An injectable hydrogel, its preparation method and application

Abstract

This invention relates to an injectable hydrogel, its preparation method, and its applications. It solves the technical problems of short half-life, rapid degradation, and inability to directly form bone by direct systemic injection of disulfiram in existing technologies. The hydrogel contains catecholized calcium alginate, disulfiram, and water. The catecholized calcium alginate contains calcium alginate, a catechol group, and calcium ions. The catechol group is coupled to the side chain of the calcium alginate. The mass percentage content of the catecholized calcium alginate is 0.5-5%; the mass percentage content of the disulfiram is 0.25-2%; and the balance is water. This invention can be used to prepare materials that promote alveolar bone repair and regeneration.

Description

Technical Field

[0001] This invention relates to a medical material, its preparation method, and its application; more specifically, it relates to an injectable hydrogel, its preparation method, and its application. Background Technology

[0002] Periodontitis is a disease characterized by the progressive destruction of periodontal soft and hard tissues due to an abnormal immune inflammatory response triggered by a complex microbial environment. Besides causing tooth loss, it is also a risk factor for various chronic diseases. Research has found that GSDMD-mediated pyroptosis, which induces a severe inflammatory response and promotes osteoclast formation, is a crucial step in the bone loss process caused by periodontitis. Previous studies have shown that GSDMD is significantly upregulated in periodontitis patients, and knocking out the gsdmd gene significantly inhibits bone resorption in periodontitis mice, suggesting that GSDMD is an important target for treating periodontitis. Therefore, inhibiting pyroptosis to regulate osteogenic processes and promote periodontal and bone tissue regeneration represents a novel therapeutic approach.

[0003] Traditional guided tissue regeneration has become a routine procedure for alveolar bone regeneration in clinical practice. However, the osteogenic potential of mesenchymal stem cells is suppressed under oral inflammation conditions, posing a significant challenge to the repair and regeneration of damaged alveolar bone. Disulfiram (DSF) is an FDA-approved and inexpensive drug that has recently been proven to be an effective inhibitor of GSDMD pore formation against pyroptosis. However, the short half-life and rapid degradation of direct systemic injection of disulfiram, along with the drug's lack of direct osteogenic efficacy, may limit its application in the treatment of periodontitis. Summary of the Invention

[0004] This invention aims to address the technical problems of short half-life, rapid degradation, and inability to directly form bone in the direct systemic injection of disulfiram in existing technologies. It provides an injectable hydrogel loaded with disulfiram and calcium ions, its preparation method, and its application. This injectable hydrogel can effectively promote alveolar bone repair and regeneration.

[0005] Therefore, the present invention provides an injectable hydrogel containing catecholized calcium alginate, disulfiram, and water; wherein the catecholized calcium alginate contains calcium alginate, catechol groups, and calcium ions; the catechol groups are coupled to the side chains of the calcium alginate; the mass percentage content of the catecholized calcium alginate is 0.5% to 5%; the mass percentage content of the disulfiram is 0.25% to 2%; and the balance is water.

[0006] Preferably, the catecholized calcium alginate has a mass percentage content of 2%; the disulfiram has a mass percentage content of 0.5%; and the balance is water.

[0007] This invention also provides a method for preparing an injectable hydrogel, comprising the following steps: (1) dissolving disulfiram in N,N-dimethylformamide solvent to obtain a disulfiram solution; (2) dissolving bovine serum albumin (BSA) in deionized water to obtain a BSA aqueous solution; (3) adding the disulfiram solution obtained in step (1) to the BSA aqueous solution obtained in step (2), mixing until the solution presents a semi-transparent suspension state to obtain a DSF@BSA nano-formulation solution; (4) dissolving sodium alginate in distilled water and stirring to obtain a sodium alginate solution; (5) adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the sodium alginate solution obtained in step (4); (6) adding multiple Dopamine hydrochloride and sodium alginate were added to the solution in step (5) to obtain a reaction mixture; (7) the reaction mixture obtained in step (6) was dialyzed with acidified deionized water and freeze-dried to obtain catecholized sodium alginate hydrogel; (8) nano-calcium carbonate powder and gluconolactone were taken and added to the catecholized sodium alginate hydrogel obtained in step (7) and mixed well; (9) the DSF@BSA nano-formulation solution prepared in step (3) was added to the product obtained in step (8) and dispersed evenly to obtain an injectable hydrogel loaded with DSF and calcium ions; in the injectable hydrogel, the mass percentage content of the catecholized calcium alginate was 0.5% to 5%; the mass percentage content of the disulfiram was 0.25% to 2%; and the remainder was water.

[0008] Preferably, in step (1), the mass-volume concentration of the disulfiram solution is 50-200 mg / ml; in step (2), the mass-volume concentration of the BSA aqueous solution is 1-20 mg / ml.

[0009] Preferably, in step (3), the volume ratio of the disulfiram solution to the bovine serum albumin aqueous solution is (0.05-0.5):(0.5-2.5).

[0010] In the preferred step (4), the mass-volume concentration of the sodium alginate solution is 5-50 g / L.

[0011] Preferably, in step (5), 1-ethyl-(3-dimethylaminopropyl) and N-hydroxysuccinimide are added to the sodium alginate solution in step (4) at a molar ratio of 1:1.

[0012] Preferably, in step (6), dopamine hydrochloride and sodium alginate are added to the solution in step (5) at a molar ratio of (1-3):1, and the molar mass of sodium alginate is based on sodium alginate monomer.

[0013] Preferably, in step (7), the pH of the acidified deionized water is 5 to 6.

[0014] Preferably, in step (8), the amount of nano-calcium carbonate powder is 0.5 to 7.5 parts by weight, the amount of gluconolactone is 1 to 15 parts by weight, and the amount of catecholized sodium alginate hydrogel is 0.2 to 2 parts by volume; the unit of weight is mg, and the unit of volume is ml.

[0015] This invention also provides the application of injectable hydrogels in the preparation of materials that promote alveolar bone repair and regeneration.

[0016] The present invention has the following beneficial effects:

[0017] (1) This invention designs immunogenic osteogenic materials from the perspective of inhibiting pyroptosis, and introduces DSF small molecule drugs to regulate the periodontal inflammatory microenvironment, Ca 2+ This invention can induce the expression of osteogenic genes such as OPN and Runx-2 in periodontal ligament stem cells (hPDLCs) and bone marrow mesenchymal stem cells (BMSCs), promoting the regeneration of periodontal and bone tissues. This invention achieves the integration of DSF drugs and Ca... 2+ With effective loading and release, it has both "immunogenic osteogenesis" and "direct osteogenesis" functions, realizing a technological upgrade in the treatment of alveolar bone defects in the inflammatory microenvironment;

[0018] (2) The hydrogel designed in this invention has injectability and certain mucosal adhesion, and can adapt well to the space in the periodontal pocket, thereby realizing local drug delivery and local sustained release of drugs;

[0019] (3) The present invention uses nanoprecipitation method to load DSF drugs that are difficult to dissolve in water, realizes local periodontal sustained-release delivery of DSF drugs, prevents them from rapidly degrading in vitro, and thus improves the therapeutic effect. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of a scanning electron microscope image of the DSF / CA hydrogel in this invention;

[0021] Figure 2A and Figure 2B This is a schematic diagram of the 1H-NMR spectrum of the hydrogel, where, Figure 2A This is the 1H NMR spectrum of sodium alginate hydrogel. Figure 2B The 1H NMR spectrum of sodium alginate-catechol;

[0022] Figure 3 This is a schematic diagram of the hydrated particle size of DSF@BSA in the DSF / CA hydrogel of the present invention;

[0023] Figure 4This is a schematic diagram of the surface potential of DSF@BSA in the DSF / CA hydrogel of the present invention;

[0024] Figure 5 This is a schematic diagram of the Ca2+ release curve of the DSF / CA hydrogel in this invention;

[0025] Figure 6 This is a schematic diagram of the release curve of DSF / CA hydrogel in Example 4 of the present invention;

[0026] Figure 7 This is a micro-CT schematic diagram of the osteogenic effect of DSF / CA hydrogel in this invention;

[0027] Figure 8 This is a schematic diagram of the DSF / CA hydrogel immunohistochemistry results in this invention;

[0028] Figure 9 This diagram illustrates the bone volume to total volume (BV / TV) ratios in the PBS group, DSF dissolved in BSA group, CA hydrogel group, and DSF / CA hydrogel group of this invention. The ratios are: BV / TV (n = 5, mean ± SD). The groups are: PBS = PBS group, DSF = DSF dissolved in BSA group, CA = CA hydrogel group, and D / C = DSF / CA hydrogel group. **, ***, and **** represent P < 0.01, P < 0.001, and P < 0.0001, respectively. The # symbol for comparisons between two groups indicates P < 0.05. Detailed Implementation

[0029] The present invention will be further described below with reference to embodiments.

[0030] To verify the drug sustained-release effect of the DSF / CA injectable hydrogel, this invention first conducted in vitro experiments on disulfiram and Ca... 2+ Release experiments were conducted, and a mouse periodontitis animal model was established. DSF / CA injectable hydrogel was then used for prevention and treatment to observe the osteogenic effect and bone immune regulation of the hydrogel in animals.

[0031] Example 1: Material Preparation

[0032] (1) Dissolve DSF in N,N-dimethylformamide solvent to obtain a DSF solution with a mass-volume concentration of 50 mg / ml;

[0033] (2) Bovine serum albumin (BSA) was dissolved in 0.5 ml of deionized water to obtain a BSA aqueous solution with a mass-volume concentration of 1 mg / ml;

[0034] (3) Take 50 μl of the solution in step (1) and add it to the solution in step (2). Use a vortex mixer to mix at a constant speed for 20 seconds until the solution is in a semi-transparent suspension state to obtain DSF@BSA nano-formulation solution.

[0035] (4) Dissolve sodium alginate in distilled water at a concentration of 0.5% (w / v) and stir overnight;

[0036] (5) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in an equal molar ratio to the sodium alginate solution in step (4);

[0037] (6) Add dopamine hydrochloride and sodium alginate to the solution from step (5) in a 1:1 molar ratio; the reaction is carried out overnight at room temperature;

[0038] (7) The reaction mixture was dialyzed with acidified deionized water (pH 5) for 48 hours, with the dialysate being replaced every 12 hours. After 48 hours, the mixture was taken out and freeze-dried for 24 hours to obtain sodium alginate hydrogel freeze-dried product.

[0039] (8) Weigh 0.5 mg of nano calcium carbonate powder and 1 mg of gluconolactone, place them in a 1.5 mL EP tube, add 0.5 mL of catecholized sodium alginate aqueous solution, and mix the solution with the powder by pipetting or shaking.

[0040] Add 0.5 ml of DSF@BSA nano-formulation solution to step (8) and disperse it evenly to obtain DSF / CA injectable hydrogel.

[0041] The prepared hydrogel contains 0.5% by mass of catecholized calcium alginate, 0.25% by mass of disulfiram, and the remainder is water.

[0042] Figure 1 Scanning electron microscope (SEM) images of the DSF / CA injectable hydrogel prepared in Example 1 of this invention, from... Figure 1 As can be seen, the gel has a loose and porous structure, which is beneficial for drug loading and release;

[0043] Figure 2 shows the 1H-NMR spectrum of the DSF / CA injectable hydrogel prepared in Example 1 of the present invention. As can be seen from Figure 2, the peaks with chemical shifts in the range of 6.8-7.5 ppm represent catechol groups.

[0044] Figure 3 The DSF@BSA hydration particle size distribution of the DSF / CA injectable hydrogel prepared in Example 1 of this invention is shown in the figure. Figure 3As can be seen from the dynamic light scattering diagram, a single peak at 566 nm represents the hydrated particle size of the DSF@BSA nanoparticles, while the PDI is 0.209, indicating that the nanoparticles have good uniformity.

[0045] Figure 4 The surface potential of the DSF / CA injectable hydrogel prepared in Example 1 of this invention is determined from... Figure 4 As can be seen from the Zeta potential diagram, the surface potential of the DSF@BSA nanoparticles is -29.2mV, indicating that the nanoparticles are negatively charged and have good stability.

[0046] Table 1 shows the DSF loading efficiency of the DSF / CA injectable hydrogel prepared in Example 1 of this invention under different BSA concentrations. As can be seen from Table 1, when the BSA concentration is less than 1%, the DSF loading rate increases with the increase of BSA concentration, while when the BSA concentration is greater than 1%, the DSF loading rate no longer increases.

[0047] Table 1

[0048] sample BSA (0.1%) BSA (0.5%) BSA (1%) BSA (2%) DSF load efficiency 32.5％ 65.8％ 92.6％ 91.3％

[0049] Example 2

[0050] (1) Dissolve DSF in N,N-dimethylformamide solvent to obtain a DSF solution with a mass-volume concentration of 100 mg / ml;

[0051] (2) Dissolve BSA in 1 ml of deionized water to obtain a BSA aqueous solution with a mass-volume concentration of 10 mg / ml;

[0052] (3) Take 100 μl of the solution in step (1) and add it to the solution in step (2). Use a vortex mixer to mix at a constant speed for 20 seconds until the solution is in a semi-transparent suspension state to obtain DSF@BSA nano-formulation solution.

[0053] (4) Dissolve sodium alginate in distilled water at a concentration of 10% (w / v) and stir overnight;

[0054] (5) Add EDC and NHS in an equal molar ratio to the sodium alginate solution in step (4);

[0055] (6) Add dopamine hydrochloride and sodium alginate to the solution from step (5) in a molar ratio of 2:1; the reaction is carried out overnight at room temperature;

[0056] (7) Dialyze the reaction mixture with acidified deionized water (pH 6) for 48 hours, changing the dialysate every 12 hours. After 48 hours, remove the mixture and freeze-dry it for 24 hours to obtain the sodium alginate hydrogel freeze-dried product.

[0057] (8) Weigh 2.5 mg of nano calcium carbonate powder and 5 mg of gluconolactone, place them in a 5 mL EP tube, add 1 mL of catecholized sodium alginate aqueous solution, and mix the solution with the powder by pipetting or shaking.

[0058] Add 1 ml of DSF@BSA nano-formulation solution to step (8) and disperse it evenly to obtain DSF / CA injectable hydrogel.

[0059] The prepared hydrogel contains 2% by mass of catecholized calcium alginate, 0.5% by mass of disulfiram, and the remainder is water.

[0060] Example 3

[0061] (1) Dissolve DSF in N,N-dimethylformamide solvent to obtain a DSF solution with a mass-volume concentration of 200 mg / ml;

[0062] (2) Dissolve BSA in 2 ml of deionized water to obtain a BSA aqueous solution with a mass-volume concentration of 20 mg / ml;

[0063] (3) Take 500 μl of the solution in step (1) and add it to the solution in step (2). Use a vortex mixer to mix at a constant speed for 20 seconds until the solution is in a semi-transparent suspension state to obtain DSF@BSA nano-formulation solution.

[0064] (4) Dissolve sodium alginate in distilled water at a concentration of 5% (w / v) and stir overnight;

[0065] (5) Add EDS and NHS in an equimolar ratio to the sodium alginate solution in step (4);

[0066] (6) Add dopamine hydrochloride and sodium alginate to the solution from step (5) in a molar ratio of 3:1; the reaction is carried out overnight at room temperature;

[0067] (7) The reaction mixture was dialyzed with acidified deionized water (pH 5.5) for 48 hours, with the dialysate being replaced every 12 hours. After 48 hours, the mixture was taken out and freeze-dried for 24 hours to obtain sodium alginate hydrogel freeze-dried product.

[0068] (8) Weigh 7.5 mg of nano calcium carbonate powder and 15 mg of gluconolactone, place them in a 5 mL EP tube, add 2.5 mL of catecholized sodium alginate aqueous solution, and mix the solution with the powder by pipetting or shaking.

[0069] Add 2.5 ml of DSF@BSA nano-formulation solution to step (8) and disperse it evenly to obtain DSF / CA injectable hydrogel.

[0070] The prepared hydrogel contains 5% by mass of catecholized calcium alginate, 2% by mass of disulfiram, and the remainder is water.

[0071] Example 4: Drug Release Detection

[0072] The prepared hydrogel was placed in a dialysis bag and dialyzed in PBS solution. The dialysate was collected at 0.5 / 2 / 4 / 8 / 12 / 24 / 48 hours and replenished with fresh PBS. A calcium ion kit was used to measure the concentration of calcium ions in the dialysate, and release curves were plotted. Figure 5 The DSF / CA hydrogel Ca in Example 4 of this invention 2+ The release curve was determined by high-performance liquid chromatography (HPLC). Figure 6 The release curve of DSF in the DSF / CA hydrogel in Example 4 of this invention is shown.

[0073] Example 5: Osteogenesis Detection

[0074] In this invention, the animal experimental method for treating periodontitis with composite materials is as follows: (1) Male C57BL mice (Beijing Vital River, China) were selected as experimental animals to construct a mouse periodontitis model. The left maxillary second molar was ligated with 5-0 silk thread. The experiment was divided into two groups: DSF / CA hydrogel (experimental group) and calcium alginate hydrogel (positive control group) were administered locally for 14 days, repeated 3 times. The animal experiment was approved by the Animal Welfare Ethics Committee of Peking University, license number (LA2022275). (2) After 14 days, the mice were sacrificed, the maxilla was collected, and micro-CT was taken to compare the alveolar bone resorption of the two groups of mice (qualitative / semi-quantitative analysis). Tissue sections were prepared to observe the difference in OPG expression in the periodontal tissues of the two groups.

[0075] C57BL mice aged 6-8 weeks were used as experimental animals. A mouse periodontitis model was established by ligating the left maxillary second molar (6-0 suture). Simultaneously, CuCl2 was injected intraperitoneally to synergize with DSF treatment. Intervention was performed at the time of model establishment (day 0) and at successful model establishment (day 14 after ligation), with DSF / CA hydrogel injection. Micro-CT analysis was used to assess the repair of the damaged alveolar bone, and immunohistochemistry was used to detect the expression levels of osteogenic-related proteins (OPN, Runx2) to verify the osteogenic properties of DSF / CA.

[0076] Figure 7The image shows a micro-CT image of the osteogenic effect of DSF / CA injectable hydrogel in Example 5 of this invention. As can be seen from the image, compared with the control group, the DSF / CA group showed significant bone repair in micro-CT imaging. Quantitative analysis showed a significant difference between the two groups, indicating that the DSF / CA injectable hydrogel has a significant osteogenic effect in vivo.

[0077] Figure 8 The image shows the immunohistochemical staining of the DSF / CA injectable hydrogel in Example 5 of this invention. The staining results indicate that the immunohistochemical staining of OPG expression in the maxillary second molar section of mice also shows significant OPG expression in the DSF / CA group, while expression is not obvious in the control group. This result suggests that the DSF / CA injectable hydrogel may promote osteoogenesis through the OPG / RANKL / RANK pathway.

[0078] Figure 9 This diagram illustrates the bone volume / tear volume (BV / TV) of the PBS group, DSF dissolved in BSA group, CA hydrogel group, and DSF / CA hydrogel group in this invention. As can be seen from the diagram, the differences between the DSF dissolved in BSA group, CA hydrogel group, and DSF / CA hydrogel group and the PBS group are all statistically significant, with the DSF / CA group showing the most significant difference. Furthermore, there is a statistically significant difference between the CA group and the DSF / CA group. This result suggests that the osteogenic effect of the DSF / CA group is more significant than that of the CA group, indicating that DSF and CA have a synergistic effect.

[0079] However, the above description is merely a specific embodiment of the present invention and should not be construed as limiting the scope of the present invention. Therefore, any substitution of equivalent components or equivalent changes and modifications made in accordance with the scope of protection of the present invention should still fall within the scope of the claims of the present invention.

Claims

1. The application of an injectable hydrogel in the preparation of materials that promote alveolar bone repair and regeneration, characterized in that, The injectable hydrogel contains catecholized calcium alginate, disulfiram, and water; the catecholized calcium alginate contains calcium alginate, catechol groups, and calcium ions; the catechol groups are coupled to the side chains of the calcium alginate; the mass percentage content of the catecholized calcium alginate is 0.5% to 5%; the mass percentage content of the disulfiram is 0.25% to 2%; the balance is water. The injectable hydrogel is prepared using a method comprising the following steps: (1) Dissolve disulfiram in N,N-dimethylformamide solvent to obtain disulfiram solution; (2) Bovine serum albumin was dissolved in deionized water to obtain a bovine serum albumin aqueous solution; (3) Take the disulfiram solution obtained in step (1) and add it to the bovine serum albumin aqueous solution obtained in step (2), mix well until the solution is a suspension that is uniformly half transparent, and obtain DSF@BSA nano-preparation solution. (4) Dissolve sodium alginate in distilled water and stir to obtain sodium alginate solution; (5) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the sodium alginate solution obtained in step (4); (6) Add dopamine hydrochloride and sodium alginate to the solution of step (5) to obtain a reaction mixture; (7) Dialyze the reaction mixture obtained in step (6) using acidified deionized water and freeze-dry it to obtain catecholized sodium alginate hydrogel; (8) Take nano-calcium carbonate powder and gluconolactone, add the catecholized sodium alginate hydrogel obtained in step (7) to it, and mix well; (9) Add the DSF@BSA nano-formulation solution prepared in step (3) to the product obtained in step (8), disperse it evenly, and obtain an injectable hydrogel loaded with DSF and calcium ions.

2. The application of the injectable hydrogel according to claim 1 in the preparation of materials that promote alveolar bone repair and regeneration, characterized in that, The catecholized calcium alginate has a mass percentage content of 2%; the disulfiram has a mass percentage content of 0.5%; and the balance is water.

3. The application of the injectable hydrogel according to claim 1 in the preparation of materials that promote alveolar bone repair and regeneration, characterized in that, In step (1), the mass-volume concentration of the disulfiram solution is 50–200 mg / ml; in step (2), the mass-volume concentration of the bovine serum albumin (BSA) aqueous solution is 1–20 mg / ml.

4. The application of the injectable hydrogel according to claim 1 in the preparation of materials that promote alveolar bone repair and regeneration, characterized in that, In step (3), the volume ratio of the disulfiram solution to the bovine serum albumin aqueous solution is (0.05-0.5):(0.5-2.5).

5. The application of the injectable hydrogel according to claim 1 in the preparation of materials that promote alveolar bone repair and regeneration, characterized in that, In step (4), the mass-volume concentration of the sodium alginate solution is 5-50 g / L.

6. The application of the injectable hydrogel according to claim 1 in the preparation of materials that promote alveolar bone repair and regeneration, characterized in that, In step (5), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added to the sodium alginate solution in step (4) at a molar ratio of 1:

1.

7. The application of the injectable hydrogel according to claim 1 in the preparation of materials that promote alveolar bone repair and regeneration, characterized in that, In step (6), dopamine hydrochloride and sodium alginate are added to the solution in step (5) at a molar ratio of (1-3):

1.

8. The application of the injectable hydrogel according to claim 1 in the preparation of materials that promote alveolar bone repair and regeneration, characterized in that, In step (7), the pH of the acidified deionized water is 5-6; in step (8), the amount of nano-calcium carbonate powder is 0.5-7.5 parts by weight, the amount of gluconolactone is 1-15 parts by weight, and the amount of catecholized sodium alginate hydrogel is 0.2-2 parts by volume; the unit of weight is mg, and the unit of volume is ml.

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