CN107469137B

CN107469137B - Injectable hemostatic hydrogel material and preparation method and application thereof

Info

Publication number: CN107469137B
Application number: CN201710812876.XA
Authority: CN
Inventors: 王桦; 蔡园园; 姜耀; 龙大成; 华玥; 刘欢; 冯路平
Original assignee: Qufu Normal University
Current assignee: Qufu Normal University
Priority date: 2017-09-11
Filing date: 2017-09-11
Publication date: 2020-04-07
Anticipated expiration: 2037-09-11
Also published as: CN107469137A

Abstract

The invention relates to an injectable hemostatic hydrogel material and a preparation method and application thereof. The hemostatic hydrogel can realize rapid change from a sol state to a gel state by utilizing the catalytic function of hemoglobin in blood. The material of the invention has the functions of rapid hemostasis, antibiosis, drug-loading sustained release, injectability and the like, and has great application value in the fields of biomedical materials and tissue engineering.

Description

Injectable hemostatic hydrogel material and preparation method and application thereof

Technical Field

The invention belongs to the technical field of preparation of functional materials for hemostasis, antibiosis, drug loading and the like in biomedicine, and relates to an injectable hemostatic hydrogel material, a preparation method and application thereof.

Background

Common hemostatic materials mainly include dressing bandages, gauze, medical cotton and the like, and these physical hemostatic materials achieve wound hemostasis mainly through compression, but are difficult to keep the wound dry and prevent harmful bacteria from entering the wound. In the last two decades, many modern new hemostatic materials have been used for clinical wound hemostasis and antisepsis. The novel hemostatic materials are different from physical materials, mainly comprise gel, alginate, hydrogel and the like, and are basically characterized in that a mild environment is created for wounds in the hemostatic process to promote wound hemostasis and healing, but rapid hemostasis on large-area or deep wound parts cannot be realized mostly.

Hydrogels are widely used in biopharmaceutical and biomedical materials due to their high biocompatibility and low mechanical irritation, as is well known. The supramolecular hydrogel is considered to be an ideal hemostatic material because the supramolecular hydrogel can provide a moist environment for wounds and form a protective barrier at the wound interface. In addition, the hydrogel has high biocompatibility, biodegradability and self-healing characteristics, so that the hemostatic process is convenient and painless, and secondary trauma can be reduced. In particular, the synthesis of in situ hydrogel has attracted much attention in drug delivery and tissue engineering applications, and injectable hydrogel has a wide range of applications in medical materials and drug release, and has become a novel research direction in biomedical materials in recent years. In addition, compared with other porous scaffold materials, the injectable hydrogel material has better superiority, and the hydrogel biomaterial with fluidity is implanted into a living body by using an injection method, so that irregular-shaped wound parts can be easily filled, and the wound in the operation is smaller. The injectable hydrogel three-dimensional network has a large amount of water due to high water absorption, is similar to organism tissues in morphological structure, and has high biocompatibility. In particular, injectable hydrogels allow the encapsulation of hemostatic or anti-inflammatory drugs, do not require surgical implantation, and can be injected into deep wounds to achieve hemostasis, but there has been no technical report of combining hydrogels with hemoglobin having peroxidase catalytic activity in blood and using the hydrogels to catalyze the aggregation of hydrosols to achieve rapid hemostasis. In addition, hydrogen peroxide has been used in various fields as a classic antibacterial substance. It has been reported that hydrogen peroxide has significant bacteriostatic effects on fungi, such as escherichia coli, gram-positive bacteria, gram-negative bacteria, viruses, and the like; recently, numerous studies have demonstrated the in vivo antimicrobial activity of hydrogen peroxide over other materials. Therefore, the invention develops a preparation and application technology of an injectable hemostatic hydrogel material, gelatin and tyrosine are covalently combined by adopting a cross-linking agent, then hydrogen peroxide is introduced to prepare the injectable functional hydrosol which is used for large-area or deep wound bleeding parts, and hemoglobin with peroxidase catalytic activity in wound blood is utilized to catalyze tyrosine peroxide packaged in the hydrosol to oxidize tyrosine, so that high-strength hydrogel is formed to achieve rapid hemostasis. The material and the technology have the functions of rapid hemostasis, antibiosis, drug loading, injectability and the like, are suitable for rapid hemostasis and subsequent antibacterial treatment of large-area or deep wounds, and have great application value in the fields of biomedical materials and tissue engineering.

Disclosure of Invention

Aiming at the defects in the prior art, the invention develops an injectable hemostatic hydrogel material and a preparation method thereof, wherein gelatin and tyrosine are covalently bonded by adopting a cross-linking agent, and then hydrogen peroxide is introduced to prepare the hydrogel material with high antibacterial property and drug slow-release capacity, and the hydrogel material has the functions of rapid hemostasis, antibiosis, drug loading, injectability and the like.

The invention also provides application of the injectable hemostatic hydrogel material, wherein the hemoglobin is used for catalyzing tyrosine peroxide packaged in the hydrogel to oxidize tyrosine, so that high-strength hydrogel is formed. Meanwhile, the antibacterial agent can also be used for antibiosis and has great application value in the fields of biomedical materials and tissue engineering.

The technical scheme of the invention is as follows:

a preparation method of an injectable hemostatic hydrogel material comprises the following steps: firstly, adopting EDC/NHS as a cross-linking agent to covalently bond gelatin and tyrosine, and then introducing hydrogen peroxide to prepare the injectable hemostatic hydrogel material.

The preparation method of the injectable hemostatic hydrogel material comprises the following steps:

(1) adding powdered gelatin into 2- (N-morpholine) ethanesulfonic acid Monohydrate (MES) at a concentration of 20-40mg/mL

Heating to 60 ℃ in the buffer solution to completely dissolve gelatin, and cooling to room temperature to obtain a gelatin solution;

(2) adding tyrosine (Tyr), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into the gelatin solution obtained in the step (1) in a stirring manner, wherein the mass ratio of the tyrosine to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide is 50-200: 37.0-140: 12.5-50, wherein the concentration of the tyrosine added is 2.5-10mg/mL, and after complete dissolution, the tyrosine is stirred and reacts in a water bath at 37 ℃ for 8 hours to ensure complete crosslinking;

(3) adding 10-15 mg/mL powdered sodium phosphate into the reaction solution prepared in the step (2), stirring for 30min, and purifying by using a dialysis membrane with the molecular weight cutoff of 10000 KD until the ultraviolet absorption peak of the solution is 275 nm to prepare a gelatin-tyrosine solution;

(4) and (4) adding 1-7% of hydrogen peroxide by mass into the gelatin-tyrosine solution prepared in the step (3), and stirring to uniformly disperse the hydrogen peroxide in the solution to obtain the hydrogen peroxide-encapsulated injectable hemostatic hydrogel material.

Further, in the step (1), gelatin is added at a concentration of preferably 40 mg/mL.

Further, the 2- (N-morpholine) ethanesulfonic acid-water (MES) buffer solution in the step (1) is an aqueous solution of 2- (N-morpholine) ethanesulfonic acid-water (MES) with the concentration of 20-50mM and the pH of 6.5; the concentration of the 2- (N-morpholine) ethanesulfonic acid-water (MES) buffer solution is preferably 50 mM.

Further, in the step (2), tyrosine is added at a concentration of preferably 5 mg/ml.

Further, in the step (2), the mass ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is preferably 3: 1.

Further, in the step (3), sodium phosphate is preferably added at a concentration of 15 mg/mL.

Further, in the step (4), the mass fraction of the added hydrogen peroxide is preferably 3%.

The solvent in the above steps is secondary distilled water.

The invention also provides an injectable hemostatic hydrogel material prepared by the method, and the injectable hemostatic hydrogel material has a compact porous structure.

The application of the injectable hemostatic hydrogel material can be used as a material for treating wound in-situ rapid hemostasis and can also be used as a drug carrier, so that the effects of drug slow release, bacterial growth inhibition and tissue secondary injury reduction are realized.

The injectable hemostatic hydrogel material is prepared by covalently bonding gelatin and tyrosine through a cross-linking agent and further introducing hydrogen peroxide, and is used for large-area or deep wound bleeding parts, and the hemoglobin with peroxidase catalytic activity in wound blood is used for catalyzing tyrosine peroxide packaged in the hydrogel to form high-strength hydrogel so as to achieve rapid hemostasis (as shown in figure 1). The hemostatic hydrogel can realize rapid change from a sol state to a gel state by utilizing the catalytic function of hemoglobin in wound blood. Meanwhile, hydrogen peroxide in the hydrogel can inhibit the growth of bacteria at the wound, the formed gel with a compact porous structure provides a moist healing environment for the wound and forms a protective barrier, so that the secondary damage of tissues is reduced, and the rapid hemostasis function of the hydrogel is verified through a live mouse test. The hemostatic water-soluble adhesive material with the functions of quick hemostasis, drug slow release, bacteriostasis and injection is suitable for quick hemostasis and subsequent antibacterial treatment of large-area or deep wounds, and has great application value in the fields of biomedical materials and tissue engineering.

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

(1) the injectable hemostatic hydrogel can have blood coagulation change within 20 s (as shown in figure 3), and achieves rapid hemostasis effect;

(2) hydrogen peroxide with a certain concentration is encapsulated in the injectable hemostatic hydrogel, so that the injectable hemostatic hydrogel has a certain function in the aspect of bacteriostasis;

(3) the gel with a compact porous structure formed by the injectable hemostatic hydrogel provides a moist healing environment for the wound and forms a protective barrier, so that the secondary injury of the tissue can be reduced;

(4) the injectable hemostatic hydrogel can coat a certain amount of functional drugs, and can control the release of the drugs while stopping bleeding;

(5) the injectable hemostatic hydrogel can be injected, and is more convenient to use.

Drawings

FIG. 1 is a schematic illustration of an injectable hemostatic hydrogel material for rapid in situ hemostasis;

FIG. 2 is a UV spectrum and an IR spectrum of gelatin and gelatin-tyrosine; (A) the ultraviolet spectrogram and the corresponding photo are shown, and the (B) is the infrared spectrogram;

FIG. 3 is a gel state of an injectable hemostatic hydrogel material formed as a hemostatic hydrogel in a non-blood (A) and blood (B) coagulated state (C);

FIG. 4 is a graph showing the effect of different concentrations of (A) gelatin, (B) tyrosine, (C) hydrogen peroxide, and (D) hemoglobin on the gelation process of an injectable hemostatic hydrogel material;

FIG. 5 is a photograph of the mouse showing hemostasis on the outer thigh (A, B) and inner thigh (C, D); (E) blocking blood coagulation of mouse skin vessels;

FIG. 6 is a photograph of inhibition zones of (A) gelatin, (B) gelatin-tyrosine, (c) gelatin encapsulating hydrogen peroxide, (d) gelatin-tyrosine encapsulating hydrogen peroxide, (e) hydrogen peroxide, for (A) Escherichia coli and (B) Staphylococcus aureus;

FIG. 7 is a microscopic image of cultured yeast cells in different culture media (A) gelatin, (B) gelatin-tyrosine, and (C) gelatin-tyrosine encapsulated with hydrogen peroxide; (D) a map of disrupted yeast cells;

FIG. 8 is a graph of the controlled release of amoxicillin drug by hemoglobin catalyzed injectable hemostatic hydrogel material, showing the change in absorbance of the solution (a) at the beginning and (b) one hour later;

FIG. 9 is a scanning electron micrograph of an injectable hemostatic hydrogel material, (A, B) no blood is present, and (C, D) blood is present.

Detailed Description

The present invention will be further described with reference to specific embodiments, and various substitutions and alterations made by those skilled in the art and by conventional means without departing from the technical idea of the invention are included in the scope of the present invention.

Example 1

A preparation method of an injectable hemostatic hydrogel material comprises the following steps:

(1) 400 mg of gelatin powder was added to 20 mL of 2- (N-morpholine) ethanesulfonic acid Monohydrate (MES) buffer solution having a concentration of 50mM and pH 6.5, and heated to 60 ℃ to completely dissolve the gelatin. The gelatin solution was then cooled to room temperature;

(2) adding 100 mg of tyrosine (Tyr), 75.0 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 25.0 mg of N-hydroxysuccinimide (NHS) into the gelatin solution obtained in the step (1) by stirring, and after complete dissolution, reacting for 8 hours in a water bath at 37 ℃ by stirring to complete crosslinking;

(3) adding 300mg/mL powdered sodium phosphate into the reaction solution obtained in the step (2), stirring for 30min, purifying with a dialysis membrane with molecular weight cutoff of 10000 KD until the ultraviolet absorption peak of the solution is 275 nm (as shown in figure 2), and obtaining gelatin-tyrosine solution;

(4) and (3) adding hydrogen peroxide with the mass fraction of 3% into the gelatin-tyrosine solution prepared in the step (3), and stirring to uniformly disperse the hydrogen peroxide in the solution to obtain the hydrogen peroxide-encapsulated injectable hemostatic hydrogel material (as shown in figure 4).

Observing an injectable hemostatic hydrogel material (gelatin-tyrosine material) encapsulating hydrogen peroxide and a hemostatic hydrogel catalyzed by hemoglobin by using a scanning electron microscope, and clearly observing that the materials before and after catalysis of the hemoglobin have obvious porous structures according to an SEM picture (as shown in figure 9).

Compared with an injectable hemostatic hydrogel material before hemoglobin catalysis, the porous structure of the gel material is more compact, so that the hemostatic hydrogel after hemoglobin catalysis is easier to protect and encapsulate hydrogen peroxide, prevent the hydrogen peroxide from decomposing and slowly release through pores. The porous hydrogel structure can provide more obvious oxygen permeation for wounds while stopping bleeding, slowly release hydrogen peroxide and anti-inflammatory drugs packaged in the porous hydrogel structure, and is beneficial to rapid healing of the wounds.

Example 2

The preparation method of the injectable hemostatic hydrogel material comprises the following specific steps:

(1) 600 mg of gelatin powder was added to 20 mL of 2- (N-morpholine) ethanesulfonic acid Monohydrate (MES) buffer solution having a concentration of 50mM and pH 6.5, and heated to 60 ℃ to completely dissolve the gelatin, and then the gelatin solution was cooled to room temperature;

(3) adding 300mg of powdered sodium phosphate into the reaction solution prepared in the step (2), stirring for 30min, and purifying by using a dialysis membrane with the molecular weight cutoff of 10000 KD until the ultraviolet absorption peak of the solution is 275 nm to prepare a gelatin-tyrosine solution;

(4) and (4) adding hydrogen peroxide with the mass fraction of 3% into the gelatin-tyrosine solution prepared in the step (3), and stirring to uniformly disperse the hydrogen peroxide in the solution to obtain the hydrogen peroxide-encapsulated injectable hemostatic hydrogel material.

Example 3

(1) 800 mg of gelatin powder was added to 20 mL of 2- (N-morpholine) ethanesulfonic acid Monohydrate (MES) buffer solution having a concentration of 20 mM and pH 6.5, and heated to 60 ℃ to completely dissolve the gelatin, and then the gelatin solution was cooled to room temperature;

(2) adding 200 mg of tyrosine (Tyr), 75.0 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 25.0 mg of N-hydroxysuccinimide (NHS) into the gelatin solution obtained in the step (1) by stirring, and after complete dissolution, reacting for 8 hours in a water bath at 37 ℃ by stirring to complete crosslinking;

Example 4

(1) 800 mg of gelatin powder was added to 20 mL of 2- (N-morpholine) ethanesulfonic acid Monohydrate (MES) buffer solution having a concentration of 50mM and pH 6.5, and heated to 60 ℃ to completely dissolve the gelatin, and then the gelatin solution was cooled to room temperature;

(2) adding 50 mg of tyrosine (Tyr), 75.0 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 25.0 mg of N-hydroxysuccinimide (NHS) into the gelatin solution obtained in the step (1) by stirring, and after complete dissolution, reacting for 8 hours in a water bath at 37 ℃ by stirring to complete crosslinking;

(4) and (4) adding 1% hydrogen peroxide by mass into the gelatin-tyrosine solution prepared in the step (3), and stirring to uniformly disperse the hydrogen peroxide in the solution to obtain the hydrogen peroxide-encapsulated injectable hemostatic hydrogel material.

Example 5

(2) adding 100 mg of tyrosine (Tyr), 75.0 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 50.0 mg of N-hydroxysuccinimide (NHS) into the gelatin solution obtained in the step (1) by stirring, and after complete dissolution, reacting for 8 hours in a water bath at 37 ℃ by stirring to complete crosslinking;

(4) and (4) adding 7% hydrogen peroxide by mass into the gelatin-tyrosine solution prepared in the step (3), and stirring to uniformly disperse the hydrogen peroxide in the solution to obtain the hydrogen peroxide-encapsulated injectable hemostatic hydrogel material.

Application and performance verification of injectable hemostatic hydrogel material

Firstly, hemostatic effect verification:

the experimental rats were weighed and recorded, and then anesthetized, and 1 mL of pentobarbital sodium hydrate with a concentration of 60.0 mg/mL was injected per 100 g of the body weight of the rats. The hydrogel prepared in example 1 was applied to anesthetized mice, 100 μ L of hemostatic hydrogel was injected into the skin vessels of the mice, and the blood coagulation state was recorded by photographing.

Further experiments are carried out to explore the hemostasis condition of the hemostatic hydrogel on the mouse wound. 1 cm-long wounds were made on the inner and outer sides of the mouse thigh, and a fixed amount of hydrogen peroxide-encapsulated gelatin-tyramine hydrogel was injected into the wounds.

The blood clotting stopping conditions of the different parts of the mice were recorded by photographing, and the results are shown in fig. 5. In experiments, the hemostatic hydrogel can realize rapid wound blood coagulation after being injected into the inner and outer side wounds of the thigh of the old mouse, and can inhibit blood outflow in a short time. In addition, in the process of injecting the hemostatic hydrogel into the skin vessel of the mouse, the hemostatic hydrogel can inhibit the blood flow in the vessel.

Secondly, verifying the bacteriostatic and bactericidal effects:

the injectable hemostatic hydrogel material is applied to bacteriostasis and sterilization, two bacterial colonies of escherichia coli and staphylococcus aureus are adopted to explore the bacteriostasis property, and the specific steps are as follows:

(1) 10.0 g peptone, 5.0g yeast extract, 5.0g sodium chloride were added to the beaker and dissolved with ultra pure water. Then, this mixed solution was diluted to 1L and the pH was adjusted to about 7.0. Thus, the preparation of the bacteria culture medium is completed;

(2) the preparation process of the culture medium is strictly carried out under aseptic conditions, and after the preparation is finished, the culture medium is sterilized under high pressure at 110 ℃ for 30 min;

(3) the bacteriostatic test of the hemostatic hydrogel adopts an oxford cup method. 15 mL of the culture medium was added to the petri dish and after spreading evenly, it was allowed to solidify. And then, vertically placing the oxford cup on a culture medium of a culture dish under an aseptic condition, respectively adding gelatin, gelatin-tyrosine and gelatin doped with hydrogen peroxide into the oxford cup, and encapsulating the gelatin-tyrosine and hydrogen peroxide solution of the hydrogen peroxide. Then, the culture is carried out for 24 hours in an incubator at 37 ℃, and after being taken out, the size of the inhibition zone is measured by using a ruler.

As shown in the photograph in FIG. 6, the inhibition effect is explored by the Oxford cup method, and the inhibition effect is intuitively reflected by measuring the diameter of the inhibition zone. In fig. 6A, the inhibition effect on escherichia coli is shown, and observation shows that pure gelatin and gelatin-tyrosine material which are not added with hydrogen peroxide have no inhibition effect on escherichia coli, and in addition, compared with a hemostatic hydrogel material which is only added with hydrogen peroxide, the prepared hemostatic hydrogel material has a smaller inhibition zone, so that the dual effects of hydrogen peroxide are further verified, on one hand, the hemostatic hydrogel material participates in the oxidation of the gel material, and on the other hand, the hemostatic hydrogel material has an inhibition effect. The inhibitory effect on staphylococcus aureus shown in fig. 6B, the inhibitory effect on staphylococcus aureus of the different materials was the same trend as the above inhibitory effect on escherichia coli. By the inhibition effect of the hemostatic hydrogel on two different bacterial colonies, the prepared material has a good hemostatic function on wounds on one hand, and has excellent performances such as wound infection inhibition and the like on the other hand.

Third, cytotoxicity test

The cytotoxicity test of the injectable hemostatic hydrogel material is carried out by adopting yeast cells, and the specific steps are as follows:

(1) dispersing quantitative yeast cells into 50 muL of buffer solution, and putting the buffer solution into a 96-well plate for cultivation;

(2) respectively adding 50 mu L of ultrapure water, gelatin and Gel-Tyr hydrogel encapsulating hydrogen peroxide into a 96-well plate, and culturing for 6 h in an incubator at 37 ℃;

(3) the morphology of the yeast cells was observed under a microscope.

The result is shown in fig. 7, the cell morphology of the yeast cells incubated with different materials in the experiment is shown under the microscope, in a, B, and C of fig. 7, it can be clearly observed that the yeast cells are not affected by the materials used, and still have better cell morphology, and this result directly indicates that the prepared hemostatic hydrogel has less toxic effect on the cells. However, it was found that too high a concentration of hydrogen peroxide greatly affects yeast cells, which are disrupted and thus not observed in a microscope, and the result is shown in fig. 7D. It can thus be confirmed that the aforementioned hydrogen peroxide cannot be used in too high a concentration, even though it plays a great role in the hemostasis process.

Fourthly, testing the drug loading and sustained release performance:

the invention relates to an injectable hemostatic hydrogel material loaded with amoxicillin serving as an anti-inflammatory drug and a drug sustained-release effect, which comprises the following specific steps:

(1) adding an amoxicillin drug solution to the Gel-Tyr hydrogel encapsulated with hydrogen peroxide, prepared in the above embodiment 1, and adding a certain amount of blood to coagulate the Gel-Tyr hydrogel;

(2) and then, the condensed hydrogel mixed with the amoxicillin medicine is put into ultrapure water, the amoxicillin medicine is slowly released into the aqueous solution along with the time, and the concentration of the amoxicillin medicine in the aqueous solution is determined by measuring the ultraviolet absorbance value of the amoxicillin medicine.

The slow release behavior of the hemostatic hydrogel to amoxicillin drugs is explored through experiments, and the result is shown in fig. 8. The hemostatic hydrogel containing amoxicillin medicine forms a gel state under the catalysis of hemoglobin, and the drug slow release degree is determined by using the drug absorbance value in ultrapure water. The invention utilizes the dense pores of the drug in the gel state formed by the hemostatic hydrogel catalyzed by hemoglobin and the gradient of the concentration difference of the drug inside and outside the gel to release amoxicillin into ultrapure water, thereby achieving the effect of drug controlled release.

Claims

1. A preparation method of an injectable hemostatic hydrogel material is characterized by comprising the following steps: firstly, adopting EDC/NHS as a cross-linking agent to covalently bond gelatin and tyrosine, and then introducing hydrogen peroxide to prepare an injectable hemostatic hydrogel material;

the method comprises the following steps:

(1) adding powdered gelatin into 2- (N-morpholine) ethanesulfonic acid-MES buffer solution with pH of 6.5 at concentration of 20-40mg/mL, heating to 60 deg.C to completely dissolve gelatin, and cooling to room temperature to obtain gelatin solution;

(2) adding tyrosine (Tyr), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into the gelatin solution obtained in the step (1) in a stirring manner, wherein the mass ratio of the tyrosine to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide is 50-200: 37.0-140: 12.5-50, wherein the tyrosine is added with the concentration of 2.5-10mg/mL, and after the tyrosine is completely dissolved, the tyrosine is stirred and reacts in water bath at 37 ℃ for 8 hours to ensure that the tyrosine is completely crosslinked;

2. The method of claim 1, wherein: in the step (1), gelatin is added with the concentration of 40 mg/mL; the 2- (N-morpholine) ethanesulfonic acid-water (MES) buffer solution is an aqueous solution of 2- (N-morpholine) ethanesulfonic acid-water (MES), the concentration is 20-50mM, and the pH value is 6.5.

3. The method of claim 1, wherein: the concentration of the 2- (N-morpholine) ethanesulfonic acid-water (MES) buffer solution is 50 mM.

4. The method of claim 1, wherein: in the step (2), tyrosine is added with the concentration of 5 mg/ml; the mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide is 3: 1.

5. The method of claim 1, wherein: in the step (3), sodium phosphate is added at a concentration of 15 mg/mL.

6. The method of claim 1, wherein: in the step (4), the mass fraction of the added hydrogen peroxide is 3%.