CN114920956A

CN114920956A - Hemostatic, antibacterial and healing-promoting hydrogel with real-time monitoring function and preparation method thereof

Info

Publication number: CN114920956A
Application number: CN202210558775.5A
Authority: CN
Inventors: 戴红莲; 杨二康; 李锋; 杨鹤; 侯雯; 刘坤; 康红磊
Original assignee: Wuhan University of Technology WUT
Current assignee: Wuhan University of Technology WUT
Priority date: 2022-05-20
Filing date: 2022-05-20
Publication date: 2022-08-19

Abstract

The invention relates to a hemostatic and antibacterial healing-promoting hydrogel with a real-time monitoring function and a preparation method thereof.

Description

Hemostatic, antibacterial and healing-promoting hydrogel with real-time monitoring function and preparation method thereof

Technical Field

The invention belongs to the technical field of wound dressing, and particularly relates to an injectable hemostatic antibacterial healing-promoting hydrogel with a real-time monitoring function and a preparation method thereof.

Background

Bleeding is inevitably caused in war, traffic accident or operation process, and complications caused by excessive bleeding or excessive blood loss are still one of the main causes of trauma death. Therefore, the rapid hemostatic material has important research and development and application values in the aspect of emergency hemostasis.

The hemostatic materials commonly used in clinic at present comprise gauze, hemostatic sponge and hemostatic microspheres, while the hemostatic powder is more used in battlefield, and the hemostatic powder can achieve the purpose of closing the wound by physically pressing the bleeding part or realize hemostasis by rapidly absorbing blood and enriching blood coagulation factors. However, these hemostatic methods such as gauze can stick to the wound after hemostasis, and hemostatic powder and hemostatic microspheres are prone to cause secondary damage during cleaning, and are often not effective in time to achieve hemostasis especially when facing irregular and deep bleeding points. Moreover, active inflammatory reaction is caused in the early healing period of the wound, and the inorganic nanoparticles can promote the inflammatory reaction to generate a large amount of ROS. A controlled inflammatory response may accelerate wound healing, but excessively high ROS levels may inhibit wound healing and lead to other complications.

Compared with hemostasis, the hydrogel serving as a dressing, particularly an injectable hydrogel, can be well attached to a bleeding part when facing a deep and irregular wound, and closed hemostasis is realized. The cement gel has good adsorption performance, can quickly absorb blood, enrich blood cells and blood platelets, promote hemostasis, maintain a moist environment of a wound, promote material exchange and promote wound healing, and the hydrogel serving as a dressing is developed quickly and comprises sodium alginate, gelatin, silk fibroin, acrylamide, chitosan and the like. The antimicrobial and hemostatic properties of these hydrogels have yet to be further improved.

The natural polysaccharide material has good biocompatibility, low immunological rejection and good biodegradability, can be subjected to functional design through modification, and is an ideal matrix material.

Disclosure of Invention

The invention provides an injectable antibacterial healing-promoting hydrogel and a preparation method thereof, aiming at the defects of the prior art, the prepared hydrogel precursor solution is directly injected to a bleeding part through an injector after being added with a photoinitiator, can be directly gelled in situ after being illuminated, seals a wound, realizes hemostasis, and has the advantages of good antibacterial performance, adhesion performance and the like. Meanwhile, in order to regulate and control the inflammatory reaction process, polyphenol with ROS eliminating capacity is introduced through a grafting modification method, excessive generation of ROS at a wound part is inhibited, and wound healing is facilitated.

The scheme adopted by the invention for solving the technical problems is as follows:

a preparation method of injectable antibacterial healing-promoting hydrogel comprises the following steps:

1) heating, stirring and dissolving polysaccharide to prepare a polysaccharide solution;

2) adding a quaternary ammonium salt compound into the polysaccharide solution obtained in the step (1) to carry out grafting reaction to obtain quaternary ammonium polysaccharide, and purifying and storing for later use;

3) preparing the quaternary amination polysaccharide obtained in the step (2) into a quaternary amination polysaccharide solution, adding a double-bond compound for grafting reaction to obtain double-bond quaternary amination polysaccharide, and purifying and storing for later use;

4) preparing the double-bond quaternized polysaccharide obtained in the step (3) into a double-bond quaternized polysaccharide solution, adding a polyphenol compound and a crosslinking agent, carrying out grafting reaction under acidic conditions and inert gas protection to obtain the polyphenol double-bond quaternized polysaccharide, and purifying and storing for later use;

5) and (4) preparing inorganic nano particles into a dispersion solution, adding a photoinitiator and the polyphenol double-bond quaternary amination polysaccharide obtained in the step (4), and uniformly mixing to obtain the injectable antibacterial healing-promoting hydrogel dressing.

Preferably, the polysaccharide is one or more of sodium alginate, gelatin, cellulose and derivatives thereof, chitin, chitosan and derivatives thereof, starch, agarose, dextran, silk fibroin, hyaluronic acid, fructose, aloe polysaccharide, tea polysaccharide, mannan, and xylan; in the step (1), the mass fraction of the polysaccharide in the obtained polysaccharide solution is 0.1-20 wt%; the temperature of the polysaccharide solution is 30-100 ℃.

Preferably, the quaternary ammonium salt compound is one or more of alkyl trimethyl quaternary ammonium alkyl, dodecyl trimethyl ammonium chloride, dodecyl dimethyl benzyl ammonium chloride, octadecyl dimethyl hydroxyethyl ammonium nitrate, octadecyl dimethyl hydroxyethyl ammonium perchlorate, tetrabutyl ammonium bromide, hexadecyl trimethyl ammonium bromide, dodecyl dimethyl benzyl ammonium chloride, dioctadecyl dimethyl ammonium bromide, 2, 3-epoxypropyl trimethyl ammonium chloride and tributyl methyl ammonium chloride; in the step (2), the mass fraction of the quaternary ammonium salt compound in the mixed solution is 0.01-10 wt%; the reaction time is 6-24 hours.

Preferably, the double-bond compound is one or more of maleimide, methacrylic anhydride, acrylic acid, itaconic acyl, fumaric acyl, hydroxypropyl methacrylate, undecylenic acyl, ethyl acrylate and propyl acrylate; in the step (3), the mass fraction of the double-bond compound in the mixed solution is 0.01-10 wt%; the reaction time is 2-18 hours.

Preferably, the polyphenol is one or more of gallic acid, tannic acid, ferulic acid, caffeic acid, dopamine, procyanidine, resveratrol, protocatechuic acid, rhein, catechol, and pyrogallol; in the step (4), the mass fraction of polyphenol in the mixed solution is 0.01-10%; the reaction time is 6-24 hours; the pH value of the solution is 1-6.

Preferably, the cross-linking agent in the step (4) is one or more of glutaraldehyde, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide/N-N-hydroxysuccinimide (EDC/NHS), genipin, EDC/1-Hydroxybenzotriazole (HOBT), O-benzotriazol-tetramethyluronium Hexafluorophosphate (HBTU), 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU), 1H-benzotriazol-1-yl oxo tris (dimethylamino) phosphonium hexafluorophosphate (BOP); the mass fraction of the cross-linking agent in the mixed solution is 0.1-10 wt%.

Preferably, the inorganic nano particles are one or more of hydroxyapatite, beta-TCP, alpha-TCP, doped TCP, whitlockite, doped whitlockite, bioglass, clay, montmorillonite, kaolin, mesoporous silica and doped mesoporous silica; in the step (5), the mass fraction of the inorganic nano particles in the mixed solution is 0.01-10 wt%, and the mass fraction of the polyphenol double-bond quaternized polysaccharide is 0.1-10 wt%.

Preferably, in the step (5), the inorganic nanoparticles are added and the color developing substance is added at the same time; the chromogenic substance is one or more of phenol red, rhodamine B, rhodamine derivatives, benzimidazole, morpholine, cyanine, lysosome derivatives, nitroxyl, 1, 8-naphthalimide and N-perylene; in the step (5), the mass fraction of the chromogenic substance in the mixed solution is 0.001-0.1 wt%.

Preferably, in the step (5), the photoinitiator is one or more of 2,4,6 (trimethylbenzoyl) diphenylphosphine oxide, ethyl 2,4, 6-trimethylbenzoylphosphonate, 1-hydroxy-cyclohexyl-phenyl ketone, 4-dimethylamino-ethyl benzoate, 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone, and phenyl bis (2,4, 6-trimethylbenzoyl) phosphine oxide; the mass fraction of the cross-linking agent in the mixed solution is 0.01-10 wt%.

The invention also provides an injectable antibacterial healing-promoting hydrogel prepared by the method.

Preferably, the injectable antibacterial healing-promoting hydrogel can be gelatinized in situ within 15 seconds by illumination after being injected into a wound surface part. Can be used as wound dressing and hemostatic material.

For the hydrogel containing the chromogenic substance, the inflammation level can be observed in real time through the fluorescence intensity displayed by ultraviolet illumination.

The invention firstly quaternizes the polysaccharide, and the antibacterial property of the quaternized polysaccharide is improved; then double-bonding is carried out on the basis of quaternization, so that the hydrogel can be endowed with a photo-crosslinking characteristic, and the hydrogel is convenient to be directly injected to a wound surface part to form light-irradiated gel in subsequent use and is more convenient and faster to use; then, on the basis of double-bond quaternization, polyphenol groups are introduced to the polymer, so that the hydrogel can be endowed with adhesive property, can be better adhered to the wound surface to realize hemostasis, and meanwhile, the ROS (reactive oxygen species) can be eliminated by utilizing the characteristics of the polyphenol groups, so that the inflammation level near the wound can be regulated, controlled and reduced; the real-time monitoring of the inflammatory reaction of the wound surface can be realized through the fluorescence effect of the chromogenic substance; the introduction of the inorganic nano particles can further improve the hemostatic and antibacterial effects of the hydrogel. The prepared injectable antibacterial hydrogel dressing can initiate gelling under the irradiation of light, has injectability and adhesion, has good antibacterial effect and hemostatic effect, can conveniently monitor and regulate the inflammation level of a wound in the using process, promotes wound healing, and is an ideal hydrogel dressing.

Compared with the prior art, the invention has the beneficial effects that:

1) the polyphenol double-bond quaternized polysaccharide prepared by the invention not only improves the antibacterial performance of the polysaccharide, but also has photocrosslinking performance due to double bonding, and can be injected in situ to rapidly gelatinize, so that the operation is more convenient when the polysaccharide is used.

2) The inorganic nano particles released by the injectable antibacterial polysaccharide hydrogel prepared by the invention can activate an intrinsic coagulation pathway to accelerate hemostasis, and the particles have good antibacterial performance in a synergistic manner.

3) The polyphenol double-bond quaternary amination polysaccharide prepared by the invention has good adhesion performance after polyphenol modification, can be adhered to a bleeding part to seal a wound, and simultaneously utilizes the oxidation resistance to regulate the ROS level of the wound, inhibit excessive inflammatory reaction and promote wound healing.

4) The injectable antibacterial polysaccharide hydrogel prepared by the invention can realize real-time monitoring of the inflammation of the wound surface through the composite chromogenic substance.

5) The polyphenol double-bond quaternized polysaccharide prepared by the invention realizes the functional modification of grafting the quaternary ammonium salt group, the double-bond group and the polyphenol group onto the same polysaccharide molecular chain, and endows the polysaccharide with multiple functions.

6) The injectable antibacterial polysaccharide hydrogel prepared by the invention has simple preparation process and is easy to popularize.

Drawings

FIG. 1 is an FTIR spectrum of a polyphenol double-bonded quaternized polysaccharide prepared in example 1 of the present invention;

FIG. 2 shows the H of the poly (phenol) double-bonded quaternized polysaccharide prepared in example 1 ¹ -NMR spectrum;

FIG. 3 is an XRD pattern of the inorganic nanoparticles used in example 2 of the present invention;

FIG. 4 is an SEM image of the hydrogel obtained after in-situ gelation of the injectable antibacterial healing-promoting hydrogel obtained in example 2 of the present invention, wherein it can be seen that inorganic nanoparticles are uniformly distributed therein;

FIG. 5 is a graph showing the injection effect of the injectable antibacterial healing-promoting hydrogel obtained in example 1 of the present invention, which shows that the obtained material has very good fluidity;

fig. 6 shows the adhesive strength of the hydrogel obtained by in-situ gelling the hydrogel with the polyphenol double-bond quaternized gelatin and the composite inorganic nanoparticles obtained in example 1 of the present invention, which indicates that the composite inorganic nanoparticles have a small influence on the adhesive strength of the hydrogel;

fig. 7 shows the antibacterial effect of the hydrogel formed by the polyphenol double-bond quaternized polysaccharide and the inorganic nanoparticles in situ, which is obtained in example 3 of the present invention, and it can be seen that the number of colonies in the blank control group is the largest, the number of colonies in the polyphenol double-bond quaternized polysaccharide group is the next, and the number of colonies in the polyphenol double-bond quaternized chitosan + inorganic nanoparticles group is the smallest;

fig. 8 is a comparison of hemostatic effects of the polyphenol double-bond quaternized hyaluronic acid and the hydrogel formed by compounding inorganic nanoparticles in situ, which are obtained in example 3 of the present invention, wherein the left graph is blank control, the middle graph is hemostatic of the polyphenol double-bond quaternized hyaluronic acid as hydrogel dressing, and the right graph is hemostatic of the polyphenol double-bond quaternized hyaluronic acid + inorganic nanoparticles as hydrogel dressing, which indicates that the blank control group bleeds most, and then the polyphenol double-bond quaternized hyaluronic acid, and the polyphenol double-bond quaternized hyaluronic acid + inorganic nanoparticles group bleeds least;

FIG. 9 is a schematic view of the test procedure of the adhesion test;

FIG. 10 is a graph of HE staining of wound tissue on day 15, wherein there was a significant amount of inflammation in the commercial hemostatic gel group, and less inflammation in the hydrogel groups obtained in examples 4, 5 and 6;

FIG. 11 shows the degree of inflammatory response of tissue by testing fluorescence intensity after treatment of wound tissue at days 4, 7 and 15, wherein the commercial hemostatic gel group showed strong fluorescence effect at days 4, 7 and 15, indicating severe inflammation, while example 4 showed strong fluorescence intensity only at day 4, and the fluorescence intensity at days 7 and 15 was low, indicating substantial disappearance of inflammation.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the following examples. It should be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not limiting.

Example 1

The preparation process of the injectable antibacterial healing-promoting hydrogel dressing in the embodiment is as follows:

1) weighing 20g of gelatin, adding into 200ml of deionized water, stirring and heating at 50 ℃ to dissolve, and preparing into a gelatin solution;

2) adding 2g of dodecyl dimethyl epoxy propyl ammonium chloride into the gelatin solution prepared in the step (1), continuously heating, stirring and reacting for 12 hours, dialyzing, freeze-drying to obtain quaternary ammonium gelatin, and storing for later use;

3) weighing 10g of quaternary amination gelatin obtained in the step (2), dissolving in 100ml of deionized water, adding 2g of maleimide, continuing to react for 10 hours, dialyzing, freeze-drying to obtain double-bond quaternary amination gelatin, and storing for later use;

4) weighing 5g of double-bond quaternized gelatin obtained in the step (3), dissolving in 50ml of deionized water, adding 1g of dopamine and 1.5g of EDC/NHS, reacting for 20 hours under the protection of inert gas at the pH value of 3, dialyzing, freeze-drying to obtain polyphenol double-bond quaternized gelatin, and storing for later use;

5) weighing 0.1g of beta-TCP and 0.005g of phenol red, dispersing in 10ml of deionized water, adding 1g of the polyphenol double-bond quaternized gelatin obtained in the step (4) and 0.05g of 2,4,6 (trimethylbenzoyl) diphenylphosphine oxide, and uniformly mixing to form a uniform hydrogel precursor solution, namely the antibacterial healing-promoting hydrogel dressing can be injected;

6) the injectable antibacterial healing-promoting hydrogel dressing obtained in the step (5) is directly injected to a bleeding part under the action of a 25G injector, the hydrogel dressing has good fluidity, and can form gel in situ within 15s after ultraviolet illumination, thereby achieving the hemostatic effect.

Example 2

1) weighing 5g of chitosan, adding the chitosan into 200ml of deionized water, stirring and heating at 50 ℃ to dissolve the chitosan, and preparing a chitosan solution;

2) adding 1g of 2, 3-epoxypropyltrimethylammonium chloride into the chitosan solution prepared in the step (1), continuously heating, stirring and reacting for 18 hours, dialyzing, freezing and drying to obtain quaternized chitosan, and storing for later use;

3) weighing 3g of quaternized chitosan obtained in the step (2), dissolving in 100ml of deionized water, adding 0.5g of methacrylic anhydride, continuing to react for 6 hours, dialyzing, freeze-drying to obtain double-bond quaternized chitosan, and storing for later use;

4) weighing 2g of double-bond quaternized chitosan obtained in the step (3), dissolving in 50ml of deionized water, adding 0.1g of dihydrocaffeic acid and 0.05g of EDC/1-Hydroxybenzotriazole (HOBT), reacting for 16 hours under the protection of inert gas and pH 5, dialyzing, freeze-drying to obtain polyphenol double-bond quaternized chitosan, and storing for later use;

5) weighing 0.1g of zinc-doped whitlockite and 0.01g of rhodamine B, dispersing in 10ml of deionized water, adding 0.1g of the polyphenol double-bond quaternized gelatin obtained in the step (4) and 0.005g of 2-hydroxy-2-methyl-1-phenyl-1-acetone, and uniformly mixing to form a uniform precursor solution;

Example 3

1) weighing 10g of hyaluronic acid, adding into 200ml of deionized water, stirring and heating at 30 ℃ to dissolve, and preparing into a hyaluronic acid solution;

2) adding 4g of hexadecyl trimethyl ammonium bromide into the hyaluronic acid solution prepared in the step (1), continuously heating, stirring and reacting for 12 hours, dialyzing, freezing and drying to obtain quaternary ammonium hyaluronic acid, and storing for later use;

3) weighing 5g of quaternary amination hyaluronic acid obtained in the step (2), dissolving in 100ml of deionized water, adding 2g of maleimide, continuing to react for 10 hours, dialyzing, freeze-drying to obtain double-bond quaternary amination hyaluronic acid, and storing for later use;

4) weighing 3g of double-bond quaternized hyaluronic acid obtained in the step (3), dissolving in 50ml of deionized water, adding 1g of gallic acid and 2g of EDC/NHS, reacting for 20 hours under the protection of inert gas and pH 4, dialyzing, freeze-drying to obtain polyphenol double-bond quaternized hyaluronic acid, and storing for later use;

5) weighing 0.5g of bioglass and 0.005g of benzimidazole, dispersing the bioglass and the benzimidazole into 10ml of deionized water, adding 1g of the polyphenol double-bond quaternized hyaluronic acid obtained in the step (4) and 0.5g of phenyl bis (2,4, 6-trimethylbenzoyl) phosphine oxide, and uniformly mixing to form a uniform precursor solution;

Example 4

1) weighing 10g of gelatin, adding into 100ml of deionized water, stirring and heating at 50 ℃ to dissolve, and preparing into a gelatin solution;

2) adding 1g of dodecyl dimethyl epoxy propyl ammonium chloride into the gelatin solution prepared in the step (1), continuously heating, stirring and reacting for 12 hours, dialyzing, freeze-drying to obtain quaternary ammonium gelatin, and storing for later use;

3) weighing 1g of quaternary amination gelatin obtained in the step (2), dissolving in 10ml of deionized water, adding 0.2g of maleimide, continuing to react for 15 hours, dialyzing, freeze-drying to obtain double-bond quaternary amination gelatin, and storing for later use;

4) weighing 5g of double-bond quaternized gelatin obtained in the step (3), dissolving in 50ml of deionized water, adding 1g of dopamine and 1.5g of EDC/NHS, reacting for 16 hours under the protection of inert gas and pH 4, dialyzing, freeze-drying to obtain polyphenol double-bond quaternized gelatin, and storing for later use;

5) weighing 0.2g of beta-TCP and 0.01g of phenol red, dispersing in 10ml of deionized water, adding 2g of the polyphenol double-bond quaternized gelatin obtained in the step (4) and 0.005g of 2,4,6 (trimethylbenzoyl) diphenylphosphine oxide, and uniformly mixing to form a uniform hydrogel precursor solution, namely the antibacterial healing-promoting hydrogel dressing can be injected;

6) the injectable antibacterial healing-promoting hydrogel dressing obtained in the step (5) is directly injected to an infected wound under the action of a 25G injector, the hydrogel dressing has good fluidity, and can form gel in situ within 15s after ultraviolet illumination, thereby having the effect of promoting repair.

Example 5

1) weighing 15g of hyaluronic acid, adding into 200ml of deionized water, stirring and heating at 50 ℃ to dissolve, and preparing into a hyaluronic acid solution;

2) adding 4g of 2, 3-epoxypropyltrimethylammonium chloride into the hyaluronic acid solution prepared in the step (1), continuously heating, stirring and reacting for 18 hours, dialyzing, freezing and drying to obtain quaternized hyaluronic acid, and storing for later use;

3) weighing 10g of quaternary amination hyaluronic acid obtained in the step (2), dissolving in 100ml of deionized water, adding 4g of methacrylic anhydride, continuing to react for 5 hours, dialyzing, freeze-drying to obtain double-bond quaternary amination hyaluronic acid, and storing for later use;

4) weighing 5g of double-bond quaternized hyaluronic acid obtained in the step (3), dissolving in 100ml of deionized water, adding 1g of dopamine and 10g of EDC, reacting for 10 hours under the protection of inert gas and pH 2, dialyzing, freeze-drying to obtain polyphenol double-bond quaternized hyaluronic acid, and storing for later use;

5) weighing 0.5g of whitlockite and 0.01g of benzimidazole, dispersing in 10ml of deionized water, adding 1g of the polyphenol double-bond quaternized hyaluronic acid obtained in the step (4) and 0.05g of phenyl bis (2,4, 6-trimethylbenzoyl) phosphine oxide, and uniformly mixing to form a uniform precursor solution;

6) the injectable antibacterial healing-promoting hydrogel dressing obtained in the step (5) is directly injected to the infected wound surface part under the action of a 25G injector, the hydrogel dressing has good fluidity, and can form gel in situ within 15s through ultraviolet illumination, so that the repairing-promoting effect is achieved.

Example 6

1) weighing 5g of chitosan, adding the chitosan into 200ml of deionized water, stirring and heating at 60 ℃ to dissolve the chitosan, and preparing a chitosan solution;

2) adding 2g of dodecyl dimethyl epoxy propyl ammonium chloride into the chitosan solution prepared in the step (1), continuously heating, stirring and reacting for 10 hours, dialyzing, freezing and drying to obtain quaternary ammonium chitosan, and storing for later use;

3) weighing 2g of quaternary amination chitosan obtained in the step (2), dissolving the quaternary amination chitosan in 50ml of deionized water, adding 0.05g of maleimide, continuing to react for 10 hours, dialyzing, freezing and drying to obtain double-bond quaternary amination chitosan, and storing for later use;

4) weighing 1g of double-bond quaternized chitosan obtained in the step (3), dissolving in 20ml of deionized water, adding 0.01g of dihydrocaffeic acid and 0.05g of EDC/1-Hydroxybenzotriazole (HOBT), reacting for 12 hours under the protection of inert gas and at the pH value of 3, dialyzing, freeze-drying to obtain polyphenol double-bond quaternized chitosan, and storing for later use;

5) weighing 0.1g of zinc-doped whitlockite and 0.05g of rhodamine, dispersing in 10ml of deionized water, adding 0.1g of the polyphenol double-bond quaternized gelatin obtained in the step (4) and 0.01g of 2-hydroxy-2-methyl-1-phenyl-1-acetone, and uniformly mixing to form a uniform precursor solution;

6) the injectable antibacterial healing-promoting hydrogel dressing obtained in the step (5) is directly injected to the infected wound surface part under the action of a 25G injector, the hydrogel dressing has good fluidity, and can be in-situ gelatinized within 15s by ultraviolet illumination, so that the repairing promoting effect is achieved.

Description of adhesion test:

as shown in fig. 9, the adhesion performance of the hydrogel to the tissue was simulated by adhesion on the pig skin. A layer of 20% gelatin solution with a length of 10mm and a width of 10mm is coated on a glass sheet (with a length of 30mm and a width of 10mm) prepared in advance, and is naturally dried overnight for standby, and then the pigskin is cut into strips with a length of 30mm and a width of 10mm for standby. Respectively smearing 200 mu L of the hydrogel precursor solution of the polyphenol double-bond quaternized polysaccharide or the composite inorganic nanoparticles on the pigskin, then covering a glass sheet coated with gelatin, and illuminating for 15s by using ultraviolet light. Tensile properties were tested on a universal tester.

And (3) testing antibacterial performance:

the experimental materials were placed in 24-well plates, and 0.9mL of sterile PBS and 0.1mL of bacterial culture were added, followed by shaking culture at 37 ℃ for 18 hours. When incubation was complete, the bacterial suspension of the mixed sample was diluted 10 with sterile PBS ⁵ After that, 0.1mL of the diluted liquid was dropped on the LB solid medium and evenly spread on a petri dish using a sterilized triangular spreading bar. The bacteria-coated culture dish was then placed in a 37 ℃ incubator and incubated for 18 hours before being counted as shown in the following formula. Wherein C is the colony number on the culture dish after dilution, V is the bacterial suspension volume on the culture dish, and N is the PBS dilution factor.

CFU＝C/(V×N)×100％

And (3) testing the hemostatic performance:

after anesthetizing the mice, the abdominal skin was shaved off and fixed on a special wood board with the abdomen facing up and the back facing down. The surgical site was sterilized with medical iodophor and alcohol cotton balls. The skin of the lower part of the thoracic cavity of the mouse is cut open by using an ophthalmic scissors to expose the liver, the liver is gently led out to avoid the damage of the liver due to extrusion, and the mouse is placed under an un-started ultraviolet lamp to wait for operation.

Clean filter paper is placed below the liver of a mouse, a linear wound with the length of 10mm and the depth of 3mm is formed on the liver by using a scalpel, and the hemostatic hydrogel is injected to a bleeding part immediately and simultaneously an ultraviolet lamp is turned on to irradiate the gel for 15 s. After hemostasis was completed, the blood on the filter paper was recorded by taking a picture.

Mouse inflammation model and experiment for promoting wound healing and biological self-degradation

A mouse back injury model is selected as a degradation model in the healing promotion and biological self-degradation experiments, and the specific operation is that 10 wt% chloral hydrate is injected into an abdominal cavity to anaesthetize the mouse, and circular wound surfaces with the diameter of 15mm are respectively formed on the back epidermis on the left side and the right side of a spine of the mouse. After simple wound cleaning, the wound surface was covered with a 15mm diameter circular hydrogel (and commercial hemostatic gel) and fixed after hemostasis, the gel residual size was counted after 7 days and 15 days, and the gel residual wound surface area was recorded. The degradation rate of the gel and the healing rate of the wound are represented by the percentage value of the area of the initial wound surface to the area of the gel reduced after treatment. The results of the experiment are shown in the following table.

Numbering	Commercial hemostatic gels	Example 4	Example 5	Example 6
					Degradation ratio in 7 days (%)	27±8	49±5	38±7	30±6
Degradation Rate in 15 days (%)	68±16	87±12	90±8	91±7

Note that commercial hemostatic gels are obtained from domestic commercial products (trade name:

chitosan gel, inner mongolia east silver science co ltd).

The test result shows that the degradable hemostatic gel can be degraded by more than 85% in 15 days, and is superior to the commercial hemostatic gel product. The hydrogel prepared has good biological absorption performance. Meanwhile, the injectable antibacterial degradable hydrogel dressing can effectively reduce inflammatory reaction and promote wound healing in the same time.

Color development experiment:

at a set time point, the mouse is anesthetized, tissues of a wound surface part with certain quality are taken for differentiation by using ophthalmic scissors and a scalpel, and after dilution, the tissue is photographed under the excitation of a specific ultraviolet light source to record the fluorescence intensity. The results of comparing the chitosan hemostatic gel with example 4 show that the chitosan hemostatic gel group shows high fluorescence intensity, and example 4 shows low fluorescence intensity. The injectable antibacterial hydrogel dressing can effectively reduce the inflammation of the wound surface.

While the foregoing is directed to the preferred embodiment of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Claims

1. A preparation method of injectable antibacterial healing-promoting hydrogel is characterized by comprising the following steps:

2) adding a quaternary ammonium salt compound into the polysaccharide solution obtained in the step (1) for grafting reaction to obtain quaternary ammonium polysaccharide, and purifying and storing for later use;

5) and (4) preparing inorganic nano particles into a dispersion solution, adding a photoinitiator and the polyphenol double-bond quaternary amination polysaccharide obtained in the step (4), and uniformly mixing to obtain the injectable antibacterial healing-promoting hydrogel.

2. The preparation method according to claim 1, wherein the polysaccharide is one or more of sodium alginate, gelatin, cellulose and derivatives thereof, chitin, chitosan and derivatives thereof, starch, agarose, dextran, silk fibroin, hyaluronic acid, fructose, aloe polysaccharide, tea polysaccharide, mannan, and xylan; in the step (1), the mass fraction of the polysaccharide in the obtained polysaccharide solution is 0.1-20 wt%; the temperature of the polysaccharide solution is 30-100 ℃.

3. The preparation method according to claim 1, wherein the quaternary ammonium salt compound is one or more of alkyl trimethyl quaternary ammonium alkyl, dodecyl trimethyl ammonium chloride, dodecyl dimethyl benzyl ammonium chloride, octadecyl dimethyl hydroxyethyl ammonium nitrate, octadecyl dimethyl hydroxyethyl ammonium perchlorate, tetrabutyl ammonium bromide, hexadecyl trimethyl ammonium bromide, dodecyl dimethyl benzyl ammonium chloride, dioctadecyl dimethyl ammonium bromide, 2, 3-epoxypropyl trimethyl ammonium chloride and tributyl methyl ammonium chloride; in the step (2), the mass fraction of the quaternary ammonium salt compound in the mixed solution is 0.01-10 wt%; the reaction time is 6-24 hours.

4. The preparation method according to claim 1, wherein the double bond compound is one or more of maleimide, methacrylic anhydride, acrylic acid, itaconic acid, fumaric acid, hydroxypropyl methacrylate, undecylenic acid, ethyl acrylate and propyl acrylate; in the step (3), the mass fraction of the double-bond compound in the mixed solution is 0.01-10 wt%; the reaction time is 2-18 hours.

5. The preparation method according to claim 1, wherein the polyphenol in step (4) is one or more of gallic acid, tannic acid, ferulic acid, caffeic acid, dopamine, procyanidin, resveratrol, protocatechuic acid, rhein, catechol, and pyrogallol; the mass fraction of polyphenol in the mixed solution is 0.01-10%; the reaction time is 6-24 hours; the pH value of the solution is 1-6.

6. The preparation method according to claim 1, wherein the inorganic nanoparticles in step (5) are one or more of hydroxyapatite, β -TCP, α -TCP, doped TCP, whitlockite, doped whitlockite, bioglass, clay, montmorillonite, kaolin, mesoporous silica, and doped mesoporous silica; the mass fraction of the inorganic nano particles in the mixed solution is 0.01-10 wt%; the mass fraction of the polyphenol double-bond quaternized polysaccharide in the mixed solution is 0.1-10 wt%.

7. The method according to claim 1, wherein the step (5) is carried out by adding a coloring material simultaneously with the addition of the inorganic nanoparticles; the chromogenic substance is one or more of phenol red, rhodamine B, rhodamine derivatives, benzimidazole, morpholine, cyanine, lysosome derivatives, nitroxyl, 1, 8-naphthalimide and N-perylene; the mass fraction of the color substance in the mixed solution is 0.001-0.1 wt%.

8. The method according to claim 1, wherein the crosslinking agent in the step (4) is one or more selected from glutaraldehyde, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide/N-N-hydroxysuccinimide (EDC/NHS), genipin, EDC/1-Hydroxybenzotriazole (HOBT), O-benzotriazol-tetramethyluronium Hexafluorophosphate (HBTU), 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU), 1H-benzotriazol-1-yl oxo tris (dimethylamino) phosphonium hexafluorophosphate (BOP); the mass fraction of the cross-linking agent in the mixed solution is 0.1-10 wt%.

9. The method according to claim 1, wherein in the step (5), the photoinitiator is one or more selected from 2,4,6 (trimethylbenzoyl) diphenylphosphine oxide, ethyl 2,4, 6-trimethylbenzoylphosphonate, 1-hydroxy-cyclohexyl-phenyl ketone, ethyl 4-dimethylamino-benzoate, 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone, and phenylbis (2,4, 6-trimethylbenzoyl) phosphine oxide; the mass fraction of the cross-linking agent in the mixed solution is 0.01-10 wt%.

10. An injectable antimicrobial healing-promoting hydrogel prepared by the method of any one of claims 1 to 9.