Chemical cross-linking preparation method and application of an anti-inflammatory, antioxidant, anti-adhesion hydrogel

Abstract

The application discloses a chemical cross-linking preparation method and application of an anti-inflammatory and anti-oxidation anti-adhesion hydrogel, and belongs to the technical field of biological medical material preparation. The method is to cross-link and construct a gel body by using disulfide bonds and amidation reaction, so that the stability of the gel body is enhanced, the residence time of the gel body in tissues is further prolonged, the anti-inflammatory and anti-oxidation performance of the gel body is increased due to the fact that the gel body contains mercapto groups, and finally the anti-adhesion effect of the product is enhanced. The anti-adhesion hydrogel product is prepared by the chemical cross-linking method. The application of the anti-adhesion hydrogel product in preparing and treating postoperative peritoneal tissue adhesion products is proved by in-vitro ROS scavenging experiments, and the free radical scavenging rate of the anti-adhesion hydrogel prepared by the technical scheme of the application can reach more than 50%.

Description

Technical Field

[0001] This invention belongs to the field of biomedical material preparation technology, specifically relating to a chemical crosslinking preparation method and application of an anti-inflammatory, antioxidant, and anti-adhesion hydrogel. Background Technology

[0002] Postoperative peritoneal adhesions are a common clinical complication, primarily caused by the pathological connection between surgical trauma and adjacent organs or tissues (such as the injured peritoneum, omentum, or serosa). Currently, the mainstream treatment methods remain open surgery or laparoscopy. However, invasive and traumatic adhesiolysis inevitably carries a high risk of peritoneal adhesion recurrence, leading to treatment failure. Therefore, non-surgical treatment strategies are currently a hot topic of research. On the other hand, the accumulation of reactive oxygen species (ROS) after tissue injury can lead to vascular dysfunction and remodeling through oxidative damage, hindering endothelium-dependent vasodilation and endothelial cell growth, inducing apoptosis, activating adhesion molecules, and ultimately strengthening tissue adhesions and scar tissue formation.

[0003] Clinically, the most common non-surgical methods for preventing postoperative adhesions are drug therapy and biomaterial barrier administration. However, local or systemic drug therapy, including anti-inflammatory drugs and anticoagulants, has been shown to be rapidly metabolized in the peritoneal cavity, which greatly reduces their effectiveness. Moreover, the efficacy of existing biomaterial barrier products in clinical practice is also relatively limited. For example, artificial membrane barrier products are difficult to cover irregular wounds and are inconvenient to use in practice; injectable polymer solutions (such as polysaccharides) have a short retention time in local peritoneal injuries. Therefore, there is an urgent need to develop novel anti-adhesion gel products.

[0004] Existing technologies utilize methacrylate-modified hyaluronic acid and N-(2-hydroxypropyl)methacrylamide for free radical polymerization to construct anti-adhesion hydrogel materials with injectability, self-fusion properties, and suitable abdominal metabolic time. These materials can rapidly promote peritoneal regeneration and prevent peritoneal adhesions and adhesion recurrence after adhesiolysis (Bioactive Materials, 16(2022) 27-46, https: / / doi.org / 10.1016 / j.bioactmat.2022.02.015). Wang et al. constructed injectable biocompatible hydrogel materials using carboxymethyl chitosan and dialdehyde-functionalized PEG via Schiff base reaction, and verified the anti-adhesion effect of the hydrogel using a rat cecal lateral wall abrasion model (Chemical Engineering Journal). 463(2023)142283, https: / / doi.org / 10.1016 / j.cej.2023.142283); A research group abroad reported that they constructed nanocomposite hydrogels using PEO and silicate nanosheets. The prepared hydrogel materials are injectable and sprayable, thus providing a powerful platform for preventing postoperative adhesions in various surgeries (Nano-Micro). Lett. (2021) 13:212, https: / / doi.org / 10.1007 / s40820-021-00712-5); Liu et al. modified hyaluronic acid with epicatechin and prepared microgels, and then blended them with polyvinyl alcohol to prepare composite hydrogel materials based on dynamic borate ester bonds. The materials have good antioxidant and anti-inflammatory activities, and can significantly reduce inflammation, oxidative stress and fibrosis. In mouse models, they can effectively reduce the formation of postoperative abdominal adhesions (ACSNano. 2023, 17(4):3847–3864. doi:10.1021 / acsnano.2c12104).

[0005] Currently, the main anti-adhesion gel products on the market include medical sodium hyaluronate gel, carboxymethyl chitosan anti-adhesion liquid, carboxyaminoglucan, polylactic acid anti-adhesion gel, and polyethylene glycol. The ingredients are relatively simple, and the gel is in a non-chemical cross-linked state, so it stays in the body for a short time. On the other hand, the products on the market do not have significant anti-inflammatory functions, which is not conducive to reducing the formation of fibrosis and adhesions. Summary of the Invention

[0006] Currently, the main anti-adhesion products used in clinical practice are gel-based and film-based products. For gel-based products, the effect is usually mediocre due to insufficient adhesion or short residence time. For film-based products, it is not easy to cover irregular wounds, and there are also technical problems in practical operation.

[0007] The primary objective of this invention is to provide a chemical crosslinking preparation method for an anti-inflammatory, antioxidant, and anti-adhesion hydrogel. This method utilizes disulfide bonds and amidation reactions to crosslink and construct the gel matrix, which not only enhances the stability of the gel matrix but also further strengthens the residence time of the gel matrix in tissues. Simultaneously, the gel matrix contains thiol groups, which increases the anti-inflammatory and antioxidant properties, ultimately enhancing the anti-adhesion effect of the product.

[0008] The second objective of this invention is to provide an anti-inflammatory, antioxidant, and anti-adhesion hydrogel, which is prepared by a chemical cross-linking method.

[0009] This invention is achieved through the following technical solution:

[0010] A method for preparing an anti-inflammatory, antioxidant, and anti-adhesion hydrogel through chemical crosslinking, comprising the following steps:

[0011] S1. Dissolve hyaluronic acid in deionized water and stir to obtain solution I.

[0012] S2. Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and buffer solution to solution I, and continue stirring to carry out activation treatment; then add L-cysteine ​​solution to carry out reaction, dialyze and freeze-dry to obtain product A;

[0013] S3. Select water-soluble carboxymethyl chitosan and dissolve it in deionized water. Stir to dissolve and obtain solution II.

[0014] S4. Dissolve product A in deionized water to obtain solution III;

[0015] S5. Add solution II and four-arm polyethylene glycol succinimide to solution III, stir evenly, and crosslink at room temperature to form an anti-adhesion hydrogel product.

[0016] Preferably, in step S2, the activation treatment time is 40–45 min;

[0017] The reaction time for adding L-cysteine ​​solution is 22–24 h;

[0018] The dialysis duration is 2-3 days;

[0019] The freeze-drying process involves transferring the dialyzed solution into a plastic beaker, freezing it in a low-temperature freezer for more than 12 hours, and then placing it in a freeze dryer at -30°C to -45°C for at least 3 days.

[0020] Preferably, the mass concentration of each raw material is:

[0021] Hyaluronic acid 0.001~0.02g / mL, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride 1~10mg / mL, N-hydroxysuccinimide 1~10mg / mL, L-cysteine ​​1~10mg / mL, carboxymethyl chitosan 10~100mg / mL.

[0022] Preferably, the mass concentration of product A is 0.01–0.2 g / mL.

[0023] Preferably, in step S2, the buffer solution is 0.02M 2-morpholine ethanesulfonic acid buffer.

[0024] Preferably, the mass fraction of the four-arm polyethylene glycol succinimide ester is 5 wt% to 20 wt%.

[0025] The added four-arm polyethylene glycol succinimide ester is a solid, which can also be converted to a mass concentration of 0.05-0.2 g / mL, which is the mass concentration in the total solution after addition.

[0026] Preferably, the L-cysteine ​​solution is prepared by mixing L-cysteine ​​with 0.02M 2-morpholine ethanesulfonic acid buffer.

[0027] An anti-inflammatory, antioxidant, and anti-adhesion hydrogel is obtained by the preparation method described above.

[0028] Application of an anti-inflammatory, antioxidant, and anti-adhesion hydrogel in the preparation of products for preventing and treating postoperative peritoneal tissue adhesions.

[0029] Compared with the prior art, the present invention has at least the following technical effects:

[0030] (I) This invention provides a chemical cross-linking preparation method for an anti-inflammatory, antioxidant, and anti-adhesion hydrogel. The method utilizes disulfide bonds and amidation reaction to cross-link and construct the gel body, which not only enhances the stability of the gel body but also further strengthens the residence time of the gel body in the tissue. At the same time, the gel body contains thiol groups, which increases the anti-inflammatory and antioxidant properties, and ultimately enhances the anti-adhesion effect of the product.

[0031] (ii) This anti-inflammatory, antioxidant and anti-adhesion hydrogel product is prepared by chemical cross-linking.

[0032] (III) The application of this anti-inflammatory, antioxidant and anti-adhesion hydrogel in the preparation of products for preventing and treating postoperative peritoneal tissue adhesions. Through in vitro ROS scavenging experiments, it was demonstrated that the free radical scavenging rate of the anti-adhesion hydrogel prepared by the technical solution of this application can reach more than 50%. Attached Figure Description

[0033] Figure 1This is a schematic diagram showing the end of the DPPH scavenging experiment in Experiment Example 1;

[0034] Figure 2 This is a schematic diagram of the results of the rat cecal adhesion experiment in Experiment Example 2. Detailed Implementation

[0035] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0036] Glossary

[0037] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: EDC

[0038] N-hydroxysuccinimide: NHS

[0039] 2-Morpholine ethanesulfonic acid buffer: MES buffer solution

[0040] Water-soluble carboxymethyl chitosan: CMC

[0041] Four-arm polyethylene glycol succinimide ester: Tera-PEG-SS

[0042] Product HA-C, also known as product A, is named product HA-C in the following examples: based on the English name of cysteine ​​and the abbreviation of hyaluronic acid HA, the product is named HA-C.

[0043] Example 1:

[0044] A method for preparing an anti-inflammatory, antioxidant, and anti-adhesion hydrogel through chemical crosslinking, comprising the following steps:

[0045] S1. In a 100 mL beaker, add 0.5 g hyaluronic acid, 0.4 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 0.25 g N-hydroxysuccinimide and 50 mL of 0.02 M MES buffer solution (pH = 5.5), stir to dissolve to form solution 1, and transfer to a 100 mL two-necked flask. Continue stirring to activate for 45 min.

[0046] S2. In another 100mL beaker, add 0.2g L-cysteine ​​and 20mL of 0.02M MES buffer solution (pH=5.5), stir to dissolve and form solution 2;

[0047] S3. Finally, transfer solution 2 to a two-necked flask and react at 25°C with stirring for 24 hours. Then, collect the reaction solution and dialyze it for 3 days. After lyophilization, the product is obtained and named HA-C.

[0048] S4. Dissolve water-soluble carboxymethyl chitosan in deionized water and stir to form a water-soluble carboxymethyl chitosan solution with a mass fraction of 8% or a mass concentration of 0.08 g / mL.

[0049] S5. Dissolve HA-C in deionized water and stir to form a HA-C solution with a mass fraction of 5% or a mass concentration of 0.05 g / mL. Then, mix 1 mL of water-soluble carboxymethyl chitosan solution with 1 mL of HA-C solution, and finally add 0.1 g of tetra-armed polyethylene glycol succinimide ester and mix well to obtain gel product 1.

[0050] Example 2:

[0051] A method for preparing an anti-inflammatory, antioxidant, and anti-adhesion hydrogel through chemical crosslinking, comprising the following steps:

[0052] S1. In a 100 mL beaker, add 0.5 g hyaluronic acid, 0.4 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 0.25 g N-hydroxysuccinimide and 50 mL of 0.02 M MES buffer solution (pH = 5.5), stir to dissolve to form solution 1, and transfer to a 100 mL two-necked flask. Continue stirring to activate for 45 min.

[0053] S2. In another 100mL beaker, add 0.2g L-cysteine ​​and 20mL of 0.02M MES buffer solution (pH=5.5), stir to dissolve and form solution 2;

[0054] S3. Finally, transfer solution 2 to a two-necked flask and react at 25°C with stirring for 24 hours. Then, collect the reaction solution and dialyze it for 3 days. After lyophilization, the product is obtained and named HA-C.

[0055] S4. Dissolve water-soluble carboxymethyl chitosan in deionized water and stir to form a water-soluble carboxymethyl chitosan solution with a mass fraction of 8% or a mass concentration of 0.08 g / mL.

[0056] S5. Dissolve HA-C in deionized water and stir to form a HA-C solution with a mass fraction of 10% or a mass concentration of 0.10 g / mL. Then, mix 1 mL of water-soluble carboxymethyl chitosan solution with 1 mL of HA-C solution, and finally add 0.1 g of tetra-arm polyethylene glycol succinimide ester and mix well to obtain gel product 2.

[0057] Example 3:

[0058] A method for preparing an anti-inflammatory, antioxidant, and anti-adhesion hydrogel through chemical crosslinking, comprising the following steps:

[0059] S1. In a 100 mL beaker, add 0.5 g hyaluronic acid, 0.4 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 0.25 g N-hydroxysuccinimide and 50 mL of 0.02 M MES buffer solution (pH = 5.5), stir to dissolve to form solution 1, and transfer to a 100 mL two-necked flask. Continue stirring to activate for 45 min.

[0060] S2. In another 100mL beaker, add 0.4g L-cysteine ​​and 20mL of 0.02M MES buffer solution (pH=5.5), stir to dissolve and form solution 2;

[0061] S3. Finally, transfer solution 2 to a two-necked flask and react at 25°C with stirring for 24 hours. Then, collect the reaction solution and dialyze it for 3 days. After lyophilization, the product is obtained and named HA-C.

[0062] S4. Dissolve water-soluble carboxymethyl chitosan in deionized water and stir to form a water-soluble carboxymethyl chitosan solution with a mass fraction of 8% or a mass concentration of 0.08 g / mL.

[0063] S5. Dissolve HA-C in deionized water and stir to form a HA-C solution with a mass fraction of 5% or a mass concentration of 0.05 g / mL. Then, mix 1 mL of water-soluble carboxymethyl chitosan solution with 1 mL of HA-C solution, and finally add 0.1 g of tetra-arm polyethylene glycol succinimide ester and mix well to obtain gel product 3.

[0064] Example 4:

[0065] A method for preparing an anti-inflammatory, antioxidant, and anti-adhesion hydrogel through chemical crosslinking, comprising the following steps:

[0066] S1. In a 100 mL beaker, add 0.5 g hyaluronic acid, 0.4 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 0.25 g N-hydroxysuccinimide and 50 mL of 0.02 M MES buffer solution (pH = 5.5), stir to dissolve to form solution 1, and transfer to a 100 mL two-necked flask. Continue stirring to activate for 45 min.

[0067] S2. In another 100mL beaker, add 0.4g L-cysteine ​​and 20mL of 0.02M MES buffer solution (pH=5.5), stir to dissolve and form solution 2;

[0068] S3. Finally, transfer solution 2 to a two-necked flask and react at 25°C with stirring for 24 hours. Then, collect the reaction solution and dialyze it for 3 days. After lyophilization, the product is obtained and named HA-C.

[0069] S4. Dissolve water-soluble carboxymethyl chitosan in deionized water and stir to form a water-soluble carboxymethyl chitosan solution with a mass fraction of 8% or a mass concentration of 0.08 g / mL.

[0070] S5. Dissolve HA-C in deionized water and stir to form a HA-C solution with a mass fraction of 10% or a mass concentration of 0.10 g / mL. Then, mix 1 mL of water-soluble carboxymethyl chitosan solution with 1 mL of HA-C solution, and finally add 0.1 g of tetra-armed polyethylene glycol succinimide ester and mix well to obtain gel product 4.

[0071] Example 5:

[0072] A method for preparing an anti-inflammatory, antioxidant, and anti-adhesion hydrogel through chemical crosslinking, comprising the following steps:

[0073] S1. In a 100 mL beaker, add 0.5 g hyaluronic acid, 0.4 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 0.25 g N-hydroxysuccinimide and 50 mL of 0.02 M MES buffer solution (pH = 5.5), stir to dissolve to form solution 1, and transfer to a 100 mL two-necked flask. Continue stirring to activate for 45 min.

[0074] S2. In another 100mL beaker, add 0.2g L-cysteine ​​and 20mL of 0.02M MES buffer solution (pH=5.5), stir to dissolve and form solution 2;

[0075] S3. Finally, transfer solution 2 to a two-necked flask and react at 25°C with stirring for 24 hours. Then, collect the reaction solution and dialyze it for 3 days. After lyophilization, the product is obtained and named HA-C.

[0076] S4. Dissolve water-soluble carboxymethyl chitosan in deionized water and stir to form a water-soluble carboxymethyl chitosan solution with a mass fraction of 8% or a mass concentration of 0.08 g / mL.

[0077] S5. Dissolve HA-C in deionized water and stir to form a HA-C solution with a mass fraction of 10% or a mass concentration of 0.10 g / mL. Then, mix 1 mL of water-soluble carboxymethyl chitosan solution with 1 mL of HA-C solution, and finally add 0.2 g of tetra-arm polyethylene glycol succinimide ester and mix well to obtain gel product 5.

[0078] The DPPH scavenging experiments were used to evaluate the comparative examples 1-5, and the conclusion was that the DPPH free radical scavenging rates were approximately 58%, 75%, 72%, 90%, and 60%, respectively.

[0079] Experimental Example 1: Evaluation of the antioxidant properties of materials using the DPPH scavenging test.

[0080] Experimental Principle: DPPH (2,2-diphenyl-1-picrichydrazine) is a stable free radical that appears deep purple in ethanol solution and has a maximum absorption peak at 517 nm. When an antioxidant (scavenger) is added, the electrons or hydrogen atoms provided by the antioxidant pair with the unpaired electrons of DPPH, causing the DPPH molecule to change to a non-radical form, and the solution color gradually changes from purple to pale yellow. The degree of color change is directly proportional to the antioxidant capacity. The scavenging rate is calculated by measuring the decrease in absorbance at 517 nm using a spectrophotometer (formula: scavenging rate % = [(blank absorbance - sample absorbance) / blank absorbance] × 100).

[0081] Experimental design: Prepare a DPPH ethanol solution with a concentration of 0.1 mg / mL and a volume of 1 mL. Then, take the gel material prepared above and load it into a 2.5 mL syringe. Add 0.2 mL of gel material to the DPPH ethanol solution and react at room temperature for 4 h. Then, centrifuge and take the supernatant to test its absorbance at 517 nm and calculate the clearance rate.

[0082] like Figure 1 The diagram shown illustrates the end of the DPPH scavenging experiment reaction.

[0083] After the reaction is complete, as follows Figure 1 As shown, from left to right, they are the blank group, gel 2, and gel 4.

[0084] Experimental results:

[0085] Calculations show that the prepared gel materials have a DPPH removal rate of over 50%, with the highest reaching around 90%.

[0086] Experimental Example 2: The anti-adhesion effect of the material was evaluated using a rat cecal abrasion and adhesion model.

[0087] Experimental procedure: Male SD rats weighing approximately 300g were anesthetized, and the abdominal villi were removed. A 3-5cm incision was made in the lower left position. The cecum tissue was then located and gently removed. The cecum was then abraded with sterile gauze until pinpoint bleeding appeared on the surface. Subsequently, the peritoneum was abraded away from the abdominal wall in the same manner, forming a 1×1cm area. 2The defect was then tightly connected and fixed with sutures. PBS (control group) and gel 3 sample were then applied around the defect area, and finally the wound was sutured.

[0088] like Figure 2 The diagram shows the results of a rat cecal adhesion experiment. A represents the control group, i.e., the PBS solution treatment group; B is a magnified view of the adhesion site (green box) in Figure A, clearly showing a large area of ​​adhesion; C represents the gel 3 treatment group; D is a magnified view of the adhesion site (green box) in Figure C, clearly showing a smaller adhesion area, and also showing the initial suture location and suture line.

[0089] Experimental results:

[0090] like Figure 2 As shown, compared with the control group (PBS solution treatment group), the gel 3 treatment group has a significant anti-adhesion effect.

[0091] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing an anti-inflammatory, antioxidant, and anti-adhesion hydrogel through chemical cross-linking, characterized in that, Includes the following steps: S1. Dissolve hyaluronic acid in deionized water and stir to obtain solution I. S2. Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and 0.02M 2-morpholine ethanesulfonic acid buffer to solution I, and continue stirring for activation treatment for 40-45 min; then add L-cysteine ​​solution and react for 22-24 h, dialyze for 2-3 days, and freeze dry to obtain product A with a mass concentration of 0.01-0.2 g / mL; The freeze-drying process involves transferring the dialyzed solution into a plastic beaker, freezing it in a low-temperature freezer for more than 12 hours, and then placing it in a freeze dryer at -30°C to -45°C for at least 3 days. The L-cysteine ​​solution was prepared by mixing L-cysteine ​​with 0.02M 2-morpholine ethanesulfonic acid buffer. S3. Select water-soluble carboxymethyl chitosan and dissolve it in deionized water. Stir to dissolve and obtain solution II. S4. Dissolve product A in deionized water to obtain solution III; S5. In the solution III, add solution II and four-arm polyethylene glycol succinimide ester with a mass fraction of 5wt%~20wt%, stir evenly, and crosslink at room temperature to form an anti-adhesion hydrogel product.

2. An anti-inflammatory, antioxidant, and anti-adhesion hydrogel, characterized in that, It is obtained by the preparation method described in claim 1.

3. The application of the anti-inflammatory, antioxidant, and anti-adhesion hydrogel as described in claim 2 in the preparation of products for preventing and treating postoperative peritoneal tissue adhesions.

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