A crosslinked hydrogel, its preparation method and application

Abstract

The present invention belongs to the technical field of polymer materials, and provides a crosslinked hydrogel, a preparation method thereof and an application. The present invention uses hydroxypropyl chitosan deacetylate, 2,3,4-trihydroxybenzaldehyde and an iron salt to undergo a crosslinking reaction to prepare the crosslinked hydrogel, which can complete rapid crosslinking within five minutes. The crosslinked hydrogel prepared by the present invention has a high water content, good moisture retention, antibacterial properties, biocompatibility and procoagulant properties, can stop bleeding efficiently and rapidly, and can be locally injected. It can not only quickly fill the entire wound to achieve the purpose of hemostasis, but also inhibit the growth of bacteria, and can be widely used in the preparation of wound hemostatic and antibacterial products.

Description

Technical Field

[0001] The present invention relates to the technical field of polymer materials, and more specifically, to a cross-linked hydrogel and its preparation method and application. Background Art

[0002] Commonly used hemostatic materials on the market include tourniquets, hemostatic powders, hemostatic gauzes, etc. However, there are still many problems when they are used for hemostasis, such as poor moisturizing effect, easy tissue adhesion, poor biocompatibility, etc. In addition, the delayed wound healing caused by irregular wounds after hemostasis will also increase the mortality rate. At present, hydrogels have received extensive attention due to their good moisturizing and degradability. In particular, in-situ injectable hydrogels can closely adhere to irregular wounds through minimally invasive injection techniques and perform well in humid and highly dynamic environments. At present, some hydrogel materials for hemostasis and wound healing have emerged. For example, Lu Yapeng synthesized a hydrogel for promoting wound healing using 2,3,4-trihydroxybenzaldehyde and tilapia skin gelatin (Lu Y, Zhao M, Peng Y, He s, Zhu X, Hu C, Xia G, Zuo T, Zhang X, Yun Y, Zhang W, Shen X. A physicochemical double-cross-linked gelatin hydrogel with enhanced antibacterial and anti-inflammatory capabilities for improving wound healing. J Nanobiotechnology. 2022 Sep 24;20(1):426.), as well as fibrin glue and cyanoacrylate glue, etc. These hydrogels can effectively seal wounds by adhering to the tissue surface. However, the hydrogel for promoting wound healing synthesized by 2,3,4-trihydroxybenzaldehyde and tilapia skin gelatin takes a long time to form. Fibrin glue is mainly composed of fibrinogen, thrombin and calcium chloride, and has problems such as insufficient biosafety and high infection risk, so it is restricted in emergency treatment applications. Although cyanoacrylate glue has strong adhesion, due to the presence of toxic degradation products such as formaldehyde, there are biosafety problems. Chitosan, as a good biocompatible reagent, has good antibacterial properties and can also solve wound hemostasis and promote wound healing. However, as an insoluble substance, chitosan needs to be dissolved under acidic conditions, which greatly limits its application.

[0003] Therefore, there is an urgent need to develop a hydrogel with good biocompatibility, hemostatic ability and antibacterial properties. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a cross-linked hydrogel, a preparation method and an application thereof. The present invention uses hydroxypropyl chitosan with good biocompatibility and 2,3,4-trihydroxybenzaldehyde to form a cross-linked network structure under mild conditions. The cross-linking is rapid and can be completed within five minutes. It also has antibacterial effects and can stop bleeding.

[0005] The first aspect of the present invention provides a preparation method of a cross-linked hydrogel.

[0006] Specifically, a preparation method of a cross-linked hydrogel includes the following steps:

[0007] First, dissolve hydroxypropyl chitosan in a solvent, add an iron salt, and then add 2,3,4-trihydroxybenzaldehyde to carry out a cross-linking reaction to obtain the cross-linked hydrogel.

[0008] 2,3,4-trihydroxybenzaldehyde (TBA) contains phenolic hydroxyl groups and aldehyde groups. In the present invention, the phenolic hydroxyl groups of 2,3,4-trihydroxybenzaldehyde form a complex with iron ions in the iron salt. Three molecules of 2,3,4-trihydroxybenzaldehyde are complexed with one molecule of iron ions. The aldehyde groups of 2,3,4-trihydroxybenzaldehyde on the complex can also undergo a Schiff base reaction with the amino groups on hydroxypropyl chitosan (CS), thereby rapidly cross-linking to form a cross-linked hydrogel with a multi-cross-linked network structure. Among them, hydroxypropyl chitosan not only retains the excellent properties of chitosan, but also does not need to be dissolved under acidic conditions and can be dissolved under mild conditions, having good water solubility and not causing strong irritation to the human body. Hydroxypropyl chitosan also has the function of promoting wound healing. The cross-linked hydrogel prepared by the present invention can be used as an injectable hydrogel, which can change according to the shape of the wound and just cover the wound, not only can stop bleeding for the wound, but also can inhibit the growth of bacteria in the wound and better promote the healing of the wound.

[0009] Preferably, the mass ratio of the hydroxypropyl chitosan, the iron salt, and the 2,3,4-trihydroxybenzaldehyde is (5-15):(0.1-3):(1-10).

[0010] More preferably, the mass ratio of the hydroxypropyl chitosan, the iron salt, and the 2,3,4-trihydroxybenzaldehyde is (10-15):(1.11-1.5):(5-10).

[0011] Even more preferably, the mass ratio of the hydroxypropyl chitosan, the iron salt, and the 2,3,4-trihydroxybenzaldehyde is 10:1.11:5.

[0012] Preferably, after dissolving the hydroxypropyl chitosan in the solvent, adding polylysine is further included.

[0013] The aldehyde group of 2,3,4-trihydroxybenzaldehyde can also undergo a Schiff base reaction with the amino group on polylysine (EPL). Polylysine has good biocompatibility and broad-spectrum antibacterial properties, which can stop bleeding while inhibiting the growth of bacteria in the wound and promoting wound healing.

[0014] Preferably, the mass ratio of the hydroxypropyl chitosan, polylysine, iron salt, and 2,3,4-trihydroxybenzaldehyde is (5 - 15):(1 - 10):(0.1 - 3):(1 - 10).

[0015] More preferably, the mass ratio of the hydroxypropyl chitosan, polylysine, iron salt, and 2,3,4-trihydroxybenzaldehyde is (10 - 15):(4 - 10):(1.11 - 1.5):(5 - 10).

[0016] Even more preferably, the mass ratio of the hydroxypropyl chitosan, polylysine, iron salt, and 2,3,4-trihydroxybenzaldehyde is 10∶4∶1.11:5.

[0017] Preferably, the temperature of the cross-linking reaction is 60 - 80 °C, and the time of the cross-linking reaction is 1 - 5 minutes.

[0018] Preferably, the solvent is phosphate (PBS) buffer solution.

[0019] Preferably, the pH value of the solvent is 7 - 8.

[0020] More preferably, the pH value of the solvent is 7.4 - 8.

[0021] Even more preferably, the pH value of the solvent is 7.5 - 8.

[0022] Preferably, the iron salt is ferric chloride.

[0023] Preferably, after adding the iron salt, it further includes heating at 70 - 90 °C for 1 - 5 minutes. The optimal reaction temperature of 2,3,4-trihydroxybenzaldehyde is 70 - 90 °C, and heating helps to accelerate the dissolution of polylysine and chitosan.

[0024] The second aspect of the present invention provides a cross-linked hydrogel.

[0025] A cross-linked hydrogel, and the water content of the cross-linked hydrogel is 70 - 90%.

[0026] The third aspect of the present invention provides an application of the cross-linked hydrogel.

[0027] An application of the cross-linked hydrogel in the preparation of hemostatic and antibacterial products.

[0028] An injectable hemostatic and antibacterial dressing, which is prepared from the crosslinked hydrogel as a raw material.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] (1) The present invention utilizes the crosslinking reaction of hydroxypropyl chitosan deacetylate, 2,3,4-trihydroxybenzaldehyde and iron salt to prepare a crosslinked hydrogel. The reaction conditions are mild, without acidic conditions, and the hydrogel can be rapidly formed within 5 minutes. The water content reaches 81.71 - 85.52%, with a relatively high water content. Moreover, the moisture retention rate can still reach more than 90% after 48 hours, showing good moisture retention. In the antibacterial experiment, it has good antibacterial performance against Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli). In the hemolytic experiment, it shows good biocompatibility, and in the in vitro hemostasis experiment, it shows the ability of efficient and rapid hemostasis. The crosslinked hydrogel prepared by the present invention can effectively inhibit the growth of wound bacteria and promote the healing of wounds;

[0031] (2) The crosslinked hydrogel provided by the present invention can not only be rapidly crosslinked, but also has injectability, hemostatic and antibacterial functions. It can quickly fill the entire wound through local injection to achieve the purpose of hemostasis, and can also inhibit the growth of bacteria, and can be widely used in the preparation of wound hemostatic and antibacterial products. Description of the Drawings

[0032] Figure 1 Synthesis schematic diagram of the crosslinked hydrogel of Example 2 of the present invention;

[0033] Figure 2 Graph obtained by injecting the crosslinked hydrogel of Example 2 of the present invention;

[0034] Figure 3 Water content test result diagram of the crosslinked hydrogels prepared in Examples 1 - 3 of the present invention;

[0035] Figure 4 Swelling property test result diagram of the crosslinked hydrogels prepared in Examples 1 - 3 of the present invention;

[0036] Figure 5 Moisture retention test result diagram of the crosslinked hydrogels prepared in Examples 1 - 3 of the present invention;

[0037] Figure 6 Hemolysis rate test result diagram of the crosslinked hydrogels prepared in Examples 1 - 3, 5 of the present invention against red blood cells;

[0038] Figure 7 Hemostasis situation diagram after applying the crosslinked hydrogels prepared in Examples 1, 3 of the present invention to rabbit liver;

[0039] Figure 8The bleeding volume graph after applying the crosslinked hydrogels prepared in Examples 1 and 3 of the present invention to rabbit livers;

[0040] Figure 9 The antibacterial ability test of the crosslinked hydrogels prepared in Examples 1-3 of the present invention. Detailed implementation manners

[0041] To make the technical solutions described in the present invention clearer and more understandable to those skilled in the art, the following examples are listed for illustration. It should be noted that the following examples do not limit the scope of protection required by the present invention.

[0042] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial channels or can be obtained by existing known methods.

[0043] Example 1

[0044] A preparation method of a crosslinked hydrogel, comprising the following steps:

[0045] (1) Add 240 mg of hydroxypropyl chitosan (CS) to 4 mL of PBS solution (pH value 7.5), and dissolve it by shaking;

[0046] (2) Add 800 μL of ferric chloride solution with a concentration of 33.33 mg / mL to the solution in step (1), mix well and heat at 80 °C for 1 minute;

[0047] (3) Add 120 mg of 2,3,4-trihydroxybenzaldehyde (TBA) to the solution in step (2), heat to 80 °C to dissolve, and wait for five minutes to obtain a crosslinked hydrogel (the crosslinked hydrogel product is named CS-TBA-EPL-0, where the content of EPL is 0, and the hydrogel can be formed without adding polylysine).

[0048] Example 2

[0049] A preparation method of a crosslinked hydrogel, comprising the following steps:

[0050] (1) Add 240 mg of hydroxypropyl chitosan (CS) to 4 mL of PBS solution (pH value 7.5), and dissolve it by shaking;

[0051] (2) Immediately add 96 mg (i.e., 5 mM) of polylysine (EPL) to the solution in step (1) and dissolve it;

[0052] (3) Add 800 μL of ferric chloride solution with a concentration of 33.33 mg / mL to the solution in step (2), mix well and heat at 80 °C for 1 minute;

[0053] (4) Add 120 mg of 2,3,4-trihydroxybenzaldehyde (TBA) to the solution in step (3), heat it to 80 °C to dissolve, and wait for five minutes to obtain a cross-linked hydrogel (the cross-linked hydrogel product is named CS-EPL-TBA-5).

[0054] The preparation process of the cross-linked hydrogel in Example 2 above is as Figure 1 shown Figure 1 A is a physical picture of the CS-EPL-TBA hydrogel prepared in Example 2. It can be seen from Figure 2 that after adding 2,3,4-trihydroxybenzaldehyde in step (4) of Example 2, the cross-linked hydrogel can be prepared in 5 minutes. Figure 1 B is a schematic diagram of the Schiff base reaction after adding polylysine. Figure 1 C is a schematic diagram of the structure of the cross-linked hydrogel prepared in Example 2. Among them Figure 1 TBA is 2,3,4-trihydroxybenzaldehyde, SciffBase reaction is the Schiff base reaction, ion coordination bond is the ion coordination bond, and Hydrogen bond is the hydrogen bond.

[0055] Example 3

[0056] The difference from Example 2 is that in step (2), 192 mg (i.e., 10 mM) of polylysine (EPL) is immediately added to the solution in step (1) to dissolve (the cross-linked hydrogel product is named CS-EPL-TBA-10).

[0057] Example 4

[0058] The difference from Example 2 is that in step (2), 286 mg (i.e., 15 mM) of polylysine (EPL) is immediately added to the solution in step (1) to dissolve (the cross-linked hydrogel product is named CS-EPL-TBA-15).

[0059] Example 5

[0060] The difference from Example 2 is that in step (2), 384 mg (i.e., 20 mM) of polylysine (EPL) is immediately added to the solution in step (1) to dissolve (the cross-linked hydrogel product is named CS-EPL-TBA-20).

[0061] Comparative Example 1

[0062] A preparation method of a bacteriostatic wound-healing hydrogel includes the following steps:

[0063] (1) Add 2,3,4-trihydroxybenzaldehyde (TBA) to deionized water to dissolve;

[0064] (2) Add anhydrous ferric chloride (0.1 M), where the molar ratio of ferric chloride to TBA = 1:3;

[0065] (3) Add 5 M NaOH to the solution and stir for 3 hours;

[0066] (4) Mix 5 g of tilapia gelatin (Tsg) with deionized water (100 mL) heated at 50 °C to obtain a 5% Tsg solution;

[0067] (5) Mix the solution obtained in step (3) and the Tsg solution and stir to dissolve. Heat the mixed solution at 50 °C for 20 minutes to prepare a pre-gel. Finally, form a hydrogel at 25 °C.

[0068] Comparative Example 2

[0069] A method for preparing a chitosan-rich hydrogel, comprising the following steps:

[0070] (1) Weigh 10.0 g of chitosan and place it in a 50 mL round-bottom flask. Then add 10 mL of an aqueous acetic acid solution containing 0.3% (volume fraction) to the reaction flask and stir to dissolve the chitosan completely;

[0071] (2) Add 33.33 mg of 2,4,5-trihydroxybenzaldehyde and 33.33 mg of 2,3,4-trihydroxybenzaldehyde to the reaction flask respectively. Stir the reaction system at room temperature (RT) for 1 h. After the reaction is completed, a light yellow viscous liquid is obtained, which is the product benzaldehyde-chitosan;

[0072] (3) Let it stand for about 24 hours to form a chitosan-rich hydrogel.

[0073] Application Example

[0074] A hemostatic and antibacterial dressing is prepared from the cross-linked hydrogel prepared in Example 1 above.

[0075] Product Effect Test

[0076] 1. Injectability of the hydrogel

[0077] As can be seen from Figure 2 injection of the cross-linked hydrogel prepared in Example 2 into a syringe can write the capital letters "ZMU" through injection, which proves that the cross-linked hydrogel prepared by the present invention has injectability and can obtain different shapes by injection.

[0078] 2. Water content test

[0079] As a wound dressing, a higher water content can ensure a moist environment around the wound, which can better promote wound healing. The following method was used for testing. After the hydrogel formed into a gel, its initial mass was weighed. Then, the cross-linked hydrogels prepared in Examples 1-3 were placed in a refrigerator at -80 °C overnight. After freeze-drying for 24 h, the mass after drying was weighed. The moisture content of the hydrogel was calculated according to the following formula:

[0080]

[0081] Where: W0 is the initial mass of the hydrogel before drying, and W1 is the mass of the hydrogel after drying.

[0082] From Figure 3 it can be seen that the moisture contents of the cross-linked hydrogels prepared in Examples 1-3 of the present invention are 85.52%, 83.88%, and 81.71% respectively, having a relatively high moisture content.

[0083] 3. Swelling test

[0084] The cross-linked hydrogels prepared in Examples 1-3 were placed in a refrigerator at -80 °C overnight. After freeze-drying for 24 h, the initial mass after drying was weighed. Then, they were respectively placed in aqueous solutions at 25 °C, 37 °C, and 45 °C. Every 5 minutes, the hydrogels were taken out, the surface moisture was blotted dry with filter paper, and the mass was weighed. The swelling degree was calculated according to the following formula:

[0085]

[0086] Where: W2 is the mass of the hydrogel after drying, and W3 is the mass of the hydrogel after water absorption.

[0087] The results are as Figure 4 shown. The cross-linked hydrogels prepared in Examples 1-3 reached swelling equilibrium in about 40 minutes, and the swelling degree of CS-EPL-TBA-10 was the highest, indicating that the cross-linked hydrogels prepared in the present invention can exhibit good swelling performance within a relatively wide temperature range, which can broaden the applications. For example, the cross-linked hydrogels prepared in the present invention can absorb the exudate around the wound, thereby reducing the suppuration of the wound.

[0088] 4. Moisture retention test

[0089] After the hydrogel was formed, its initial mass was weighed. Then, the cross-linked hydrogels prepared in Examples 1-3 were placed in an incubator at 37 °C, and the mass after being placed in the incubator for 0 h, 6 h, 12 h, 24 h, and 48 h was weighed. The water holding ratio of the hydrogel was calculated according to the following formula:

[0090]

[0091] Wherein: W4 is the initial mass of the hydrogel, and W5 is the mass of the hydrogel after being placed for a period of time.

[0092] Figure 5 a - c are respectively the test result diagrams of the moisture retention performance of the crosslinked hydrogels prepared in Examples 1 - 3. It can be seen from Figure 5 that after 48 h, the moisture retention rates of the three hydrogels can still reach more than 90%. The good moisture retention rate ensures that the wound is in a moist environment, and such an environment can promote wound healing.

[0093] 5. Hemolysis test

[0094] Add 200 μL of 4% (volume concentration) red blood cells to the crosslinked hydrogels prepared in Examples 1 - 3. Use physiological saline as the negative control (BLANK), and Triton X - 100 as the positive control group, measure the absorbance at 540 nm, and calculate the hemolysis rate (Hemolysis) according to the following formula:

[0095]

[0096] Wherein: A0 is the absorbance of the sample, A1 is the absorbance of the negative control, and A2 is the absorbance of the positive control.

[0097] The results are as Figure 6 shown. The hemolysis rates of the negative control group, positive control group, CS - EPL - TBA - 0, CS - EPL - TBA - 5, CS - EPL - TBA - 10, and CS - EPL - TBA - 20 are 0%, 100%, 2.28%, 3.36%, 5.01%, and 7.23% respectively, indicating that the CS - EPL - TBA hydrogel prepared by the present invention has a relatively appropriate hemolysis rate.

[0098] 6. Hemostasis test

[0099] Use a scalpel to make a wound nearly 1 cm on the non - adhering surface between the rabbit liver and the filter paper (ensure that the lowest end of the liver is ruptured to facilitate blood flow on the filter paper). Group A (Blank) does not receive any treatment, Group B uses the hydrogel CS - TBA - EPL - 0 prepared in Example 1, and Group C uses CS - TBA - EPL - 10 prepared in Example 3 to cover the bleeding area (the amount of hydrogel should not be too much, just cover it). Record the bleeding conditions of the rabbit liver at 0 s, 20 s, 40 s, and 60 s after the above - mentioned treatment. The results are as Figure 7, Group A was not treated with anything. As time went by, the bleeding volume increased. However, after applying the CS-TBA-EPL-0 hydrogel to Group B, the bleeding area significantly shrank and the bleeding volume gradually decreased. After applying the CS-TBA-EPL-10 hydrogel to Group C, the bleeding volume further decreased. The bleeding volumes (Massing Bleeding) of each group after 60 s were as Figure 8 shown. The bleeding volumes of Group A (control group, Control), Group B, and Group C were 910.67 mg, 346.67 mg, and 83.33 mg respectively. This indicates that after applying the hydrogel prepared by the present invention, it helps to shrink the bleeding area and reduce the bleeding volume.

[0100] 7. Bacteriostatic ability test

[0101] 1 mL of the hydrogels prepared in Examples 1-3 was taken separately into glass bottles, and then 400 μL of bacteria with a concentration of 1×10 6 CFU / mL was added to incubate with the hydrogels. The absorbance (OD 600 ) of different bacteria at 600 nm was measured at 0 h, 6 h, 12 h, and 24 h. The results were as Figure 9 shown. The synthesized CS-EPL-TBA hydrogel showed good bacteriostatic performance against both Gram-positive bacteria (Staphylococcus aureus, S. aureus) and Gram-negative bacteria (Escherichia coli, E. coli). The absorbance of bacteria basically did not change after 24 hours. In addition, after spreading the bacteria on the plate, it was found that there was no bacterial growth on the agar plate incubated with the hydrogel, indicating that the bacteriostatic performance of the hydrogel was excellent.

[0102] Compared with Example 1, the bacteriostatic performance of the tilapia skin gelatin used in Comparative Example 1 was poor, and a strong base (NaOH) needed to be added during the preparation of the hydrogel in Comparative Example 1, and the process of forming the hydrogel took 180 minutes. However, the preparation process of the hydrogel of the present invention not only does not require the addition of a strong base, but also can crosslink quickly and can be completed within 5 minutes.

[0103] Compared with Example 1, the chitosan used in Comparative Example 2 needed to be dissolved under acidic conditions, and the reaction conditions were not mild. Moreover, since the formed hydrogel did not have fluidity, the gel could not be injected, and it took 24 hours to form the hydrogel, which could not achieve the purpose of quickly healing the wound.

Claims

1. Use of a crosslinked hydrogel in the preparation of a hemostatic and antibacterial product, wherein the water content of the crosslinked hydrogel is 70 - 90%, and the crosslinked hydrogel is prepared by a preparation method comprising the following steps: First, dissolve hydroxypropyl chitosan in a solvent, add an iron salt, and then add 2,3,4-trihydroxybenzaldehyde to carry out a crosslinking reaction to obtain the crosslinked hydrogel.

2. The application according to claim 1, characterized in that, The mass ratio of the hydroxypropyl chitosan, the iron salt, and the 2,3,4-trihydroxybenzaldehyde is (5 - 15):(0.1 - 3):(1 - 10).

3. The application according to claim 1, wherein First, dissolve hydroxypropyl chitosan in a solvent, and then it further includes adding polylysine.

4. The application according to claim 3, wherein The mass ratio of the hydroxypropyl chitosan, polylysine, iron salt, and 2,3,4-trihydroxybenzaldehyde is (5 - 15):(1 - 10):(0.1 - 3):(1 - 10).

5. The application according to claim 1, characterized in that The temperature of the crosslinking reaction is 60 - 80°C, and the time of the crosslinking reaction is 1 - 5 minutes.

6. The application according to claim 1, characterized in that, The pH value of the solvent is 7 - 8.

7. The application according to claim 1, characterized in that, After adding the iron salt, it further includes heating at 70 - 90°C for 1 - 5 minutes.

8. An injectable hemostatic and antibacterial dressing, characterized in that, It is prepared by using a crosslinked hydrogel as a raw material, wherein the water content of the crosslinked hydrogel is 70 - 90%, and the crosslinked hydrogel is prepared by a preparation method comprising the following steps: First, dissolve hydroxypropyl chitosan in a solvent, add an iron salt, and then add 2,3,4-trihydroxybenzaldehyde to carry out a crosslinking reaction to obtain the crosslinked hydrogel.

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