CN110448721B

CN110448721B - Antibacterial adhesive conductive hemostatic and antioxidant injectable composite hydrogel and preparation method and application thereof

Info

Publication number: CN110448721B
Application number: CN201910652099.6A
Authority: CN
Inventors: 郭保林; 梁永平; 史梦婷; 赵鑫
Original assignee: Xian Jiaotong University
Current assignee: Xian Jiaotong University
Priority date: 2019-07-18
Filing date: 2019-07-18
Publication date: 2020-10-27
Anticipated expiration: 2039-07-18
Also published as: CN110448721A

Abstract

The invention relates to an injectable composite hydrogel with antibacterial adhesion, conductivity, hemostasis and antioxidation, and a preparation method and application thereof, wherein dopamine is grafted to a gelatin main chain to generate gelatin grafted dopamine GT-DA; preparing a polydopamine coating on the surface of the CNT by utilizing self-polymerization of dopamine under an alkaline condition to obtain the CNT-PDA; preparing GT-DA and CS into GT-DA/CS solution, and preparing CNT-PDA into CNT-PDA dispersion solution; under the action of an initiator, dopamine in a GT-DA/CS solution and a CNT-PDA dispersion liquid is subjected to self-polymerization to obtain the injectable composite hydrogel with the functions of antibiosis, adhesion, electric conduction, hemostasis and antioxidation. The hydrogel prepared by grafting dopamine to the gelatin main chain has good oxidation resistance, and also has the characteristics of good biocompatibility, adhesion, hemostasis, photo-thermal antibiosis, drug slow release and the like.

Description

Antibacterial adhesive conductive hemostatic and antioxidant injectable composite hydrogel and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to an injectable composite hydrogel with antibacterial adhesion, conductivity, hemostasis and oxidation resistance, and a preparation method and application thereof.

Background

Regeneration of damaged tissue is the most fundamental requirement to reestablish tissue integrity and restore normal function to the tissue. It is well known that infection is one of the major obstacles to wound healing and has become an ever-increasing factor in the cause of death in critically ill patients. Treatment of infections places a significant burden on the medical system and even the entire society, and currently, the main strategy for clinically dealing with infections is still the use of antibiotics. Therefore, it remains challenging to develop antimicrobial drug delivery systems that not only can effectively and accurately deliver antibiotics to a wound site, but can also regulate the release of active factors for wound healing. In order to find wound dressings that not only avoid wound infection, but also have an enhancing effect on the wound repair process, many modern wound dressings have been developed that have sustained drug release properties, such as semipermeable membranes, thin permeable foams, hydrocolloids, hydrogels, and the like. Among them, thanks to excellent hydrophilicity, hydrogels can achieve the effect of reducing the risk of infection by absorbing wound exudate and keeping the wound environment moist, exhibiting good potential for improving the repair of damaged tissues.

Gelatin (GT), a protein derivative, has been used in the manufacture of wound dressings in combination with a large number of synthetic or natural macromolecules due to its non-immunogenic, cell adhesion behaviour and clotting properties. However, gelatin-based hydrogels also suffer from certain drawbacks. For example: weak mechanical strength, fast degradation speed and the like.

Excess Reactive Oxygen Species (ROS) during wound repair often affect the repair by altering or degrading ECM proteins, destroying dermal fibroblasts and reducing keratinocyte function.

Researchers have found that the human body has endogenous bioelectrical systems. The intact human skin surface carries more negative charges than the deeper skin. However, when a defect or wound occurs in the skin, cells deeper in the epidermis and cells at the wound site are positively charged. The positive charge at the wound site and the negative charge of the surrounding intact skin combine to create a skin cell. This bioelectric current facilitates wound healing when the wound tissue is wetted. The promoting effect of electrically conductive materials in wound healing has also been demonstrated in previous studies.

Carbon Nanotubes (CNTs) have led to a hot tide of research in nanotechnology in the past few decades due to their excellent mechanical, thermal and electronic properties. However, the strong hydrophobic interaction between individual CNTs makes it difficult for unmodified CNTs to disperse in water. Thereby limiting its application in the synthesis of conductive bioscaffolds.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide an injectable composite hydrogel with antibacterial adhesion, conductivity, hemostasis and oxidation resistance, and a preparation method and application thereof; the method has the advantages of wide raw material source, simple preparation process and low preparation cost; the hydrogel dressing prepared by the method has good biocompatibility, adhesiveness, hemostatic property, mechanical property, conductivity, oxidation resistance, antibacterial property and effect of promoting infected wound repair, and can be applied to the field of skin injury treatment.

The preparation method is realized by the following technical scheme:

the method comprises the following steps:

(1) grafting dopamine to a gelatin main chain to generate gelatin grafted dopamine GT-DA;

(2) preparing a polydopamine coating on the surface of the CNT by utilizing self-polymerization of dopamine under an alkaline condition to obtain the CNT-PDA;

(3) preparing GT-DA and CS into GT-DA/CS solution, and preparing CNT-PDA into CNT-PDA dispersion solution; under the action of an initiator, dopamine in a GT-DA/CS solution and a CNT-PDA dispersion liquid is subjected to self-polymerization to obtain the injectable composite hydrogel with the functions of antibiosis, adhesion, electric conduction, hemostasis and antioxidation.

Further, in the step (1), the preparation of gelatin grafted dopamine comprises the following steps:

(1A) dissolving gelatin in a buffer solution, and stirring at 50-60 ℃ to fully dissolve the gelatin to obtain a gelatin solution, wherein the mass concentration of the gelatin is 3-6%;

(1B) adding dopamine hydrochloride into the gelatin solution obtained in the step (1A), stirring at room temperature to dissolve the dopamine hydrochloride to obtain a mixed solution, wherein the concentration of the dopamine hydrochloride in the mixed solution is 44-88 mg/mL;

(1C) adding EDC and NHS into the mixed solution obtained in the step (1B) to obtain a reaction solution, and continuously stirring the reaction solution at 30-50 ℃ for reacting for 6-18 hours, wherein the concentration of EDC and the concentration of NHS in the reaction solution are 44-89 mg/mL and 27-54 mg/mL respectively;

(1D) and (3) after the reaction in the step (1C) is finished, dialyzing the obtained solution, and freeze-drying to obtain the gelatin grafted dopamine.

Further, in the step (1A), the buffer solution is phosphate buffer solution or 2-morpholinoethanesulfonic acid buffer solution, and the pH value of the buffer solution is 5-6; in the step (1D), deionized water is adopted for dialysis for 48-72 hours; and (3) regulating the pH values of the reaction liquid in the step (1C) and the deionized water in the step (1D) to be 5-6 all the time by using hydrochloric acid and an aqueous solution of sodium hydroxide.

Further, in the step (2), the specific preparation steps of the CNT-PDA include:

(2A) dispersing CNT in a buffer solution to obtain a CNT dispersion solution with the concentration of 0.5-2 mg/mL;

(2B) adding dopamine hydrochloride into the CNT dispersion liquid obtained in the step (2A) to obtain a reaction liquid, and stirring the reaction liquid at room temperature for 12-48 hours, wherein the concentration of the dopamine hydrochloride in the reaction liquid is 0.5-2 mg/mL;

(2C) and (3) carrying out post-treatment on the reaction solution after the reaction in the step (2B) to obtain the CNT-PDA.

Further, dispersing the CNT in the step (2A) in a Tris buffer solution, and carrying out ultrasonic treatment at the temperature of 0-4 ℃ for 0.5-2 h, wherein the pH value of the Tris buffer solution is 8-9; and (2C) centrifuging at 5000-10000 rpm for 5-20 minutes, discarding the supernatant, washing with distilled water and ethanol for three times respectively, and drying at 30-60 ℃ for 24-72 hours.

Further, in the step (3), the specific preparation steps for preparing the injectable composite hydrogel with antibacterial adhesion, conductivity, hemostasis and oxidation resistance comprise:

(3A) dissolving GT-DA/CS in a buffer solution to prepare a GT-DA/CS solution with the mass concentration of 5-20%;

(3B) dispersing CNT-PDA in a buffer solution to prepare CNT-PDA dispersion liquid with the mass concentration of 1-4%;

(3C) uniformly mixing 1mL of CNT-PDA dispersion liquid and 7.5mL of LGT-DA/CS solution, adding 0.25-0.65 mol of initiator, and reacting to obtain the antibacterial adhesive conductive hemostatic antioxidant injectable composite hydrogel; and (3C) reacting for 10-200 s.

Further, in the step (3A) and the step (3B), the buffer solution is PBS; the mass ratio of CS in GT-DA/CS is 5-10%.

Further, the initiator is characterized by comprising H with the concentration of 0.1-0.3 mol/mL₂O₂And horseradish peroxidase with the concentration of 0.2-0.5 mg/mL, and 0.5mL of H is added into each 1mLCNT-PDA dispersion liquid₂O₂And 1mL of horseradish peroxidase.

The injectable composite hydrogel prepared by the preparation method has the advantages of antibacterial adhesion, conductivity, hemostasis and antioxidation.

The application of the antibacterial adhesive conductive hemostatic antioxidant injectable composite hydrogel in preparation of wound dressings.

Compared with the prior art, the invention has the following beneficial technical effects:

the preparation method disclosed by the invention is based on protein derivative Gelatin (GT), and endows the tissue adhesion performance and the oxidation resistance of the protein derivative gelatin by chemically grafting dopamine to a main chain; chitosan (CS) is added into a hydrogel system to improve the mechanical property of the hydrogel and make up for the defect of fast degradation; the introduction of CNTs into hydrogel systems imparts electrical conductivity thereto; meanwhile, the polydopamine coating is carried out on the surface of the CNT by utilizing the self-polymerization of the dopamine under the alkaline condition, so that the water solubility, the conductivity and the biocompatibility of the CNT are enhanced.

Further, the invention uses H₂O₂The HRP is used as a catalytic system, so that the biosafety problem caused by the traditional oxidation method is reduced, and the product has mechanical properties similar to or relatively better than those of the traditional chemical method.

The hydrogel prepared by the method has the following advantages:

(1) the hydrogel prepared by grafting dopamine to the gelatin main chain has good oxidation resistance.

(2) Based on the hemostatic property of chitosan, the chitosan is introduced into hydrogel, so that the hemostatic property of the hydrogel is improved, the mechanical property of the hydrogel is improved, and the defect of quick degradation of the hydrogel is overcome.

(3) The addition of catechol moieties (derived from dopamine) to GT also enhances the bioadhesive properties of GT-DA/CS based hydrogels due to the physical binding and chemical cross-linking of dopamine or polydopamine moieties to the damaged tissue. In addition, the good adhesion performance can also enable the hydrogel to rapidly seal the wound, thereby achieving a good hemostatic effect.

(4) The introduction of CNTs into the hydrogel increases the electrical conductivity of the hydrogel, thereby helping to facilitate the wound repair process.

(5) The dopamine is utilized to be self-polymerized in an alkaline solution to form a Polydopamine (PDA) coating on the surface of the CNT, so that the defects caused by the traditional oxidation modification CNT strategy are avoided. Enhancing the water solubility, conductivity and biocompatibility of the CNT.

The experimental results show that: the porosity, rheological property, mechanical property, conductivity, swelling property, degradability and the like of the hydrogel prepared by the invention can be adjusted by changing the content of the CNT-PDA in the hydrogel. Experimental results prove that the CNT-PDA endows the hydrogel with broad-spectrum photo-thermal antibacterial activity, and the dopamine endows the hydrogel with the properties of tissue adhesion, hemostasis, oxidation resistance and the like. In addition, drug release and zone of inhibition experiments of the doxycycline-encapsulated hydrogel show that the hydrogel has sustained drug release characteristics and good antibacterial performance. Hemolysis experiment and L929 cell co-culture experiment prove that the protein has good in vitro biocompatibility. Histological results: the collagen metabolism, granulation tissue thickness, epidermal regeneration, hair follicle and blood vessel regeneration and the results of TGF-beta and CD31 immunofluorescence staining confirm the good effect of the hydrogel in promoting wound healing of infected skin. Therefore, the series of multifunctional hydrogel dressings have good application prospect in the field of promoting the healing of infectious skin wounds.

Drawings

FIG. 1(a) is a graph of the swelling curve of a hydrogel; FIG. 1(b) is a graph of the degradation profile of a hydrogel; fig. 1(c) is SEM images of hydrogel before and after swelling, scale bar: 50 μm; FIG. 1(d) is a graph of the rheological properties of a hydrogel.

FIG. 2(a) is a graph of bioadhesive properties of different hydrogels; FIG. 2(b) is the hemostatic capacity of the GT-DA/CS/CNT2 hydrogel; FIG. 2(c) is the conductivity of the hydrogel; FIG. 2(d) is the DPPH clearance of GT-DA/CS/CNT2 hydrogel at various concentrations; figure 2(e) is a doxycycline release profile.

FIG. 3(a) is a graph showing a light intensity of 1.0W/cm²A plot of Δ T-NIR illumination time for the hydrogel; FIG. 3(b) is an in vitro antimicrobial activity of NIR illumination enhanced hydrogels against Staphylococcus aureus; FIG. 3(c) is the in vitro antibacterial activity against E.coli; FIG. 3(d) is the bacterial survival rate of an in vivo antibacterial activity assay for Staphylococcus aureus;

figure 4(a) is a cytocompatibility assessment, P < 0.05; FIG. 4(b) is quantitative data of the hemolysis rate.

FIG. 5(a) is Tegaderm^TMStatistical plots of wound healing for films, GT-DA/CS/CNT0, GT-DA/CS/CNT2, and GT-DA/CS/CNT2/Doxy hydrogel at 3 days, 7 days, and 14 days. P<0.05; fig. 5(b) is hydroxyproline content in neonatal skin tissue. P<0.05; FIG. 5(c) is granulation tissue thickness for different groups on day 7; FIG. 5(d) is the regenerated epidermal thickness at the wound site after 7 days; FIG. 5(e) is a statistics of revascularization after 7 and 14 days; fig. 5(f) is a relative number statistics of hair follicles after 14 days.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings.

The preparation method of the injectable composite hydrogel with the functions of antibiosis, adhesion, electric conduction, hemostasis and oxidation resistance comprises the following steps:

(1) grafting dopamine onto a gelatin backbone under the action of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to form gelatin grafted dopamine (GT-DA);

the preparation method of the gelatin grafted dopamine comprises the following steps:

(1A) dissolving gelatin in a buffer solution, and stirring for 10-30 min at 50-60 ℃ to fully dissolve the gelatin, wherein the concentration of the gelatin is 3-6 wt%, the buffer solution can be any one of Phosphate Buffer Solution (PBS) and 2-morpholinoethanesulfonic acid (MES) buffer solution, and the pH value of the buffer solution is 5-6;

(1B) adding dopamine hydrochloride into the solution obtained in the step (1A), and stirring at room temperature for 10-30 min to fully dissolve the dopamine hydrochloride, wherein the concentration of the dopamine hydrochloride is 44-88 mg/mL;

(1C) adding EDC and NHS into the solution obtained in the step (1B), and continuously stirring for 6-18 hours at 30-50 ℃, wherein the concentrations of EDC and NHS are 44-89 mg/mL and 27-54 mg/mL respectively; after addition of EDC and NHS, the reaction pH should be maintained at 5-6 using hydrochloric acid (1M) and aqueous sodium hydroxide (1M).

(1D) And (3) after the step (1C) is finished, dialyzing the obtained solution for 48-72 hours, and freeze-drying to obtain the gelatin grafted dopamine (GT-DA). The pH of the deionized water during dialysis should be kept at 5-6 using hydrochloric acid (1M) and aqueous sodium hydroxide (1M).

(2) Preparing Poly Dopamine (PDA) coated CNT (CNT-PDA) by utilizing self-polymerization of dopamine under alkaline condition;

the specific preparation steps for synthesizing the functional CNT with the PDA coating comprise:

(2A) dispersing CNT in Tris buffer solution and carrying out ultrasonic treatment at 0-4 ℃ for 0.5-2 h, wherein the concentration of the CNT is 0.5-2 mg/mL, and the pH value of the Tris buffer solution is 8-9;

(2B) adding dopamine hydrochloride into the solution obtained in the step (2A), and stirring at room temperature for 12-48 hours, wherein the concentration of the dopamine hydrochloride is 0.5-2 mg/mL;

(2C) and (3) centrifuging the solution obtained in the step (2B) at 5000-10000 rpm for 5-20 minutes, discarding the supernatant, washing the solution with distilled water and ethanol for three times respectively, and drying the solution for 24-72 hours at 30-60 ℃ to obtain the product, namely the CNT (CNT-PDA) with the PDA coating.

(3) Dissolving a certain amount of GT-DA and Chitosan (CS) in a solvent to prepare a GT-DA/CS solution, and carrying out self-polymerization on the GT-DA/CS solution and dopamine in the CNT-PDA dispersion liquid under the action of an initiator to obtain the injectable composite hydrogel dressing with antibacterial property, conductivity and oxidation resistance. The preparation method comprises the following specific steps:

(3A) dissolving GT-DA/CS in PBS to prepare a solution with a certain concentration, wherein the mass ratio of CS in GT-DA/CS is 5-10 wt%, and the concentration range of GT-DA/CS is 5-20 wt%;

(3B) dispersing CNT-PDA in PBS to prepare dispersion liquid, wherein the concentration range of the CNT-PDA is 1 wt% -4 wt%;

(3C) adding 1mL of CNT-PDA dispersion liquid into 7.5mL of GT-DA/CS solution, fully and uniformly mixing, sequentially adding 0.5mL of initiator A and 1mL of initiator B, and carrying out sol-gel conversion for 10-200 s to obtain the hydrogel; the initiator A is H₂O₂The concentration is 0.1-0.3 mol/mL; the initiator B is horseradish peroxidase (HRP), and the concentration of the initiator B is 0.2-0.5 mg/mL.

The prepared injectable composite hydrogel with the functions of antibiosis, adhesion, electric conduction, hemostasis and oxidation resistance can be applied to the repair of the injury of the infected skin tissue, such as the aspect of wound dressing.

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

Example 1

(1) Grafting dopamine onto a gelatin backbone under the action of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to form gelatin grafted dopamine (GT-DA):

gelatin (0.5g, type a from pig skin, Sigma-Aldrich) was dissolved in 10mL of phosphate buffer (PBS, pH 5.0) with stirring at 50 ℃. Dopamine hydrochloride (0.741g, Sigma-Aldrich) was then added thereto, and stirred at room temperature for 30min to be sufficiently dissolved. EDC (0.749g, Sigma-Aldrich) and NHS (0.45g, Sigma-Aldrich) were added. Stirring was continued for 12 hours at 37 ℃. And finally dialyzing the reaction solution in deionized water for 48 hours, and freeze-drying to obtain the gelatin-grafted-dopamine. After addition of EDC and NHS, and deionized water for dialysis, the pH was maintained at 5 using hydrochloric acid (1M) and aqueous sodium hydroxide (1M).

(2) Preparation of Polydopamine (PDA) coated CNTs (CNT-PDA) by auto-polymerization of dopamine under alkaline conditions:

30mg of CNT were dispersed in 30mL of Tris buffer at pH 8.5 and sonicated at 2 ℃ for 2 hours. Then, dopamine hydrochloride (30mg) was added to the above solution, and stirred at room temperature for 48 hours to allow sufficient reaction and adhesion to the CNT surface. And centrifuging the solution at 8000rpm for 10 minutes, discarding the supernatant, washing the precipitate with distilled water and ethanol for three times respectively, and drying in an oven at 40 ℃ for 12 hours to obtain the product, namely the CNT with the PDA coating (CNT-PDA).

(3) Under the action of an initiator, the injectable composite hydrogel dressing with antibacterial property, conductivity and oxidation resistance is obtained through the self-polymerization of dopamine in GT-DA/CS and CNT-PDA:

(3A) GT-DA/CS (95: 5) was dissolved in PBS to prepare a 13.3 wt% solution.

(3B) CNT-PDA was dispersed in PBS to make up a 1 wt% CNT-PDA dispersion.

(3C) 1mL of the CNT-PDA dispersion described above was added to 7.5mL of GT-DA/CS solution. After mixing completely, 0.5mL of H was added₂O₂(0.1mol/mL) and 1mL HRP (0.25mg/mL) were subjected to sol-gel conversion for 50s to prepare a hydrogel of 10 wt% GT-DA/CS and 0.1 wt% CNT-PDA, which was named GT-DA/CS/CNT 1.

Example 2

In contrast to example 1, the hydrogel obtained by replacing 1 wt% CNT-PDA in step (3) of step (3) at step (3B) with 2 wt% was also named GT-DA/CS/CNT 2.

Example 3

In contrast to example 1, the hydrogel obtained in step (3) by replacing 13.3 wt% GT-DA/CS in step (3A) by 20 wt% was also designated (GT-DA/CS)15/CNT 1.

Example 4

In contrast to example 1, the hydrogel obtained in step (3) at step (3A) was replaced by GT-DA/CS (95: 5) to GT-DA/CS (90: 10) and was also designated GT-DA 90/CS/CNT 1.

Example 5

gelatin (0.3g, type a from pig skin, Sigma-Aldrich) was dissolved in 10ml mes buffer (pH 5.25) with stirring at 55 ℃. Dopamine hydrochloride (0.44g, Sigma-Aldrich) was then added thereto, and stirred at room temperature for 20min to dissolve it sufficiently. EDC (0.44g, Sigma-Aldrich) and NHS (0.27g, Sigma-Aldrich) were added. Stirring was continued for 6 hours at 30 ℃. And finally dialyzing the reaction solution in deionized water for 60 hours, and freeze-drying to obtain the gelatin-grafted-dopamine. After addition of EDC and NHS, and deionized water for dialysis, the pH was maintained at 5.5 using hydrochloric acid (1M) and aqueous sodium hydroxide (1M).

15mg of CNT were dispersed in 30mL of Tris buffer at pH 9 and sonicated at 4 ℃ for 1 hour. Then, dopamine hydrochloride (15mg) was added to the above solution, and stirred at room temperature for 24 hours to react sufficiently and attach to the CNT surface. And centrifuging the solution at 5000rpm for 5min, discarding the supernatant, washing the precipitate with distilled water and ethanol for three times respectively, and drying in an oven at 30 ℃ for 24 h to obtain the product, namely the CNT with the PDA coating (CNT-PDA).

(3A) GT-DA/CS (95: 5) was dissolved in PBS to prepare a 13.3 wt% solution.

(3B) CNT-PDA was dispersed in PBS to make up a 1 wt% CNT-PDA dispersion.

(3C) 1mL of the CNT-PDA dispersion described above was added to 7.5mL of GT-DA/CS solution. After mixing completely, 0.5mL of H was added₂O₂(0.2mol/mL) and 1mL HRP (0.5mg/mL) were subjected to sol-gel conversion for 35 seconds to prepare a hydrogel of 10 wt% GT-DA/CS and 0.1 wt% CNT-PDA.

Example 6

gelatin (0.6g, type a from pig skin, Sigma-Aldrich) was dissolved in 10mL MES buffer (pH 6) with stirring at 60 ℃. Dopamine hydrochloride (0.88g, Sigma-Aldrich) was then added thereto, and stirred at room temperature for 10min to dissolve it sufficiently. EDC (0.89g, Sigma-Aldrich) and NHS (0.54g, Sigma-Aldrich) were added. Stirring was continued for 18 hours at 50 ℃. And finally dialyzing the reaction solution in deionized water for 72 hours, and freeze-drying to obtain the gelatin-grafted-dopamine. After addition of EDC and NHS, and deionized water for dialysis, the pH was maintained at 6 using hydrochloric acid (1M) and aqueous sodium hydroxide (1M).

60mg of CNT were dispersed in 30mL of Tris buffer at pH 8 and sonicated at 0 ℃ for 0.5 hours. Then, dopamine hydrochloride (60mg) was added to the above solution, and stirred at room temperature for 12 hours to react sufficiently and attach to the CNT surface. And centrifuging the solution at 10000rpm for 20 minutes, removing the supernatant, washing and precipitating the solution by using distilled water and ethanol for three times respectively, and drying the solution in an oven at 60 ℃ for 72 hours to obtain the product, namely the CNT (CNT-PDA) with the PDA coating.

(3A) GT-DA/CS (92: 8) was dissolved in PBS to prepare a 5 wt% solution.

(3B) CNT-PDA was dispersed in PBS to make up a 4 wt% CNT-PDA dispersion.

(3C) 1mL of the CNT-PDA dispersion described above was added to 7.5mL of GT-DA/CS solution. After mixing completely, 0.5mL of H was added₂O₂(0.3mol/mL) and 1mL HRP (0.2mg/mL) were subjected to sol-gel conversion for 40 seconds to prepare a hydrogel of 3.75 wt% GT-DA/CS and 0.4 wt% CNT-PDA.

The injectable hydrogel dressing with adhesion, hemostasis, conductivity and oxidation resistance has stable performance, the antibacterial performance of the injectable hydrogel dressing is good in-vitro and in-vivo antibacterial tests, the mechanical performance and the conductivity of the injectable hydrogel dressing are excellent in tests, blank gel and gel loaded with doxycycline have a healing promotion effect superior to that of a commercial dressing (Tegaderm) on a mouse skin wound, the hydrogel prepared by the method can remarkably promote adhesion and proliferation of fibroblast (L929) and shows good biocompatibility, and the injectable hydrogel dressing is analyzed in detail by combining the attached drawings and experimental data as follows:

in the experiment of the attached figure part, the GT-DA/CS concentration is 10 wt%, and the mass ratio of the GT-DA to the CS is 95: 5, H₂O₂Samples were designated GT-DA/CS/CNT0, GT-DA/CS/CNT1, GT-DA/CS/CNT2, and GT-DA/CS/CNT4 at once, based on the concentration of CNT-PDA in the hydrogel from 0 wt% to 1 wt%, 2 wt%, 4 wt%, as represented by a concentration of 0.2mol/mL and a concentration of HRP of 0.5 mg/mL.

FIG. 1(a) swelling behavior test results of hydrogel show that the swelling ratio of hydrogel gradually increases with increasing CNT-PDA content in hydrogel.

FIG. 1(b) degradation behavior test results of hydrogels show that the degradation rate of hydrogels gradually slows down as the CNT-PDA content in the hydrogel increases.

Figure 1(c) shows SEM images of the pre-and post-swelling lyophilized hydrogels, showing interconnected uniform pore size microstructures.

The time of the intersection of the storage modulus (G') and the loss modulus (G ") obtained by rheological testing in fig. 1(d) is considered to be the gel formation time. When the mass percentage of CNT-PDA was increased from 0 wt% to 1 wt%, 2 wt% and 4 wt%, the gel forming time was gradually decreased from 120s to 60s, 36s and 17s, indicating that higher concentration of CNT-PDA in the hydrogel network is more advantageous for improving the gelation efficiency. Meanwhile, as the content of CNT-PDA in the hydrogel increases, the final strength (storage modulus) of the hydrogel gradually increases.

Fig. 2(a) is a result of an adhesion test experiment of the hydrogel dressing prepared according to the present invention, and the adhesion property of these hydrogels to the skin was estimated through the experiment. Test results show that the hydrogel prepared by the invention has good biological adhesion performance. And increasing the concentration of CNT-PDA from 1 wt% to 2 wt% to 4 wt% improves the adhesion properties of the synthetic hydrogel.

Fig. 2(b) test results of hemostatic properties of the hydrogel dressing prepared according to the present invention, the experiment shows that the hydrogel has a significant effect of reducing blood loss compared to the control group without any measures.

FIG. 2(c) is a graph showing the results of conductivity tests on hydrogel dressings made according to the present invention, the GT-DA/CS/CNT0 hydrogel having a minimum conductivity of 2.5X 10-2S/m. When CNT-PDA was added, the conductivity of GT-DA/CS/CNT1, GT-DA/CS/CNT2 increased from 6.2X 10-2 to 6.7X 10-2 and 7.2X 10-2S/m. The addition of the CNT-PDA is shown to significantly improve the conductivity of the hydrogel.

FIG. 2(d) is a graph showing the results of radical scavenging experiments for hydrogel dressings made according to the present invention, in which 3mg/mL of copolymer scavenged nearly 86.5% of radicals, and almost all radicals were scavenged when the hydrogel concentration was increased to 5 mg/mL. The antioxidant activity of the GT-DA/CS/CNT hydrogel was demonstrated.

Fig. 2(e) is a test result of drug release of the hydrogel dressing prepared according to the present invention, at the beginning, doxycycline of four hydrogel groups was relatively rapidly released, and almost half of the drug was released within the first 20 hours. Thereafter, despite the slow release rate of doxycycline, the entire drug release process lasted for nearly 100 hours, eventually releasing nearly 80% of doxycycline from the hydrogel species. The whole experiment shows that the hydrogel has good drug slow-release performance.

FIG. 3(a) shows the results of photothermal test of hydrogel dressings prepared according to the present invention, irradiated in NIR for 10 minutes (1.0W/cm)²) Thereafter, the GT-DA/CS/CNT0 hydrogel temperature was raised by about 10 deg.C, which resulted from the oxidation of catecholThe quinone formed by the group has certain photo-thermal capability. When the concentration of CNT-PDA added to the hydrogel was increased from 1 wt% to 2 wt% and 4 wt%, Δ Ts was increased from 17.2 ℃ to 21.3 ℃ and 23.3 ℃ after 10min of light irradiation, respectively. All results demonstrate the excellent photothermal effect of the GT-AD/CS/CNT hydrogel with CNT-PDA.

FIGS. 3(b) and 3(c) show the photothermal antibacterial effect test of the hydrogel dressing prepared by the present invention on Staphylococcus aureus and Escherichia coli, and the results show that 3min irradiation can significantly reduce the survival rate of bacteria, 5min almost kills most of bacteria, and 10min can kill 100% of all bacteria. The experimental result shows that the hydrogel has good in-vitro photo-thermal antibacterial performance.

FIG. 3(d) shows that the hydrogel dressing prepared by the invention can significantly reduce the survival rate of bacteria at a wound part by 10min irradiation, and the hydrogel dressing has good in-vivo photothermal antibacterial performance.

FIG. 4(a) is the result of in vitro cell compatibility of the hydrogel dressing prepared according to the present invention, and the cell survival rate did not show significant difference between all groups when cultured for 1 day and 3 days, indicating the non-toxicity of GT-DA/CS/CNT hydrogel. After 5 days of co-culture, only the GT-DA/CS/CNT4 hydrogel showed significant differences from TCP (P <0.05), although the average cell proliferation was lower than that of TCP for the entire hydrogel group. The good cell compatibility of the hydrogel was demonstrated.

Fig. 4(b) is an in vitro hemolysis test result of the hydrogel dressing prepared according to the present invention, and when the hemolysis ratio of the positive group (0.1% triton) is set as 100%, the hemolysis ratio of all hydrogel groups is less than 4%, showing good blood compatibility.

FIG. 5(a) is a statistical result of wound closure rate in the infected skin wound repair experiment of the hydrogel dressing prepared in the present invention, after 3 days of treatment, with Tegaderm^TMThe wound closure was significantly higher for the GT-DA/CS/CNT0, GT-DA/CS/CNT2, and GT-DA/CS/Doxy hydrogels compared to the thin film group (P)<0.05). The GT-DA/CS/CNT hydrogel is shown to have better wound healing effect. In addition, the GT-DA/CS/CNT2/Doxy hydrogel also significantly reduced the wound area over GT-DA/CS/CNT0 and GT-DA/CS/CNT2 hydrogelsSmall (P)<0.05) which demonstrates a positive effect of the antibiotic on the repair of infected wounds.

Fig. 5(b) is a statistical result of hydroxyproline content in an infected skin wound repair experiment of the hydrogel dressing prepared by the present invention. Collagen deposition is an indispensable process in the healing process of skin wounds and is an important index for evaluating the healing effect. Therefore, we examined the repair effect of neogenetic skin tissue as a representative of collagen deposition. As shown in FIG. 5(b), the collagen deposition level increased greatly from day 3 to day 7, and slightly increased at day 14. In addition, with Tegaderm^TMAll hydrogel groups showed higher collagen levels on both day 3 and day 7 of the repair process compared to the membrane group. And the collagen deposition at 7 days of the GT-DA/CS/CNT2/Doxy and GT-DA/CS/CNT2 groups is obviously higher than that of the GT-DA/CS/CNT0 group (p)<0.05), showing that the hydrogel group, especially the hydrogel added with the conductive component has remarkable function of promoting the collagen metabolism.

Fig. 5(c) is a granulation tissue thickness statistic in an infected skin wound repair experiment of the hydrogel dressing prepared according to the present invention. The thickness of granulation tissue can reflect the quality of wound repair. As shown in fig. 5(c), the granulation tissue was thicker in the three hydrogel groups than in the control group (P < 0.05). In addition, the GT-DA/CS/CNT2/Doxy hydrogel granulation tissue is thicker than GT-DA/CS/CNT0 and GT-DA/CS/CNT2 hydrogel granulation tissue, which indicates that the wound healing effect is better.

Fig. 5(d) is a skin thickness statistic in infected skin wound repair experiment of the hydrogel dressing prepared in the present invention. The results show that all hydrogel groups exhibited a specific Tegaderm at day 7^TMThicker film skin (P)<0.05), especially the GT-DA/CS/CNT2 and GT-DA/CS/CNT2/Doxy groups. The GT-DA/CS/CNT2 group exhibited a thicker skin thickness than the GT-DA/CS/CNT0 group due to the addition of CNT-PDA.

Fig. 5(e) is a statistics of angiogenesis in infected skin wound repair experiments of hydrogel dressings made in accordance with the present invention. The results show an increasing trend of vascular regeneration in the defect skin wounds from day 7 to day 14 after treatment. And the GT-DA/CS/CNT2 and GT-DA/CS/CNT2/Doxy hydrogel group ratio Tegaderm^TMMembrane modules and GT-DA/CS/CNT0The group has better effect of improving the vascularization speed (P)<0.05)。

Fig. 5(f) is a statistics of hair follicle regeneration in infected skin wound repair experiments with hydrogel dressings made in accordance with the present invention. And Tegaderm^TMThe number of hair follicles in the dermal tissue of the hydrogel group was greater than that of the membrane group, showing a better repairing effect.

The invention discloses a preparation method of an injectable composite hydrogel with antibacterial adhesion, conductivity, hemostasis and oxidation resistance and application of the injectable composite hydrogel in wound dressing. Firstly, grafting dopamine on gelatin under the action of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to generate gelatin grafted dopamine (GT-DA); secondly, preparing Poly Dopamine (PDA) coated CNT (CNT-PDA) by polymerization of dopamine under alkaline condition; finally, the GT-DA/CS solution and the CNT-PDA dispersion are mixed using H₂O₂The hydrogel is obtained by polymerization of dopamine by using HRP as an initiator. The porosity, degradation, swelling, rheology, mechanical and electrical conductivity properties of the hydrogel can be fine tuned by varying the concentration of CNT-PDA. The hydrogel dressing has the characteristics of good biocompatibility, adhesion, hemostasis, oxidation resistance, photo-thermal antibiosis, drug slow release and the like.

Claims

1. A preparation method of an injectable composite hydrogel with antibacterial adhesion, conductivity, hemostasis and oxidation resistance is characterized by comprising the following steps:

in the step (1), the preparation of the gelatin grafted dopamine comprises the following steps:

(1D) after the reaction in the step (1C) is finished, dialyzing the obtained solution, and freeze-drying to obtain gelatin grafted dopamine;

the process is based on protein derivative gelatin, and endows the tissue with adhesion performance and oxidation resistance by chemically grafting dopamine to a main chain;

2. The preparation method of the injectable composite hydrogel with antibacterial adhesion, conductivity, hemostasis and antioxidation functions according to claim 1, characterized in that in the step (1A), the buffer is phosphate buffer or 2-morpholinoethanesulfonic acid buffer, and the pH value of the buffer is 5-6; in the step (1D), deionized water is adopted for dialysis for 48-72 hours; and (3) regulating the pH values of the reaction liquid in the step (1C) and the deionized water in the step (1D) to be 5-6 all the time by using hydrochloric acid and an aqueous solution of sodium hydroxide.

3. The preparation method of the injectable composite hydrogel with antibacterial, adhesive, conductive, hemostatic and antioxidant effects as claimed in claim 1, wherein in the step (2), the specific preparation steps of the CNT-PDA include:

4. The preparation method of the antibacterial adhesive conductive hemostatic antioxidant injectable composite hydrogel according to claim 3, wherein the CNT in the step (2A) is dispersed in Tris buffer solution, and is subjected to ultrasound at 0-4 ℃ for 0.5-2 h, and the pH of the Tris buffer solution is 8-9; and (2C) centrifuging at 5000-10000 rpm for 5-20 minutes, discarding the supernatant, washing with distilled water and ethanol for three times respectively, and drying at 30-60 ℃ for 24-72 hours.

5. The preparation method of the injectable composite hydrogel with antibacterial adhesion, conductivity, hemostasis and antioxidation functions according to claim 1, wherein in the step (3), the specific preparation steps for preparing the injectable composite hydrogel with antibacterial adhesion, conductivity, hemostasis and antioxidation functions comprise:

(3C) and (3) uniformly mixing 1mL of CNT-PDA dispersion liquid and 7.5mL of GT-DA/CS solution, adding 0.25-0.65 mol of initiator, and reacting to obtain the antibacterial adhesive conductive hemostatic antioxidant injectable composite hydrogel.

6. The preparation method of the injectable composite hydrogel with antibacterial, adhesive, conductive, hemostatic and antioxidant effects according to claim 5, wherein in the step (3A) and the step (3B), the buffer solution is PBS; the mass ratio of CS in GT-DA/CS is 5-10%.

7. The preparation method of the injectable composite hydrogel with antibacterial, adhesive, conductive, hemostatic and antioxidant effects according to claim 1 or 5, wherein the initiator comprises H with a concentration of 0.1-0.3 mol/mL₂O₂And horseradish peroxidase with the concentration of 0.2-0.5 mg/mL, and 0.5mL of H is correspondingly added into each 1mL of CNT-PDA dispersion liquid₂O₂And 1mL horseradish peroxidaseAn oxidase enzyme; and (3C) reacting for 10-200 s.

8. The injectable composite hydrogel with antibacterial, adhesion, conductivity, hemostasis and antioxidation prepared by the preparation method of claim 1.

9. Use of an antimicrobial adherent electrically conductive hemostatic oxidation resistant injectable composite hydrogel of claim 8 in the preparation of a wound dressing.