co-culture, the chamber was removed, washed with PBS, and replaced with α-MEM complete medium for continued culture. After 24 hours, CCK-8 reagent (10μl per well) was added. After incubation at 37°C for 2 hours, 100μL of supernatant was transferred to a 96-well plate, and the absorbance (OD value) at a wavelength of 450nm was measured by an enzyme marker.

[0061] Cytotoxicity assay ( Figure 8 ) illustrates its safety: the 24-hour cell viability of MC3T3-E1 cells was measured after CD / BSA / GEL hydrogel and CD solution with the same ClO2 content (1 / 4MIC, 1 / 2MIC and MIC) acted on them for 4 hours.

[0062] In the experimental group, 0.25 mm orthodontic ligature wire was wrapped around the cervical area of ​​the second maxillary molars on both sides of the rats and fixed on the buccal side. The wire was bent back to avoid damaging the buccal mucosa. P. gingivalis suspension (diluted to 1×10 8CFU / ml), the normal control group was not ligated or inoculated with P.gingivalis; after 3 weeks, the ligature wire was removed, and the rat periodontitis model was established. The starting point of treatment was determined by taking Micro-CT of small animals in vivo, and normal saline (control group and periodontitis group), CD solution (800ppm), BSA / GEL hydrogel and CD / BSA / GEL hydrogel (Example 1) were injected into the periodontal pockets of the second molars on both sides of the rats according to the groups, twice a week. Small animal in vivo Micro-CT was taken every week to monitor the progress of periodontitis treatment in rats. A total of 4 weeks of treatment. After the treatment, the 1st, 2nd, and 3rd molars and the connected maxillary bones on both sides of the rats were sampled, Micro-CT was taken in vitro, and three-dimensional reconstruction was performed using Mimics Medical 21.0 to compare and evaluate the repair of alveolar bone defects in each group.

[0063] Animal experiments Fig. 9 ) illustrate its anti-inflammatory and osteogenic effects: as shown in the figure, the normal control group, the periodontitis group, the CD solution group, and the CD / BSA / GEL hydrogel group. It can be seen that the CD / BSA / GEL hydrogel group has a good therapeutic effect compared with the periodontitis group and the CD group.

[0064] 4. The protein hydrogel can be completely degraded and release protein small molecules under the dual response of body temperature and protease.

[0065] The CD / BSA / GEL hydrogel prepared in Example 1 was immersed in PBS buffer and trypsin solution (Enzyme) to evaluate the degradability of the hydrogel at a constant temperature of 37°C. 1 ml of hydrogel + 3 ml of PBS buffer / trypsin solution was taken and placed in a 37°C constant temperature box. Every 12 hours, 2 ml of the supernatant of the degradation system was carefully aspirated and an equal amount of PBS buffer / trypsin solution was added. The degradation time of the hydrogel was determined by recording the time when the hydrogel lost its semi-solid form in the PBS buffer / trypsin solution.

[0066] The results are as follows Fig.10 As shown: the CD / BSA / GEL hydrogel immersed in protease can be seen that the solution is completely clear on the fifth day and can be completely degraded in the enzyme (under 37°C conditions); the CD / BSA / GEL hydrogel immersed in PBS cannot be completely degraded in PBS (partially degraded), which is the enzyme responsiveness of the hydrogel.

[0067] The volatility of ClO2 gas in CD / BSA / GEL hydrogel was evaluated, and the CD group (ClO2 solution with the same initial concentration) was used as a control. 500ul CD solution and CD / BSA / GEL hydrogel were placed in vials and placed in a 25℃ room temperature environment and a 37℃ constant temperature box, respectively, to observe the volatility (color and volume changes) of CD solution and CD / BSA / GEL hydrogel in different temperature environments. Fig.11 Schematic diagram of the different volatilization rates of CD / BSA / GEL hydrogel and CD solution with the same initial concentration stored without a cover at room temperature (A) and in a 37°C incubator (B). It can be seen that the drug in the CD / BSA / GEL hydrogel evaporates over time. At room temperature, it evaporates completely in 14 days, and the bottom of the bottle (indicated by the arrow) can be clearly seen, and the residual protein carrier components can be seen on the bottle wall.

[0068] The process of exploring the gelation of different ratios of gelatin and protein: Gelatin solutions of different concentrations (15%, 7.5%, 3.75%, 1.875%, 0.9375%, 0.46875%, 0.2344%, 0%) were added to four eight-tube strips containing BSA solutions of different concentrations (3%, 1.5%, 0.75% and 0.375%), and then polymerized at 95°C and refrigerated at 4°C overnight. A combination of gels with a gel state below 37.5°C and a semi-fluid state above 37.5°C was selected. The results are as follows Fig.12 shown.

[0069] Bacterial experiment (ie, actual use) of the CD / BSA / GEL hydrogel prepared by the present invention: 10 to the 8th power bacteria were cultured for 1 day, and the amount of hydrogel added was 10 vt%, based on the total amount of liquid in the culture solution.

[0070] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A protein hydrogel capable of sustained release of chlorine dioxide, characterized in that: The protein hydrogel capable of sustained-release of chlorine dioxide comprises chlorine dioxide and an embedding material for embedding chlorine dioxide, wherein the embedding material is a hydrogel obtained by thermal polymerization of bovine serum albumin and gelatin; wherein, in the protein hydrogel capable of sustained-release of chlorine dioxide, the mass fraction of bovine serum albumin accounts for 2.4%, the mass fraction of gelatin accounts for 15%, and the mass fraction of chlorine dioxide accounts for 0.020-0.030%.

2. A method for preparing a protein hydrogel capable of sustained-release of chlorine dioxide as claimed in claim 1, characterized in that: The following steps are included: (1) adding chlorine dioxide solution to bovine serum albumin stock solution and mixing, letting it stand until its color no longer changes, then adding chlorine dioxide solution and mixing, letting it stand until its color no longer changes, and then adding gelatin stock solution and mixing evenly; (2) The solution obtained after being mixed evenly in step (1) is subjected to protein thermal polymerization until the solution becomes completely opaque, and then frozen overnight to form a CD / BSA / GEL hydrogel, which is a protein hydrogel capable of sustained release of chlorine dioxide.

3. The method for preparing the protein hydrogel capable of sustained release of chlorine dioxide according to claim 2, characterized in that: The mass concentration of the bovine serum albumin stock solution in step (1) is 24%, which is obtained by dissolving bovine serum albumin powder in deionized water; the gelatin stock solution is obtained by dissolving gelatin in deionized water, and the mass concentration of the gelatin stock solution is 30%.

4. The method for preparing the protein hydrogel capable of sustained release of chlorine dioxide according to claim 2, characterized in that: In step (1), the total volume ratio of the chlorine dioxide solution, the bovine serum albumin stock solution and the gelatin is chlorine dioxide solution: bovine serum albumin stock solution: gelatin = 4:1:5; wherein the chlorine dioxide solution is added in two times, firstly in a volume ratio of chlorine dioxide: bovine serum albumin stock solution = 10%:10%, and the second addition of chlorine dioxide is 30% of the total volume.

5. The method for preparing the protein hydrogel capable of sustained release of chlorine dioxide according to claim 2, characterized in that: The concentration of the chlorine dioxide solution in step (1) is 1800-2500ppm.

6. The method for preparing the protein hydrogel capable of sustained release of chlorine dioxide according to claim 2, characterized in that: The protein thermal polymerization described in step (2) is polymerization at 95° C. for 2-3 minutes.

7. The method for preparing the protein hydrogel capable of sustained release of chlorine dioxide according to claim 2, characterized in that: The overnight freezing in step (2) is to place the mixture in a refrigerator at 4°C overnight.

8. Use of the protein hydrogel capable of sustained-release of chlorine dioxide as claimed in any one of claims 1 to 7 in a drug for treating periodontitis.

Images