Injectable biodegradable hydrogel with in-situ mechanical self-reinforcement characteristics and preparation method thereof

Abstract

The invention discloses an injectable biodegradable hydrogel with in-situ mechanical self-reinforcement characteristics and a preparation method thereof. The invention adopts KH560 to modify a hydroxyl-containing natural substance into a hydroxyl-containing natural substance modified with an epoxy group, uses a solvent to dissolve a cross-linking agent containing succinimide groups at both ends and the hydroxyl-containing natural substance modified with an epoxy group, which are recorded as component A, and uses a solvent to dissolve an amino-containing natural macromolecule, which is recorded as component B. Component A and component B are respectively filled into a double-barreled syringe, and mixed and extruded to obtain the injectable hydrogel. The hydrogel of the invention has the properties of rapid gelation after in-situ injection, self-reinforcement, degradability, self-healing, good biocompatibility, antibacterial, hemostasis and the like, and is suitable for the fields of surgical bleeding blocking, in vivo and in vitro inflammation treatment, in vivo and in vitro tissue damage repair, and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials and hydrogels, and relates to an injectable biodegradable hydrogel with in-situ mechanical self-reinforcement characteristics and a preparation method thereof. Background Art

[0002] Daily accidents, diseases and other factors affect human safety and health, and will inevitably cause varying degrees of damage to human tissues. Although human tissues can self-repair to a certain extent, their repair capacity is very limited, and using some injectable materials to promote tissue healing is the current frontier research field. Injectable silicone liquid materials are injectable materials that were promoted and applied in the United States in the early days, but silicone materials have poor biocompatibility. After being implanted in the human body for a period of time, there is a high probability that an immune response will occur, causing derivative safety hazards to the patient's health.

[0003] The development of a material that can adapt to the human body, promote wound repair, and can be completely degraded to avoid the pain of secondary surgery is of great significance for human implant tissue engineering research. Injectable hydrogels have received widespread attention due to their similar properties to biological tissues and good biocompatibility. They have strong designability, and through molecular structure design, multifunctional hydrogels with antibacterial, anti-inflammatory and hemostatic functions can be prepared. However, the mechanical properties of existing injectable hydrogels are usually too hard or too weak. If the mechanical strength of the gel is too weak, it will be difficult to support the wound, and if the gel is too strong, it will cause discomfort to the patient. Therefore, how to make the gel quickly gel but weak during implantation to ensure patient comfort; then gradually self-enhance the mechanical properties over a period of time to adapt to the surrounding biological environment and realize the support function is very critical.

[0004] Before this invention, we designed a hydrogel dressing that can quickly gel and slowly enhance after injection (Yang Jintao, Yuan Jingfeng, Zheng Siyu, Zhang Dong, a method for preparing a hydrogel dressing that can quickly gel and slowly enhance after injection, CN202110636898.1). The invention uses the rapid gelation reaction of Schiff bases between amino groups and aldehyde groups and the slow gelation reaction of amino groups and epoxy groups to construct an in-situ self-reinforced gel network, but the Schiff base gel system is lacking in biocompatibility, and most of the current Schiff base compounds have the disadvantages of poor hydrophobicity and stability. In view of this, the present invention integrates the advantages of amino and succinimide cross-linked gel systems, such as better biocompatibility and a wider range of applications, and proposes a more innovative method for preparing an in-situ enhanced injectable gel network. Summary of the invention

[0005] The purpose of the present invention is to provide an injectable biodegradable hydrogel with in-situ mechanical self-reinforcement characteristics and a preparation method thereof.

[0006] The technical solution of the present invention is as follows:

[0007] An injectable biodegradable hydrogel with in-situ mechanical self-reinforcement characteristics, mainly made of the following raw materials: a cross-linking agent containing succinimide groups at both ends, KH560 (γ-glycidyloxypropyltrimethoxysilane), a natural substance containing hydroxyl groups, and a natural macromolecule containing amino groups;

[0008] Among them, the cross-linking agent containing succinimide groups at both ends includes but is not limited to: one or more of double-terminal succinimide ester polyethylene glycol (NHS-PEG-NHS), succinimide-disulfide bond active ester-succinimide (NHS-SS-NHS), and N,N'-disuccinimidyl carbonate;

[0009] The natural substances containing hydroxyl groups include, but are not limited to: one or more of hydroxyapatite (HAP), montmorillonite (MMT), and nanocrystalline cellulose (CNC);

[0010] The amino-containing natural macromolecules include, but are not limited to, one or more of chitosan (CS), chitosan oligosaccharide (COS), polylysine (EPL), and gelatin.

[0011] A method for preparing an injectable biodegradable hydrogel with in-situ mechanical self-reinforcement characteristics comprises the following steps:

[0012] (1) adding a hydroxyl-containing natural substance to a solvent for dissolution to obtain a hydroxyl-containing natural substance solution; adding KH560 to a solvent for dissolution, and adjusting the pH to 1 to 5 to obtain a KH560 solution; adding the KH560 solution to the hydroxyl-containing natural substance solution, stirring and reacting at 25 to 40° C. for 6 to 12 hours, and then post-treating to obtain a hydroxyl-containing natural substance modified with an epoxy group;

[0013] The preferred mass ratio of the hydroxyl-containing natural substance to KH560 is 3-8:2-9;

[0014] The solvent for dissolving the hydroxyl-containing natural substance includes, but is not limited to, one or more of water, ethanol, a mixed solvent of ethanol and water, acetic acid, isopropanol, and acetone; the volume mass ratio of the solvent to the hydroxyl-containing natural substance is 30-100 (mL): 3-8 (g); preferably, ultrasonic dispersion is performed while dissolving the hydroxyl-containing natural substance, and the ultrasonic time is 10-100 minutes;

[0015] The solvent for dissolving KH560 includes but is not limited to: one or more of water, ethanol, a mixed solvent of ethanol and water, acetic acid, isopropanol, and acetone; the volume mass ratio of the solvent to KH560 is 20-100 (mL): 2-9 (g); glacial acetic acid is used to adjust the pH after KH560 is dissolved;

[0016] Preferably, the KH560 solution is added to the hydroxyl-containing natural substance solution and then ultrasonically dispersed, and the ultrasonic time is 10 to 100 minutes;

[0017] The specific post-treatment method is as follows: after the reaction is completed, the mixed system is centrifuged, washed, and dried to obtain the natural substance containing hydroxyl groups modified with epoxy groups; wherein the centrifugal speed is set to 3000-9000 rpm, and the centrifugal time is set to 5-10 minutes; the number of washing times is 3-6 times, and the washing liquid includes but is not limited to: one or more of ethanol, water, a mixed solvent of ethanol and water, acetic acid, isopropanol, and acetone; the drying temperature is 60-90° C., and the drying time is 6-24 hours;

[0018] (2) adding a cross-linking agent having succinimide groups at both ends and the natural substance containing hydroxyl groups modified with epoxy groups obtained in step (1) into a solvent and mixing them evenly, which is recorded as component A; dissolving the natural macromolecule containing amino groups in a solvent, which is recorded as component B; mixing components A and B and extruding them to obtain the injectable biodegradable hydrogel with in-situ mechanical self-reinforcement characteristics;

[0019] Preferably, the feed ratio of the cross-linking agent containing succinimide groups at both ends, the natural substance containing hydroxyl groups modified with epoxy groups and the natural macromolecule containing amino groups is 100-800 (μL): 10-20 (mg): 30-120 (mg);

[0020] Preferably, the total concentration of the cross-linking agent containing succinimide groups at both ends and the natural substance containing hydroxyl groups modified with epoxy groups in component A is 6 to 20 wt %; the solvent in component A includes but is not limited to: one or more of water, PBS solution, and ethanol;

[0021] Preferably, the concentration of the amino-containing natural macromolecule in component B is 6 to 20 wt %; the solvent in component B includes, but is not limited to, one or more of water, PBS solution, and ethanol;

[0022] Preferably, component A and component B are respectively filled into a double-barreled syringe and mixed and extruded, and the injection temperature is 25-40°C.

[0023] The beneficial effects of the present invention are:

[0024] 1. Injectable gel can quickly gel within a few seconds after injection.

[0025] 2. All raw materials are natural materials with good biocompatibility and excellent biodegradability.

[0026] 3. The prepared injectable gel has the properties of antibacterial, hemostatic, self-healing, and promoting tissue repair.

[0027] 4. The prepared injectable gel has a dual gelling mechanism. Specifically, the gel is extruded by a double-barreled syringe, one barrel contains amino-containing natural macromolecules, and the other barrel contains a cross-linking agent containing succinimide groups at both ends and a hydroxyl-containing natural substance modified with epoxy groups. After mixed and extruded by a double-barreled mixing syringe, the amino groups in the amino-containing natural macromolecules will react quickly with the succinimide groups in the cross-linking agent containing succinimide groups at both ends to form a gel network, which plays a role in quickly supporting the tissue defect cavity in a short time. At the same time, the epoxy groups in the hydroxyl-containing natural substance modified with epoxy groups will react with the remaining amino groups in the system, slowly strengthening the gel network, and achieving the effect of in-situ self-enhancement of the gel. After the weak network is quickly cross-linked, the strong network is slowly cross-linked to give the damaged part of the human tissue time to adapt to the modulus of the implant.

[0028] 5. The prepared injectable gel can be widely used in surgical bleeding blockage, in vivo and in vitro inflammation treatment, and in vivo and in vitro tissue damage repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Self-reinforcing properties of the injectable gel in Example 1.

[0030] Figure 2 Biocompatibility of the injectable gel in Example 1.

[0031] Figure 3 Antibacterial properties of the injectable gel in Example 1.

[0032] Figure 4 Hemostatic properties of the injectable gel of Example 1. DETAILED DESCRIPTION

[0033] The present invention is further described below by means of specific embodiments, but the protection scope of the present invention is not limited thereto.

[0034] Example 1 Preparation of gel

[0035] 5g of HAP was dissolved by ultrasonication in 60ml of ethanol for 30min. At the same time, 2g of KH560 was fully dissolved in 100mL of a mixed solution of water and ethanol (water: ethanol = 1:3, volume ratio). The pH of the KH560 solution was adjusted to 4 with glacial acetic acid, and then the HAP suspension was added and ultrasonicated again for 30min. The suspension was stirred at 25℃ for 8h. The product was centrifuged (9000rpm, 7min), washed with ethanol three times and centrifuged again. The treated product was dried in a vacuum oven at 80℃ for later use.

[0036] 400 μL of NHS-PEG-NHS and 20 mg of hydroxyapatite (mHAP) modified with epoxy groups were dissolved in 1 ml of 0.99% PBS solution (sodium chloride: disodium hydrogen phosphate: sodium dihydrogen phosphate = 85:13.22:0.6, mass ratio), which was recorded as component A; 100 mg of CS was dissolved in 1 ml of PBS solution, which was recorded as component B. Components A and B were filled into double-barreled syringes respectively, injected into the site of action, and quickly formed gel.

[0037] Example 2 Preparation of gel

[0038] Use 60ml ethanol to ultrasonically dissolve 6g of CNC for 30min. At the same time, use 100mL of a mixed solution of water and ethanol (water: ethanol = 2:3) to fully dissolve 2.5g of KH560. Use glacial acetic acid to adjust the pH of the KH560 solution to 5, then add the CNC suspension and ultrasonicate again for 30min. Stir the suspension at 25℃ for 8h. Centrifuge the product (9000rpm, 7min), wash the product with ethanol three times and centrifuge it again. The treated product is dried in a vacuum oven at 80℃ for use.

[0039] 300 μL of NHS-SS-NHS and 20 mg of CNC modified with epoxy groups (mCNC) were dissolved in 1 ml of 0.99% PBS solution (sodium chloride: disodium hydrogen phosphate: sodium dihydrogen phosphate = 85:13.22:0.6, mass ratio), which was recorded as component A; 100 mg of COS was dissolved in 1 ml of PBS solution, which was recorded as component B. Components A and B were filled into double-barreled syringes respectively, injected into the action site, and quickly formed gel.

[0040] Example 3 Preparation of gel

[0041] 5 g of HAP was dissolved by ultrasonication in 60 ml of ethanol for 40 min. Meanwhile, 2 g of KH560 was fully dissolved in 100 ml of ethanol solution. The pH of KH560 solution was adjusted to 3.5 with glacial acetic acid, and then HAP suspension was added and ultrasonicated again for 30 min. The suspension was stirred at 25 °C for 8 h. The product was centrifuged (9000 rpm, 7 min), washed with ethanol three times and centrifuged again. The treated product was dried in a vacuum oven at 80 °C for later use.

[0042] Dissolve 450 μL of NHS-PEG-NHS and 20 mg of mHAP in 1 ml of 0.99% PBS solution (sodium chloride: disodium hydrogen phosphate: sodium dihydrogen phosphate = 85:13.22:0.6, mass ratio), which is recorded as component A; dissolve 100 mg of gelatin in 1 ml of PBS solution, which is recorded as component B. Fill components A and B into double-barreled syringes respectively, inject them into the site of action, and quickly form gel.

[0043] Example 4 Preparation of gel

[0044] 5g of HAP was dissolved by ultrasonication in 80ml of ethanol for 30min. At the same time, 2g of KH560 was fully dissolved in 100ml of a mixed solution of water and ethanol (water: ethanol = 1:1). The pH of the KH560 solution was adjusted to 4 with glacial acetic acid, and then the HAP suspension was added and ultrasonicated again for 30min. The suspension was stirred at 25℃ for 8h. The product was centrifuged, washed with ethanol three times and centrifuged again. The treated product was dried in a vacuum oven at 80℃ for later use.

[0045] Dissolve 450 μL of NHS-SS-NHS and 20 mg of mHAP in 1 ml of deionized water, which is called component A; dissolve 100 mg of EPL in 1 ml of deionized water, which is called component B. Fill components A and B into double-barreled syringes respectively, inject them into the site of action, and quickly form gel.

[0046] Example 5 Self-reinforcement performance test of injectable gel

[0047] The hydrogel in Example 1 was subjected to compression testing using a high and low temperature dual column tester. Due to the dual gel crosslinking mechanism of rapid amino group-succinimide reaction and slow epoxy group-amino group reaction, the modulus of the hydrogel in Example 1 continued to rise within 168 hours after injection ( Figure 1 ).

[0048] Example 6 Biocompatibility testing of injectable gel

[0049] The hydrogel in Example 1 was completely dissolved in PBS solution. The hydrogel sample was then co-cultured with RPMI 1640 culture medium containing 10% fetal bovine serum and 1% penicillin at 37°C. The extracts on the 1st, 2nd and 3rd days were diluted with cell culture medium to 50 and 100 mg / mL, respectively. At the same time, calvarial osteoblasts (COB) were added to the well plate and cultured in a cell culture incubator for 24 hours. The cell suspension was replaced with hydrogel extracts of different concentrations and continued to be cultured in a cell culture incubator for 24 hours. Then, the culture medium was removed again and replaced with MTT solution. Subsequently, the absorbance of the solution was detected. The cell viability of the experimental group was obtained by comparing with the normal proliferating cell density of the control group. Similarly, COB was added to the well plate and cultured with the hydrogel extract. Use paraformaldehyde solution, discard the culture medium, and fix at room temperature for 20 minutes. After rinsing with PBS several times, live / dead staining was performed on each well. After culturing for a period of time at 37°C, it was observed using a fluorescence microscope. It can be seen that ( Figure 2) On the first day, the density of COB cells was ~107.5% (50 mg / mL) and ~112.4% (100 mg / mL), respectively, and on the third day it reached nearly 124.5% (50 mg / mL) and 125.5% (100 mg / mL), reflecting that the hydrogel in Example 1 has excellent biocompatibility and the ability to promote cell proliferation.

[0050] Example 7 Antibacterial Performance Test of Injectable Gel

[0051] Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) were used to detect the bactericidal properties of the hydrogel in Example 1. First, Escherichia coli and Staphylococcus aureus were cultured on agar medium at 37°C overnight. Then a single colony LB liquid culture medium was inoculated and cultured overnight with shaking. The bacterial solution was diluted to a standard concentration using LB liquid culture medium. The pre-sterilized hydrogel was placed in a well plate containing a standard bacterial solution and co-cultured in a constant temperature gas bath. The LIVE / DEAD backlight survival kit was used to stain and observe 24h (Escherichia coli) and 12h (Staphylococcus aureus). The morphology of live / dead bacteria adhering to the surface of the hydrogel was observed using a fluorescence microscope. The results are as follows. Figure 3 As shown, after a certain period of incubation, red dead bacteria can be observed in both groups. Since CS itself has antibacterial properties, the hydrogel in Example 1 has a certain degree of bactericidal ability against Escherichia coli and Staphylococcus aureus.

[0052] Example 8 Hemostatic Performance Test of Injectable Gel

[0053] The in vivo hemostatic ability of the hydrogel described in Example 1 was tested by establishing a SD rat liver bleeding model. The abdomen of the rat was cut open to expose the liver. Two identical wounds on the rat liver were cut with a scalpel, and the hydrogel was immediately injected into one of the bleeding sites. The control group was not treated. Filter paper was used to absorb the blood flowing out of the wound for a certain period of time. The change in the weight of the filter paper was recorded as the amount of bleeding from the wound. Figure 4 It can be seen that the wound injected with the hydrogel described in Example 1 has a smaller amount of bleeding, reflecting its good hemostatic performance.

Claims

1. An injectable biodegradable hydrogel with in situ mechanical self-reinforcement characteristics, characterized in that: It is mainly made of the following raw materials: a cross-linking agent containing succinimide groups at both ends, KH560, natural substances containing hydroxyl groups, and natural macromolecules containing amino groups; Wherein, the cross-linking agent containing succinimide groups at both ends is selected from one or more of: double-terminal succinimide ester polyethylene glycol, succinimide-disulfide bond active ester-succinimide, and N,N'-disuccinimide carbonate; The hydroxyl-containing natural substance is selected from: one or more of hydroxyapatite, montmorillonite, and nanocrystalline cellulose; The amino-containing natural macromolecule is selected from one or more of chitosan, chitosan oligosaccharide, polylysine and gelatin; The method for preparing the injectable biodegradable hydrogel with in-situ mechanical self-reinforcement characteristics comprises the following steps: (1) adding a hydroxyl-containing natural substance to a solvent for dissolution to obtain a hydroxyl-containing natural substance solution; adding KH560 to a solvent for dissolution, and adjusting the pH to 1-5 to obtain a KH560 solution; adding the KH560 solution to the hydroxyl-containing natural substance solution, stirring and reacting at 25-40° C. for 6-12 hours, and then post-treating to obtain a hydroxyl-containing natural substance modified with an epoxy group; (2) adding a cross-linking agent having succinimide groups at both ends and the natural substance containing hydroxyl groups modified with epoxy groups obtained in step (1) into a solvent and mixing them evenly, which is recorded as component A; dissolving the natural macromolecule containing amino groups in a solvent, which is recorded as component B; mixing components A and B and extruding them, thereby obtaining the injectable biodegradable hydrogel having in-situ mechanical self-reinforcement characteristics; The feeding ratio of the cross-linking agent containing succinimide groups at both ends, the natural substance containing hydroxyl groups modified with epoxy groups and the natural macromolecule containing amino groups is 100~800 (μL): 10~20 (mg): 30~120 (mg).

2. The injectable biodegradable hydrogel with in situ mechanical self-reinforcement characteristics according to claim 1, characterized in that: In step (1) of the preparation method, the mass ratio of the hydroxyl-containing natural substance to KH560 is 3-8:2-9.

3. The injectable biodegradable hydrogel with in situ mechanical self-reinforcement characteristics according to claim 1, characterized in that: In step (1) of the preparation method, the solvent for dissolving the hydroxyl-containing natural substance is selected from: one or more of water, ethanol, a mixed solvent of ethanol and water, acetic acid, isopropanol, and acetone; the volume mass ratio of the solvent to the hydroxyl-containing natural substance is 30~100 (mL): 3~8 (g).

4. The injectable biodegradable hydrogel with in situ mechanical self-reinforcement characteristics according to claim 1, characterized in that: In step (1) of the preparation method, the solvent for dissolving KH560 is selected from one or more of water, ethanol, a mixed solvent of ethanol and water, acetic acid, isopropanol, and acetone; the volume mass ratio of the solvent to KH560 is 20-100 (mL): 2-9 (g).

5. The injectable biodegradable hydrogel with in situ mechanical self-reinforcement characteristics according to claim 1, characterized in that: In step (1) of the preparation method, the post-treatment method is: after the reaction is completed, the mixed system is centrifuged, washed, and dried to obtain a hydroxyl-containing natural substance modified with an epoxy group; wherein the centrifugal speed is set to 3000~9000 rpm, and the centrifugal time is set to 5~10 min; the number of washing times is 3~6 times, and the washing liquid is selected from: one or more of ethanol, water, a mixed solvent of ethanol and water, acetic acid, isopropanol, and acetone; the drying temperature is 60~90°C, and the drying time is 6~24 h.

6. The injectable biodegradable hydrogel with in situ mechanical self-reinforcement characteristics according to claim 1, characterized in that: In step (2) of the preparation method, the total concentration of the cross-linking agent containing succinimide groups at both ends and the natural substance containing hydroxyl groups modified with epoxy groups in component A is 6-20wt%; the solvent in component A is selected from one or more of water, PBS solution, and ethanol.

7. The injectable biodegradable hydrogel with in situ mechanical self-reinforcement characteristics according to claim 1, characterized in that: In step (2) of the preparation method, the concentration of the amino-containing natural macromolecule in component B is 6-20wt%; the solvent in component B is selected from: one or more of water, PBS solution, and ethanol.

8. Use of the injectable biodegradable hydrogel with in situ mechanical self-reinforcement characteristics as claimed in claim 1 in the preparation of surgical bleeding blocking materials, in vivo and in vitro inflammation treatment materials, and in vivo and in vitro tissue damage repair materials.

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