Injectable antibacterial hydrogel, preparation method and application thereof

Abstract

The application discloses an injectable antibacterial hydrogel, a preparation method and application thereof, and belongs to the technical field of high polymer materials. The structure shown in formula I is used as antibacterial components and a gel skeleton, and is mixed with aldehyde group functionalized hyaluronic acid in a weak alkaline water medium to obtain the hydrogel. The hydrogel uses polyhydrogenated pyrimidine heterocyclic polymer as antibacterial skeleton, cooperates with aldehyde group functionalized polysaccharide hyaluronic acid, and is crosslinked based on Schiff-base reaction to have a C=N double bond dynamic bond, has the properties of fast gelation and self-antibacterial, and can be applied to the treatment material of purulent subcutaneous infection, and has wide application in the field of biological medical materials.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of high polymer materials, and particularly relates to an injectable antibacterial hydrogel, a preparation method and application thereof. BACKGROUND

[0002] Pus bacterial infection is one of the clinical difficulties, and the pus bacteria has less diffusion after invading the tissue, but the damage to the local tissue is much more serious than other strains. Daily pus infection is mostly caused by sharp instrument injury carrying pus bacteria. Once the lesion forms a pustule, the resistance to antibiotics will increase exponentially, and it is difficult to treat except for incision and drainage.

[0003] In recent years, hydrogel as a drug carrier has attracted widespread attention. Due to its wet and soft characteristics, it has been widely used as an open wound dressing. The injectable hydrogel is generally cross-linked by dynamic chemical bonds, usually has self-healing and viscous flow, can self-adaptively fill the tissue gap of the wound, and release the loaded drug in the wound surface to prevent bacterial infection and accelerate wound healing. However, subcutaneous pus infection cannot be easily cleaned and changed like open wound, and surgery will also cause secondary damage to the lesion skin.

[0004] At present, most of the antibacterial gels are used as carriers of antibiotics. Some gel carriers do not have cross-linked chemical structure, and are only composed of flocculants, water retaining agents and thickening agents, and have rapid degradation and irregular drug release. Therefore, in view of the above problems, it is a good strategy to develop an injectable hydrogel that can resist bacteria itself. However, at present, there are few antibacterial gel injections that can play a role in the clinic, and basically in the blank stage. The development of new antibacterial injections has strategic significance. SUMMARY

[0005] In order to overcome the above-mentioned defects in the prior art, the application provides an injectable antibacterial hydrogel, a preparation method and application thereof. The hydrogel uses polyhydrogenated pyrimidine heterocyclic polymer as an antibacterial skeleton, cooperates with aldehyde-functionalized hyaluronic acid, and is cross-linked by C=N double bond dynamic bond based on Schiff-base reaction, has the properties of rapid gel formation and self-antibacterial, and can be applied to the treatment material of pus subcutaneous infection, and has wide application in the field of biological and medical materials.

[0006] The application is realized by the following technical scheme:

[0007] An injectable antibacterial hydrogel, which uses the structure of formula I as an antibacterial component and a gel skeleton, and is mixed with aldehyde-functionalized hyaluronic acid in a weak alkaline aqueous medium to obtain the hydrogel;

[0008]

[0009] Wherein, m is the polyether amine D-230 monomer repeat unit, n is a positive integer, 10≤n≤60.

[0010] Further, the aldehyde group functionalized hyaluronic acid has a structure shown in formula II:

[0011]

[0012] Wherein, n is the degree of polymerization, 50≤n≤1000, m is the structural unit modified by oxidation in n, and m accounts for k proportion in n, 20%≤k≤60%.

[0013] In another aspect, the application also provides a preparation method of the injectable antibacterial hydrogel, which specifically comprises the following steps:

[0014] First, the hydrogenated pyrimidine antibacterial polymer is dissolved in a weak alkaline aqueous medium, and then the aldehyde group functionalized hyaluronic acid solution is added to obtain a hydrogel material.

[0015] Further, the mass ratio of the hydrogenated pyrimidine antibacterial polymer and the aldehyde group functionalized hyaluronic acid is 1:0.5-5; and the molar ratio of the amino group in the hydrogenated pyrimidine antibacterial polymer to the aldehyde group in the aldehyde group functionalized hyaluronic acid is 1:0.5-5.

[0016] Further, the solvent is weak alkaline water, physiological saline or a buffer solution; wherein, the pH of the weak alkaline water is 7.4-8.5; the mass-volume concentration of the aldehyde group functionalized hyaluronic acid is 5%-25%; the mass-volume concentration of the hydrogenated pyrimidine antibacterial polymer is 5%-30%; and the mixing temperature is 4-37℃.

[0017] Further, the aldehyde group functionalized hyaluronic acid is obtained by oxidizing natural polysaccharide hyaluronic acid with a periodate salt under the conditions of light shielding and room temperature, and has a structure shown in formula II; wherein, the solvent used is water, and the periodate salt includes but is not limited to sodium salt, potassium salt, etc.

[0018] In a third aspect, the application also provides the use of the injectable antibacterial hydrogel, which is used as an antibacterial dressing for wound healing and has anti-inflammatory and antioxidant effects.

[0019] Compared with the prior art, the application has the following advantages:

[0020] The application provides an injectable antibacterial hydrogel, a preparation method and application thereof, the hydrogel is quickly obtained by mixing polyhydroxy pyrimidine antibacterial polymer and high-iodic acid oxidized hyaluronic acid macromolecule in a weak alkaline aqueous medium, the gelation is rapid, the reaction condition is mild, and the cell compatibility is good; the hydrogel can be slowly degraded in response to acidic metabolites of pyogenic bacteria, the hydrogel with the properties of rapid gelation and self-antibacterial property can be applied as a drug in the field of biomedical materials. Compared with traditional surgical intervention for pyogenic infection, the application blocks the contact between bacteria and normal skin tissue in the upper layer through a non-specific membrane-breaking sterilization method, and destroys the pimple tissue, while the acidic metabolites of pyogenic bacteria at the pimple trigger local degradation of the gel, and free antibacterial polymers are released in the tissue, so that a sterilization microenvironment is created at the lesion, and the infection is completely blocked from worsening. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the drawings needed to be used in the specific embodiments or prior art description will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale.

[0022] Figure 1 Nuclear magnetic resonance hydrogen spectrum of the polyhydroxy pyrimidine antibacterial polymer prepared in Example 1 of the present application;

[0023] Figure 2 Mechanical strength test results of the hydrogel with a mass concentration of 10% prepared in Example 5 of the present application;

[0024] Figure 3 Test results of the self-repairing performance of the hydrogel with a mass concentration of 10%;

[0025] Figure 4 Scanning electron microscope image of the hydrogel with a mass concentration of 10%;

[0026] Figure 5 Toxicity test results of the gel material with different concentrations on L929 cells;

[0027] Figure 6 Contact sterilization effect of the surface of the 10% hydrogel;

[0028] Figure 7 Treatment effect of the 10% hydrogel on a subcutaneous infection model on the back of a mouse after 5 days of treatment. DETAILED DESCRIPTION

[0029] In order to clearly and completely describe the technical solutions of the present application and the specific working process thereof, in combination with the drawings of the specification, the specific embodiments of the present application are as follows:

[0030] In the following examples, dimethyl acetylene dicarboxylate, formaldehyde solution, polyether amine D-230, glacial acetic acid, hyaluronic acid, sodium periodate were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. The hyaluronic acid has a molecular weight of 80-100 million and an oxidation degree of 35%.

[0031] An injectable antibacterial hydrogel, which is obtained by mixing an antibacterial component and a gel skeleton described by formula I with an aldehyde-functionalized hyaluronic acid in a weakly alkaline aqueous medium.

[0032]

[0033] wherein m is a polyether amine D-230 monomer repeating unit, and n is a positive integer, 10≤n≤60.

[0034] The aldehyde-functionalized hyaluronic acid has a structure shown in formula II:

[0035]

[0036] wherein n is a polymerization degree, 50≤n≤1000, m is a structure unit modified by oxidation in n, and m accounts for k in n, 20%≤k≤60%.

[0037] Example 1 Preparation of polyhydroxy pyrimidine antibacterial polymer

[0038] The present example provides a preparation method of a polyhydroxy pyrimidine antibacterial polymer, which specifically includes the following contents:

[0039] 1.42 g of dimethyl acetylene dicarboxylate and 2.3 g of polyether amine D-230 were respectively dissolved in 20 mL of methanol, then the polyether amine solution was slowly added dropwise to the dimethyl acetylene dicarboxylate solution under stirring, 2.43 g of formaldehyde solution and 1.2 g of glacial acetic acid were added dropwise to the reaction system after 20 minutes of reaction, and the mixture was reacted at room temperature for 48 hours. After distillation under reduced pressure, the mixture was washed with 50 mL of saturated sodium bicarbonate solution for 3 times, then the organic phase was collected, and excess anhydrous magnesium sulfate was added for drying overnight. The mixture was filtered and concentrated, and rotary evaporation was performed under oil pump reduced pressure for 3 hours to obtain a brown viscous sample.

[0040] The polyhydroxy pyrimidine obtained above was subjected to amination in a 1:2.5 equivalent ratio with DMF as the reaction solution, and after 48 hours of reaction at 50°C, the mixture was dialyzed with deionized water for 3 days, and then freeze-dried to obtain a brown viscous liquid, which was subjected to nuclear magnetic resonance analysis. Figure 1 The nuclear magnetic resonance hydrogen spectrum of the polyhydroxy pyrimidine prepared in Example 1 of the present application.

[0041] Example 2

[0042] Polyhydroxypyrimidine prepared in Example 1 (100 mg) was dissolved in PBS solution (2 mL) with pH 7.4 to obtain solution 1, and hyaluronic acid (50 mg) was dissolved in deionized water (2 mL) to obtain solution 2. Solution 1 (50 μL) and solution 2 (200 μL) were mixed thoroughly to obtain a polymer solution, which was gelled at room temperature. The gelling time was 10 seconds, which was observed by the inverted method.

[0043] Example 3

[0044] Polyhydroxypyrimidine prepared in Example 1 (100 mg) was dissolved in PBS solution (2 mL) with pH 7.4 to obtain solution 1, and hyaluronic acid (50 mg) was dissolved in deionized water (2 mL) to obtain solution 2. Solution 1 (50 μL) and solution 2 (150 μL) were mixed thoroughly to obtain a polymer solution, which was gelled at room temperature. The gelling time was 10 seconds, which was observed by the inverted method.

[0045] Example 4

[0046] Polyhydroxypyrimidine prepared in Example 1 (100 mg) was dissolved in PBS solution (2 mL) with pH 7.4 to obtain solution 1, and hyaluronic acid (50 mg) was dissolved in deionized water (2 mL) to obtain solution 2. Solution 1 (50 μL) and solution 2 (100 μL) were mixed thoroughly to obtain a polymer solution, which was gelled at room temperature. The gelling time was 10 seconds, which was observed by the inverted method.

[0047] Example 5

[0048] Polyhydroxypyrimidine prepared in Example 1 (100 mg) was dissolved in PBS solution (2 mL) with pH 7.4 to obtain solution 1, and hyaluronic acid (50 mg) was dissolved in deionized water (2 mL) to obtain solution 2. Solution 1 (200 μL) and solution 2 (200 μL) were mixed thoroughly to obtain a polymer solution, which was gelled at room temperature. The gelling time was 10 seconds, which was observed by the inverted method.

[0049] The prepared hydrogel was tested for performance, and the results were as follows:

[0050] After the gel was formed, it was transferred to a rotational rheometer to measure the changes in storage modulus and loss modulus over time. The results are shown in Table 1. Figure 2 The elastic modulus was always higher than the loss modulus, indicating that a three-dimensional network had been formed and the structure was stable.

[0051] After the gel was formed, it was transferred to a rotational rheometer to measure the changes in storage modulus and loss modulus over time under 1% and 1000% strain alternation. The results are shown in Table 2. Figure 3At 1% strain, the storage modulus of the gel material is always higher than the loss modulus, which proves that the high elasticity of the gel is maintained by the polymer network. At 1000% strain, the loss modulus is greater than the storage modulus, which proves that the gel has been transformed from high elasticity to viscous flow state, and the cross-linked network has been destroyed. The high elastic deformation can still be maintained after multiple strain cycles, which proves the ability of the gel to encapsulate pustules after subcutaneous injection.

[0052] The obtained gel material was frozen in liquid nitrogen and subjected to shear force brittle fracture, followed by freeze-drying. The brittle fracture surface was sputtered with gold, and the microstructure was observed by scanning electron microscopy. The results are shown in Figure 4 The gel exhibits regular three-dimensional mesh structures, and the structures forming the mesh exhibit a silk screen shape.

[0053] Different masses of the gel were co-incubated with L929 cells for 24 hours, as shown in Figure 5 The cell viability was tested by CCK-8 method, and it can be seen that the gel does not produce obvious toxicity to the cells.

[0054] The gel material was placed in a sterile 24-well plate, and after the surface was flat, Staphylococcus aureus liquid was added to the surface. After 2 hours of incubation, the surface bacteria were collected and spread on LB solid medium, and the growth of the colonies was observed after overnight incubation, as shown in Figure 6 The hydrogel needle completely killed Staphylococcus aureus after 2 hours of contact with the bacteria.

[0055] The hydrogel prepared in this example was applied to a mouse Staphylococcus aureus subcutaneous infection model by subcutaneous injection, as shown in Figure 7 The antibacterial hydrogel can block the invasion of Staphylococcus aureus infection to the skin.

[0056] Example 6

[0057] 100 mg of polyhydrogenated pyrimidine prepared by Example 1 was dissolved in a PBS solution (2 mL) with a pH of 7.4 to obtain solution 1. Hyaluronic acid (50 mg) was dissolved in deionized water (2 mL) to obtain solution 2. Solution 1 (200 μL) and solution 2 (50 μL) were mixed uniformly to obtain a polymer solution, which was gelled at room temperature. The gelation time was 10 seconds, which was observed by the inverted method.

[0058] Example 7

[0059] The polyhydroxypyrimidine prepared in Example 1 (100 mg) was dissolved in a PBS solution (2 mL) with pH 7.4 to obtain solution 1, and hyaluronic acid (50 mg) was dissolved in deionized water (2 mL) to obtain solution 2. Solution 1 (150 μL) and solution 2 (50 μL) were mixed thoroughly to obtain a polymer solution, which was gelled at room temperature. The gelling time was 10 seconds, which was observed by the inverted method.

[0060] Example 8

[0061] The polyhydroxypyrimidine prepared in Example 1 (100 mg) was dissolved in a PBS solution (2 mL) with pH 7.4 to obtain solution 1, and hyaluronic acid (50 mg) was dissolved in deionized water (2 mL) to obtain solution 2. Solution 1 (100 μL) and solution 2 (50 μL) were mixed thoroughly to obtain a polymer solution, which was gelled at room temperature. The gelling time was 10 seconds, which was observed by the inverted method.

[0062] The preferred embodiments of the present application are described in detail above with reference to the drawings, but the present application is not limited to the specific details of the above-described embodiments. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

[0063] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present application will not describe various possible combinations again.

[0064] Furthermore, various different embodiments of the present application can be combined in any manner, as long as they do not deviate from the technical concept of the present application, and they should be considered as disclosed content of the present application.

Claims

1. An injectable antibacterial hydrogel, characterized in that: The structure described in Formula I is used as the antibacterial component and gel skeleton, and is mixed with aldehyde-functionalized hyaluronic acid in a weakly alkaline aqueous medium; Wherein, m is a repeating unit of polyetheramine D-230 monomer, n is a positive integer, 10≤n≤60; The aldehyde-functionalized hyaluronic acid has a structure shown in Formula II: Wherein, n is the degree of polymerization, 50≤n≤1000, m is the structural unit modified by oxidation in n, and the proportion of m in n is measured as k, 20%≤k≤60%.

2. The method for preparing an injectable antibacterial hydrogel according to claim 1, wherein: The specific steps include: First, the hydrogenated pyrimidine antibacterial polymer is dissolved in a weakly alkaline aqueous medium, and then an aldehyde-functionalized hyaluronic acid solution is added to obtain a hydrogel material; The hydrogenated pyrimidine antibacterial polymer has a structure shown in Formula I, and the aldehyde-functionalized hyaluronic acid solution has a structure shown in Formula II.

3. The method for preparing an injectable antibacterial hydrogel according to claim 2, wherein: The mass ratio of the hydrogenated pyrimidine antibacterial polymer to the aldehyde-functionalized hyaluronic acid is 1:0.5-5; the molar ratio of the amino groups in the hydrogenated pyrimidine antibacterial polymer to the aldehyde groups in the aldehyde-functionalized hyaluronic acid is 1:0.5-5.

4. The method for preparing an injectable antibacterial hydrogel according to claim 2, wherein: in, The pH of the weakly alkaline water is 7.4-8.5; the mass-volume concentration of the aldehyde-functionalized hyaluronic acid is 5%-25%; and the mixing temperature is 4-37°C.

5. The method for preparing an injectable antibacterial hydrogel according to claim 2, wherein: The aldehyde-functionalized hyaluronic acid is obtained by oxidation reaction of natural polysaccharide hyaluronic acid with periodate under light-proof and room temperature conditions to obtain a polymer represented by formula II; wherein the periodate is a sodium salt or a potassium salt.

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