CN115991883B - Hydrogel for digestive tract ESD and preparation method and application thereof - Google Patents

Abstract

The present invention provides a hydrogel for digestive tract ESD and a preparation method and use thereof, belonging to the field of medical materials. The hydrogel of the present invention is obtained by dissolving the following raw materials in a weight ratio in a solvent and mixing them into a gel: 1 to 10 parts of oxidized alginate modified by grafting of unsaturated carbon-carbon double bonds, and 1 to 10 parts of carboxymethyl chitosan modified by grafting of thiol groups. The hydrogel of the present invention has both the function of an ESD mucosal pad and a "dual function" material for sealing, hemostasis, and repairing postoperative wounds. At the same time, it solves the problems of poor padding effect of submucosal liquid pad (SFC) materials in existing ESD, intraoperative/postoperative complications, etc., and has very good clinical application prospects.

Description

A hydrogel for digestive tract ESD and its preparation method and use

Technical Field

The invention belongs to the field of medical materials, and in particular relates to a hydrogel for digestive tract ESD and a preparation method and application thereof.

Background technique

Endoscopic submucosal dissection (ESD) is an effective method for the treatment of early gastrointestinal tumors, with the characteristics of small surgical trauma and complete preservation of pathological tissues. However, ESD is often accompanied by risks such as bleeding and perforation during and after the operation. Therefore, liquid preparations need to be injected submucosally before ESD to form a buffer zone between the mucosal layer and the submucosal muscular layer, which can avoid damage to the submucosal muscular layer during mucosal dissection and cause serious complications such as bleeding and perforation. Existing submucosal injection materials have defects such as easy diffusion, short maintenance time, high price, and easy tumorigenesis.

After ESD surgery, due to the loss of mucosal barrier function, the wound is exposed to the open lumen environment, which is prone to infection, ulcers and even necrosis. Therefore, other methods or materials are often needed to assist wound closure and hemostasis after ESD surgery. At present, the commonly used mechanical hemostasis (hemostatic clips, ligation, etc.) methods in clinical practice have limited effects on postoperative wound closure and hemostasis, and are prone to postoperative bleeding and perforation. The existing research materials have the problem that the degradation of the covering does not match the healing of ulcers, and the effect of promoting ulcer healing is limited. In addition, due to the strong peristalsis and complex physiological structure of the digestive tract, simple medical adhesives have a short retention time in situ, and there are problems such as inconvenient delivery, cumbersome surgical operations, and long operation time. Therefore, it is urgent to develop a "dual-function" material that can be used for submucosal injection and postoperative wound closure to meet the requirements of clinical ESD.

Sodium alginate is an anionic polysaccharide with non-toxicity, good biocompatibility and gelation mechanism. The aqueous solution of sodium alginate has non-Newtonian and shear-thinning properties and is a cheap high-viscosity solution. Sodium alginate has strong water retention capacity and can form a low-density viscous gel in the acidic environment of the stomach. Sodium alginate solution can also be used as a submucosal injection material for endoscopic treatment. Carboxymethyl chitosan is a derivative of chitosan, composed of glucosamine and N-acetylglucosamine. Glucosamine has active hydroxyl and amino groups, which can be modified by grafting to obtain new or improved properties and expand the scope of application. The carboxylated chitosan not only improves solubility, but also retains the unique biological properties of chitosan, such as good biocompatibility, biodegradability, non-toxicity and excellent antibacterial properties. The patent application with publication number CN114159586A discloses a submucosal injection marker carrier gel for endoscopy, which is prepared from sodium alginate and carboxymethyl chitosan as raw materials. The gel can be used as a mucosal pad to separate the mucosal layer from the muscle layer, reduce surgical complications such as bleeding and perforation during surgery, and facilitate wound healing. However, from the perspective of the patent, it is made by physical mixing of sodium alginate and carboxymethyl chitosan solution, that is, the two solutions cannot gel through covalent bonds or intermolecular forces. Strictly speaking, it is not a hydrogel, but only a high-viscosity mixed solution; the mixed solution cannot react with groups in the tissue to play an adhesion and tissue sealing role, so it can only simply meet the role of a submucosal pad in ESD or EMR, and cannot play a role in sealing the postoperative wound and preventing wound infection and ulcers. And because the mixed solution has no tissue adhesion ability, even if growth factors that promote tissue repair are added, they will be rapidly lost with tissue peristalsis, causing the wound to continue to be exposed to the environment of an open cavity.

Existing studies are limited to finding ideal alternative materials for submucosal injections, such as thermosensitive hydrogels, photocrosslinked hydrogels, shear-thinning hydrogels, etc., or materials that can close wounds after surgery, inhibit delayed postoperative bleeding, promote ulcer healing, and prevent fibrosis and scar formation, such as gelatin membranes, fibrin glue, and medical adhesives. There are no reports on materials that can be used as submucosal injections and can close wounds, stop bleeding, and promote wound healing after ESD. Moreover, the existing research on submucosal injection materials for ESD or materials that can close wounds after surgery has corresponding problems, such as inconvenient material delivery, cumbersome surgical operations, long surgical time, and tissue damage.

In view of the shortcomings of existing mucosal injection materials in ESD and the problems of intraoperative/postoperative complications, it is necessary to develop a material that has both mucosal lining and the "dual functions" of closing, stopping bleeding, promoting repair and inhibiting fibrosis of postoperative wounds, while solving the defects of existing submucosal injection materials in ESD and the problem of postoperative wound closure.

Summary of the invention

The purpose of the present invention is to provide a hydrogel for digestive tract ESD and its preparation method and use. The hydrogel is a "dual-function" material that has both the function of ESD mucosal pad and the functions of sealing, hemostasis and promoting repair of postoperative wounds, while solving the problems of poor padding effect of liquid pad materials in existing ESD and complications during/after surgery.

The present invention provides a hydrogel, which is obtained by dissolving the following raw materials in weight ratio in a solvent respectively and then mixing them into a gel:

1 to 10 parts of oxidized alginate modified by grafting unsaturated carbon-carbon double bonds and 1 to 10 parts of carboxymethyl chitosan modified by grafting thiol groups.

Furthermore, the aforementioned hydrogel is obtained by dissolving the following raw materials in a weight ratio in a solvent and then mixing them into a gel:

3-7 parts of oxidized alginate modified by grafting unsaturated carbon-carbon double bonds, and 3-7 parts of carboxymethyl chitosan modified by grafting thiol groups;

Preferably, 7 parts of oxidized alginate modified by grafting unsaturated carbon-carbon double bonds and 3 parts of carboxymethyl chitosan modified by grafting thiol groups are used.

Furthermore, the degree of alginate oxidation in the oxidized alginate modified by grafting unsaturated carbon-carbon double bonds is 14% to 50%; and/or the grafting rate of unsaturated carbon-carbon double bonds in the oxidized alginate modified by grafting unsaturated carbon-carbon double bonds is 24% to 37%;

And/or, the thiol grafting rate of the thiol-grafted modified carboxymethyl chitosan is 24% to 52%.

Further, the unsaturated carbon-carbon double bond grafted modified oxidized alginate is maleimide grafted modified oxidized alginate; and/or, the thiol group grafted modified carboxymethyl chitosan is cysteamine hydrochloride grafted modified carboxymethyl chitosan;

Preferably, the alginate is sodium alginate; and/or the carboxymethyl chitosan is O-carboxymethyl chitosan with a degree of substitution less than 1.

Furthermore, the unsaturated carbon-carbon double bond grafted modified oxidized alginate is maleimide grafted modified oxidized sodium alginate, and the preparation method of the maleimide grafted modified oxidized sodium alginate comprises the following steps:

(1) dissolving oxidized sodium alginate in a solvent to obtain an oxidized sodium alginate solution;

(2) adding EDC and NHS to the oxidized sodium alginate solution, and then adding maleimide hydrochloride to react;

(3) After the reaction, dialyzing and drying are performed to obtain maleimide-grafted modified oxidized sodium alginate;

Preferably,

In step (1), the solvent is MES solution, PBS solution or deionized water; and/or, in step (1), the mass percentage of the oxidized sodium alginate solution is 1% to 3%;

and/or, in step (2), the ratio of the amount of the oxidized sodium alginate substance to the total amount of EDC and NHS is 1:1.5-3; and/or, in step (2), the molar ratio or mass ratio of EDC and NHS is 1:1-1.5; and/or, in step (2), the ratio of the amount of the oxidized sodium alginate substance to the amount of maleimide hydrochloride substance is 1:1-2; and/or, in step (2), the reaction is carried out at room temperature for 12-24 hours;

And/or, in step (3), the dialysis is first placed in a 0.01-0.05M HCl deionized water solution for 3-5 days, and then placed in deionized water for 12-24 hours;

More preferably,

In step (2), during the reaction, the pH of the reaction solution is adjusted every 6 hours to maintain the pH value at 5.0 to 5.5;

And/or, in step (3), the dialysis bag has a cutoff of 3.5 to 8 kDa; and/or, in step (3), the water is changed every 6 to 12 hours during the dialysis.

Furthermore, the synthesis method of the thiol-grafted modified carboxymethyl chitosan comprises the following steps:

1) dissolving carboxymethyl chitosan in a solvent to obtain a carboxymethyl chitosan solution;

2) adding EDC to the carboxymethyl chitosan solution, and then adding cysteamine hydrochloride to react;

3) After the reaction, dialyze, freeze-dry, and alcohol separate to obtain mercaptocarboxymethyl chitosan;

Preferably,

In step 1), the solvent is deionized water or PBS solution; and/or, in step 1), the mass percentage of the carboxymethyl chitosan solution is 2-5%;

and/or, in step 2), the ratio of the amount of the carboxymethyl chitosan substance to the amount of the EDC substance is 1:1.5-3; and/or, in step 2), the ratio of the amount of the carboxymethyl chitosan substance to the amount of the cysteamine hydrochloride substance is 1:1-2; and/or, in step 2), the reaction is carried out at room temperature for 12-24 hours;

And/or, in step 3), the dialysis is performed in a 0.005-0.05M borax solution for 3-5 days, and then dialyzed in deionized water for 12-24 hours; and/or, in step 3), DTT is added to stir the reaction before freeze drying; and/or, in step 3), anhydrous ethanol is used for the alcohol analysis;

More preferably,

In step 3), the dialysis bag has a cutoff of 3.5 to 8 kDa; and/or, in step (3), the water is changed every 12 hours during the dialysis.

Furthermore, the mass volume ratio of the unsaturated carbon-carbon double bond grafted modified oxidized alginate to the solvent is (1-10) g:100 mL; and/or: the mass volume ratio of the thiol group grafted modified carboxymethyl chitosan to the solvent is (1-10) g:100 mL;

Preferably, the mass volume ratio of the unsaturated carbon-carbon double bond grafted modified oxidized alginate to the solvent is (3-7) g:100 mL; and/or: the mass volume ratio of the thiol group grafted modified carboxymethyl chitosan to the solvent is (3-7) g:100 mL;

More preferably, the mass volume ratio of the oxidized alginate modified by grafting unsaturated carbon-carbon double bonds to the solvent is 7 g:100 mL; and/or the mass volume ratio of the carboxymethyl chitosan modified by grafting thiol groups to the solvent is 3 g:100 mL.

Further, the solvent for dissolving the oxidized alginate modified by grafting unsaturated carbon-carbon double bonds is water or PBS buffer; and/or, the solvent for dissolving the carboxymethyl chitosan modified by grafting thiol groups is a borax aqueous solution with a concentration of 0.005 to 0.05 M;

Preferably, the solvent for dissolving the oxidized alginate modified by grafting unsaturated carbon-carbon double bonds is PBS buffer; and/or, the solvent for dissolving the carboxymethyl chitosan modified by grafting thiol groups is a 0.01M borax aqueous solution.

Furthermore, during the mixing, the volume ratio of the oxidized alginate solution modified by grafting unsaturated carbon-carbon double bonds to the carboxymethyl chitosan solution modified by grafting thiol groups is 1:(1-10);

Preferably, during the mixing, the volume ratio of the oxidized alginate solution modified by grafting unsaturated carbon-carbon double bonds to the carboxymethyl chitosan solution modified by grafting thiol groups is 1:1.

Furthermore, the gelling time is 3 to 10 seconds;

And/or, the mixing is to directly mix the oxidized alginate solution modified by grafting unsaturated carbon-carbon double bonds and the carboxymethyl chitosan solution modified by grafting thiol groups, or to spray the carboxymethyl chitosan solution modified by grafting thiol groups on the oxidized alginate solution modified by grafting unsaturated carbon-carbon double bonds;

Preferably, the gelling time is 5 s.

The present invention also provides a method for preparing the aforementioned hydrogel, which comprises the following steps:

(1) weighing unsaturated carbon-carbon double bond graft-modified oxidized alginate and thiol group graft-modified carboxymethyl chitosan according to a weight ratio, and dissolving them in a solvent respectively;

(2) Mix the two solutions into a gel.

The present invention also provides the use of the aforementioned hydrogel in preparing a submucosal pad and/or a tissue hemostatic material;

Preferably, the submucosal pad is a submucosal pad used for endoscopic submucosal dissection; and/or, the tissue hemostatic material is a material used for wound hemostasis during endoscopic submucosal dissection.

Explanation of terms in this invention:

The "grafting modification" of the present invention refers to the reaction of connecting a specific branched structure or functional side group on the polymer main chain through chemical bonds. The "grafting rate" of the present invention refers to: grafting rate (%) = (the amount of the branched structure or functional side group connected to the polymer main chain / the amount of the substance in the polymer main chain) * 100%.

The "oxidized alginate" mentioned in the present invention refers to: oxidative modification of algae salt, so that the hydroxyl groups at the C2 and C3 positions of some uronic acid units in the algae salt structure are converted into aldehyde groups, and the schematic structural formula is: The carboxylate group is combined with a cation with one positive charge (such as Na ⁺ , K ⁺ ).

The alginate of the present invention is preferably sodium alginate, and the oxidized alginate is preferably oxidized sodium alginate, and the schematic structural formula is:

The "oxidized alginate containing unsaturated carbon-carbon double bonds grafted and modified" mentioned in the present invention refers to: the uronic acid unit of the alginate molecule is oxidized by _NaIO4 to generate an aldehyde group, and a structure containing an unsaturated carbon-carbon double bond group is grafted to form a polymer.

The "oxidized alginate modified by grafting unsaturated carbon-carbon double bonds" mentioned in the present invention refers to a polymer of modified oxidized alginate (modified oxidized sodium alginate) formed by grafting a maleimide group "containing unsaturated carbon-carbon double bonds" on the oxidized alginate molecular chain; the structural formula of the maleimide group is:

The "carboxymethyl chitosan grafted with thiol groups" mentioned in the present invention refers to a polymer formed by grafting cysteamine hydrochloride containing thiol groups onto the molecular chain of carboxymethyl chitosan. The structural formula of the thiol group is:

The "digestive tract ESD" mentioned in the present invention refers to: endoscopic submucosal dissection of lesioned tissues of the digestive tract performed with the assistance of endoscope.

The "ESD postoperative spraying solution" described in the present invention refers to spraying a thiol-grafted modified carboxymethyl chitosan solution on the wound surface after ESD surgery of the digestive tract.

The "room temperature" mentioned in the present invention refers to: 25±5°C.

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

1. The modified sodium alginate solution provided by the present invention has good tissue matching and can adapt to irregular wounds of any shape;

2. The modified sodium alginate solution provided by the present invention can maintain a lasting cushion height under the esophageal mucosa;

3. The modified sodium alginate solution provided by the present invention is easy to deliver and can be delivered to any part and shape of the lesion through an endoscopic needle, and can be gelled in situ by spraying the modified carboxymethyl chitosan solution. The gelling time can be controlled within 3 to 10 seconds, preferably 5 seconds.

4. The in situ hydrogel provided by the present invention has good tissue adhesion and is not easy to fall off with tissue creeping; the bursting strength of the in situ hydrogel is 9.51±1.20 kPa, which is not significantly different from the bursting strength of fibrin glue of 10.16±0.78 kPa;

5. The in situ hydrogel provided by the present invention has the function of quickly sealing wounds and achieving rapid hemostasis; the hemostasis time of the in situ hydrogel is 91.33±16.65s, which is not significantly different from the hemostasis time of fibrin glue, which is 78.00±29.21s;

6. The in situ hydrogel provided by the present invention has good antibacterial properties, which can prevent bacterial infection of the wound surface and reduce the incidence of inflammation and ulcers; the hydrogel can seal the surgical wound surface after ESD surgery of the esophagus in dogs, accelerate wound healing, and inhibit the formation of esophageal fibrosis.

Whether it is the submucosal injection material disclosed in the patent application with publication number CN114159586A or fibrin glue, they can only play a unilateral role in ESD; either a submucosal padding role (material disclosed in the patent application with publication number CN114159586A) or a tissue sealing role (fibrin glue). The "dual-function" hydrogel of the present invention aims to solve the above two problems at the same time. Component a in the hydrogel, modified sodium alginate (AM solution), can play a submucosal padding role before ESD surgery. After ESD surgery, component b, modified carboxymethyl chitosan (CS solution), is sprayed on the wound. After the CS solution contacts the AM solution exposed after ESD surgery, it quickly forms a gel. The formed hydrogel has a sealing effect on the wound surface, which can effectively prevent the wound surface from contacting with the open environment of the cavity and causing infection or ulcer formation, and plays a role in hemostasis and wound healing.

In summary, the present invention has developed a "dual-function" material that has both the function of an ESD mucosal pad and the functions of sealing, hemostasis, and repairing postoperative wounds. It also solves the problems of poor padding effect, intraoperative/postoperative complications, and the like of the existing submucosal liquid pad (SFC) material in ESD, and has very good clinical application prospects.

Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the synthesis of ADA-Mal and CMCS-SH.

Figure 2 shows the FTIR images of maleimide-oxidized sodium alginate (ADA-Mal) and mercaptocarboxymethyl chitosan (CMCS-SH) before and after modification.

FIG. 3 is the ¹ H NMR spectra (400 MHz) of maleimide-oxidized sodium alginate (ADA-Mal) and mercaptocarboxymethyl chitosan (CMCS-SH) before and after modification.

FIG4 is a viscosity rheological analysis of ADA-Mal and CMCS-SH with different mass percentages: A is ADA-Mal (AM); B is CMCS-SH (CS).

FIG5 shows the ultrasonic detection results of the subcutaneous pad effect of ADA-Mal solution with different mass percentages: A is an ultrasonic image; B is the statistical result; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 6 shows the mechanical test results of ADA-Mal and CMCS-SH hydrogels (AxCy) with different mass percentages: A is the stress-strain curve; B is the compressive strength; C is the Young's modulus; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

FIG. 7 shows the swelling and degradation performance test results of ADA-Mal and CMCS-SH hydrogels (AxCy) with different mass percentages: A is the swelling rate; B is the residual mass percentage.

Figure 8 shows the gelation time after mixing ADA-Mal and CMCS-SH with different mass percentages; wherein, the hydrogel combination is represented by AxCy, A represents ADA-Mal, C represents CMCS-SH, x and y represent the mass percentages of ADA-Mal and CMCS-SH, respectively; *P＜0.05, **P＜0.01, ***P＜0.001, ****P＜0.0001.

Figure 9 is an evaluation of the tissue adhesion of the hydrogel; A is the adhesion behavior of A7C3 hydrogel on the pig skin surface; B is the adhesion behavior of the simulated hydrogel to an irregular wound.

Figure 10 shows the bursting strength test of A7C3 hydrogel (A7C3 Gel) and fibrin glue group (Fibrin Glue, Fib Gel in the figure); A is a hydrogel bursting strength test device; B is a schematic diagram of the working principle of the bursting strength test device; C is the statistics of the bursting strength of fibrin glue and A7C3 hydrogel group.

Figure 11 is an evaluation of the hemostatic effect of A7C3 group hydrogel as a tissue sealant in a rabbit liver bleeding model (in vitro); A is a picture of the hemostatic effect of the untreated control group and the sample group treated with fibrin glue and A7C3 group hydrogel within 0 to 120 seconds; B and C are the bleeding volume and bleeding time of the rabbit liver after intervention with A7C3 hydrogel compared with the fibrin glue group and the control group (without any treatment), respectively (*P<0.05).

Figure 12 shows the procedure for canine esophageal ESD.

Figure 13 shows the ESD surgical procedure and the evaluation of the effect of AM7 solution on the submucosal pad of the dog's esophagus; A is the portable endoscope system and ESD surgical procedure; B is the ESD submucosal injection process, including lesion localization, electrosurgical marking, and submucosal injection of solution; C is a comparison of the time-dependent changes (within 30 minutes) in the submucosal pad height of glycerol-fructose injection (GFI) and AM7 solution in the dog's esophagus.

Figure 14 shows the application of hydrogel in canine esophageal ESD; A is endoscopic submucosal injection of AM7 solution to cushion the lesion; B is resection of the lesion; C is cleaning the wound to expose the lesion (for submucosal tumors); D is the AM7 solution exposed after resection of the surface mucosa (or the AM7 solution backfilled after resection of the submucosal tumor); E is the in situ hydrogel morphology after spraying CS3 solution on the AM7 solution; F is the morphology of the in situ hydrogel after stabilization on the wound for 10 minutes; ("Δ" indicates AM7 solution; "→" indicates hydrogel on the wound surface).

Figure 15 shows the ESD groups and the observation of wound healing at various time points after surgery: A is the ESD surgical group: two surgical sites were performed at the proximal end (the site close to the heart is defined as the proximal end) and the distal end (the surgical site close to the mouth is defined as the distal end) of the esophagus of each dog, and the treatment methods of the two surgical sites of each esophagus were shown in the groups in Figure A (n=5); B is the observation of wound healing in each experimental group at 0, 3, 9, 14, 21, and 28 days (the yellow "△" indicates the gland).

Figure 16 shows the esophageal morphology of each experimental group after sampling 28 days after ESD surgery; A is the esophageal morphology of the A7C3 Gel group and the AM7 solution group after surgery; B is the esophageal morphology of the AM7 solution group and the GFI group after surgery; C is the esophageal morphology of the GFI group and the GFI+CS3 group after surgery; D is the esophageal morphology of the GFI+CS3 solution group and the A7C3 Gel group after surgery; E is the esophageal morphology of the A7C3 Gel group and the GFI+CS3 solution group after surgery; F is the morphology of the normal esophagus; (in the figure, ① represents the first experimental site at the distal end; ② represents the second surgical site).

Detailed ways

Unless otherwise specified, the raw materials and equipment used in the present invention are all known products obtained by purchasing commercially available products.

The oxidized sodium alginate (ADA) used in the present invention is a commercially available product or synthesized according to the following method:

1. Weigh 10 g of sodium alginate and disperse it in 50 mL of anhydrous ethanol. Use a magnetic stirrer to disperse it evenly to prepare an ethanol dispersion system of sodium alginate with a mass percentage of 20%.

2. Weigh 2.5 g of NaIO ₄ , dissolve it in 50 mL of deionized water in a dark environment, and stir until it is completely dissolved to prepare a NaIO ₄ solution with a mass percentage of 5%;

3. In a dark environment, slowly add the NaIO ₄ solution dropwise to the sodium alginate ethanol dispersion solution under magnetic stirring; continue stirring and reacting for 6 hours at room temperature in a dark environment;

4. After 6 hours of reaction in a dark environment, add 10 mL of ethylene glycol dropwise to the reaction system in a dark environment and continue to stir the reaction for 30 minutes to terminate the reaction;

5. After the reaction is terminated, stop stirring, let it stand at room temperature for 10 minutes, and wait for the precipitate to precipitate;

6. Carefully pour out the upper solution, collect the precipitate and put it into a dialysis bag with a molecular weight cutoff of 3.5 kDa, and dialyze it in 5L 0.01M HCl deionized water solution for 3 days, changing the dialysate every 12 hours;

7. Collect the dialyzed oxidized sodium alginate solution and freeze-dry it to obtain oxidized sodium alginate (ADA).

The "modified alginate containing an aldehyde group and an unsaturated carbon-carbon double bond structure" used in the present invention, namely maleimide oxidized sodium alginate (ADA-Mal), is synthesized according to the following method:

1. Weigh 2 g of ADA and dissolve it in 200 mL of MES solution. Stir until completely dissolved to prepare a 1% ADA solution.

2. Add EDC/NHS to the ADA solution so that the molar ratio of ADA to EDC/NHS is 1:1.5 (the molar ratio of EDC to NHS is 1:1); add maleimide hydrochloride (Mal-HCl) to the reaction system so that the molar ratio of ADA to Mal-HCl is 1:1, stir the reaction at room temperature for 24 hours, adjust the pH of the reaction solution every 6 hours, and maintain the pH value at 5.0-5.5;

3. After 24 hours of reaction, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 3.5 kDa, and dialyzed in 5L 0.01M HCl deionized water solution for 5 days, with the dialysate replaced every 12 hours. On the fifth day of dialysis, the dialysis bag was transferred to 5L deionized water and the dialysis was continued for 12 hours.

4. Take out the dialyzed sodium maleimide oxidized alginate solution and freeze-dry it to obtain sodium maleimide oxidized alginate (ADA-Mal).

The "modified carboxymethyl chitosan containing thiol groups" used in the present invention, namely thiol carboxymethyl chitosan (CMCS-SH), is synthesized according to the following method:

1. Weigh 4 g of CMCS (carboxymethyl chitosan) and dissolve it in 200 mL of deionized water. Stir until completely dissolved to prepare a 2% CMCS solution.

2. Add EDC to the CMCS solution so that the molar ratio of CMCS to EDC is 1:1.5; add cysteamine hydrochloride (CSA-HCl) to the reaction system so that the molar ratio of CMCS to CSA-HCl is 1:1.5, and stir the reaction at room temperature for 24 hours;

3. After 24 hours of reaction, the reaction solution was transferred to a 3.5 kDa molecular weight cutoff and dialyzed in 5 L 0.01 M borax solution for 5 days, with the dialysate replaced every 12 hours;

4. Pour the dialyzed CMCS-SH solution into a beaker, add 2 g DTT and continue stirring at room temperature for 1 to 2 hours, then take it out and freeze-dry it;

5. Add freeze-dried CMCS-SH to anhydrous ethanol, seal and place in a low-temperature shaker (4°C) at 60 rpm for 1 hour, filter out the ethanol with a suction flask, and repeat this operation 5 times;

6. After alcohol precipitation, the residual ethanol is removed from the CMCS-SH by a rotary evaporator to obtain mercaptocarboxymethyl chitosan (CMCS-SH).

The schematic diagram of the synthesis of ADA-Mal and CMCS-SH is shown in Figure 1.

After the preparation, the products of sodium alginate and carboxymethyl chitosan before and after modification were characterized by Fourier transform infrared absorption spectrometer (FTIR) and nuclear magnetic hydrogen spectrometer (1HNMR), which confirmed that maleimide oxidized sodium alginate (ADA-Mal) was successfully prepared after grafting modification of sodium alginate; and mercapto carboxymethyl chitosan (CMCS-SH) was successfully prepared after grafting modification of carboxymethyl chitosan.

Example 1. Preparation of in-situ hydrogel of the present invention

1. Weigh the freeze-dried ADA-Mal and dissolve it in PBS buffer to prepare a 7% ADA-Mal solution;

2. Weigh the freeze-dried CMCS-SH and dissolve it in a 0.01M borax aqueous solution to prepare a 3% CMCS-SH solution. The preparation method of the 0.01M borax aqueous solution is as follows: weigh 19.05g _{Na2B4O7 · 10H2O} (normal temperature, _borax ) and dissolve it in 5L deionized water, stirring until completely dissolved;

3. The solutions in step (1) and (2) are uniformly mixed at a volume ratio of 1:1 to prepare the A7C3 hydrogel.

Example 2: Preparation of in situ hydrogel of the present invention

1. Weigh the freeze-dried ADA-Mal and dissolve it in PBS buffer to prepare a 7% ADA-Mal solution;

2. Weigh the freeze-dried CMCS-SH and dissolve it in a 0.01M borax aqueous solution to prepare a 7% CMCS-SH solution; the preparation method of the 0.01M borax aqueous solution is the same as in Example 1;

3. The solutions in step (1) and (2) are uniformly mixed at a volume ratio of 1:1 to prepare the A7C7 hydrogel.

Example 3: Preparation of in-situ hydrogel of the present invention

1. Weigh the freeze-dried ADA-Mal and dissolve it in PBS buffer to prepare a 3% ADA-Mal solution;

2. Weigh the freeze-dried CMCS-SH and dissolve it in a 0.01M borax aqueous solution to prepare a 7% CMCS-SH solution; the preparation method of the 0.01M borax aqueous solution is the same as in Example 1;

3. The solutions in step (1) and (2) are uniformly mixed at a volume ratio of 1:1 to prepare the A3C7 hydrogel.

Example 4: Preparation of in-situ hydrogel of the present invention

1. Weigh the freeze-dried ADA-Mal and dissolve it in PBS buffer to prepare a 4% ADA-Mal solution;

2. Weigh the freeze-dried CMCS-SH and dissolve it in a 0.01M borax aqueous solution to prepare a 6% CMCS-SH solution; the preparation method of the 0.01M borax aqueous solution is the same as in Example 1;

3. The solutions in step (1) and (2) are uniformly mixed at a volume ratio of 1:1 to prepare the A4C6 hydrogel.

Example 5: Preparation of in-situ hydrogel of the present invention

1. Weigh the freeze-dried ADA-Mal and dissolve it in PBS buffer to prepare a 5% ADA-Mal solution;

2. Weigh the freeze-dried CMCS-SH and dissolve it in a 0.01M borax aqueous solution to prepare a 5% CMCS-SH solution; the preparation method of the 0.01M borax aqueous solution is the same as in Example 1;

3. The solutions in step (1) and (2) are uniformly mixed at a volume ratio of 1:1 to prepare the A5C5 hydrogel.

Example 6: Preparation of in situ hydrogel of the present invention

1. Weigh the freeze-dried ADA-Mal and dissolve it in PBS buffer to prepare a 6% ADA-Mal solution;

2. Weigh the freeze-dried CMCS-SH and dissolve it in a 0.01M borax aqueous solution to prepare a 4% CMCS-SH solution; the preparation method of the 0.01M borax aqueous solution is the same as in Example 1;

3. The solutions in step (1) and (2) are uniformly mixed in a volume ratio of 1:1 to prepare the A6C4 hydrogel.

The beneficial effects of the present invention are demonstrated below through specific experimental examples.

Experimental Example 1: Structural Characterization of ADA-Mal and CMCS-SH

1. Experimental methods

FTIR detection of ADA-Mal/CMCS-SH: The infrared absorption spectrum of the material was detected by potassium bromide tablet method. The specific operation was as follows: 5-10 mg of SA, ADA, ADA-Mal, CMCS and CMCS-SH 5 kinds of freeze-dried samples were weighed respectively, the materials were cut into small pieces, and 50-60 mg of KBr powder was added and mixed and ground evenly, and then the ground mixed powder was pressed into thin sheets by a tablet press under a pressure of 10 kPa, taken out, and infrared spectrum test was performed. The specific conditions of the test were set as follows: in ATR detection mode, the detection spectrum range was 400-4000 cm ^-1 , the spectral resolution was 4 cm ^-1 /time, and the scanning frequency was 0.0625 Hz.

¹ HNMR detection of ADA-Mal/CMCS-SH: ¹ HNMR sample preparation and detection are as follows: 5 mg of SA, ADA, ADA-Mal, CMCS and CMCS-SH lyophilized samples were weighed and placed in NMR tubes, 0.55 mL of deuterated water (D ₂ O) was added to each tube, and the materials were shaken to fully dissolve. The absorption spectrum of the sample chemical shift in the range of 0-12 ppm was measured by NMR (400 MHz, Bruker AV II-400, Switzerland), and the integrated area of the ¹ H peak in the main chain of the polysaccharide was set as the reference peak. The grafting degree (DS) of the maleimide and thiol groups was calculated based on the ratio of the ¹ H peak area in the measured -Mal and -SH spectra to the reference peak area.

2. Experimental results

The stretching vibration absorption peak at 1734cm ^-1 in the FTIR spectrum of oxidized sodium alginate indicates the generation of aldehyde groups, generating oxidized sodium alginate (ADA); the vibration peaks at 1657cm ^-1 and 1542cm ^-1 in the FTIR spectrum of ADA modified with maleimide hydrochloride are amide bonds I and amide II (Figure 2). The two peaks at δ = 2.88ppm and δ = 2.95ppm in the ¹ HNMR spectrum are the methylene proton peaks introduced by the maleimide group (-CH ₂ CH ₂ -); the proton peak at δ = 6.88ppm is the proton peak introduced into the -C = C- in the -Mal functional group (Figure 3). The degree of substitution (DS) of maleimide groups in ADA-Mal is calculated by the ratio of the peak area corresponding to the hydrogen atoms in the carbon-carbon double bond (-C=C-) (δ=6.88ppm) in the maleimide group to the peak area corresponding to the hydrogen atoms on the main chain (δ=3.2-4.0ppm) in ADA, and the DS is about 27.29%. FTIR and ¹ HNMR results show that the present invention successfully prepares ADA-Mal.

It can be seen from the FTIR spectra of CMCS and CMCS-SH that absorption peaks appear at 1657 and 1542 cm ^-1 in CMCS-SH, which are the stretching vibration peaks of amide I (-C=O) and the bending vibration peaks of amide II (-CN), indicating that the amino group (-NH ₂ ) in the cysteamine thiol group reacts with the carboxyl group (-COO-) on CMCS to form an amide bond (Figure 2). From the hydrogen spectrum of CMCS, it can be seen that δ=2.02ppm is the methyl proton peak (-CO-CH ₃ ) in the acetyl group in CMCS. Compared with CMCS, δ=2.86ppm and δ=2.91ppm appearing in the hydrogen spectrum of CMCS-SH belong to the methylene proton peaks (-CH ₂ CH ₂ SH) of the cysteamine thiol group, among which the peak at δ=2.91ppm belongs to the methylene hydrogen adjacent to the amide bond (Figure 3). The degree of substitution (DS) of the mercapto group was calculated based on the peak area corresponding to the hydrogen atoms at the double bond positions (δ=2.91ppm and (δ=2.86-2.91ppm) and the peak area corresponding to the hydrogen atoms at the methylene peak (δ=2.56ppm). The DS was about 48.2%. The results of FTIR and ¹ HNMR showed that CMCS-SH was successfully prepared in the present invention.

Experimental Example 2: Viscosity test of ADA-Mal and CMCS-SH solutions

1. Experimental methods

The viscosity of ADA-Mal and CMCS-SH solutions with different mass percentages was tested, where ADA-Mal was abbreviated as AMx, x = 3, 4, 5, 6, 7, 8, representing 3%, 4%, 5%, 6%, 7%, 8% of ADA-Mal respectively; CMCS-SH was abbreviated as CSy, y = 3, 4, 5, 6, 7, representing 3%, 4%, 5%, 6%, 7% of CMCS-SH respectively. The specific operation was as follows: AMx solution and CSy solution with different mass percentages (w/v%) were prepared respectively, and glycerol fructose injection (GFI), a mucosal injection commonly used in clinical ESD, was used as a control. A rotational rheometer (MCR302) was used, and a 50 mm flat plate rotor (PP50) was used as the test component. The instrument test parameters were set as follows: the gap between the upper and lower plates was set to 0.25 mm, and the platform temperature was set to 37 °C. The initial value of the shear rate in the initial test was set to 0.1 rad/s, and the final value was set to 100 rad/s. The viscosity curves of 12 liquids, namely GFI, AM3, AM4, AM5, AM6, AM7, AM8, and CS3, CS4, CS5, CS6, and CS7, were tested in the rotation mode.

2. Experimental results

The viscosity curves of the two modified solutions of AMx and CSy are shown in Figures 4A and 4B. Figure 4A is the viscosity test curve of GFI and different concentrations of AMx solutions; Figure 4B is the viscosity test curve of different concentrations of CSy solutions. Figures 4A and 4B reflect the trend of the viscosity of solutions of different concentrations changing with shear rate. The vertical scales intersecting Figures 4A and 4B are the viscosity values when the horizontal axis is 0.1s. The static viscosity values of the solutions of GFI, AM3, AM4, AM5, AM6, AM7, and AM8 were detected to be approximately 13.409, 3783.7, 21663, 52487, 69562, 88476, and 168940mPs, respectively; the static viscosity values of the solutions of CS3, CS4, CS5, CS6, and CS7 were approximately 136, 137.07, 148.44, 148.28, and 184.96mPs, respectively. The viscosity of the two solutions decreases as the shear rate of the rheometer rotor increases, showing shear thinning characteristics, which is a typical non-Newtonian fluid. It can be seen that both ADA-Mal solution and CMCS-SH solution are injectable.

From the static viscosity test results of the two solutions, it can be found that the viscosity of AMx solution is much higher than that of CSy solution and GFI solution. The viscosity of the solution is crucial to maintain the pad height. Therefore, in order to achieve a lasting submucosal pad effect, AMx solution is preferred as the submucosal injection solution. From the AMx static viscosity curve, it can be seen that as the mass percentage of AM solution increases, the static viscosity of the solution increases, and when x=8, the viscosity increases suddenly. The possible reason is that as the AM percentage increases, the solution viscosity increases. The high viscosity prevents water molecules from entering the material and combining with the undissolved sugar chains in the AM, resulting in the sugar chains in the solute not being fully unfolded and existing in the solution in a curled form, further increasing the viscosity of the solution system. During endoscopic submucosal injection, the needle will be blocked due to excessive viscosity, affecting the quality of the operation. Therefore, when selecting the AMx concentration as the submucosal injection solution, the AM solution in the x=8 group is not considered.

Experimental Example 3: Ultrasonic testing results of the subcutaneous pad effect of ADA-Mal solution

1. Experimental methods

As described in Experimental Example 2, five different concentrations of AM solutions, AM3, AM4, AM5, AM6, and AM7, were prepared. SD rats were anesthetized with chloral hydrate (300 g/mL), the back skin was prepared, the skin on both sides of the back was exposed, and the injection points were marked. 100 μL of the above-mentioned AM solutions of each concentration was drawn with a 1 mL syringe, and the syringe was placed at a small angle to the back to inject the AM solution into the subcutaneous part of the rat from the marked point. Ultrasonic detection was performed immediately after the bulge was observed, and the detection height of the detection point was used as the initial height, and the time of the detection point was used as the initial time (T ₀ = 0 min). Normal saline and GFI were used as control groups, and three parallel injection points were set for each group of injections (n = 3). The subcutaneous padding height of each group of solutions was detected at three time points of 0 min, 15 min, and 30 min, respectively, and the changes in the subcutaneous padding height of each group of AM solutions over time were recorded and counted.

2. Experimental results

Figure 5 is a morphological diagram of the change of the subcutaneous pad height of the solution detected by ultrasound over time after the normal saline (NS), glycerol fructose injection (GFI) and AM solution with a mass percentage of 3%, 4%, 5%, 6% and 7% were injected into the rat subcutaneously. It can be clearly seen from the figure that under the premise of injecting the same volume of injection solution, as the percentage of AM increases, the initial (T ₀ = 0min) pad height detected increases, and when the concentration is greater than 4% (pad height is 0.48±0.08cm), the subcutaneous pad height of the AM solution is significantly higher than that of the NS and GFI groups (0.32±0.05cm and 0.34±0.05cm, respectively) (Figure 5B). 30min after injection, the AM6 and AM7 groups still maintain obvious pad heights, which are 0.36±0.03cm and 0.42±0.06cm, respectively, which are higher than the initial detection heights of NS and GFI. Ultrasonic detection of the subcutaneous pad effect of AM solution showed that compared with the commonly used NS and GFI in clinical practice, a certain concentration of AM solution can maintain a better pad height and pad time in rats, and the pad effect can be adjusted with the concentration. The results of ultrasonic detection of the subcutaneous pad effect of AM solution in rats showed that AM solution can be used as a potential alternative product for ESD submucosal injection. From the subcutaneous pad effect of AMx solution in rats, the higher the concentration, the higher the initial pad height, and the higher the subcutaneous maintenance height in the same time. Therefore, in order to maintain a stable submucosal pad effect, AM7 solution is proposed as the esophageal submucosal injection material.

Experimental Example 4: Mechanical properties of AxCy hydrogel

1. Experimental methods

The compressive strength and Young's modulus of 6 groups of hydrogels A3C7, A4C6, A5C5, A6C4, A7C3, and A7C7 prepared in Examples 1 to 6 were tested using an electronic universal material testing machine (Instron 5967). The specific operation is as follows: The hydrogel is prepared into a cylindrical shape with a diameter of 0.8 cm and a height of 1.0 cm with flat ends, and the hydrogel column is placed upright at the center of the pressure head of the testing machine. The pressure head of the testing machine is connected to a sensor with a range of 30 kN. The parameters of the testing machine are set to a running speed of 5 mm/min and a compression strain set to 80% of the length of the test object. The testing machine is run to record the stress-strain curve of the hydrogel (n=5), and the maximum value of the gel compression stress and the maximum compression strain corresponding to the maximum value are recorded. The compression modulus is the slope of the strain of the test object occurring in the compression stage of 15% to 25% (n=5).

The Young's modulus of the hydrogel can be calculated by the following formula:

Where Δδ is the Young's modulus of the hydrogel; ΔF is stress, whose physical meaning is the force per unit cross-sectional area; ΔL strain, whose physical meaning is the elongation per unit length; F is the pressure on the hydrogel; S is the cross-sectional area of the hydrogel; L is the height of the hydrogel; ΔL' is the compressed height of the hydrogel, and r is the radius of the hydrogel cylinder.

2. Experimental results

Figure 6 shows the relationship curves between the compressive stress and strain of the six groups of hydrogels. It can be seen from the figure that the hydrogel collapses at the maximum compressive stress, and the corresponding compression distance at this time is the strain. The compressive stress and corresponding strain of different hydrogels are different. The strain of collapse of A7C7 hydrogel is the smallest, and the strain of collapse of A3C7 hydrogel is the largest. In terms of the magnitude of compressive stress, the maximum stress that the A5C5 group hydrogel can withstand is 50.30±5.54kPa, and the maximum stress that the A7C3 group can withstand is 76.82±4.93kPa. The difference between the maximum stresses that the two groups can withstand is significant (P<0.01); the maximum compressive stress that the A7C7 group hydrogel can withstand is 54.57±4.72kPa, which is significantly lower than that of the A7C3 group (P<0.05). Among the six groups of hydrogels, the A7C7 group hydrogel has the largest compression modulus, followed by the A7C3 group, and the smallest is the A3C7 group. There was no significant difference in compression modulus among A4C6, A5C5 and A6C4 (P＞0.05). From the mechanical results, A7C3 hydrogel had greater compression strength and Young's modulus, and the gelation time of A7C3 hydrogel was shorter.

Experimental Example 5: Evaluation of Swelling and Degradation Performance of AxCy Hydrogel

1. Experimental methods

(1) Hydrogel swelling: Six groups of columnar hydrogels prepared in Examples 1 to 6 were taken from each group, and the surface moisture was absorbed with filter paper and weighed (W ₀ ); each group of weighed columnar hydrogels was immersed in 10 mL PBS buffer and placed in a constant temperature shaker at 37°C and 60 rpm. After a certain period of time, the hydrogel was taken out, the surface moisture was absorbed with filter paper, and then weighed (Wt). The above process was repeated until the weight of each group of hydrogels remained unchanged. Three parallels were set for each group of hydrogels (n=3). The hydrogel swelling rate calculation formula is:

Swelling rate (%) = (Wt-Wo)/Wo×100%

Where: Wt is the mass of the hydrogel block after swelling at time t, and _W0 is the initial mass of the hydrogel block.

(2) Hydrogel degradation: The hydrogels prepared in Examples 1 to 6 were taken, freeze-dried, and freeze-dried hydrogels were weighed (W ₀ ), and placed in 10 mL of PBS buffer respectively, placed in a constant temperature shaker at 37°C and 60 rpm, taken out after a certain period of time, and the surface was rinsed with deionized water. After freeze-drying, the hydrogels were weighed (Wt), and 3 parallels were set for each group of hydrogels (n=3). The residual mass percentage was calculated. The degradation performance of the hydrogel can be expressed by the formula:

Residual mass percentage (%) = Wt/Wo × 100%

Where: Wt, _W0 are the weight of the hydrogel freeze-dried scaffold at time t and the initial weight, respectively.

2. Experimental results

The swelling and degradation performance test results of the six groups of hydrogels are shown in Figures 7A and 7B. Figure 7A is the swelling curve of the hydrogel. It can be seen from the swelling curve that the six groups of different hydrogels almost tend to swelling equilibrium in PBS buffer at around 30 hours. Among them, the swelling rates of A7C3 and A3C7 groups reached 101.9±14.63% and 94.34±10.75%, respectively; the A7C7 group was the smallest at 55.77±6.39%; the swelling rates of the hydrogels in the A4C6, A5C5, and A6C4 groups were close, at 85.38±6.94%, 81.28±9.06%, and 82.87±4.10%, respectively. After 30 hours, under the action of water molecules and ions, the hydrogel began to degrade, among which the degradation of the A3C7 group was the most obvious.

The degradation of the six groups of freeze-dried hydrogels in PBS buffer is shown in Figure 7B. It can be seen from the figure that the slope of the degradation curve is the largest and the degradation rate is the largest in the first 3 days of degradation; then the degradation rate slows down and tends to balance. Among them, the hydrogel in the A3C7 group degrades the fastest, followed by A4C6, A5C5, A6C4 and A7C7, and the A7C3 group degrades the slowest.

The A7C3 group hydrogel has a greater swelling rate than the other groups of hydrogels, with a swelling rate of about 100%. The rich hydrophilic groups (such as -CHO, -CONH, -C＝C-, etc.) in the A7C3 hydrogel increase the hydrophilicity of the hydrogel, resulting in an increase in the equilibrium swelling rate of the hydrogel (Figure 7A). In addition, the hydrophilic groups in the A7C3 hydrogel can hydrate with water through hydrogen bonds to form a large molecular network, which can slow down the degradation rate of the A7C3 hydrogel (Figure 7B). Therefore, the A7C3 group hydrogel has a greater swelling rate and a smaller degradation rate than the other groups of hydrogels.

From the comprehensive perspective of the mechanical properties, swelling and degradation properties of each group of hydrogels, the A7C3 hydrogel has better performance; therefore, subsequent experiments plan to use the A7C3 group of hydrogels for further research.

Experimental Example 6: AxCy hydrogel gelation time

1. Experimental methods

The gelation time detection operation is as follows: 500 μL AMx (x = 3, 4, 5, 6, 7) solution is drawn and added to a 5 mL EP tube, a magnetic stirrer (C type, 0.5 cm, olive type) is added, and the tube is placed on a magnetic stirrer at 120 rpm; after the stirring speed of the stirrer is stable, 500 μL CSy (y = 3, 4, 5, 6, 7) solution is drawn and added to the EP tube, and a stopwatch is used to time the mixture. The EP tube is inverted every 3 seconds, and the timing is stopped at the same time. When the mixed solution is observed to be stably gelled at the bottom of the EP tube and does not slide down from the bottom of the EP tube, the time is recorded; if there is gel that cannot stay stably at the bottom and slides down when inverted, the magnetic stirring reaction is continued until the gel does not slide down when the EP tube is inverted, and the final gelation time is recorded. Each group is repeated 5 times (n = 5), and the final gelation time of each group of gels is counted. The hydrogel combination is represented by AxCy, where A represents ADA-Mal, B represents CMCS-SH, and x and y represent the mass percentage of ADA-Mal and CMCS-SH, respectively. Meanwhile, ADA-CS and ADA-CMCS hydrogels were prepared by mixing 7wt% oxidized sodium alginate (ADA) with 3wt% CMCS-SHCS (mercaptocarboxymethyl chitosan) and 3wt% CMCS (carboxymethyl chitosan), and the gelation time of the two groups of hydrogels was recorded.

2. Experimental results

Figure 8 shows the statistical results of the gelation time of each group of gels, A3C7, A4C6, A5C5, A6C4, A7C3, and A7C7, measured by the stirring-inversion method. As can be seen from the figure, the gelation time of the gel prepared in this study is short, and it can be gelled within 3 to 10 seconds. Among the six groups of gels, the A3C7 group has the longest gelation time, with an average gelation time of 8.3±1.5 seconds; the A7C7 group has the fastest gelation time, with an average gelation time of 3.7±0.56 seconds. The stable gelation time of both ADA-CS and ADA-CMCS hydrogels is greater than 30 minutes, which does not meet the requirements of shortening the surgical operation time.

Experimental Example 7: Tissue Adhesion Performance of A7C3 Hydrogel

1. Experimental methods

Take fresh pig skin with a thickness of about 0.3 cm in the dermis, peel off the subcutaneous fat layer, and clean the grease of the epidermis. Cut the pig skin into strips of 5 cm × 3 cm. Pipette 100 μL AM7 solution (add a small amount of red tissue marker liquid in advance, and experiments have shown that the red tissue marker liquid will not affect the gelation) and drop it on the center of the pig skin, then take 100 μL CS3 solution, evenly drop it on the AM7 solution, and gently stir and mix it with a gun tip, let it stand at room temperature for 30 minutes to allow the gel to stabilize. After the gel is stable, use tweezers to clamp the two ends of the pig skin, and repeatedly stretch, bend, squeeze, and twist it in turn to observe the adhesion of the gel to the pig skin.

Take fresh pig skin without fat, cut it into 10cm×6cm pieces, and carve a "☆" about 0.1-0.15cm deep in the epidermis of the pig skin. Pipette 60μL AM7 solution and add it to the "☆" groove to fill the groove with AM7 solution. Pipette 60μL CS3 solution and drop it into the groove. Stir gently with the tip of the gun to mix the two solutions thoroughly and cover the entire groove. Let it stand at room temperature for 30 minutes to allow the hydrogel to gel and stabilize. After the gel is stable, use a washing bottle to flush water around the gel and observe whether the gel falls off.

2. Experimental results

FIG9A shows that after in-situ gelation on the pig skin surface, the pig skin is repeatedly stretched, bent, squeezed, and twisted to simulate the peristalsis of the gel when used on the skin on the body surface and the soft tissue in the body (such as the digestive tract). As can be seen from FIG9A, the hydrogel can still adhere firmly to the pig skin after repeated action. FIG9B shows the in-situ gelation on the irregular wound surface of the pig skin to evaluate the adhesion of the gel on the irregular wound. After in-situ gelation and stabilization, the irregular wound edge is rinsed with running water, and it can be seen that the gel is still firmly adhered without falling off. The above results show that the hydrogel prepared by the present invention has good adhesion properties.

Experimental Example 8: Bursting Strength of A7C3 Hydrogel

1. Experimental methods

The device has a three-way valve connection, one end of the three-way valve is connected to a 50mL syringe, one end is connected to an electronic pressure valve (16kPa, MD-S280), and the other end is connected to a cylindrical water storage tank (φ3cm). The lower end of the small tank is connected to the water inlet pipe, and the upper end is open. There are four screw holes evenly distributed on the small tank for water storage and fixing pigskin (Figure 10A, 10B). The fresh pigskin that has been degreased is cut into a circular piece with a diameter of about 5cm and fixed on the top of the device by screws to seal the water storage tank with a diameter of 3cm. There is a hole with a diameter of 0.2cm×0.1cm in the center of the pigskin. 100μL AM7 solution is drawn and dripped around the central hole of the pigskin, and 100μL CS3 solution is dripped at the same time. The two solutions are gently mixed with the tip of a gun and left to stand at room temperature for 30min to allow the gel to stabilize into a gel. After the hydrogel is stabilized into gel, the pressure gauge value is adjusted to zero. When the pressure gauge is stable, slowly push the syringe, and water flows to the pressure gauge and the sink through the three-way valve at the same time. Continue to slowly push the syringe, and observe the reading of the pressure gauge head at the same time. Record the pressure gauge reading when the gel bursts. Repeat the test 3 times in the same way (n=3). Commercial porcine fibrin glue was used as a control (n=3), and the pressure gauge reading is the bursting strength of the gel (unit: kPa).

2. Experimental results

The bursting strength of the hydrogel was tested to simulate the damage of the sealant to the wound by bleeding, and commercial porcine fibrin glue was used as a control. Figure 10A is the designed bursting strength detection device; Figure 10B is the working principle diagram of the bursting strength detection device; Figure 10C shows the bursting strength of fibrin glue (Fibrin Glue, Fib Gel in the figure) and A7C3 group hydrogel, which are 10.16±0.78kPa and 9.51±1.20kPa respectively. The bursting strength of the two groups of gels was compared, and the difference was not significant (P＞0.05). The experimental results show that A7C3 hydrogel has similar tissue adhesion ability to commercial fibrin glue, proving that the hydrogel can seal the wound after ESD surgery, thereby preventing wound infection or ulcer formation after ESD surgery.

Experimental Example 9: Hemostatic performance of A7C3 hydrogel

1. Experimental methods

Construction of liver bleeding model: 15 New Zealand male rabbits were randomly divided into three groups, with five rabbits in each group (n=5): blank control group (Blank), fibrin glue group (Fibrin glue), and A7C3 hydrogel group. 3% sodium pentobarbital was injected into the ear vein for anesthesia (3mL/kg), and the rabbits were fixed on the operating table in a supine position. The abdomen was prepared and the abdominal cavity was opened layer by layer after disinfection. The tissue fluid and blood on the abdominal cavity and abdominal wall were absorbed with gauze to fully expose the right liver lobe, and the liver lobe was lifted out of the abdominal cavity. The tissue fluid and blood on the surface of the liver lobe were absorbed with gauze, and two layers of sterile filter paper (10 cm in diameter) were placed under the liver lobe. A 1-shaped incision with a length of 1 cm and a depth of 0.5 cm was made with a scalpel at the same position of the right liver lobe to establish the bleeding model;

Liver hemostasis: The blank group was not treated until natural hemostasis occurred; the fibrin glue group had pig fibrin glue dripped at the bleeding point to seal the bleeding site; the experimental group first dripped AM7 solution at the bleeding site, and then dripped CS3 solution on the AM7 solution to form gel in situ. The blank group recorded the bleeding time after injury; the fibrin glue group and the experimental group recorded the bleeding time after intervention; the filter paper absorbed the outflowing blood until the blood stopped flowing out. The total mass of the filter paper after blood absorption was weighed to calculate the amount of bleeding.

2. Experimental results

FIG11A is a rabbit liver bleeding model, from which it can be seen that the liver bleeding is obvious after modeling. The A7C3 hydrogel is gelled in situ at the wound, and the hydrogel covers the bleeding site to act as a physical barrier to prevent blood from continuing to seep out. The untreated group and commercially available fibrin glue were used as controls. As can be seen from FIG11A, a larger blood stain area appeared on the filter paper of the untreated group, and the blood loss was 0.945±0.161 g (FIG11B). The blood stain area on the filter paper of the fibrin glue group was smaller, and the bleeding amount was 0.603±0.040 g. The blood stain area of the A7C3 hydrogel group was slightly larger than that of the fibrin glue group, and the bleeding amount was 0.658±0.075 g. The bleeding amount of the A7C3 hydrogel group was less than that of the untreated group (P<0.05); slightly larger than that of the fibrin glue group, but there was no significant statistical difference (P>0.05). This shows that the hydrogel of the present invention has good tissue adhesion. From the intuitive diagram of the hemostasis process, it can be seen that after the use of fibrin glue and hydrogel on the bleeding site, a layer of hydrogel film can be formed on the wound surface, which plays a role in wound closure and can prevent wound bleeding. The in vitro hemostasis experiment of rabbit liver showed that A7C3 in situ hydrogel has good wound closure and hemostasis properties.

Experimental Example 10: Canine esophageal ESD and postoperative care and examination

1. Experimental methods

(1) Preoperative preparation: Healthy adult Beagle dogs were selected and fasted without water for 6 hours before surgery;

(2) Experimental groups: The experiment was divided into four groups: ① AM7 was used as a submucosal injection solution, and the postoperative wound surface was sealed with CS3 solution (A7C3 Gel group); ② AM7 was used as a submucosal injection solution, and the postoperative wound surface was not intervened (AM7 group); ③ Glycerol fructose injection (GFI) was used as a submucosal injection solution, and the postoperative wound surface was sealed with CS3 solution (GFI+CS3 group); ④ GFI was used as a submucosal injection solution, and the postoperative wound surface was not intervened (GFI group);

(2) Anesthesia and fixation: 3% sodium pentobarbital was injected intraperitoneally (1 mL/kg) for anesthesia. After the dog was fully anesthetized, 1 mL of sudamixin was injected intramuscularly. The dog was fixed in a supine position on the operating table, the back of the neck was raised, and the head position was adjusted so that the mouth, throat and trachea were in the same longitudinal direction;

(3) Tracheal intubation: Hold the laryngoscope and slowly insert it along the curvature of the tongue dorsum. Gently lift the epiglottis cartilage at the root of the tongue to expose the glottis. When the glottis opens during inspiration, quickly insert the tracheal tube into the trachea. Pull out the tube core, place a dental pad, remove the laryngoscope, and secure the tracheal tube and dental pad.

(4) Submucosal injection: Use a visual endoscope system (4.0-mm, VLS2-01) to enter the esophagus through the oral epiglottis and pyriform sinus. Under the guidance of the endoscope, use an aspirator to remove excess secretions remaining in the oral cavity and esophagus. Use a biopsy forceps to grasp a sterile rubber band with an outer diameter of about 1.5 cm and insert it into the esophagus. Place it at 1.5 cm and 4 cm from the entrance of the esophagus (simulating lesion positioning), and use an electrocoagulation knife to mark the tissue around the rubber band to construct a simulated lesion area with a diameter of about 1.5 cm, as shown in the "marking" process in Figure 12. Use a disposable endoscopic injection needle (1650mm, 23G) to inject 400-500 μL of AM7 solution under the marked mucosa. Obvious bulges can be seen under the endoscope. The control group was injected with GFI;

(5) Circumcision and peeling: Use an electrocoagulation hook to cut along the marked points at the base of the bulge and peel off the upper layer of the mucosa at the bulge to create a defect area with a diameter of about 1.5 cm. For the resection of submucosal tumors, it is necessary to use normal saline to flush out the exuded blood and residual mucosal surface injection fluid to obtain a clean field of view;

(6) Sealing: Replace the disposable endoscopic injection needle with a disposable endoscopic drug delivery tube (1200 mm, φ1.8), and spray the CS3 solution on the wound. The CS3 solution will form a gel after contacting with the AM7 solution remaining in the wound or supplemented after the submucosal tumor is removed, and will have a sealing effect on the wound;

(7) Gastric tube insertion: After surgery, a gastric tube is inserted through the nose with the assistance of endoscope.

The procedure for canine esophageal ESD is shown in Figure 12 .

In the first three days after surgery, the dog was fed by nasogastric feeding, and the gastric tube was removed after three days to feed normally. Postoperative observation indicators: a. General observation: observe the dog's eating and weight changes; b. Esophageal endoscopy: on the 3rd, 9th, 14th, 21st, and 28th days after surgery, esophageal endoscopy was performed after anesthesia (Sutane, intramuscular injection, 0.1 mg/kg) to observe the mucosal inflammation, granulation growth, and closure of the local defect area of the esophagus.

2. Experimental results

(1) Pad effect: AM solution was used as the submucosal pad material for canine esophageal ESD to evaluate its submucosal pad effect. Figure 13A shows the endoscopic system and the actual operation process of canine esophageal ESD. Figure 13B shows the marking and submucosal injection process of canine esophageal ESD, which is divided into lesion (simulation) positioning, lesion marking, and submucosal injection. The position of the rubber ring (φ1.5cm) in the figure is the simulated lesion area. After positioning, the electrocoagulation knife is used to mark the area around the lesion. AM7 solution is injected into the submucosal layer of the marked area to isolate the normal or lesion (such as polyps) mucosal layer from the submucosal muscle layer, making it easier to peel the mucosal layer with an electrosurgery knife (resection of polyps or exposure of submucosal tumors). Figure 13C shows the evaluation of the submucosal pad effect of AM7 solution and GFI in the esophagus. As can be seen from the figure, after GFI injection, it presents a broad-based peak-shaped bulge under the mucosa, while after AM7 solution injection, it presents a sharp peak-shaped bulge. After 15 minutes, the peak surface of the GFI group tended to be flat, accompanied by the appearance of mucosal wrinkles; while the bulge shape of the AM7 group remained basically unchanged; after 30 minutes, the peak surface of the GFI group basically disappeared, and obvious folds appeared in the mucosa at the bulge, indicating that GFI could not maintain a good cushioning effect under the mucosa within 30 minutes; while the height of the bulge in the AM7 group after 30 minutes was not significantly different from the initial injection height. The results of the evaluation of the effect of esophageal submucosal injection show that AM7 solution, as a submucosal injection solution, has the characteristics of maintaining a high cushioning height, a long cushioning time, and is not easy to lose.

(2) Postoperative closure effect of A7C3 hydrogel: FIG14 is an operation diagram of the use process of the "dual-function" in-situ hydrogel developed by the present invention for submucosal padding and postoperative wound closure. FIG14A is the submucosal injection process of AM7 solution under the assistance of endoscopy, and the white "Δ" in the figure indicates the AM7 solution that flows back after the endoscopic needle is withdrawn. FIG14B is a general view after mucosal stripping, and the white "Δ" in the figure indicates the AM7 residual liquid exposed after stripping. FIG14C is a postoperative treatment for submucosal tumors, in which the blood and submucosal injection liquid remaining in the postoperative wound are washed away with physiological saline, which is conducive to providing a clear submucosal field of vision and facilitating the localization of the tumor. FIG14D is a wound backfilled with AM7 solution after mucosal stripping or tumor resection, and CS3 solution is sprayed on the wound surface at the same time. FIG14E is a gel-like form of AM7 and CS3 solution after contact with the wound surface after spraying CS3 solution (0min). FIG14F shows the morphology of the in situ hydrogel after 10 minutes of gelation. It can be seen from the figure that the hydrogel completely matches the irregular morphology of the wound and effectively isolates the submucosal muscle layer from the external environment.

(3) Effect of A7C3 hydrogel on repair after ESD surgery:

GFI and AM7 solutions were injected submucosally into the esophagus, and the postoperative wounds were treated in different ways (as shown in Figure 15A). The inflammation, granulation growth, and closure of the wounds in each group were observed 28 days after surgery, as shown in Figure 15B. As can be seen from the healing process diagram (Figure 15B), the wounds in the GFI group, GFI+CS3 group, and AM7 group were not closed after surgery. The wounds had obvious redness, swelling, and inflammation 3 days after surgery, and the granulation tissue did not completely fill the wounds. During the healing process from 9 to 28 days after surgery, obvious scar tissue formed on the wounds, and the scars were still obvious at 28 days. After the in situ hydrogel was formed on the wounds in the A7C3 Gel group, the wounds were closed and isolated from the digestive tract lumen environment. Microscopic examination revealed that there was no obvious redness or swelling in the wound 3 days after surgery, and obvious granulation tissue filling was visible; on the 9th day after surgery, the granulation tissue shrank; on the 14th day, the granulation tissue matured and the wound surface tended to be flat; on the 21st day, the wound surface was basically healed; on the 28th day, the wound surface was flat, and there was no significant difference in morphology from normal tissue. Figure 16 is a morphological diagram of the esophagus after sampling 28 days after ESD surgery. It can be seen from Figure 16 that compared with normal esophageal tissue (Figure 16F), the wound surface of the A7C3 hydrogel group (Figures 16A-①, 16D-②, 16E-①) was flat and had no obvious scars, while the other groups had varying degrees of scar formation. The healing effect of canine esophageal ESD surgery shows that the postoperative wound closure of the hydrogel of the present invention is beneficial to wound healing.

In summary, the present invention has developed a "dual-function" material that has both the function of an ESD mucosal pad and the functions of sealing, hemostasis and repairing postoperative wounds. It also solves the problems of poor padding effect of SFC materials in existing ESD and intraoperative/postoperative complications, and has very good clinical application prospects.

Claims (20)

1. A hydrogel, characterized in that it is obtained by dissolving the following raw materials in weight ratio in a solvent and mixing them into a gel: 3-7 parts of oxidized alginate modified by grafting unsaturated carbon-carbon double bonds, and 3-7 parts of carboxymethyl chitosan modified by grafting thiol groups; The oxidation degree of alginate in the unsaturated carbon-carbon double bond grafted modified oxidized alginate is 14% to 50%; The grafting rate of unsaturated carbon-carbon double bonds in the oxidized alginate modified by grafting unsaturated carbon-carbon double bonds is 24% to 37%; The thiol grafting rate of the thiol-grafted modified carboxymethyl chitosan is 24% to 52%; The unsaturated carbon-carbon double bond grafted modified oxidized alginate is maleimide grafted modified oxidized alginate; The thiol-grafted modified carboxymethyl chitosan is cysteamine hydrochloride-grafted modified carboxymethyl chitosan. 2. The hydrogel according to claim 1 is characterized by: 7 parts of oxidized alginate modified by grafting unsaturated carbon-carbon double bonds and 3 parts of carboxymethyl chitosan modified by grafting thiol groups. 3. The hydrogel according to claim 1 or 2, characterized in that: The alginate is sodium alginate; and/or the carboxymethyl chitosan is O-carboxymethyl chitosan with a substitution degree less than 1. 4. The hydrogel according to claim 1 or 2, characterized in that: the unsaturated carbon-carbon double bond grafted modified oxidized alginate is maleimide grafted modified oxidized sodium alginate, and the preparation method of the maleimide grafted modified oxidized sodium alginate comprises the following steps: (1) dissolving oxidized sodium alginate in a solvent to obtain an oxidized sodium alginate solution; (2) adding EDC and NHS to the oxidized sodium alginate solution, and then adding maleimide hydrochloride to react; (3) After the reaction, the product is dialyzed and dried to obtain maleimide-grafted modified oxidized sodium alginate. 5. The hydrogel according to claim 4, characterized in that: In step (1), the solvent is MES solution, PBS solution or deionized water; and/or, in step (1), the mass percentage of the oxidized sodium alginate solution is 1% to 3%; and/or, in step (2), the ratio of the amount of the oxidized sodium alginate substance to the total amount of EDC and NHS is 1:1.5-3; and/or, in step (2), the molar ratio or mass ratio of EDC and NHS is 1:1-1.5; and/or, in step (2), the ratio of the amount of the oxidized sodium alginate substance to the amount of maleimide hydrochloride substance is 1:1-2; and/or, in step (2), the reaction is carried out at room temperature for 12-24 hours; And/or, in step (3), the dialysis is first placed in a 0.01-0.05M HCl deionized water solution for 3-5 days, and then placed in deionized water for 12-24 hours. 6. The hydrogel according to claim 5, characterized in that: In step (2), during the reaction, the pH of the reaction solution is adjusted every 6 hours to maintain the pH value at 5.0 to 5.5; And/or, in step (3), the dialysis bag has a cutoff of 3.5 to 8 kDa; and/or, in step (3), the water is changed every 6 to 12 hours during the dialysis. 7. The hydrogel according to claim 1 or 2, characterized in that: the synthesis method of the thiol-grafted modified carboxymethyl chitosan comprises the following steps: 1) dissolving carboxymethyl chitosan in a solvent to obtain a carboxymethyl chitosan solution; 2) adding EDC to the carboxymethyl chitosan solution, and then adding cysteamine hydrochloride to react; 3) After the reaction, dialyze, freeze-dry and alcohol separate to obtain mercaptocarboxymethyl chitosan. 8. The hydrogel according to claim 7, characterized in that: In step 1), the solvent is deionized water or PBS solution; and/or, in step 1), the mass percentage of the carboxymethyl chitosan solution is 2-5%; and/or, in step 2), the ratio of the amount of the carboxymethyl chitosan substance to the amount of the EDC substance is 1:1.5-3; and/or, in step 2), the ratio of the amount of the carboxymethyl chitosan substance to the amount of the cysteamine hydrochloride substance is 1:1-2; and/or, in step 2), the reaction is carried out at room temperature for 12-24 hours; And/or, in step 3), the dialysis is performed in a 0.005-0.05M borax solution for 3-5 days, and then dialyzed in deionized water for 12-24 hours; and/or, in step 3), DTT is added to stir the reaction before freeze-drying; and/or, in step 3), anhydrous ethanol is used for the alcohol analysis. 9. The hydrogel according to claim 8, characterized in that: In step 3), the dialysis bag has a cutoff of 3.5 to 8 kDa; and/or, in step (3), the water is changed every 12 hours during the dialysis. 10. The hydrogel according to claim 1 or 2, characterized in that: The mass volume ratio of the unsaturated carbon-carbon double bond grafted modified oxidized alginate to the solvent is (3-7) g:100 mL; and/or: the mass volume ratio of the thiol group grafted modified carboxymethyl chitosan to the solvent is (3-7) g:100 mL. 11. The hydrogel according to claim 10, characterized in that: the mass volume ratio of the oxidized alginate modified by grafting unsaturated carbon-carbon double bonds to the solvent is 7 g:100 mL; and/or: the mass volume ratio of the carboxymethyl chitosan modified by grafting thiol groups to the solvent is 3 g:100 mL. 12. The hydrogel according to claim 10, characterized in that: the solvent for dissolving the oxidized alginate modified by grafting unsaturated carbon-carbon double bonds is water or PBS buffer; and/or the solvent for dissolving the carboxymethyl chitosan modified by grafting thiol groups is a borax aqueous solution with a concentration of 0.005 to 0.05 M. 13. The hydrogel according to claim 12, characterized in that: the solvent for dissolving the oxidized alginate modified by grafting unsaturated carbon-carbon double bonds is PBS buffer; and/or the solvent for dissolving the carboxymethyl chitosan modified by grafting thiol groups is a 0.01M borax aqueous solution. 14. The hydrogel according to claim 1 or 2, characterized in that: during the mixing, the volume ratio of the oxidized alginate solution modified by grafting unsaturated carbon-carbon double bonds to the carboxymethyl chitosan solution modified by grafting thiol groups is 1:(1-10). 15. The hydrogel according to claim 14, characterized in that: during the mixing, the volume ratio of the oxidized alginate solution modified by grafting unsaturated carbon-carbon double bonds to the carboxymethyl chitosan solution modified by grafting thiol groups is 1:1. 16. The hydrogel according to claim 1 or 2, characterized in that: the gelation time is 3 to 10 seconds; And/or, the mixing is to directly mix the oxidized alginate solution modified by unsaturated carbon-carbon double bonds grafting and the carboxymethyl chitosan solution modified by thiol groups grafting, or to spray the carboxymethyl chitosan solution modified by thiol groups grafting on the oxidized alginate solution modified by unsaturated carbon-carbon double bonds grafting. 17. The hydrogel according to claim 16, characterized in that the gelation time is 5 seconds. 18. The method for preparing the hydrogel according to any one of claims 1 to 17, characterized in that it comprises the following steps: (1) weighing unsaturated carbon-carbon double bond graft-modified oxidized alginate and thiol group graft-modified carboxymethyl chitosan according to a weight ratio, and dissolving them in a solvent respectively; (2) Mix the two solutions into a gel. 19. Use of the hydrogel according to any one of claims 1 to 17 in the preparation of a submucosal pad and/or a tissue hemostatic material. 20. The use according to claim 19, characterized in that: the submucosal pad is a submucosal pad used for endoscopic submucosal dissection; and/or the tissue hemostatic material is a material used for wound hemostasis during endoscopic submucosal dissection.