CN113174063B

CN113174063B - Preparation and application of bioadhesive enhanced temperature-sensitive chitosan-based postoperative adhesion prevention hydrogel

Info

Publication number: CN113174063B
Application number: CN202110481835.3A
Authority: CN
Inventors: 王学清; 王柏裕; 张强; 何冰; 张华�; 代文兵
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2022-03-18
Anticipated expiration: 2041-04-30
Also published as: CN113174063A

Abstract

The invention belongs to the technical field of medicine and health, and particularly relates to preparation and application of a bioadhesive enhanced temperature-sensitive chitosan-based postoperative adhesion-prevention hydrogel, which comprises the following steps: firstly, grafting a hydroxybutyl group on chitosan to prepare hydroxybutyl chitosan with temperature-sensitive property. Secondly, thioglycollic acid is grafted on the chitosan to prepare the thiolated chitosan with the enhanced biological adhesion performance. And thirdly, physically mixing the hydroxybutyl chitosan solution and the thiolated chitosan solution according to a certain proportion to obtain the bioadhesive enhanced temperature-sensitive chitosan-based postoperative adhesion-prevention hydrogel. The product of the invention has the following advantages: the wound surface treatment liquid has reversible temperature sensitive phase change capacity, is liquid with good fluidity at low temperature, is convenient to inject and use, can quickly flow and fit to cover irregular wound surfaces after being injected to the wound surfaces, quickly forms a hydrogel barrier at physiological temperature to isolate the surfaces of adjacent tissues or organs, and prevents postoperative adhesion formation.

Description

Preparation and application of bioadhesive enhanced temperature-sensitive chitosan-based postoperative adhesion prevention hydrogel

Technical Field

The invention relates to a preparation method of a bioadhesive enhanced temperature-sensitive chitosan-based anti-adhesion hydrogel and an application of the bioadhesive enhanced temperature-sensitive chitosan-based anti-adhesion hydrogel in prevention of postoperative adhesion of surgical operations, belonging to the technical field of medicines and sanitation.

Background art:

postoperative adhesion is a common postoperative complication of clinical surgery, and most of the postoperative adhesions occur after surgical operations such as abdominal cavity, joint, pelvic cavity and the like, and the incidence rate is different from 50% to 97%. The adhesion after the surgical operation often seriously harms the health of the patient, greatly reduces the postoperative life quality of the patient, and brings huge mental and emotional stress and heavy economic burden to the patient. Therefore, prevention of post-surgical adhesions is of great importance to patients undergoing surgery.

The existing prevention methods for postoperative adhesion are mainly divided into three categories: (1) improving surgical techniques to reduce surgical trauma; (2) use of anti-inflammatory agents or agents that affect the fibrin formation/degradation balance; (3) the barrier material is used to physically isolate the wound surface, preventing direct contact between adjacent wound tissues and organs, and further preventing the formation of post-operative adhesions. The improved surgical operation technology can reduce the incidence rate of postoperative adhesion to a certain extent, reduce the scale of postoperative adhesion and reduce the morbidity degree of postoperative adhesion, but cannot completely prevent the formation of adhesion; the anti-adhesion medicament has high metabolism and absorption speed in vivo and short retention time, and can influence the normal healing process of the wound; the development and improvement of the current anti-adhesion physical barrier products are the key points for preventing postoperative adhesion.

The anti-adhesion barrier products currently on the market have certain drawbacks: the biological safety is poor, and adverse reactions are reported more after clinical use; the degree of fitting and covering the postoperative wound surface, especially the irregular wound surface, is poor; the biological adhesion is insufficient, and the biological adhesion is easy to move or fall off from an action part, so that the anti-adhesion barrier effect is influenced, or extra suture is needed for fixation, so that extra burden is brought to a patient; the retention time of the action part is too short, and the action part is degraded within 7 days of the key period of postoperative adhesion formation; expensive materials, and difficult burden for patients.

Chitosan is a research hotspot in the field of biomedical polymer materials in recent years. The chitosan-based material has good tissue biocompatibility, biodegradability, appropriate in-vivo retention time, antibacterial activity and anti-inflammatory activity. Meanwhile, the chitosan has better chemical modification potential, and the raw materials are cheap and easy to obtain.

-NH on chitosan molecular chain₂、-C₆And (3) introducing a hydroxybutyl group to the-OH to prepare a chitosan derivative with temperature-sensitive property, namely hydroxybutyl chitosan (HBC). HBC is liquid with good fluidity at the storage temperature slightly lower than room temperature, and is convenient for injectionWhen the hydrogel is injected into a wound, the hydrogel can rapidly flow to cover an irregular wound surface, can rapidly generate phase change at a physiological temperature to form a hydrogel barrier, isolates the surfaces of adjacent tissues or organs near the wound surface, and prevents postoperative adhesion.

-NH on chitosan molecular chain₂Introducing sulfhydryl group through amidation reaction to obtain thiolated chitosan (CS-SH). Sulfydryl on the sulfhydrylation chitosan can form a disulfide bond with protein containing abundant cysteine in a biological mucous membrane through oxidation reaction or sulfydryl-disulfide bond exchange, so that the biological adhesion capability of a gel system is obviously enhanced, the gel can be firmly adhered to a wet and irregular wound surface after operation, and the gel is not easy to fall off and move.

The chitosan-based hydrogel such as hydroxybutyl chitosan, thiolated chitosan and the like is good in mixing compatibility, and the prepared HBC aqueous solution and the CS-SH aqueous solution are physically mixed according to a proper proportion to prepare the bioadhesion enhanced temperature-sensitive chitosan-based anti-adhesion hydrogel HBC/CS-SH which retains the HBC temperature sensitive capability and the CS-SH enhanced bioadhesion capability. Due to the temperature-sensitive property of HBC/CS-SH, the hydrogel can rapidly flow and fit to cover irregular wound surfaces after being injected into the wound surfaces, and a hydrogel barrier is rapidly formed to isolate the surfaces of adjacent tissues or organs; the enhanced biological adhesion capability ensures that the hydrogel can be continuously adhered to the wound surface to play the role of an anti-adhesion physical barrier and is not easy to move or fall off.

In conclusion, the chitosan is subjected to chemical grafting modification to prepare hydroxybutyl chitosan (HBC) and thiolated chitosan (CS-SH), and the hydroxybutyl chitosan solution and the thiolated chitosan solution are mixed according to a certain proportion to prepare the bioadhesive enhanced temperature-sensitive chitosan-based anti-adhesion hydrogel HBC/CS-SH. The bioadhesive enhanced temperature-sensitive chitosan-based anti-adhesion hydrogel prepared by the method has excellent performance and can meet the requirement of postoperative anti-adhesion.

Disclosure of Invention

The invention aims to develop a hydrogel material with ideal postoperative anti-adhesion effect aiming at the defects of the current commercial anti-adhesion products. -NH on chitosan molecular chain₂、-C₆A hydroxybutyl group is introduced to-OH to prepare the product with temperature-sensitive propertyHydroxybutyl chitosan (HBC), the-NH group on the chitosan molecular chain₂Sulfhydryl groups are introduced to prepare thiolated chitosan CS-SH, and the prepared HBC aqueous solution and the CS-SH aqueous solution are physically mixed according to a proper proportion to prepare the bioadhesion enhanced temperature-sensitive chitosan anti-adhesion hydrogel HBC/CS-SH which retains the temperature sensitive capability of HBC and the bioadhesion capability enhanced by CS-SH.

The preparation process of the hydrogel mainly comprises the following steps:

(1) preparation of hydroxybutyl chitosan (HBC)

1) Alkalization: putting Chitosan (CS) into potassium hydroxide or sodium hydroxide aqueous solution, fully dispersing and alkalizing for 12-24h for later use; 2) isopropyl alcohol/water system dispersion: mixing isopropanol and deionized water to obtain isopropanol aqueous solution, and stirring and dispersing the alkalized chitosan with the isopropanol aqueous solution for 12-24 h; 3) grafting reaction: adding 1, 2-butylene oxide into the solution, heating and stirring in water bath to perform grafting reaction at the reaction temperature of 40-80 ℃ for 12-36h, and 4) adjusting pH and dialyzing: after the grafting reaction, adjusting the pH value to 5.0-7.0 by acid, and putting the reaction liquid into a dialysis bag with the molecular cut-off of 8000-14000Da for dialysis; 5) and (3) freeze drying: after dialysis, freeze-drying the product, and storing the freeze-dried product at low temperature for later use;

(2) preparation of thiolated chitosan (CS-SH)

1) Preparing a system I: weighing N-hydroxysuccinimide (NHS), dissolving NHS in N, N-Dimethylformamide (DMF), and dropwise adding thioglycolic acid (TGA) for later use; preparing a mixed solvent of DMF and water, and fully dissolving 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC); slowly dripping the EDAC solution into DMF (dimethyl formamide) solutions of NHS (polyethylene glycol succinate) and TGA (TGA) at a constant speed, and stirring overnight in a dark place after dripping is finished to obtain a system I;

2) preparing a system II: weighing chitosan, adding 1M HCl solution to fully acidify and swell the chitosan, adding deionized water after swelling, and continuously stirring to fully dissolve the chitosan;

3) mixing reaction of a system I and a system II: after the chitosan raw material is completely dissolved, slowly dripping the system I solution system into the system II solution at a constant speed under the condition of continuous stirring, adjusting the pH of the mixed system to 4.0-6.0 by using 1M NaOH solution after finishing dripping, and continuously stirring for reaction;

4) adjusting pH and dialyzing: putting the reaction system into a dialysis bag with the cut-off molecular weight of 8000-14000Da, firstly dialyzing by using an HCl solution system, then dialyzing by using an HCl solution system containing NaCl, and finally dialyzing by using the HCl solution system;

5) and (3) freeze drying: after dialysis, the product is freeze-dried for later use;

(3) preparation of bioadhesive enhanced temperature-sensitive chitosan-based anti-adhesion hydrogel

Mixing the HBC solution and the CS-TGA solution according to the mixing ratio v (HBC) to v (CS-TGA) of 6-1:0.5-2, carrying out vortex oscillation after mixing, and obtaining the hydrogel HBC/CS-TGA after standing and eliminating bubbles.

Wherein, the thiolation reagent used in the thiolation of the chitosan in the step (2) is thioglycolic acid, and the thiolation reagent used in the method includes, but is not limited to, thioglycolic acid (TGA), Glutathione (GSH), Mercaptoethylamine (MEA), cysteine (Cys), N-acetylcysteine (NAC), etc.

Preferably, the preparation method of the hydrogel comprises the following steps:

(1) preparation of hydroxybutyl chitosan

1) Alkalization: adding Chitosan (CS) into potassium hydroxide or sodium hydroxide aqueous solution, fully dispersing and alkalizing for 12-24h,

2) isopropyl alcohol/water system dispersion: mixing isopropanol and deionized water to obtain isopropanol water solution, and stirring to disperse alkalized chitosan at isopropanol-deionized water ratio of 6-1:0.5-2 for 12-24 hr;

3) grafting reaction: adding 1, 2-butylene oxide into the solution, heating in a water bath and stirring to perform a grafting reaction, wherein the using amount of the 1, 2-butylene oxide is 0.5-2g, 40-160mL, the reaction temperature is 40-80 ℃, and the reaction time is 12-36 h;

4) adjusting pH and dialyzing: after the grafting reaction, 1M HCl is used for adjusting the pH value to 5.0-7.0, and the reaction liquid is put into a dialysis bag with the molecular cut-off of 8000-;

5) and (3) freeze drying: after dialysis, freeze-drying the product, and storing the freeze-dried product at low temperature for later use;

(2) preparation of thiolated chitosan

1) Preparing a system I: weighing N-hydroxysuccinimide (NHS) according to the proportion of 2-8g:0.5-2g relative to the amount of chitosan, dissolving NHS in N, N-Dimethylformamide (DMF) according to the proportion of 0.5-2g:5-20mL, and dropwise adding thioglycolic acid (TGA) according to the proportion of 0.5-2g:1-4mL relative to the amount of chitosan; preparing a mixed solvent of DMF and water in a volume ratio of 1:1 to 1:6, and fully dissolving 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) by using the mixed solvent, wherein the using amount ratio of the DMF to the water is 0.5-2g:5-20 mL; slowly dripping the EDAC solution into DMF (dimethyl formamide) solutions of NHS (polyethylene glycol succinate) and TGA (TGA) at a constant speed, and stirring overnight in a dark place after dripping is finished to obtain a system I;

2) preparing a system II: weighing chitosan according to the dosage of 0.5-2g:1-4mL of thioglycolic acid (TGA), adding 1M HCl solution to fully acidify and swell the chitosan for 2-6h, adding deionized water after swelling, and continuously stirring to fully dissolve the chitosan;

3) mixing reaction of a system I and a system II: after the chitosan raw material is completely dissolved, slowly dripping the system I solution system into the system II solution at a constant speed under the condition of continuous stirring, adjusting the pH of the mixed system to 4.0-6.0 by using 1M NaOH solution after finishing dripping, and sealing and keeping away from light for continuous stirring reaction at the temperature of 4 ℃;

4) adjusting pH and dialyzing: putting the reacted reaction system into a dialysis bag with the cut-off molecular weight of 8000-14000Da, clamping by a dialysis clamp, firstly dialyzing for 1-3 times by using a 1M HCl solution system, then dialyzing for 1-3 times by using a 1mM HCl solution system containing NaCl, and finally dialyzing for 1-2 times by using a 0.2mM HCl solution system;

5) and (3) freeze drying: after dialysis, freeze-drying the product, and sealing and storing the freeze-dried product in a refrigerator for later use;

And (3) mixing the hydroxybutyl chitosan HBC solution in the step (1) with the CS-TGA solution in the step (2) according to the mixing ratio of 6-1:4-0.5, performing vortex oscillation after mixing, and standing to eliminate bubbles to obtain the hydrogel HBC/CS-TGA.

Further preferably, the preparation method of the hydrogel comprises the following steps:

(1) preparation of hydroxybutyl chitosan

1) Alkalization: adding Chitosan (CS) into 50% (w/v) potassium hydroxide or sodium hydroxide aqueous solution, fully dispersing and alkalizing for 12-24h, wherein the dosage ratio of alkali liquor is 1g:50 mL;

2) isopropyl alcohol/water system dispersion: mixing isopropanol and deionized water to obtain isopropanol aqueous solution, and stirring to disperse the alkalized chitosan at a mixing ratio of 3:1-1:1 and a dispersing ratio of 1g:80mL for 12-24 h;

3) grafting reaction: adding 1, 2-butylene oxide into the solution, heating in a water bath and stirring to perform a grafting reaction, wherein the using amount of the 1, 2-butylene oxide is 1g, 80mL, the reaction temperature is 40-80 ℃, and the reaction time is 12-36 h;

4) adjusting pH and dialyzing: after the grafting reaction, adjusting the pH value to 5.0-7.0 by using 1M HCl, and putting the reaction liquid into a dialysis bag with the molecular cut-off of 8000-;

(2) preparation of thiolated chitosan

1) Preparing a system I: weighing N-hydroxysuccinimide (NHS) according to the relative dosage of 4g:1g with chitosan, dissolving NHS by using N, N-Dimethylformamide (DMF) according to the ratio of 1g:10mL, and dropwise adding thioglycolic acid (TGA) according to the relative dosage of 1g:2mL with chitosan; preparing a mixed solvent of DMF and water in a volume ratio of 1:1 to 1:3, and fully dissolving 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) by using the mixed solvent, wherein the using amount ratio of the two is 1g:10 mL; slowly dripping the EDAC solution into DMF (dimethyl formamide) solutions of NHS (polyethylene glycol succinate) and TGA (TGA) at a constant speed, and stirring overnight in a dark place after dripping is finished to obtain a system I;

2) preparing a system II: weighing chitosan according to the dosage of 1g:2mL of thioglycollic acid (TGA), adding 1M HCl solution to fully acidify and swell the chitosan for 2-6h, wherein the addition is 1g:5mL, adding deionized water after swelling, and continuously stirring to fully dissolve the chitosan, wherein the addition of the deionized water is 1g:50 mL;

3) mixing reaction of a system I and a system II: after the chitosan raw material is completely dissolved, slowly dripping the solution system of the system one into the solution of the system two at a constant speed under the condition of continuous stirring, adjusting the pH of the mixed system to 4.0-6.0 by using a 1M NaOH solution after finishing dripping, sealing and keeping out of the sun under the condition of 4 ℃, and continuously stirring and reacting for 4-5 hours;

4) adjusting pH and dialyzing: putting the reacted reaction system into a dialysis bag with the cut-off molecular weight of 8000-14000Da, clamping by a dialysis clamp, dialyzing for 2 times by using a 1M HCl solution system, then dialyzing for 2 times by using a 1mM HCl solution system containing 1% NaCl, and finally dialyzing for 1 time by using a 0.2mM HCl solution system;

5) and (3) freeze drying: after dialysis, freeze-drying the product, and sealing and storing the freeze-dried product in a refrigerator at the temperature of-20 ℃ for later use;

Mixing an HBC aqueous solution with a certain proportion of 2.5-4.5% (w/v) and a CS-TGA aqueous solution with a certain proportion of 1.5-3% (w/v), wherein v (HBC) and v (CS-TGA) are respectively 3:1-2:1, mixing, performing vortex oscillation, and standing to eliminate bubbles to obtain the bioadhesion enhanced type temperature-sensitive chitosan-based anti-adhesion hydrogel HBC/CS-TGA.

It is another object of the present invention to provide the hydrogel as a medical material for preventing adhesion after surgery.

The hydrogel is used for preventing postoperative adhesion, including postoperative adhesion of abdominal cavity, joint, pelvic cavity, etc.

Hydrogels serve as physical barrier materials for post-surgical adhesions.

The biological adhesion enhanced temperature-sensitive chitosan-based anti-adhesion hydrogel can be used as a physical barrier material for preventing the adhesion after surgical operations such as abdominal cavity and the like.

The invention has the advantages that:

(1) no toxicity, good biological safety, no adverse reaction such as inflammation and infection;

(2) the biodegradable film is biodegradable, has proper degradation speed, and can not be degraded in the key period of adhesion formation after 0-7 days;

(3) the hydrogel has temperature-sensitive performance, is liquid with good fluidity at the storage temperature slightly lower than the room temperature, is convenient to inject and use, can quickly flow to cover irregular wound surfaces when being injected into a wound, can quickly generate phase change at the physiological temperature to form a hydrogel barrier, isolates the surfaces of adjacent tissues or organs near the wound surfaces, and prevents postoperative adhesion;

(4) has enough biological adhesion, can firmly cover the wet irregular wound surface, and is not easy to move or fall off from the action part;

(5) easy acquisition of raw materials, low cost and the like.

Description of the drawings:

FIG. 1 shows an infrared spectrum of hydroxybutyl chitosan (HBC)

FIG. 2, change in HBC solution phase change time with concentration at 37 ℃

FIG. 3 is an infrared spectrum of thiolated chitosan (CS-TGA)

FIG. 4 shows reversible temperature-sensitive phase transition capability of HBC/CS-TGA

FIG. 5 is a graph showing the change of HBC/CS-TGA phase transition time at 37 ℃ in accordance with the volume mixing ratio

FIG. 6 shows the rheological curve of HBC/CS-TGA of bioadhesive-enhanced temperature-sensitive chitosan gel

FIG. 7, maximum adhesion and adhesion energy comparison of HBC hydrogel and HBC/CS-TGA hydrogel; a is maximum adhesion force contrast, b is adhesion energy contrast; indicates that there is a significant difference (P <0.01) between HBC/CS-TGA and HBC.

FIG. 8, cytotoxicity of gel extracts on L929 cells and HUVEC cells. a is the absorbance at 450nm for each group on L929 cells and b is the absorbance at 450nm for each group on HUVEC cells.

Positive: a positive control group;

negative: a negative control group;

a: 100% HBC leach liquor group;

b: a 75% HBC leach liquor group;

c: a 50% HBC leaching liquor group;

d: 25% HBC extract group;

e: 100% HBC/CS-TGA leaching liquor group;

f: 75% HBC/CS-TGA leaching liquor group;

g: a 50% HBC/CS-TGA leach liquor group;

h: 25% HBC/CS-TGA extract panel.

FIG. 9, rat dorsal gel subcutaneous injection degradation experiment

A-E is the gel residue on the back of the rat from week 1 to week 5

FIG. 10 shows the experiment of adhesion prevention in rat cecum-abdominal wall adhesion model

A. The results of postoperative adhesion in rats in the blank group (normal feeding without laparotomy) showed no adhesion in all rats;

B. the result of the postoperative adhesion condition of rats in a negative control group (carrying out cecal-abdominal wall postoperative adhesion model modeling and injecting physiological saline) shows that only one rat in 6 rats is moderate adhesion, and other 5 rats are severe adhesion or more;

C. the results of the postoperative adhesion conditions of rats in the control group 1 (subjected to cecum-abdominal wall postoperative adhesion model modeling, and injected with 2.67% HBC hydrogel at a dose of 5 mL/kg) show that only 2 rats have no postoperative adhesion, 1 rat has moderate adhesion, and 3 rats have severe adhesion;

D. the results of the postoperative adhesion conditions of rats in the control group 2 (subjected to cecum-abdominal wall postoperative adhesion model modeling, and injected with 3.33% HBC hydrogel at a dose of 5 mL/kg) show that only 1 rat among 6 rats has no postoperative adhesion, 1 rat has mild adhesion, 2 rats has moderate adhesion, 1 rat has severe adhesion, and 1 rat has more than severe adhesion;

E. the results of the postoperative adhesion conditions of rats in the control group 3 (a model of the postoperative adhesion model of cecum-abdominal wall is made, and commercially available medical chitosan anti-adhesion solution is injected according to the dose of 5 mL/kg) show that only 1 rat among 6 rats has no adhesion, 1 rat has slight adhesion, 3 rats has moderate adhesion, and 1 rat has more than severe adhesion;

F. the results of the postoperative adhesion conditions of rats in an experimental group (carrying out cecal-abdominal wall postoperative adhesion model modeling, injecting HBC/CS-TGA hydrogel according to 5 mL/kg) show that only 1 rat in 6 rats has slight adhesion, and other 5 rats have no postoperative adhesion;

G. the results of the rats in the low-dose experimental group (subjected to cecum-abdominal wall postoperative adhesion model modeling, and injected with HBC/CS-TGA hydrogel at a dose of 2 mL/kg) show that only 2 rats have moderate adhesion, and 4 rats have no postoperative adhesion.

FIG. 11 shows the experimental group shown in FIG. 10, and the statistical results A-G of the adhesion degree of the adhesion prevention experiment on the rat cecum-abdominal wall adhesion model.

FIG. 12, rat cecum-abdominal cavity wall adhesion model anti-adhesion experiment on various groups of rat injured cecum tissue HE staining sheets

A-G refer to the experimental groups as in FIG. 10.

FIG. 13 shows the TGF- β 1 concentrations A-G in the serum of rats in each group of rat caecum-abdominal wall adhesion model experiment, which is the same as FIG. 10.

Denotes p <0.01 compared to B; # denotes p <0.05 compared to F; # indicates p <0.01 compared to F.

FIG. 14, IL-6 concentration in rat injured cecum tissue grinding fluid of each group of rat cecum-abdominal cavity wall adhesion model

A-G refer to the experimental groups as in FIG. 10.

Denotes p <0.01 compared to B.

Detailed Description

The invention is further illustrated and described by the following specific examples, which are not to be construed as limiting the invention thereto.

Example 1 Synthesis of hydroxybutyl Chitosan

Alkalization: respectively weighing 300mg of chitosan with molecular weight of 1000kD, 600kD and 100kD, placing the chitosan into a 50mL conical flask, adding 50% (w/v) potassium hydroxide aqueous solution, stirring, fully swelling for 2h, and stirring and alkalizing for 24h at room temperature.

Isopropyl alcohol/water system dispersion: and (3) filtering the alkalized chitosan to remove redundant alkali liquor, and standing for 12 hours at the temperature of 4 ℃. Mixing 10mL of isopropanol and 10mL of deionized water according to the volume ratio of 1:1 to obtain an isopropanol aqueous solution, adding the alkalized and standing chitosan into the isopropanol aqueous solution with the volume ratio of 1:1, and stirring for 24 hours at room temperature until the alkalized chitosan is completely dispersed in an isopropanol/water system.

Grafting reaction: dropwise adding 20mL of 1, 2-epoxybutane into the isopropanol/water system dispersed with the alkalized chitosan while stirring, and stirring and reacting in a water bath at 60 ℃ for 24 hours after the dropwise adding is finished.

Adjusting pH and dialyzing: heating in water bath at 60 deg.C, stirring, standing to room temperature, dropwise adding 1M HCl solution until pH of the reaction solution is neutral, loading the reaction solution into dialysis bag with molecular weight cutoff of 8000-14000Da, dialyzing with deionized water for more than 6 times at dialysis interval of 4-6 h.

And (3) freeze drying: and (3) subpackaging the product with a plurality of 50mL centrifuge tubes after dialysis is finished, wherein each tube contains about 20mL, sealing and puncturing a PALA membrane, pre-freezing at-80 ℃ for 12h, and freeze-drying for 72h to obtain the product HBC.

Example 2 Infrared Spectrometry of hydroxybutyl Chitosan

Weighing appropriate amount of HBC at relative humidity<Grinding with KBr powder at 70% room temperature, placing into sample cup, tabletting, and subjecting to 400cm with Fourier transform infrared spectrometer at 4000-^-1An in-range scan. The infrared spectrum of HBC is shown in FIG. 1. 3400cm^-1The strong absorption peak superposed by the left-OH and right-N-H vibration absorption peaks is the characteristic peak of chitosan, and the characteristic peak of chitosan is retained after the hydroxybutyl is introduced. 2870cm^-1-2980cm^-1three-C-H vibration absorption peaks and 1463cm in the hydroxybutyl carbon chain between^-1Nearby newly introduced-CH on hydroxybutyl group₃The absorption peak of (2) is a characteristic peak of HBC. Due to the newly introduced hydroxybutyl group, at 1155cm^-1nearby-C₆The characteristic band of-OH weakens and almost disappears. HBC infrared spectra prepared from chitosan with different molecular weights prove that hydroxybutyl groups are successfully grafted to a chitosan molecular chain, and HBC with different molecular weights are successfully prepared.

Example 3 determination of the concentration of hydroxybutyl Chitosan gel solution used

The HBC phase change time is measured by a test tube method, namely 1mL of hydrogel is added into a 10mL centrifuge tube and placed into a 37 ℃ water bath, whether phase change occurs or not is observed every 5s, the phase change standard is that the centrifuge tube is placed on a table upside down, HBC in the centrifuge tube does not flow down along the tube wall within 30s, and the time that the centrifuge tube is soaked in the water bath when the centrifuge tube is taken out from the water bath is the phase change time of the temperature-sensitive gel, and the phase change time is accurate to 5 s.

Preparing 1.5-5% (w/v) HBC aqueous solution, observing the condition that HBC solution with different concentrations turns into gel in water bath at 37 ℃ according to the operation of the method, recording the phase-change time, and drawing a curve of the phase-change time along with the concentration change, as shown in figure 2. With the gradual increase of the concentration of the HBC solution, the phase change time at 37 ℃ is gradually shortened, but the change amplitude is smaller and smaller, the trend of the change is toward equilibrium, and the fluidity of the hydroxybutyl chitosan solution is reduced when the concentration is increased in practical use, so that the use concentration of the HBC is determined to be 2.5-4.5% (w/v) in order to take the fluidity of the temperature-sensitive gel into consideration.

Example 4 Synthesis of thiolated Chitosan (CS-TGA)

Preparing a system I: 1.2g N-hydroxysuccinimide (NHS) was weighed into a 50mL Erlenmeyer flask, dissolved in 6mL DMF under stirring, and 600. mu.L thioglycolic acid (TGA) was added dropwise with stirring. 2.1g of EDAC are weighed into a 50mL LEP tube and the volume ratio of 10mL of DMF to water is 1:1 in ice bath, EDAC was dissolved well. And (3) after the EDAC is fully dissolved, slowly dropwise adding the EDAC solution into the mixed DMF solution of NHS and TGA at a constant speed, stirring while dropwise adding, and stirring overnight in a dark place after dropwise adding to obtain a system I.

Preparing a system II: weighing 300mg of chitosan into a 100mL conical flask, adding 5mL of 0.1M HCl solution to fully acidify and swell the chitosan for 4h, adding 25mL of deionized water after swelling, and continuously stirring to fully dissolve the chitosan.

Mixing reaction of a system I and a system II: after the chitosan raw material is completely dissolved, slowly dripping the system-solution system into the chitosan diluted acid solution at a constant speed under the condition of continuous stirring, adjusting the pH value of the mixed system to 5.0 by using 1M NaOH solution after dripping is finished, sealing and keeping away from light to continuously stir for reaction for 4-5h at the temperature of 4 ℃.

Adjusting pH and dialyzing: the reaction system after 4-5h reaction was placed in a dialysis bag with molecular cut-off of 8000-14000Da and clamped with a dialysis clamp, and was dialyzed 2 times with 1M HCl solution system, 2 times with 1mM HCl solution system containing 1% NaCl, and 1 time with 0.2mM HCl solution system. The dialysis interval is 4-6 h.

And (3) freeze drying: and (3) after dialysis, packaging the product in a plurality of 50mL centrifuge tubes, packaging each tube with about 20mL, sealing the openings of the tubes by using a PALA membrane, pricking the openings, pre-freezing the tubes at-80 ℃ for 12h, and freeze-drying the tubes for 72h to obtain a freeze-dried product, namely CS-TGA.

EXAMPLE 5 Infrared Spectroscopy of thiolated Chitosan

Vacuum drying the appropriate amount of CS-TGA overnight at relative humidity<Grinding with KBr powder at 70% room temperature, placing into a sample cup, tabletting, and subjecting to 400cm with a Fourier transform infrared spectrometer at 4000-^-1An in-range scan. From FIG. 3, it can be seen that 1650cm^-1And 1590cm^-1An amido bond characteristic peak appears nearby, which indicates that amido bonds exist in CS-TGA and CS-NAC and amidation grafting reaction occurs; 2500cm^-1The absorption peaks appearing on the left and right are stretching vibration absorption peaks of sulfydryl, and prove that sulfydryl exists in CS-TGA and the expected results are completely consistent. The infrared spectrum of CS-TGA proves that thioglycolic acid is grafted to a chitosan molecular chain by forming an amido bond, and CS-TGA is successfully prepared.

Example 6 thiolated Chitosan (CS-TGA) gel solutions determination Using concentration

The prepared CS-TGA can be dissolved in deionized water to obtain 0.5-3.5% (w/v) of gel liquid. When CS-TGA is dissolved, the CS-TGA is difficult to completely dissolve at the beginning when the concentration is too high, and the solution has relatively high viscosity and relatively poor fluidity, so that the CS-TGA can be completely dissolved in water and has good fluidity so as to be convenient for injection and flow to cover irregular wound surfaces, and the CS-TGA is determined to be used at a concentration of 1.5-3% (w/v).

Example 7 preparation of bioadhesive-enhanced temperature-sensitive chitosan-based anti-adhesion hydrogel

Mixing 2.5-4.5% (w/v) HBC solution and 1.5-3% (w/v) CS-TGA solution, vortex vibrating, standing to eliminate bubbles to obtain the bioadhesion enhanced temperature-sensitive chitosan-based anti-adhesion hydrogel HBC/CS-TGA. As shown in fig. 4.

Example 8 determination of the mixing ratio of HBC solution and CS-TGA solution

Preparation of HBC/CS-TGA gel 1mL at a mixing ratio v (HBC): v (CS-TGA) of 4:1 to 1.5:1 was put into a 10mL centrifuge tube, and three portions were prepared in parallel at each ratio, and the phase transition time in a water bath at 37 ℃ was measured. The method for measuring the phase transition time is also called a test tube method. Finally, the curve of the HBC/CS-TGA phase transition time of different v (HBC) and v (CS-TGA) mixing ratios at 37 ℃ along with the change of the volume mixing ratio is drawn according to the measured data, and is shown in figure 5.

The actual concentration of HBC is gradually reduced with the increasing CS-TGA ratio, and the phase transition time is gradually prolonged at 37 ℃, especially when the mixing ratio v (HBC)/v (CS-TGA) is changed from 2:1 to 1.5:1, the phase transition time is obviously prolonged. CS-TGA provides free sulfydryl for the HBC/CS-TGA hydrogel, the higher the CS-TGA content is, the higher the sulfydryl content in the HBC/CS-TGA gel is, the stronger the mucous membrane adhesion capability is theoretically, the temperature-sensitive phase change capability and the mucous membrane adhesion capability of the HBC/CS-TGA are comprehensively considered, and the volume mixing ratio v (4% HBC): v (2% CS-TGA) for preparing the HBC/CS-TGA is determined to be 3:1-2: 1.

Example 9 rheological measurements

The curves of the elastic modulus G 'and the viscous modulus G' of HBC/CS-TGA as a function of temperature were determined by a rotational rheometer. The rotational rheometer model was MCR301, manufactured by Thermo Fisher Scientific, Germany. During testing, 400 mu L of gel solution is dripped on a rheometer test circular platform, and the parameters of a rotary rheometer are as follows: 70 points are taken, the temperature range is 10 ℃ to 40 ℃, the temperature rise time is 2500s, the CS stress is 0.5pa, the frequency is 1Hz, and the rotating speed is 6.28 rad/s. The instrument is started and the rheometer will automatically record rheological data while running. FIG. 6 is a HBC/CS-TGA rheology curve. The elastic modulus G 'and the viscous modulus G' of the HBC/CS-TGA are gradually increased along with the increase of the temperature, and the change speed of the elastic modulus G 'is larger than that of the viscous modulus G', which shows that the curve is gradually changed from G 'larger than G' to G 'larger than G' along with the increase of the temperature, and the phase change of the HBC/CS-TGA from liquid to solid gel is proved. From FIG. 6, it can be seen that the HBC/CS-TGA phase transition temperature is around 27 ℃.

Example 10 measurement of bioadhesive force

In the experiment, the physical property instrument is used for measuring the biological adhesion capability of the HBC/CS-TGA hydrogel to the caecum and abdominal cavity wall of the SD rat, and the biological adhesion capability is compared with that of the HBC hydrogel. The physical property instrument model is TA.XTPlus, produced by Stable Micro Systems company in UK. Firstly, abdominal cavity of SD rat is opened, the inner wall of abdominal cavity of rat is cut into rectangle of 1.5cm multiplied by 2.5cm, then the outer wall of caecum of the same rat is cut into square of 1cm multiplied by 1cm, and then the rat is cleaned by PBS solution with pH value of 7.4. Secondly, placing a double-faced adhesive tape at the lower end of a probe of the physical property instrument, and firmly adhering the outer wall of the rat cecum to the double-faced adhesive tape below the probe; fixing the abdominal cavity inner wall of the rat on a culture dish with a cover filled with warm water at 37 ℃ by using a double-faced adhesive tape, and injecting a hydrogel solution on the abdominal cavity inner wall of the rat on the culture dish cover during an experiment; the petri dish filled with warm water at 37 ℃ was used to keep the temperature of the abdominal wall of the rat above the phase transition temperature of the gel. Thirdly, setting the parameters of a driving program of the physical property instrument: the loading arm is lowered at a rate of 0.1mm/s until a pressure of 2N is reached between the cecal wall and the probe, held for 2.5min, and then the measuring arm is raised at a rate of 10mm/s until the adherent reagent and the biofilm are separated. The test was started by dropping 200. mu.L of the gel solution onto the inner abdominal wall of the fixed rat. The test results are displayed on a display as an adhesion-displacement distance curve by the analysis software. The area under the curve (AUC) of the adhesion force-displacement distance curve is the adhesion energy (unit: mJ).

The maximum adhesion force and the adhesion energy of the HBC/CS-TGA hydrogel measured by the physicometer are shown in FIG. 7. We can see that the maximum adhesion of the HBC/CS-TGA hydrogel is significantly enhanced compared to the HBC hydrogel, with P < 0.01; the adhesion energy is also significantly enhanced, P < 0.01.

EXAMPLE 11 cytotoxicity of hydrogel extracts

First step, preparation of hydrogel DMEM leach solution: adding the HBC/CS-TGA hydrogel or the HBC hydrogel solution into a DMEM culture solution according to the volume ratio of 1:4, standing and incubating for 24 hours in a constant-temperature incubator at 37 ℃ to obtain hydrogel DMEM culture solution leaching liquid, and sucking supernatant of the leaching liquid as gel leaching liquid with the concentration of 100%. The gel 100% leaching liquor is diluted into three leaching liquor with different concentrations of 75%, 50% and 25% by using DMEM culture solution in a gradient manner.

Secondly, cell preparation: resuscitating mouse fiberVitamin cells (L929) and Human Umbilical Vein Endothelial Cells (HUVEC), culturing L929 cells and HUVEC cells at 37 deg.C and relative humidity of 90% and CO₂The cell culture solution was changed every two days in a 5% thermostatted cell incubator. Culturing until the confluence degree of the cells reaches about 90%, digesting and passaging the cells by using 0.25% pancreatin/0.02% EDTA, and passaging the cells until the cells which are free of pollution and grow well are obtained. Adding 5X 10 of the culture plate into each well of 96-well culture plate⁴Cell suspension 100. mu.L/mL.

Thirdly, setting the experimental group for the control group:

negative control group: adding 100 mu L of DMEM culture solution into each well;

positive control group: adding 100mL of DMEM culture solution containing 0.5% phenol into each well;

experimental groups: adding 100 μ L of 25%, 50%, 75%, 100% hydrogel leaching solution into each well;

parallel controls of 6 wells per group

Fourthly, the cell culture method comprises the following steps: putting the 96-well plate into a constant-temperature cell culture box for culture, replacing culture solution after 24h, and observing the growth conditions of the L929 cells and the HUVEC cells under a microscope after 48 h;

step five, CCK-8 detection:

(1) sucking out the culture solution in each hole of each group, and rinsing for three times by PBS;

(2) preparing 10% CCK-8 solution by using DMEM;

(3) add 100. mu.L 10% CCK-8 solution into each well;

(4) incubation was continued for 2 hours in the cell incubator;

(5) measuring absorbance of each hole of the 96-hole plate at 450nm by using an enzyme-labeling instrument;

(6) the relative proliferation rate (RGR) of the cells in each well was calculated from the measured absorbance.

Relative proliferation rate RGR is the average value of absorbance in experimental group/the average value of absorbance in negative control group × 100%

Evaluating the toxicity degree of the material according to national evaluation standards of medical instrument biology, wherein the result standard is as follows:

the material is qualified after the toxicity degree is graded to be 0-1; the grade 2 can comprehensively evaluate whether the cells are qualified or not by combining the cell morphology and the cell growth density; grade greater than 3 is rejected.

TABLE 1 grading Scale for toxicity degrees of materials

The results of CCK-8 cytotoxicity experiments on L929 cells and HUVEC cells are shown in FIG. 8, and the RGR values and material toxicity scores thereof are shown in tables 2 and 3. As can be seen from Table 2, except for the 25% HBC extract group, which was grade 1, each of the remaining groups was grade 0, and the safety of the HBC/CS-TGA hydrogel and the HBC hydrogel on the L929 cell line was confirmed to be acceptable. As can be seen from Table 3, all material groups were rated 1, demonstrating that HBC/CS-TGA gels and HBC gels were safe and acceptable on HUVEC cell lines. The results show that the HBC and the HBC/CS-TGA gel have good biocompatibility and meet the biosafety requirement of being an ideal anti-adhesion material.

TABLE 2 RGR values and grade of toxicity for groups of L929 cell CCK-8 cytotoxicity assays

TABLE 3 RGR values and grade of toxicity for various groups of HUVEC cell CCK-8 cytotoxicity experiments

Example 12 hydrogel in vivo degradation experiment

In order to verify the degradation speed of the HBC/CS-TGA hydrogel, the 2.67% HBC hydrogel and the 3.33% HBC hydrogel in a rat body, a hydrogel subcutaneous injection degradation experiment is carried out on the back of a female SD rat, and the number of injection sites 1,2, 3 and 4 are respectively numbered in the upper left part, the upper right part, the lower left part and the lower right part by taking the spinal column of the back of the rat as a central axis. We also prepared HBC/CS-NAC hydrogel by replacing CS-TGA with N-acetyl-L-cysteine grafted thiolated chitosan CS-NAC to verify whether the type of thiolated chitosan has an effect on the degradation rate in vivo. 1. No. 2, 3 and 4 injection site injection hydrogel and the dosage thereof are as follows:

position 1 was injected with 200 μ L of HBC/CS-NAC hydrogel;

position 2 was injected with 200 μ L of HBC/CS-TGA hydrogel;

position 3 was injected with 200 μ L of 2.67% HBC hydrogel;

position 4 200 μ L of 3.33% HBC hydrogel.

About 200g female SD rats 18 were divided into 6 groups of 3 animals each, and injected with corresponding hydrogel as shown in FIG. 10, and injected with 1mL syringe (needle size 0.45X 16TWLB) to ensure consistent and accurate gel amount. After the first week the first group of rats was sacrificed, the dorsal cortex was cut open and the gel degradation at each injection site was observed, and in the second week the second group of rats was sacrificed, and so on, until the hydrogels of all injection sites in the group were completely degraded.

FIG. 9 shows the skin of the back of SD rats cut off each week to observe the degradation of the hydrogel injected at injection sites No. 1,2, 3 and 4. The residual gel under the skin of the rat is circled in the figure, and we find that the injection sites No. 1,2, 3 and 4 in the first week have obvious gel block residual, the gel blocks at the injection sites No. 1,2, 3 and 4 in the second week become smaller, the gel residual quantity of 4 injection sites in the third week is smaller, 2.67 percent of HBC hydrogel of the site No. 3 in the fourth week is completely degraded, the gel residual quantity of other sites is also less, and the hydrogel injected at the sites No. 1,2, 3 and 4 in the fifth week is completely degraded. This experiment shows that the in vivo complete degradation time of HBC/CS-NAC hydrogel, HBC/CS-TGA hydrogel and 3.33% HBC hydrogel is between 28 and 35 days, and the in vivo degradation time of the lower 2.67% HBC hydrogel is between 21 and 28 days. Compared with the HBC/CS-TGA hydrogel and the HBC/CS-NAC hydrogel, the gel residue size is approximate in each week, which indicates that the difference of the thiolated chitosan species does not cause the difference of the in vivo degradation time. The key period of postoperative adhesion formation is 0-7d after operation, and the experiment shows that the HBC/CS-TGA mixed hydrogel has proper in-vivo degradation speed, can not be degraded in the key period of postoperative adhesion formation of 0-7d, and meets the requirement of the degradation time of an ideal anti-adhesion material.

Example 13 model creation of rat cecum-Abdominal wall postoperative adhesion model and HE staining observation of injured cecum tissue

Firstly, injecting a chloral hydrate solution (10 percent, w/v) into the abdominal cavity to anaesthetize the rat, wherein the anaesthetization dose is 0.3mL/100 g;

secondly, fixing the rat in a supine position after the rat is anesthetized, properly trimming the skin of the abdomen of the rat, preventing the skin from falling into the abdominal cavity during subsequent abdominal opening, and disinfecting the abdomen of the rat by 75% alcohol;

thirdly, cutting the outer skin of the rat at the midline of the lower abdomen of the rat by using a sterilized surgical scissors, cutting the inner skin of the abdominal cavity, taking out the cecum by using sterilized surgical forceps, slightly wiping the serosa on the front side surface of the cecum by using dry gauze, and slightly scraping the back of a surgical blade to ensure that the serosa surface of the cecum has extensive bleeding points and slight bleeding;

fourthly, turning over the abdominal wall at the position corresponding to the cecum of the rat by using forceps, and scraping the back of the surgical blade slightly to ensure that the surface of the abdominal wall has extensive bleeding points and slight bleeding;

fifthly, injecting a certain amount of hydrogel into the caecum and abdominal wall of the rat according to different hydrogels used by different animal groups, and then placing the caecum back into the abdominal cavity, wherein the caecum injury position faces to the abdominal wall injury position;

and sixthly, suturing layer by using surgical suture lines, and observing and recording the state, the weight and the diet condition of the rat every day within two weeks after surgery.

The grouping of female SD rats (6 weeks old, body weight 200g or so) used in this experiment, and the type and dose of administration are shown in Table 4:

table 4 rat cecum-abdominal wall postoperative adhesion model anti-adhesion effect evaluation experimental animal grouping setting

Each group of rats is sacrificed by adopting a vertebral dislocation method after 2 weeks of model building operation, the abdominal cavity of the rat is cut off along the original abdominal incision of the rat, the peritoneal adhesion condition of the rat is observed, the postoperative adhesion severity is evaluated in a grading way, and the evaluation is based on Nair five-grade grading standard, as shown in table 5:

TABLE 5 grading evaluation criteria for adhesion severity of rat cecum-abdominal wall postoperative adhesion model

The experimental results are shown in fig. 10 and fig. 11, the postoperative adhesion prevention effect of the experimental group is significantly improved compared with that of the negative control group and the control group 1, the control group 2 and the control group 3, which indicates that the HBC/CS-TGA hydrogel has better postoperative adhesion prevention performance than that of the 2.67% HBC hydrogel, the 3.33% HBC hydrogel and the commercially available medical chitosan anti-adhesion solution.

And performing HE staining observation on the damaged cecal tissues of each group of rat models, and comparing the infiltration degree of inflammatory cells of the cecal tissues and the proliferation degree of collagen fiber tissues of each group. HE stained sections of the injured cecal tissue of each group of rats were observed, as shown in fig. 12. The part enclosed by the black circular dotted line represents the hyperplastic connective tissue after the caecum is damaged, and the inside of the caecum contains a large amount of fibrin, collagen and hyperplastic blood vessels; the portion enclosed by the black square dashed box represents the proliferating blood vessels in the proliferating connective tissue after the caecum is damaged. The rat cecum tissue is respectively provided with a mucosa (fold layer), a submucosa, a muscle layer (containing longitudinal muscle, circular muscle and the like) and a serosa layer 4 layer structure from the inner side of the cecum to the abdominal cavity side from inside to outside. The blank group has no modeling operation, so that the structures of the layers of the caecum are clearly and clearly arranged and orderly arranged, and inflammatory cells are hardly infiltrated; the negative control group contains a great deal of fibrin and collagen, the connective tissue is seriously proliferated, the mucous layer and the submucosal blood vessel are greatly proliferated, the structures of each layer of the caecum are difficult to distinguish, and the collagen and the fibrin in the proliferated connective tissue are densely arranged and infiltrated by a great deal of inflammatory cells; compared with the negative control group, the control group 1, the control group 2 and the control group 3 have clearer caecum structure and also have connective tissue hyperplasia, but the hyperplasia vessels in the connective tissue are less than that of the negative control group, and inflammatory cell infiltration and collagen fiber quantity are less than that of the negative control group; the structures of the cecum layers of the experimental group and the low-dose experimental group are clear and visible, the trace of molding damage can still be seen in the muscle layer, the proliferation degree of connective tissues and the deposition degree of collagen fibers are obviously reduced compared with the negative control group, and the infiltration of inflammatory cells is obviously reduced compared with the negative control group. The anti-adhesion effect of the HBC/CS-TGA hydrogel is better than that of 3.33 percent of HBC gel, 2.67 percent of HBC gel and commercially available medical chitosan anti-adhesion solution.

Example 14 cytokine assay related to adhesion severity

The content of the rat fibrosis factor TGF-beta 1 and the content of the inflammatory factor IL-6 in each group are measured by an ELISA kit. TGF-beta 1 and IL-6 detection results are shown in figures 13 and 14, and the HBC/CS-TGA hydrogel can remarkably inhibit the expression of TGF-beta 1 and IL-6, so that postoperative inflammatory reaction and fibrin deposition can be remarkably reduced, and the inflammation degree of postoperative adhesion is reduced.

Claims

1. A bioadhesive enhanced type temperature-sensitive chitosan-based anti-postoperative adhesion hydrogel is characterized by being prepared by mixing a hydroxybutyl chitosan solution and a thiolated chitosan solution in proportion, wherein in the preparation process of the thiolated chitosan solution, a thiolated reagent is mercaptoacetic acid, the hydroxybutyl chitosan solution and the thiolated chitosan solution are mixed in a volume ratio of 2.5-4.5% (w/v) and 1.5-3% (w/v), the volume ratio of the hydroxybutyl chitosan solution to the thiolated chitosan solution is 3:1-2:1, vortex oscillation is carried out after mixing, and the bioadhesive enhanced type chitosan-based anti-adhesion hydrogel is obtained after standing and bubble elimination.

2. The hydrogel of claim 1, wherein the hydroxybutyl chitosan is prepared by a process comprising the steps of:

1) alkalization: putting Chitosan (CS) into potassium hydroxide or sodium hydroxide aqueous solution, fully dispersing and alkalizing for 12-24h for later use;

2) isopropyl alcohol/water system dispersion: mixing isopropanol and deionized water to obtain isopropanol aqueous solution, and stirring and dispersing the alkalized chitosan with the isopropanol aqueous solution for 12-24 h;

3) grafting reaction: adding 1, 2-butylene oxide into the solution, heating and stirring in water bath to perform grafting reaction at 40-80 ℃ for 12-36h,

4) adjusting pH and dialyzing: after the grafting reaction, adjusting the pH value to 5.0-7.0 by acid, and putting the reaction liquid into a dialysis bag with the molecular cut-off of 8000-14000Da for dialysis;

5) and (3) freeze drying: and (5) after dialysis is finished, freezing and drying the product, and storing the product at low temperature in a sealing way for later use after freeze-drying.

3. The hydrogel of claim 1, wherein the thiolated chitosan is prepared by a method comprising the steps of:

5) and (3) freeze drying: after dialysis, the product was freeze-dried for use.

4. The hydrogel of claim 1, wherein hydroxybutyl chitosan is prepared by the steps of:

5. The hydrogel of claim 1, wherein the thiolated chitosan is prepared by the steps of:

5) and (3) freeze drying: and (4) after dialysis is finished, freeze-drying the product, and sealing and storing the freeze-dried product in a refrigerator at the temperature of-20 ℃ for later use.

6. The hydrogel of claim 1 as a medical material for preventing adhesion after surgery.