Injectable hydrogel with anti-inflammatory and anti-oxidation functions and preparation method thereof

Abstract

The invention relates to injectable hydrogel with anti-inflammatory and anti-oxidation functions and a preparation method thereof.The preparation method comprises the steps that hyaluronic acid, EDC and NHS are dissolved in an MES buffer solution and stirred to be dissolved, and a solution I is formed; dissolving L-cysteine in an MES buffer solution to form a solution II; mixing the solution II with the solution I, reacting at room temperature, dialyzing the solution after the reaction is completed, and then freeze-drying to obtain a product; and adding the freeze-dried product into an I-type collagen solution, mixing and dissolving to prepare the hydrogel. The preparation method provided by the invention is simple to operate, does not need post-treatment and can preserve the original structure of collagen, and the obtained product can be directly injected and has remarkable anti-inflammatory and anti-oxidation functions.

Description

Technical Field

[0001] The present invention relates to an injectable hydrogel with anti-inflammatory and antioxidant functions and a preparation method thereof, belonging to the technical field of medical biomaterials. Background Art

[0002] Hydrogel is a three-dimensional network polymer material that can retain a certain amount of water. It can have characteristics such as viscoelasticity, hydrophilicity, biocompatibility, and responsiveness, and can be widely used as drug delivery, wound dressing, cartilage repair, and in vivo implant materials. The materials for constructing hydrogels include natural polymers and synthetic polymers. Among them, collagen, as a natural extracellular matrix component, is an ideal raw material for constructing hydrogel materials and is commonly used in the field of cartilage repair. For example, Huang et al. used type I collagen extracted from calf skin to prepare a collagen hydrogel with time-dependent mechanical properties by regulating the gelation process, and studied how viscoelasticity coordinates the changes of MSC cytoskeleton at different cartilage stages (Sci. Adv. 2023, 9, eade9497, https: / / doi.org / 10.1126 / sciadv.ade9497); Chen et al. prepared collagen and hyaluronic acid modified with DBCO groups and -N3 groups respectively, and used the bioorthogonal reaction (SPAAC click reaction) between the two to construct a gel network. The in vitro and in vivo test results showed that the hydrogel does not require suturing and has the function of promoting corneal repair and regeneration (Biomaterials, 2020, 255, 120176, https: / / doi.org / 10.1016 / j.biomaterials.2020.120176); Rosenquist et al. used cetivolone to modify type I collagen to construct a thiol group, and then cross-linked it with maleimide-modified PEG through Michael addition reaction to form a gel. The prepared hydrogel has good injectability and mechanical tunability, and shows great application potential in the fields of prefabricated implants, injectable fillers, corneal repair, and regenerative sealants (ACS Appl. Mater. Interfaces, 2023, 15, 34407 - 34418, https: / / doi.org / 10.1021 / acsami.3c03963); Long et al. prepared a silk fibroin / collagen composite hydrogel scaffold by ultrasonic induction method. The hydrogel scaffold has good mechanical properties and stability, and shows good repair effects in rabbit knee cartilage repair experiments (J. Mater. Chem. B, 2022, 10, 5045 - 5057, https: / / doi.org / 10.1039 / D2TB00564F).

[0003] Currently marketed hydrogel products have all been reported to have a certain repair effect. However, due to the continuous existence of factors such as joint aging and long-term mechanical load, chronic inflammation in the joint is aggravated, the intracellular redox homeostasis is disrupted, and a large amount of free radicals such as ROS gradually accumulate in the joint cavity. Simply relying on collagen materials cannot effectively reduce the ROS content, thus affecting the therapeutic effect of the materials.

[0004] For the collagen-based gel products for cartilage repair reported in the existing literature, on the one hand, most of them are blended with other polymers or used alone. Either the preparation process is relatively complex, or the component functions are relatively single. And after collagen is chemically modified, post-treatment operations are required, which easily destroys the collagen structure. On the other hand, there is currently no injectable collagen product with significant anti-inflammatory and antioxidant functions in the marketed products. Summary of the Invention

[0005] (I) Technical Problems to be Solved

[0006] To solve the above problems of the existing technology, the present invention provides an injectable hydrogel with anti-inflammatory and antioxidant functions and its preparation method.

[0007] (II) Technical Solutions

[0008] To achieve the above object, the main technical solutions adopted by the present invention include:

[0009] An injectable hydrogel with anti-inflammatory and antioxidant functions, which comprises hyaluronic acid grafted with L-cysteine coated with type I collagen.

[0010] A preparation method of an injectable hydrogel with anti-inflammatory and antioxidant functions, which comprises the following steps:

[0011] S1. Dissolve hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in MES buffer solution, and stir to dissolve to form solution I; S2. Dissolve L-cysteine in MES buffer solution to form solution II.

[0012] Then mix solution II with solution I, react at room temperature, dialyze the solution after the reaction is completed, and then freeze-dry to obtain the product.

[0013] S3. Add the freeze-dried product to the type I collagen solution, and mix and dissolve to prepare the hydrogel.

[0014] The preparation method as described above, preferably, in step S1, the final concentration of the hyaluronic acid in the MES buffer solution is 0.001 - 0.02 g / mL, and the mass ratio of the hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 15 - 5:8:5.

[0015] Through a large number of experimental studies, it is found that the mass ratio of the hyaluronic acid, N-hydroxysuccinimide, and N-hydroxysuccinimide is preferably 15 - 5:8:5. When the dosage ratio exceeds this range, it will lead to insufficient grafting degree of the hyaluronic acid or excessive remaining catalyst, affecting the product effect or excessive impurities.

[0016] The preparation method as described above, preferably, in steps S1 and S2, the concentration of the MES buffer solution is 0.01 - 0.2 mol / L, and the pH value is 5 - 7.

[0017] The preparation method as described above, preferably, in step S1, the stirring rate is 50 - 200 rpm, and the stirring time is 30 - 60 min.

[0018] The preparation method as described above, preferably, in step S2, the final concentration of the L-cysteine in the MES buffer solution is 5 - 25 mg / mL.

[0019] The preparation method as described above, preferably, in step S2, the solution I and the solution II are mixed at a volume ratio of 10 - 2:5; the reaction time is 6 - 48 h.

[0020] The preparation method as described above, preferably, in step S2, the molecular weight of the dialysis bag used for dialysis is 8 - 14 KDa, dialysis is carried out with dialysis water, and the dialysis time is 1 - 3 d.

[0021] The preparation method as described above, preferably, in step S3, the concentration of the type I collagen solution is 0.5 - 10 mg / mL; the concentration of the lyophilized product in the type I collagen solution is preferably 0.05 - 0.2 g / mL. If the concentration is too high or too low, either gel formation cannot occur or dissolution cannot occur. Therefore, the concentration of the dry product in the type I collagen solution.

[0022] The injectable hydrogel prepared by the present invention with anti-inflammatory and antioxidant functions is used for the treatment of osteoarthritis and is injected into the joint cavity.

[0023] The application of the injectable hydrogel obtained by the above-mentioned preparation method in the preparation of drugs for the treatment of osteoarthritis or cartilage repair.

[0024] (III) Beneficial effects

[0025] The beneficial effects of the present invention are:

[0026] The preparation method of the injectable hydrogel with anti-inflammatory and antioxidant functions provided by the present invention uses type I collagen and functionalized hyaluronic acid derived from pig skin as raw materials to prepare an injectable hydrogel material with anti-inflammatory and antioxidant functions. This method is simple to operate, the raw materials are easily obtained, no post-treatment is required, the original structure of collagen can be preserved, the obtained product can be directly injected, and it has significant anti-inflammatory and antioxidant functions. The prepared injectable hydrogel material can be used for cartilage repair and the treatment of knee osteoarthritis. Brief Description of the Drawings

[0027] Figure 1 It is the NMR spectrum of L-HA-2 prepared in Example 2 of the present invention;

[0028] Figure 2 It is the NMR spectrum of HA;

[0029] Figure 3 It is the injectability result of the hydrogel prepared by the present invention;

[0030] Figure 4 It is a schematic diagram of the DPPH free radical scavenging experiment results of some hydrogel materials prepared by the present invention. Detailed Embodiments

[0031] The invention principle of the present invention is to successfully graft L-cysteine methylene onto hyaluronic acid by using the amideification reaction catalyzed by EDC-NHS, and utilize the characteristics of disulfide bonds and the self-gelling of collagen to successfully prepare an injectable hydrogel material with antioxidant functions; in vitro ROS scavenging experiments prove that the scavenging rate of free radicals is more than 75%.

[0032] The injectable hydrogel prepared by the present invention is suitable for anti-inflammatory and antioxidant treatment of knee arthritis or cartilage repair at the knee. It should be noted that the technical terms: "injectable" means that the hydrogel material is usually formed by physical or chemical cross-linking, including dynamic chemical bonds and non-dynamic chemical bonds. The hydrogel material formed by dynamic chemical bonds has the phenomenon of gel-sol transformation under the action of external force shear and can be administered by injection. Therefore, the injectable hydrogel prepared by the present invention has greater application potential. "Anti-inflammatory and antioxidant": When human tissues age or are damaged, reactive oxygen species (ROS) are usually produced. If not removed in time, cells will be in a state of excessive oxidative stress, which will then lead to aggravated inflammation, further damaging tissue cells, etc. Therefore, the anti-inflammatory and antioxidant ability of materials is usually characterized by the effect of scavenging ROS.

[0033] For a better explanation and understanding of the present invention, the present invention will be described in detail below in conjunction with the accompanying drawings and through specific embodiments. Among them, the raw materials used in the present invention can be purchased commercially. For example, hyaluronic acid can be purchased from Macklin Chemical Reagent Company, N-hydroxysuccinimide (NHS) can be purchased from Macklin Chemical Reagent Company, MES buffer solution can be purchased from Macklin Chemical Reagent Company, and type I collagen is type I collagen derived from pig skin and can be purchased from Chongqing Organ Intelligent Biofabrication Engineering Research Center.

[0034] Example 1

[0035] In a beaker, add 0.5 g of hyaluronic acid, 0.4 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 0.25 g of N-hydroxysuccinimide and 50 mL of MES buffer solution with a concentration of 0.02 M (pH = 5.5). Stir to dissolve to form Solution I and transfer it to a two-necked flask. Continue stirring and activating for 45 min. In another beaker, add 0.2 g of L-cysteine and 20 mL of MES buffer solution with a concentration of 0.02 M (pH = 5.5). Stir to dissolve to form Solution II. Finally, transfer Solution II to the two-necked flask and react at 25 °C under stirring (50 rpm) for 24 h. Then collect the reacted solution and use a dialysis bag with a molecular weight of 8-14 KDa to dialyze with deionized water for 3 days, and then freeze-dry with a freeze dryer for 3 days to obtain a product, named L-HA-1.

[0036] Example 2

[0037] In a beaker, add 0.5 g of hyaluronic acid, 0.4 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 0.25 g of N-hydroxysuccinimide and 50 mL of MES buffer solution with a concentration of 0.02 M (pH = 5.5). Stir to dissolve to form Solution I and transfer it to a two-necked flask. Continue stirring and activating for 45 min. In another beaker, add 0.3 g of L-cysteine and 20 mL of MES buffer solution with a concentration of 0.02 M (pH = 5.5). Stir to dissolve to form Solution II. Finally, transfer Solution II to the two-necked flask and react at 25 °C under stirring (100 rpm) for 24 h. Then collect the reacted solution and dialyze with a dialysis bag (molecular weight 8-14 KDa) in water for 3 days, and then freeze-dry with a freeze dryer to obtain a product, named L-HA-2.

[0038] Example 3

[0039] In a beaker, add 0.5 g of hyaluronic acid, 0.4 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 0.25 g of N-hydroxysuccinimide and 50 mL of MES buffer solution with a concentration of 0.02 M (pH = 5.5), stir to dissolve to form Solution I and transfer it to a 100 mL two-necked flask, and continue stirring and activating for 45 min; in another beaker, add 0.4 g of L-cysteine and 20 mL of MES buffer solution with a concentration of 0.02 M (pH = 5.5), stir to dissolve to form Solution II; finally transfer Solution II to the two-necked flask and react for 24 h under stirring (150 rpm) at 25 °C; then collect the reacted solution and dialyze it in water with a dialysis bag (molecular weight 8-14 KDa) for 3 days, and then freeze-dry it with a freeze dryer to obtain a product named L-HA-3.

[0040] Example 4

[0041] In a 100 mL beaker, add 0.5 g of hyaluronic acid, 0.4 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 0.25 g of N-hydroxysuccinimide and 50 mL of MES buffer solution with a concentration of 0.02 M (pH = 5.5), stir to dissolve to form Solution I and transfer it to a 100 mL two-necked flask, and continue stirring and activating for 45 min; in another 100 mL beaker, add 0.5 g of L-cysteine and 20 mL of MES buffer solution with a concentration of 0.02 M (pH = 5.5), stir to dissolve to form Solution II; finally transfer Solution II to the two-necked flask and react for 24 h under stirring (200 rpm) at 25 °C; then collect the reacted solution and dialyze it in water with a dialysis bag (molecular weight 8-14 KDa) for 3 days, and then freeze-dry it with a freeze dryer to obtain a product named L-HA-4.

[0042] Example 5

[0043] Preparation of hydrogel material: Prepare a type I collagen solution with a concentration of 5 mg / mL using deionized water, and then add L-HA-1, L-HA-2, L-HA-3, and L-HA-4 to the type I collagen solution at a mass fraction of 10%, that is, a concentration of 0.1 g / mL, observe the gelation situation, and evaluate the injectability and antioxidant capacity of the material, etc. The products are named P1, P2, P3, and P4 in sequence.

[0044] The nuclear magnetic resonance (NMR) of the sample was tested using an NMR spectrometer. It was determined by the characteristic peaks of functional groups that, compared with the NMR of the original hyaluronic acid (HA), the NMR spectrum of L-HA had characteristic peaks of the methylene group of L-cysteine between 2.5 and 2.7 in chemical shift, indicating the successful preparation of L-HA (i.e., through amidation reaction, L-cysteine was successfully grafted onto the hyaluronic acid molecular chain). Among them, the NMR spectra of L-HA-1, L-HA-2, L-HA-3, and L-HA-4 were basically the same, except that the integral areas of the characteristic peaks were slightly different. Taking the NMR spectrum of L-HA-2 as an example Figure 1 as shown, the NMR spectrum of hyaluronic acid (HA) is as Figure 2 shown.

[0045] The product obtained in this invention is a hydrogel. As Figure 3 shown, it shows injectability.

[0046] The antioxidant property of the product was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging experiment. The specific experimental procedure was as follows: Ethanol was used as the solvent to prepare a DPPH solution with a concentration of 0.1 mg / mL. Then, 500 μL of the DPPH solution was taken and 200 μL of the hydrogels (P1, P2, P3, P4) were added respectively. The mixture was placed in the dark at room temperature for 4 h. In the control group, 200 μL of deionized water was added. As Figure 4 shown, then the absorbance of the solution was measured using a UV-visible spectrophotometer, and the antioxidant capacity of the sample was calculated based on the absorbance value. The results showed that the radical scavenging rates of P1, P2, P3, and P4 were 75%, 83%, 88%, and 92% in sequence, indicating that the hydrogel prepared in this invention has a high antioxidant capacity.

[0047] Many literatures have proved the anti-inflammatory effect of hydrogels on the repair of osteoarthritis and the reduction of cartilage damage. Therefore, the hydrogel product prepared in this invention not only has a repair effect on cartilage damage, but also can be used to treat osteoarthritis, has antioxidant function, and can also be treated by injection.

[0048] The above are only the preferred embodiments of the present invention, and are not limitations on the present invention in other forms. Any person skilled in the art can use the disclosed technical content above to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. An injectable hydrogel with anti-inflammatory and antioxidant functions, characterized in that: It consists of L-cysteine ​​grafted hyaluronic acid coated with type I collagen.

2. A method for preparing an injectable hydrogel with anti-inflammatory and antioxidant functions, characterized in that: It includes the following steps: S1, dissolving hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in MES buffer solution, stirring and dissolving to form solution I; S2, dissolving L-cysteine ​​in MES buffer solution to form solution II; Then, solution II is mixed with solution I, and reacted at room temperature. After the reaction is completed, the solution is dialyzed and then freeze-dried to obtain the product; S3. Add the freeze-dried product to the type I collagen solution, mix and dissolve to form a hydrogel.

3. The preparation method according to claim 2, characterized in that: In step S1, the final concentration of the hyaluronic acid in the MES buffer solution is 0.001-0.02 g / mL, and the mass ratio of hyaluronic acid to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 15-5:8:

5.

4. The preparation method according to claim 2, characterized in that: In steps S1 and S2, the concentration of the MES buffer solution is 0.01-0.2 mol / L, and the pH value is 5-7.

5. The preparation method according to claim 2, characterized in that: In step S1, the stirring rate is 50-200 rpm, and the stirring time is 30-60 min.

6. The preparation method according to claim 2, characterized in that: In step S2, the final concentration of L-cysteine ​​in the MES buffer solution is 5 to 25 mg / mL.

7. The preparation method according to claim 2, characterized in that: In step S2, the solution II and the solution I are mixed at a volume ratio of 10 to 2:5; the reaction time is 6 to 48 hours.

8. The preparation method according to claim 2, characterized in that: In step S2, the dialysis uses a dialysis bag with a molecular weight of 8 to 14 KDa, dialysis water, and a dialysis time of 1 to 3 days.

9. The preparation method according to claim 2, characterized in that: In step S3, the concentration of the type I collagen solution is 0.5-10 mg / mL; the concentration of the freeze-dried product in the type I collagen solution is 0.05-0.2 g / mL.

10. Use of the injectable hydrogel with anti-inflammatory and antioxidant functions according to claim 1 or the injectable hydrogel obtained by the preparation method according to any one of claims 2 to 8 in the preparation of drugs for treating arthritis or cartilage repair.

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