A two-stage composite gel and a preparation method and application thereof

Abstract

This invention discloses a two-stage composite gel, its preparation method, and its applications, relating to the field of medical materials technology. The gel consists of components A and B. Component A includes aldehyde-modified dextran, sodium alginate-γ-polyglutamic acid composite microgel particles, medical-grade layered silicate nanosheets, and indigo carmine. Component B includes amino-modified dextran-masked catechol grafts, carboxymethyl chitosan, cationic gelatin-tannic acid nanocomposite, ROS-responsive epithelialization-promoting nanocapsules, calcium gluconate, and magnesium chloride. During use, components A and B are mixed, and cross-linked with aldehyde groups and amino-Schiff bases to form a first gel network for intraoperative submucosal support. After exposure to gastrointestinal fluid and the wound microenvironment, further adhesiolytic activation and secondary cross-linking occur to form a second gel network for wound closure, hemostasis, and wound healing. This invention, through its two-stage network design, balances intraoperative support and postoperative repair, demonstrating promising clinical application prospects.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, specifically to a two-stage composite gel, its preparation method, and its application. Background Technology

[0002] Endoscopic submucosal dissection (ESD) is a minimally invasive endoscopic technique used to treat early-stage gastrointestinal tumors and precancerous lesions. This technique involves injecting a liquid or gel-like substance into the submucosal layer of the lesion, causing the lesion to separate from the muscularis propria and form a submucosal bulge. An endoscopic electrocautery device is then used to dissect along the submucosal layer, achieving en bloc resection of the lesion. Compared to traditional endoscopic mucosal resection, ESD offers advantages such as a higher rate of en bloc resection, more complete pathological evaluation, and a lower local recurrence rate.

[0003] During ESD procedures, the performance of submucosal injection materials directly impacts surgical safety and operational efficiency. An ideal submucosal injection material should be able to rapidly and stably form a submucosal support pad, fully elevating the diseased mucosa and maintaining this elevation for a prolonged period. This expands the safe operating space between the mucosa and muscularis propria, reducing complications such as perforation, bleeding, and thermal damage during electrocautery. Simultaneously, the material should possess good injectability, appropriate viscoelasticity, biocompatibility, visualization properties, and biodegradability, and should not obstruct the endoscopic view or hinder electrocautery cutting.

[0004] Currently, commonly used submucosal injection materials in clinical practice include saline, glucose solution, glycerol solution, hydroxyethyl starch solution, sodium hyaluronate solution, and mixed injection solutions containing dyes. Among these, saline is inexpensive and convenient to use, but it diffuses and is absorbed rapidly after entering the submucosal layer, resulting in a short duration of elevation. Repeated injections are often required during surgery, increasing operation time and the risk of bleeding and perforation. Glycerol solution and hydroxyethyl starch solution can prolong the duration of submucosal elevation to some extent compared to saline, but they still cannot meet the long-term stable support requirements of ESD for large-area lesions or complex sites. Sodium hyaluronate solution has good viscoelasticity and tissue compatibility, providing relatively durable submucosal elevation, but its main function remains focused on intraoperative physical support, with limited effects on postoperative wound protection, hemostasis, anti-inflammation, and mucosal repair.

[0005] In recent years, to address the issues of short support time and limited functionality in traditional injectable solutions, researchers have proposed various novel submucosal injection materials, such as thermosensitive hydrogels, self-assembled peptide hydrogels, hyaluronic acid-based dynamically cross-linked hydrogels, chitosan-based adhesive hydrogels, alginate gels, and other injectable biomaterials. These materials typically form gel networks through temperature changes, ionic cross-linking, dynamic covalent bonds, molecular self-assembly, or physical entanglement, thereby improving submucosal support performance. However, existing materials still have certain shortcomings.

[0006] On the one hand, some gel-based materials primarily focus on intraoperative mucosal elevation and support. While they can prolong support time, they are insufficient in protecting, stopping bleeding, and repairing the exposed wound after lesion removal. Post-ESD wounds are typically large, exposing the submucosa, making them susceptible to gastric acid, bile, intestinal fluid, digestive enzymes, mechanical friction, and local inflammatory responses. This can lead to complications such as delayed bleeding, delayed ulcer healing, local infection, scar formation, and esophageal stricture. Therefore, injectable materials with only supportive functions are insufficient to meet the needs of the entire ESD treatment process.

[0007] On the other hand, while some hydrogels with tissue adhesion can be used for wound closure or postoperative protection, they also exhibit strong adhesion during the procedure, potentially adhering to endoscopic attachments, electrocautery devices, or tissue surfaces. This can affect clear exposure of the dissection layer and instrument manipulation, and even increase cutting resistance and operational complexity. Therefore, for ESD applications, stronger adhesion is not always better. Instead, materials need to maintain a relatively low-adhesion, easily operable state during the procedure, and only after dissection is completed or the wound is exposed should they provide appropriate wound adhesion and protection.

[0008] Furthermore, existing postoperative wound covering materials typically need to be applied to the wound via spraying, spreading, or separate injection after ESD (endoscopic submucosal dissection). These methods increase the number of steps, place higher demands on the stability of endoscopic procedures and wound localization, and are prone to displacement, detachment, or uneven coverage in the moist, peristaltic, and fluid-fluid-washing environment of the gastrointestinal tract. If submucosal injection materials could provide support during the procedure and transform into wound protection materials after lesion removal and wound exposure, the additional steps could be reduced, improving treatment efficiency.

[0009] Meanwhile, the wound healing process after ESD involves multiple stages, including inflammatory response, oxidative stress, cell migration, angiogenesis, collagen deposition, and re-epithelialization. Traditional injectable materials are usually inert support materials, lacking the ability to respond to the postoperative wound microenvironment and unable to regulate the release of healing-promoting active substances according to local inflammation or oxidative stress. Especially after large-area ESD in the esophagus, excessive activation of fibroblasts and collagen deposition during wound repair may lead to scar contraction and esophageal stricture, and existing single support materials are insufficient to effectively intervene in this process.

[0010] Therefore, there is an urgent need to develop a novel submucosal injection material suitable for endoscopic submucosal dissection (ESD). This material should possess good injectability, shear-thinning properties, long-lasting submucosal support, visualization performance, and low instrument adhesion during the procedure. Simultaneously, when the wound is exposed to the gastrointestinal environment after ESD dissection, it should be able to transform in situ into a gel layer with functions of wound adhesion, hemostasis, isolation and protection, anti-inflammation, and promoting mucosal repair, thereby achieving integrated intraoperative support and postoperative wound healing. Based on this, it is necessary to provide a two-stage submucosal injection composite gel to improve the safety of ESD surgery, reduce the risk of complications, and promote postoperative wound repair. Summary of the Invention

[0011] To address the technical problems of existing submucosal injection materials having short duration of elevation and insufficient protection, hemostasis, and repair effects on exposed wounds, this invention provides a two-stage composite gel, its preparation method, and its application.

[0012] According to one aspect of the present invention, a method for preparing a two-stage composite gel is provided, comprising: S1, Preparation of aldehyde-modified dextran; Dextran is oxidized with sodium periodate to yield aldehyde-modified dextran; S2, prepare sodium alginate-γ-polyglutamic acid composite microgel particles; Using an aqueous solution of sodium alginate and γ-polyglutamic acid as the dispersion phase and liquid paraffin as the continuous oil phase, a water-in-oil microdroplet dispersion system was formed by homogenization and emulsification. After calcium ion crosslinking, demulsification, and washing, composite microgel particles were obtained. S3, preparation of amino-glucan-masked catechol grafts; After activating 3,4-dihydroxyphenylpropionic acid, it was grafted onto amino-modified dextran, and then reacted with a phenylboronic acid group donor to form a borate ester complex structure. After dialysis and drying, the amino-modified dextran-masked catechol graft was obtained. S4, preparation of cationic gelatin-tannic acid nanocomposite; Gelatin was modified with glycidyl trimethylammonium chloride to obtain cationic gelatin, and then cationic gelatin was combined with tannic acid to obtain cationic gelatin-tannic acid nanocomposite. S5, preparation of ROS-responsive epithelialization-promoting nanocapsules; 3-Mercapto-1,2-propanediol and acetone were subjected to a thioketation reaction catalyzed by trifluoroacetic acid to obtain a polyhydroxy monomer containing thioketation bonds. The obtained monomer and hexamethylene diisocyanate were polycondensed under the catalysis of dibutyltin dilaurate to obtain a ROS-responsive polymer containing thioketation bonds. The ROS-responsive polymer containing thioketation bonds was dissolved in ethyl acetate as the oil phase. The epithelialization-promoting active substance and polyvinyl alcohol were dissolved in water as the first aqueous phase. Polyvinyl alcohol was dissolved in water as the second aqueous phase. The first aqueous phase and the oil phase were mixed and ultrasonically sonicated to form a W / O primary emulsion. The W / O primary emulsion was then added to the second aqueous phase and ultrasonically sonicated to form a W / O / W complex emulsion. The ethyl acetate was removed and the precipitate was collected to obtain ROS-responsive epithelialization-promoting nanocapsules. S6, Prepare component A; Aldehyde-modified dextran, sodium alginate-γ-polyglutamic acid composite microgel particles, medical layered silicate nanosheets, and indigo carmine were dispersed in HEPES buffer and degassed to obtain component A. S7, prepare component B; Aminated dextran-masked catechol graft, carboxymethyl chitosan, cationic gelatin-tannic acid nanocomposite, ROS-responsive epithelialization nanocapsules, calcium gluconate and magnesium chloride were dispersed in HEPES buffer and degassed to obtain component B.

[0013] Optionally, in S2, Span80 is pre-added to the liquid paraffin as an emulsifier; calcium chloride aqueous solution is used as a crosslinking liquid for calcium ion crosslinking; and anhydrous ethanol is used for demulsification.

[0014] Optionally, in S3, 3,4-dihydroxyphenylpropionic acid is activated in MES buffer using EDC and NHS; 4-aminophenylboronic acid is used as a phenylboronic acid group donor.

[0015] Optionally, in S5, the preparation of the sulfur-ketal bond-containing ROS-responsive polymer specifically includes: adding 3-mercapto-1,2-propanediol to anhydrous acetone, and adding trifluoroacetic acid as an acid catalyst, and carrying out a sulfur-ketalization reaction under nitrogen protection to form a polyhydroxy monomer containing sulfur-ketal bonds and retaining hydroxyl groups. After the reaction, triethylamine is added to neutralize the trifluoroacetic acid, acetone is removed, and the crude product obtained is dissolved in ethyl acetate, washed, dried, filtered, and concentrated under reduced pressure to obtain a sulfur-ketal bond-containing polyhydroxy monomer; dissolving the sulfur-ketal bond-containing polyhydroxy monomer and hexamethylene diisocyanate in anhydrous dimethyl sulfoxide, respectively, and adding the diisocyanate solution dropwise to the sulfur-ketal bond-containing polyhydroxy monomer solution under nitrogen protection, and adding dibutyltin dilaurate as a catalyst to carry out a polycondensation reaction to form a sulfur-ketal bond-containing polyurethane ROS-responsive polymer; collecting the precipitate after the reaction to obtain the sulfur-ketal bond-containing ROS-responsive polymer.

[0016] Optionally, in S5, the epithelialization-promoting active substance is an EGFR-affinity short peptide GE11, whose amino acid sequence is YHWYGYTPQNVI.

[0017] Optionally, in S6, the A component, by mass percentage, comprises 1%–2% aldehyde-modified dextran, 2%–5% sodium alginate-γ-polyglutamic acid composite microgel particles, 0.1%–0.2% medical layered silicate nanosheets, 0.005%–0.02% indigo carmine, and the balance being HEPES buffer.

[0018] Optionally, in S7, the B component, by mass percentage, comprises: 0.5%–2.0% aminoglucan-masked catechol graft, 0.5%–1% carboxymethyl chitosan, 0.2%–1% cationic gelatin-tannic acid nanocomposite, 0.1%–0.3% ROS-responsive epithelialization nanocapsules, 0.2%–0.3% calcium gluconate, 0.01%–0.1% magnesium chloride, with the balance being HEPES buffer.

[0019] According to another aspect of the present invention, a two-stage composite gel is provided, prepared by the method described above, comprising component A and component B; component A includes aldehyde-modified dextran, sodium alginate-γ-polyglutamic acid composite microgel particles, medical layered silicate nanosheets, and indigo carmine; component B includes amino-modified dextran-masked catechol graft, carboxymethyl chitosan, cationic gelatin-tannic acid nanocomposite, ROS-responsive epithelialization nanocapsules, calcium gluconate, and magnesium chloride; when used, components A and B are mixed, first forming a first gel network through a Schiff base reaction, and then further undergoing adhesion group activation and secondary crosslinking to form a second gel network, thereby possessing two-stage characteristics.

[0020] Optionally, component A and component B are respectively filled into two independent chambers of a dual-chamber pre-filled syringe, and a static mixer is installed at the front end of the dual-chamber pre-filled syringe.

[0021] According to another aspect of the invention, there is provided the use of the two-stage composite gel as described above in the preparation of a submucosal injection material for ESD, wherein the submucosal injection material forms a first gel network for support after being injected into the submucosal layer during surgery, and forms a second gel network for closure of the wound and promotion of re-epithelialization after the wound is exposed postoperatively.

[0022] The beneficial effects of this invention are: (1) After injection into the submucosal layer, the composite gel of the present invention can quickly form a stable submucosal support pad, which can fully separate the diseased mucosa from the muscularis propria and expand the endoscopic dissection operation space. Through the combined action of dynamic gel network, resilient microgel particles and physical reinforcement components, it can effectively prolong the maintenance time of mucosal elevation and reduce the number of times of repeated injection during the operation. At the same time, the material has shear-thinning properties, which can be smoothly injected through conventional endoscopic injection needles, and maintains a relatively low adhesion state during the operation, making it less likely to adhere to electrocautery or endoscopic accessories, which is conducive to maintaining a clear operating field and smooth dissection.

[0023] (2) Unlike traditional injection solutions or ordinary hydrogels used only for submucosal lifting, this invention, after ESD dissection and exposure of the gel to the gastrointestinal lumen and wound environment, can further undergo adhesive group activation and secondary cross-linking, transforming in situ into a repair gel layer covering the wound. This repair gel layer can adhere to the moist wound surface, isolating it from stimulation by gastric acid, bile, intestinal fluid, and digestive enzymes, reducing wound bleeding and external mechanical friction, thereby reducing the risks of delayed bleeding, wound exposure, and delayed ulcer healing.

[0024] (3) The composite gel of the present invention can be loaded with hemostatic, anti-inflammatory and epithelialization-promoting functional components, and can be released in response to changes in the microenvironment such as postoperative wound oxidative stress, thereby improving the local wound repair environment and promoting epithelial cell migration and re-epithelialization. For large-area ESD wounds, especially sites such as the esophagus that are prone to scar contraction or stenosis, the present invention helps to improve the local wound repair microenvironment through wound coverage and protection, release of anti-inflammatory components and delivery of epithelialization-promoting active substances, reduce excessive inflammatory response and abnormal fibrosis tendency, and thus is expected to reduce the risk of postoperative delayed bleeding, poor wound healing and scar contraction-related complications. Attached Figure Description

[0025] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a schematic diagram of the two-stage composite gel injection of the present invention; Figure 2 This is a test diagram of the maintenance of submucosal bulge with the two-stage composite gel of the present invention; Figure 3 This is a test diagram of the ROS response release of the two-stage composite gel of the present invention; Figure 4 This is a cell viability test diagram using the two-stage composite gel CCK8 method of the present invention; Figure 5 This is a schematic diagram of the postoperative wound repair stages of the two-stage composite gel of the present invention. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. All equivalent substitutions, conventional adjustments, or parameter optimizations made based on the concept of the present invention should fall within the scope of protection of the present invention.

[0027] Unless otherwise specified, all conditions in the examples were performed under standard conditions. Reagents or instruments whose manufacturers are not specified are commercially available products. Unless otherwise stated, all technical and scientific terms herein have the meanings commonly understood by one of ordinary skill in the art.

[0028] Example 1: A two-stage composite gel.

[0029] This two-stage composite gel consists of component A and component B. Component A includes aldehyde-modified dextran, sodium alginate-γ-polyglutamic acid composite microgel particles, medical layered silicate nanosheets, and indigo carmine.

[0030] Component B includes amino-glucan-masked catechol graft, carboxymethyl chitosan, cationic gelatin-tannic acid nanocomposite, ROS-responsive epithelialization nanocapsules, calcium gluconate, and magnesium chloride.

[0031] In use, components A and B are mixed in a 1:1 volume ratio and injected into the submucosa through an endoscopic needle. The mixture forms a first gel network in situ in the submucosa for support during ESD. When some of the gel is exposed to the gastrointestinal lumen and wound environment after ESD dissection, the material further forms a second gel network for wound adhesion, hemostasis, and promoting mucosal repair.

[0032] The specific preparation steps for this two-stage composite gel are as follows: Step 1: Preparation of aldehyde-modified dextran; Step 1.1: Weigh 10.00 g of dextran with an average molecular weight of 40 kDa, add it to 500 mL of deionized water, and stir at 400 rpm for 2 hours at 25 °C until it is completely dissolved to obtain a dextran aqueous solution.

[0033] Step 1.2: Weigh out 2.00g of sodium periodate, add it to 50mL of deionized water, and stir to dissolve it under light-protected conditions to obtain an aqueous solution of sodium periodate.

[0034] Step 1.3: Place the dextran aqueous solution in an ice-water bath at 4°C, adjust the stirring speed to 300 rpm, and slowly add the sodium periodate aqueous solution dropwise to the dextran aqueous solution over 20 minutes under light-protected conditions. After the addition is complete, continue the reaction at 4°C under light-protected conditions for 4 hours.

[0035] Step 1.4: After the reaction is complete, add 2.00 mL of ethylene glycol to the reaction solution to terminate the sodium periodate oxidation reaction. Continue stirring at 4°C and 300 rpm for 30 minutes.

[0036] Step 1.5: The reaction solution was placed into a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water at 4°C for 48 hours, with the deionized water being replaced every 6 hours. After dialysis, the dialysate was pre-frozen at -80°C for 12 hours, and then dried in a freeze dryer for 48 hours to obtain aldehyde-modified dextran powder.

[0037] Step 2: Preparation of sodium alginate-γ-polyglutamic acid composite microgel particles; Step 2.1: Weigh 2.00g of sodium alginate and 1.00g of γ-polyglutamic acid, add them to 100mL of deionized water, and stir at 500rpm for 3 hours at 25℃ to completely dissolve them, thus obtaining a polymer aqueous solution.

[0038] Step 2.2: Weigh 1.50g of calcium chloride, add it to 100mL of deionized water, and stir at 300rpm for 20 minutes at 25℃ to obtain a calcium chloride crosslinking solution with a mass concentration of 1.5%.

[0039] Step 2.3: Place the aqueous polymer solution in a high-speed homogenizer and homogenize at 10,000 rpm for 2 minutes at 25°C. Then, using a peristaltic pump, add the aqueous polymer solution dropwise to 200 mL of liquid paraffin at a rate of 1.0 mL / min. 2.0 g of Span80 has been pre-added to the liquid paraffin as an emulsifier. Maintain the homogenization rate at 10,000 rpm and the emulsification temperature at 25°C during the dropwise addition. After the aqueous polymer solution has been added, continue homogenizing and emulsifying for 10 minutes to form a water-in-oil microdroplet dispersion system, i.e., a W / O emulsion system with sodium alginate-γ-polyglutamic acid aqueous phase as the dispersed phase and liquid paraffin as the continuous oil phase.

[0040] Step 2.4: Subsequently, 100 mL of 1.5% calcium chloride crosslinking solution was added dropwise to the above water-in-oil microdroplet dispersion system at a rate of 2.0 mL / min. During the addition, the stirring speed was maintained at 8000 rpm and the temperature at 25°C. After the addition was completed, stirring and crosslinking continued for 30 minutes to allow sodium alginate and γ-polyglutamic acid to form composite microgel particles under the action of calcium ions.

[0041] Step 2.5: After cross-linking is complete, add 300 mL of anhydrous ethanol to the system to break the emulsion, centrifuge at 4000 rpm for 10 minutes, and discard the supernatant. Wash the precipitate twice with anhydrous ethanol and three times with sterile phosphate buffer. Centrifuge at 4000 rpm for 10 minutes each time.

[0042] Step 2.6: Resuspend the obtained microgel particles in sterile phosphate buffer to prepare a 10% (w / w) microgel particle stock solution, and store it at 4°C for later use.

[0043] Step 3: Preparation of amino-glucan-masked catechol grafts; Step 3.1: Weigh 5.00 g of aminodextrose and add it to 200 mL of MES buffer solution with a concentration of 0.1 mol / L and a pH of 5.8. Stir at 400 rpm for 2 hours at 25 °C until it is completely dissolved to obtain an aminodextrose solution.

[0044] Step 3.2: Weigh 1.00 g of 3,4-dihydroxyphenylpropionic acid and add it to 50 mL of MES buffer, adjusting the pH to 5.8. Then add 1.20 g of EDC and 0.70 g of NHS, and activate the solution by stirring at 300 rpm for 30 minutes at 25 °C to obtain an activated catechol carboxylic acid solution.

[0045] Step 3.3: Under nitrogen protection, the activated catechol carboxylic acid solution was slowly added dropwise to the amino-modified dextran solution over a period of 30 minutes. After the addition was complete, nitrogen protection was maintained, and the reaction was carried out at 25°C and 400 rpm for 12 hours.

[0046] Step 3.4: After the reaction is complete, weigh 0.60 g of 4-aminophenylboronic acid and add it to 30 mL of 0.1 mol / L MES buffer solution (pH 7.4) to fully dissolve it, obtaining a phenylboronic acid group donor solution. Slowly add this phenylboronic acid group donor solution to the reaction solution obtained in step 3.3, and continue the reaction at 25°C and 400 rpm for 6 hours. This allows the phenylboronic acid groups to form a dynamically reversible borate ester complex structure with the catechol groups grafted onto the aminodextran molecular chain, thereby temporarily masking some of the catechol adhesion groups, resulting in a crude product solution of aminodextran-masked catechol graft. The masking structure is a dynamically reversible complex structure, exhibiting certain stability under neutral to weakly alkaline conditions. It can partially dissociate under the influence of acidic gastric juice, changes in local ionic strength, wound proteins, and an oxidative microenvironment, thus gradually releasing free catechol groups.

[0047] Step 3.5: The crude product solution was placed into a dialysis bag with a molecular weight cutoff of 8000-14000 Da and dialyzed with deionized water at 4°C for 72 hours, with the deionized water being replaced every 6 hours. After dialysis, the dialysate was pre-frozen at -80°C for 12 hours, and then freeze-dried for 48 hours to obtain the amino-glucan-masked catechol graft powder.

[0048] Step 4: Preparation of cationic gelatin-tannic acid nanocomposite; Step 4.1: Weigh 5.00 g of gelatin and add it to 100 mL of deionized water. Stir at 500 rpm for 1 hour in a 45°C water bath to fully dissolve the gelatin, obtaining a gelatin aqueous solution. The gelatin is preferably type A or type B gelatin (type A gelatin is used in this embodiment, with a molecular weight preferably between 50 kDa and 150 kDa). After the gelatin is completely dissolved, adjust the pH of the system to 9.0–9.5 using a 1 mol / L sodium hydroxide solution. Weigh 3.00 g of glycidyltrimethylammonium chloride and add it to 10 mL of deionized water. Stir at 300 rpm for 10 minutes at 25°C to fully dissolve the gelatin, obtaining a glycidyltrimethylammonium chloride aqueous solution. Slowly add the glycidyltrimethylammonium chloride aqueous solution dropwise to the above gelatin aqueous solution over 20 minutes. During the dropwise addition, maintain the system temperature at 45°C, the stirring speed at 500 rpm, and maintain the pH of the system at 9.0–9.5 using a 1 mol / L sodium hydroxide solution. After the addition was complete, the reaction continued at 45℃ and 500rpm for 6 hours to allow the epoxy groups in glycidyltrimethylammonium chloride to undergo ring-opening reactions with the amino, hydroxyl, or carboxyl groups in the gelatin molecular chain, thereby introducing quaternary ammonium salt cationic groups onto the gelatin molecular chain, yielding a cationic gelatin crude product solution. After the reaction was complete, the pH of the system was adjusted to 7.0–7.4 using 1mol / L hydrochloric acid solution. The reaction solution was then placed in a dialysis bag with a molecular weight cutoff of 8000–14000 Da and dialyzed with deionized water at 4℃ for 72 hours, with the deionized water replaced every 6 hours to remove unreacted glycidyltrimethylammonium chloride and small molecule byproducts. After dialysis, the dialysate was pre-frozen at -80℃ for 12 hours, and then freeze-dried in a freeze dryer for 48 hours to obtain cationic gelatin.

[0049] Step 4.2: Weigh 1.00 g of cationic gelatin, add it to 100 mL of deionized water, and stir at 500 rpm for 1 hour in a 37°C water bath to completely dissolve it, thus obtaining a cationic gelatin solution.

[0050] Step 4.3: Weigh 0.30g of tannic acid, add it to 20mL of deionized water, and stir at 300rpm for 20 minutes at 25℃ to obtain an aqueous solution of tannic acid.

[0051] Step 4.4: Adjust the temperature of the cationic gelatin solution to 37°C and the stirring speed to 600 rpm. Using a syringe pump, add the tannic acid aqueous solution dropwise to the cationic gelatin solution at a rate of 0.5 mL / min over 40 minutes. Maintain the pH of the system at 7.0–7.2 during the addition process.

[0052] Step 4.5: After the addition is complete, continue stirring the reaction at 37°C and 600 rpm for 2 hours to allow the cationic gelatin and tannic acid to form a nanocomposite through hydrogen bonding, hydrophobic interaction and electrostatic interaction.

[0053] Step 4.6: After the reaction is complete, cool the system to 25°C, centrifuge at 10,000 rpm for 15 minutes, and discard the supernatant. Wash the precipitate three times with sterile phosphate buffer, centrifuging at 10,000 rpm for 15 minutes each time.

[0054] Step 4.7: Resuspend the obtained precipitate in 50 mL of sterile phosphate buffer to obtain a cationic gelatin-tannic acid nanocomposite dispersion.

[0055] Step 5: Preparation of ROS-responsive epithelialization-promoting nanocapsule dispersion; This step utilizes a ROS-responsive polymer containing thioketal bonds as the nanocapsule wall material. This polymer is prepared by the condensation polymerization of a polyhydroxy monomer containing thioketal bonds and a diisocyanate monomer. The epithelialization-promoting active substance is a short peptide that promotes epithelial cell migration, adhesion, or repair, preferably the EGFR-affinity short peptide GE11, with the amino acid sequence YHWYGYTPQNVI. This short peptide can promote wound re-epithelialization by interacting with epithelial cell-related receptors or repair pathways.

[0056] Step 5.1: Weigh 10.00 g of 3-mercapto-1,2-propanediol and add it to 100 mL of anhydrous acetone. Add 0.5 mL of trifluoroacetic acid as an acid catalyst. Under nitrogen protection, react at 25 °C and 500 rpm for 24 hours to allow the carbonyl group in acetone to undergo a thioketation reaction with the mercapto group in 3-mercapto-1,2-propanediol, forming a polyhydroxy monomer containing a thioketal bond and retaining a hydroxyl group. After the reaction is complete, add 1.00 mL of triethylamine to the reaction system to neutralize the trifluoroacetic acid, and continue stirring for 30 minutes. Then, remove the acetone by rotary evaporation under reduced pressure. Dissolve the crude product in 100 mL of ethyl acetate and wash it three times each with 50 mL of saturated sodium bicarbonate aqueous solution, deionized water, and saturated sodium chloride aqueous solution. Dry the product with anhydrous sodium sulfate for 12 hours, filter, and concentrate the filtrate under reduced pressure to obtain the polyhydroxy monomer containing a thioketal bond. The obtained monomer can be stored at -20 °C in the dark for later use. The sulfur-ketal bond-containing polyhydroxy monomer is a sulfur-ketal structure monomer formed by the condensation of acetone and 3-mercapto-1,2-propanediol, and its molecule contains a sulfur-ketal bond and hydroxyl groups that can react with isocyanate groups.

[0057] Step 5.2: Further weigh 2.00 g of the thioketal bond-containing polyhydroxy monomer and add it to 30 mL of anhydrous dimethyl sulfoxide. Stir at 500 rpm for 30 minutes at 25°C until completely dissolved to obtain a solution of the thioketal bond-containing polyhydroxy monomer. Weigh 1.20 g of hexamethylene diisocyanate and add it to 10 mL of anhydrous dimethyl sulfoxide. Stir at 300 rpm for 10 minutes at 25°C to obtain a diisocyanate solution. Under nitrogen protection, slowly add the diisocyanate solution dropwise to the thioketal bond-containing polyhydroxy monomer solution over 20 minutes. Maintain the reaction temperature at 50°C and the stirring speed at 500 rpm during the dropwise addition. After the addition was complete, 20 μL of dibutyltin dilaurate was added to the reaction system as a catalyst, and the reaction continued for 8 hours at 50 °C, 500 rpm, and under nitrogen protection. This allowed the condensation reaction between the thioketal-bonded polyhydroxy monomer and the diisocyanate monomer to form a thioketal-bonded polyurethane ROS-responsive polymer. After the reaction was complete, the reaction solution was cooled to 25 °C and then slowly added dropwise to 500 mL of ice-cold anhydrous diethyl ether to precipitate the polymer, stirring at 600 rpm during the addition. After precipitation, the precipitate was collected by centrifugation at 8000 rpm for 10 minutes. The precipitate was washed three times with 100 mL of anhydrous diethyl ether each time to remove unreacted small molecules and residual solvent. After washing, the precipitate was placed in a vacuum drying oven and dried at 25 °C and -0.08 MPa for 24 hours to obtain the thioketal-bonded ROS-responsive polymer.

[0058] Step 5.3: Weigh 100 mg of the ROS-responsive polymer containing thioketal bonds, add it to 5.0 mL of ethyl acetate, and stir at 500 rpm for 30 minutes at 25 °C until it is completely dissolved to obtain an oil phase solution.

[0059] Step 5.4: Weigh 10 mg of the epithelialization-promoting active substance (EGFR-affinity short peptide GE11), add it to 10 mL of deionized water, and then add 100 mg of polyvinyl alcohol to make the polyvinyl alcohol concentration 1.0%. Stir at 600 rpm for 30 minutes at 25°C to fully dissolve it, obtaining the first aqueous phase solution. Place the first aqueous phase solution in an ice bath for later use.

[0060] Step 5.5: Slowly add the first aqueous phase solution dropwise to the oil phase solution obtained in step 5.3, and sonicate for 2 minutes at 200W using a probe-type ultrasonic instrument under ice bath conditions to form a W / O (water-in-oil) primary emulsion with water encapsulated in the oil phase. A pulse mode of 2 seconds working followed by 2 seconds of rest is used during the ultrasonication process.

[0061] Step 5.6: Take another 1.00g of polyvinyl alcohol, add it to 100mL of deionized water, stir at 600rpm for 1 hour at 60℃ to completely dissolve it, and cool it to 25℃ to obtain a second aqueous phase solution with a mass concentration of 1.0%.

[0062] Step 5.7: Slowly add the above W / O colostrum to the second aqueous phase solution, and sonicate at 300W for 3 minutes under ice bath conditions to form a W / O / W (water-in-oil-in-water) double emulsion. A pulse mode of 2 seconds on, 2 seconds off is used during the sonication process.

[0063] Step 5.8: The obtained double emulsion was placed at 25°C and magnetically stirred at 500 rpm for 6 hours to allow the ethyl acetate to evaporate completely. Then, it was centrifuged at 12000 rpm and 4°C for 20 minutes to collect the precipitate. The precipitate was washed three times with sterile deionized water, and centrifuged at 12000 rpm and 4°C for 20 minutes after each wash to obtain nanocapsules.

[0064] Step 5.9: Finally, the obtained nanocapsules are resuspended in 20 mL of sterile phosphate buffer to obtain a ROS-responsive epithelialization-promoting nanocapsule dispersion.

[0065] Step six, preparation of component A; Component A is a uniformly shear-thinned dispersion system, comprising, by mass percentage: 1.20% aldehyde-modified dextran; 3.00% sodium alginate-γ-polyglutamic acid composite microgel particles; 0.12% medical-grade layered silicate nanosheets; 0.01% indigo carmine; and the balance being sterile buffer. The sterile buffer is preferably physiological saline or HEPES buffer, with a pH of 7.0–7.4. In this embodiment, HEPES buffer is specifically used. Component A contains little or no free amino polymers that can undergo rapid cross-linking reactions with aldehyde groups to improve storage stability.

[0066] Step 6.1: Weigh 1.20 g of the aldehyde-modified dextran obtained in Step 1 and add it to 80 mL of sterile HEPES buffer.

[0067] Step 6.2: Stir at 500 rpm for 3 hours at 25°C to completely dissolve the aldehyde-modified dextran.

[0068] Step 6.3: Then add 3.00 g of the sodium alginate-γ-polyglutamic acid composite microgel particles obtained in Step 2 (dry weight). After addition, reduce the stirring speed to 300 rpm and stir at 25°C for 30 minutes to ensure uniform dispersion of the microgel particles.

[0069] Step 6.4: Weigh 0.12g of medical-grade layered silicate nanosheets and slowly add them to the above dispersion system. Maintain a stirring speed of 500rpm during the addition process, and continue stirring for 60 minutes after the addition is complete to ensure the nanosheets are fully dispersed.

[0070] Step 6.5: Weigh 0.01g of indigo carmine, dissolve it in 5mL of HEPES buffer, add it to the above dispersion system, and continue stirring at 25℃ and 300rpm for 15 minutes.

[0071] Step 6.6: Finally, bring the volume to 100 mL using HEPES buffer. Adjust the pH to 7.2 using 0.1 mol / L sodium hydroxide solution or 0.1 mol / L hydrochloric acid solution.

[0072] Step 6.7: Place the obtained dispersion system in a vacuum degassing device and degas for 10 minutes under -0.08 MPa conditions to obtain component A.

[0073] Step 7, Preparation of Component B; Component B, by mass percentage, comprises: 1.00% aminoglucan-masked catechol graft; 0.80% carboxymethyl chitosan; 0.50% cationic gelatin-tannic acid nanocomposite; 0.20% ROS-responsive epithelialization-promoting nanocapsules; 0.25% calcium gluconate; 0.05% magnesium chloride; and the balance being sterile buffer. The sterile buffer is preferably physiological saline or HEPES buffer, with a pH of 7.0–7.4. In this embodiment, HEPES buffer is specifically used to reduce the risk of precipitation of calcium and magnesium ions with phosphate. Specifically: Step 7.1: Weigh 1.00 g of the amino-glucan-masked catechol graft obtained in step 3 and 0.80 g of carboxymethyl chitosan, and add them to 70 mL of sterile HEPES buffer.

[0074] Step 7.2: Stir at 400 rpm for 2 hours at 25°C to fully dissolve the amino-glucan-masked catechol graft and carboxymethyl chitosan, and obtain the basic solution of component B containing amino crosslinking components.

[0075] Step 7.3: Subsequently, the cationic gelatin-tannic acid nanocomposite dispersion obtained in Step 4 is added. The amount added is 0.50 g based on the dry weight of the cationic gelatin-tannic acid nanocomposite. After addition, the mixture is stirred at 25°C and 300 rpm for 30 minutes.

[0076] Step 7.4: Further add the ROS-responsive epithelialization-promoting nanocapsule dispersion obtained in Step 5. The amount added is 0.20 g based on the dry weight of the nanocapsules. After addition, continue stirring at 25°C and 300 rpm for 30 minutes.

[0077] Step 7.5: Weigh 0.25 g of calcium gluconate and 0.05 g of magnesium chloride, dissolve them separately in 5 mL of sterile HEPES buffer, and then slowly add them to the above system. Maintain a stirring speed of 300 rpm during the addition process, and continue stirring for 20 minutes after the addition is complete.

[0078] Step 7.6: Finally, bring the volume to 100 mL using sterile HEPES buffer. Adjust the pH to 7.2 using 0.1 mol / L sodium hydroxide solution or 0.1 mol / L hydrochloric acid solution.

[0079] Step 7.7: Place the obtained system in a vacuum degassing device and degas for 10 minutes under -0.08 MPa conditions to obtain component B.

[0080] Step 8: Filling and Storage; Components A and B were aseptically filled into the two independent chambers of a dual-chamber pre-filled syringe, with each chamber containing 10 mL. After filling, a static mixer was installed at the tip of the dual-chamber syringe, and the outlet of the static mixer could be connected to an endoscopic injection needle.

[0081] It should be further noted that component B, containing active peptides and nanocapsules, is preferably stored at 2–8°C. Component A is also preferably stored at 2–8°C. Repeated freeze-thaw cycles should be avoided during storage. For formulations requiring long-term storage, components A and B can be lyophilized separately and reconstituted to their original volumes with sterile water for injection or sterile HEPES buffer before use. Preferably, the filling volumes of components A and B are the same.

[0082] Example 2: How to use.

[0083] During use, components A and B are simultaneously ejected using a dual-chamber syringe at a volume ratio of 1:1. After being mixed by a static mixer, they are injected into the submucosal layer through a 23G, 25G, or 26G endoscopic injection needle.

[0084] After components A and B are mixed, the aldehyde-modified dextran in component A undergoes a dynamic Schiff base reaction with the amino-modified dextran-masked catechol graft in component B and the free amino groups in carboxymethyl chitosan, forming a first gel network. This first gel network forms rapidly and serves to construct a submucosal support pad between the mucosal layer and the muscularis propria.

[0085] During ESD exfoliation, sodium alginate-γ-polyglutamic acid composite microgel particles, acting as compressible and resilient particles, fill the first gel network, providing volume support and deformation recovery. Medical-grade layered silicate nanosheets serve as a physical reinforcing phase, improving the gel's storage modulus, anti-collapse ability, and network stability, and contributing to better structural retention under electrosurgical conditions. Indigo carmine is used to visualize the injection area, facilitating endoscopic identification of the submucosal layer and exfoliation boundaries.

[0086] After the lesion mucosa is peeled off, part of the composite gel is exposed to the gastrointestinal lumen and wound environment. At this time, the masking catechol structure undergoes dynamic dissociation under the combined effects of acidic gastric juice, changes in local ionic strength, wound proteins, and the oxidative microenvironment of the wound, gradually releasing free catechol adhesive groups. The released catechol groups can further interact with tissue proteins through hydrogen bonding, hydrophobic interactions, and the ortho-quinone structures formed after oxidation, thereby enhancing the material's adhesion to the moist wound surface.

[0087] Simultaneously, calcium and magnesium ions released from calcium gluconate and magnesium chloride further form an ionic cross-linking network with sodium alginate, γ-polyglutamic acid, and carboxylated polysaccharides. Tannic acid interacts with wound proteins, gelatin, and chitosan structures through multi-point hydrogen bonding and hydrophobic interactions, further forming a second gel network.

[0088] The second gel network covers the surface of the ESD wound, providing moist protection, local hemostasis, isolation from gastrointestinal fluid irritation, and promoting mucosal repair.

[0089] ROS-responsive epithelialization nanocapsules gradually degrade in the local oxidative stress environment of the wound, releasing epithelialization-promoting active substances, promoting epithelial cell migration and re-epithelialization, thereby accelerating wound healing after ESD.

[0090] Example 3: Performance testing.

[0091] (1) Injection performance test: After mixing components A and B, the injectability of the composite gel was determined by using an injection needle to evaluate its clinical injection operability.

[0092] like Figure 1 As shown, the composite gel can be extruded normally through the injection needle without any blockage.

[0093] (2) Submucosal Elevation Maintenance Test: Ex vivo porcine gastric or esophageal tissue was used as a submucosal elevation model. Fresh porcine gastric or esophageal tissue was taken, and after removing residual contents from the surface, it was repeatedly rinsed with physiological saline and cut into tissue blocks of approximately 5cm × 5cm. The tissue blocks were laid flat on the test platform with the mucosal side facing up, and the tissue surface was kept moist. Using a 25G endoscopic injection needle, the mixed composite gel was injected into the submucosal layer, with a single injection volume of 1.0mL. After injection, it was observed whether a local elevation was formed on the mucosal surface, and the initial elevation height and elevation area were recorded.

[0094] The elevation height was measured using vernier calipers. Test time points were set immediately after injection, at 5 minutes, 10 minutes, 20 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes. The elevation height was recorded at each time point, and the elevation height retention rate was calculated. The elevation height retention rate was calculated using the following formula: Elevation height retention rate (%) = Ht / H0 × 100%. Where H0 is the elevation height immediately after injection, and Ht is the elevation height measured at different time points. Control groups were also set up, including a saline group, a sodium hyaluronate solution group, and a hydroxyethyl starch group. All groups used the same injection volume and the same tissue model for testing. By comparing the relative elevation height retention rates of different groups at each time point, the supporting stability, anti-diffusion ability, and long-term elevation maintenance ability of the composite gel of this invention in the submucosal layer were evaluated.

[0095] Test results are as follows Figure 2As shown, with the extension of time after injection, the height of the submucosal edema formed by each group of materials showed a decreasing trend to varying degrees, but the rate of decrease differed significantly among different materials. Among them, the dual-stage composite gel group of this invention exhibited the highest relative edema height retention rate throughout the entire test period, indicating that it has good submucosal support stability and anti-collapse ability. In the initial post-injection period, all groups of materials could form a certain submucosal edema, but the saline group decreased the fastest. Five minutes after injection, the relative edema height of the saline group had significantly decreased, maintaining only a low level by 30 minutes, and essentially collapsing after 60 minutes. This result indicates that saline, after entering the submucosal layer, is easily diffused and absorbed by the tissue, making it difficult to provide continuous and stable submucosal support. The hydroxyethyl starch group showed some improvement compared to the saline group, with a slower rate of edema height decrease, but still showed a significant decrease after 30 minutes, and the retention rate further decreased after 60 minutes, indicating that although it can enhance the support effect of the submucosal liquid pad in a short time, its support durability is still insufficient for longer ESD operations or large-area lesion dissection. The sodium hyaluronate group exhibited better viscoelastic support during the test, with a significantly higher relative elevation height retention rate than the saline and hydroxyethyl starch groups. Even 120 minutes after injection, the sodium hyaluronate group maintained a certain degree of submucosal elevation, indicating that its higher viscosity helps delay material diffusion and elevation collapse. However, compared to the two-stage composite gel of this invention, its later elevation retention rate was still significantly lower, indicating that a single viscoelastic solution is insufficient for long-term support. The two-stage composite gel of this invention maintained a high elevation height at 5, 10, 20, and 30 minutes after injection, and maintained a high relative elevation height retention rate even after 60 minutes, retaining approximately two-thirds or more of the initial elevation height at 120 minutes. These results demonstrate that the support structure formed by the composite gel of this invention in the submucosal layer has good stability and persistence, effectively mitigating submucosal elevation collapse. These superior properties are mainly attributed to the multiple support mechanisms of the composite gel of this invention. On the one hand, after component A and component B are mixed, the aldehyde-modified dextran undergoes dynamic Schiff base crosslinking with the amino groups in the aminated polymer and carboxymethyl chitosan, forming a first gel network with self-healing properties. This allows the material to rapidly recover its viscoelasticity and form a support pad after injection into the submucosa. On the other hand, sodium alginate-γ-polyglutamic acid composite microgel particles, as compressible and resilient particle reinforcement phases, provide volumetric support and deformation recovery capabilities within the gel network, reducing material loss and diffusion in the interstitial spaces. Furthermore, medical-grade layered silicate nanosheets, as a physical reinforcement component, can further improve the storage modulus, anti-collapse ability, and structural stability of the gel network.Therefore, the test results demonstrate that the two-stage composite gel of this invention, compared to materials such as saline, hydroxyethyl starch, and sodium hyaluronate, can maintain the submucosal bulge for a longer period, providing a more stable and durable safe operating space for ESD procedures. This property helps reduce the number of repeated intraoperative injections, lowers the risk of perforation, bleeding, and thermal damage caused by submucosal pad collapse, and improves the safety and efficiency of endoscopic submucosal dissection procedures.

[0096] (3) ROS-responsive release test: To evaluate the oxidative stress response release performance of the ROS-responsive epithelialization-promoting nanocapsules in this invention, nanocapsules containing epithelialization-promoting active substances were placed in simulated wound fluids with different oxidation levels for in vitro release tests. The simulated wound fluid could be phosphate buffer, physiological saline, or a protein-containing buffer system with a pH of 7.4 (physiological saline was used in this embodiment). To simulate the local oxidative stress environment of the postoperative wound, different concentrations of hydrogen peroxide were added to the simulated wound fluid, namely 0 μM, 50 μM, 100 μM, 500 μM, and 1 mM. A composite gel sample containing ROS-responsive epithelialization-promoting nanocapsules was taken and placed in a dialysis bag, and a predetermined volume of release medium was added. The dialysis bag was placed in a centrifuge tube or beaker containing the same release medium, and the release experiment was carried out under constant temperature shaking conditions at 37°C. The shaking speed could be set to 50 rpm. Samples were taken at 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours. After each extraction of a certain volume of the release solution, an equal volume of fresh release medium was added to maintain a constant total volume of the release system. The content of epithelialization-promoting short peptides in the release solution was determined by high-performance liquid chromatography (HPLC). The cumulative release amount and cumulative release rate at each time point were calculated based on the measured short peptide concentrations. The cumulative release rate was calculated using the following formula: Cumulative release rate (%) = Mt / Mtotal × 100%, where Mt is the cumulative amount of short peptides released up to a certain time point, and Mtotal is the total short peptide loading in the sample. The response of the nanocapsules to oxidative stress was evaluated by comparing the release curves at different hydrogen peroxide concentrations.

[0097] The results are as follows Figure 3As shown, under different hydrogen peroxide concentrations, the cumulative release rate of the epithelialization-promoting short peptides gradually increased with time, but the release rate and final release amount increased significantly with increasing hydrogen peroxide concentration, exhibiting typical oxidative stress response release characteristics. In simulated wound fluid without hydrogen peroxide, the release of the epithelialization-promoting short peptides was relatively slow. This result indicates that under conditions without significant oxidative stimulation, the capsule wall structure of the ROS-responsive nanocapsules remains relatively stable, with only a small amount of basal release occurring. This helps to avoid the excessively rapid release of active short peptides in non-wound oxidative environments, thereby improving the stability and sustained action of the delivery system. Under 50 μM and 100 μM hydrogen peroxide conditions, the release rate of the epithelialization-promoting short peptides increased compared to the 0 μM group. This indicates that low to moderate oxidative stress levels can promote a certain degree of degradation of the nanocapsule wall material, leading to the gradual release of the encapsulated epithelialization-promoting short peptides. This release behavior is consistent with the physiological characteristics of local inflammation and oxidative stress in the wound after ESD surgery, which is beneficial for the material to continuously provide epithelialization-promoting active substances in the early stage of wound repair. Under conditions of 500 μM and 1 mM hydrogen peroxide, the release of the pro-epithelialization short peptides was significantly accelerated. These results indicate that higher levels of oxidative stress can significantly accelerate the degradation of ROS-responsive nanocapsules, thereby rapidly releasing the pro-epithelialization short peptides. The overall trend of the release curves shows that none of the groups exhibited instantaneous complete release, but rather a combination of moderate early release and sustained later release. This release pattern is beneficial for timely delivery of pro-epithelialization active substances to the wound in the early stages after ESD surgery, while maintaining a certain concentration of active short peptides during the subsequent repair phase, thereby promoting epithelial cell migration, adhesion, and re-epithelialization.

[0098] Therefore, the ROS-responsive release test results demonstrate that the epithelialization-promoting nanocapsules loaded in the composite gel of this invention can achieve controlled release according to the level of oxidative stress in the wound. Under weak oxidative stress, the active short peptides are released slowly, which helps maintain system stability; under strong oxidative stress, the nanocapsules degrade rapidly and release the epithelialization-promoting short peptides quickly, which helps to responsively repair wound areas with high inflammation and oxidative stress. This performance indicates that the composite gel of this invention can not only serve as an intraoperative submucosal support material, but also exert responsive drug delivery and mucosal repair effects in the postoperative wound environment, thereby achieving the integration of intraoperative support and postoperative wound repair in ESD.

[0099] (4) Biocompatibility Test: To evaluate the biocompatibility and repair potential of the composite gel of this invention, cell viability testing was conducted. Gastrointestinal epithelial cells, esophageal epithelial cells, or fibroblasts were selected as evaluation subjects. The composite gel extract was co-cultured with the cells. The composite gel extract was prepared as follows: a certain mass of composite gel sample was added to complete culture medium and extracted at 37°C for 24 hours. The extraction ratio was set to 0.1 g / mL. After extraction, the supernatant was collected and sterilely filtered to obtain the composite gel extract. Cells were seeded in 96-well plates. After the cells adhered, different concentrations of composite gel extract were added, and the cells were cultured for 24, 48, and 72 hours. Cell viability was detected using the CCK-8 assay. Cell viability was calculated using the following formula: Cell viability (%) = Absorbance of experimental group / Absorbance of blank control group × 100%.

[0100] The results are as follows Figure 4 As shown, during the 24, 48, and 72-hour culture periods, the cell viability of each composite gel extract treatment group remained at a high level, with no significant decrease in cell viability observed. This result indicates that the extract of the two-stage composite gel of this invention has no significant cytotoxicity to epithelial cells or fibroblasts and exhibits good in vitro cell compatibility. Specifically, the 50% extract group showed good cell viability maintenance and a slight promoting effect at all time points, indicating that a medium concentration of extract is more conducive to cell growth. Although the cell viability of the 100% extract group was slightly lower than that of the control group and the low-concentration extract group, it still remained above 90%, not reaching a significant cytotoxic level, suggesting that the high-concentration extract still has good safety for cells. The above results indicate that the composite gel of this invention did not release significant harmful substances during contact with cells. The epithelialization-promoting active substances and biomolecules in the composite gel may also provide favorable conditions for cell adhesion, migration, and proliferation, thereby contributing to re-epithelialization and mucosal repair of the wound after ESD surgery. Therefore, the cell viability test results demonstrate that the two-stage composite gel of this invention possesses good in vitro biocompatibility and cell safety, meeting the basic safety requirements for use as a submucosal injection material and wound protection and repair material. Furthermore, at appropriate concentrations, this material promotes cell viability to a certain extent, and has potential healing-promoting effects during postoperative wound repair, epithelial cell migration, and re-epithelialization.

[0101] Principle of this invention: The composite gel of the present invention has a two-stage functional conversion characteristic.

[0102] The first stage is the intraoperative support stage. After components A and B are mixed using a dual-lumen syringe and a static mixer, the aldehyde-modified dextran in component A undergoes a dynamic Schiff base reaction with the amino-modified dextran-masked catechol graft in component B and the free amino groups in carboxymethyl chitosan, forming a reversible dynamic cross-linked network. This network exhibits shear-thinning and self-healing properties; its viscosity decreases during injection via the endoscopic needle, facilitating injection. Once it enters the submucosal layer, the system's viscosity and elasticity gradually recover, thus forming a stable submucosal support pad.

[0103] Simultaneously, sodium alginate-γ-polyglutamic acid composite microgel particles, as compressible and resilient particle-reinforcing phases, are embedded in the dynamic cross-linked network, enabling the gel to form a composite support structure of "molecular dynamic network-particle-reinforcing network." Medical-grade layered silicate nanosheets, as a physical reinforcing component, help improve the gel's storage modulus, anti-collapse properties, and structural stability. The combined effect of these multiple structures can slow down the diffusion and absorption of materials in the submucosa, increasing the mucosal elevation height and duration.

[0104] The second stage is the postoperative wound repair stage. After ESD removal, part of the composite gel is exposed to gastrointestinal fluid and the wound microenvironment. At this time, the dynamic complex structure of catechol-boronic acid ester in the amino-decapitated catechol graft undergoes partial dissociation under the influence of acidic gastric juice, local ionic strength changes, competitive diol molecules, wound proteins, and the oxidative microenvironment, gradually releasing free catechol groups. The free catechol groups can interact with tissue proteins through hydrogen bonding, coordination, hydrophobic interactions, and the ortho-quinone structure formed after oxidation, thereby enhancing the adhesion of the gel to the moist wound. The principle is illustrated in the diagram below. Figure 5 As shown.

[0105] Meanwhile, the calcium and magnesium ions released by calcium gluconate and magnesium chloride can undergo ionic cross-linking with sodium alginate, γ-polyglutamic acid, and carboxyl groups in carboxylated polysaccharides, forming a further enhanced ionic cross-linking network. Tannic acid can form multi-point hydrogen bonds, hydrophobic interactions, and complexation with gelatin, chitosan, and wound tissue proteins, thereby further improving the wound coverage and hemostatic protection properties of the gel layer.

[0106] ROS-responsive epithelialization nanocapsules gradually degrade under the oxidative stress environment of the wound, releasing short peptides that promote epithelialization, thereby helping to promote epithelial cell migration, wound re-epithelialization, and mucosal repair.

[0107] Therefore, the composite gel of the present invention exhibits low adhesion, injectability, resilience, and long-lasting support during surgery; after the dissected wound is exposed, it gradually transforms into a wound protection gel layer with wet adhesion, covering protection, local hemostasis, and mucosal repair promotion functions, thus achieving the integration of intraoperative support and postoperative repair in ESD.

[0108] Alternative options: In component A, the mass of aldehyde-modified dextran is 1%–2%, sodium alginate-γ-polyglutamic acid composite microgel particles are 2%–5%, medical layered silicate nanosheets are 0.1%–0.2%, and indigo carmine is in the range of 0.005%–0.02%. In component B, the mass of amino-modified dextran-masked catechol graft is 0.5%–2.0%, carboxymethyl chitosan is 0.5%–1%, cationic gelatin-tannic acid nanocomposite is 0.2%–1%, ROS-responsive epithelialization-promoting nanocapsules are 0.1%–0.3%, calcium gluconate is 0.2%–0.3%, and magnesium chloride is in the range of 0.01%–0.1%. Both of these components can achieve the purpose of this invention.

[0109] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a two-stage composite gel, characterized in that, include: S1, Preparation of aldehyde-modified dextran; Dextran is oxidized with sodium periodate to yield aldehyde-modified dextran; S2, prepare sodium alginate-γ-polyglutamic acid composite microgel particles; Using an aqueous solution of sodium alginate and γ-polyglutamic acid as the dispersion phase and liquid paraffin as the continuous oil phase, a water-in-oil microdroplet dispersion system was formed by homogenization and emulsification. After calcium ion crosslinking, demulsification, and washing, composite microgel particles were obtained. S3, preparation of amino-glucan-masked catechol grafts; After activating 3,4-dihydroxyphenylpropionic acid, it was grafted onto amino-modified dextran, and then reacted with a phenylboronic acid group donor to form a borate ester complex structure. After dialysis and drying, the amino-modified dextran-masked catechol graft was obtained. S4, preparation of cationic gelatin-tannic acid nanocomposite; Gelatin was modified with glycidyl trimethylammonium chloride to obtain cationic gelatin, and then cationic gelatin was combined with tannic acid to obtain cationic gelatin-tannic acid nanocomposite. S5, preparation of ROS-responsive epithelialization-promoting nanocapsules; 3-Mercapto-1,2-propanediol and acetone were subjected to a thioketation reaction catalyzed by trifluoroacetic acid to obtain a polyhydroxy monomer containing thioketation bonds. The obtained monomer and hexamethylene diisocyanate were polycondensed under the catalysis of dibutyltin dilaurate to obtain a ROS-responsive polymer containing thioketation bonds. The ROS-responsive polymer containing thioketation bonds was dissolved in ethyl acetate as the oil phase. The epithelialization-promoting active substance and polyvinyl alcohol were dissolved in water as the first aqueous phase. Polyvinyl alcohol was dissolved in water as the second aqueous phase. The first aqueous phase and the oil phase were mixed and ultrasonically sonicated to form a W / O primary emulsion. The W / O primary emulsion was then added to the second aqueous phase and ultrasonically sonicated to form a W / O / W complex emulsion. The ethyl acetate was removed and the precipitate was collected to obtain ROS-responsive epithelialization-promoting nanocapsules. S6, Prepare component A; Aldehyde-modified dextran, sodium alginate-γ-polyglutamic acid composite microgel particles, medical layered silicate nanosheets, and indigo carmine were dispersed in HEPES buffer and degassed to obtain component A. S7, prepare component B; Aminated dextran-masked catechol graft, carboxymethyl chitosan, cationic gelatin-tannic acid nanocomposite, ROS-responsive epithelialization nanocapsules, calcium gluconate and magnesium chloride were dispersed in HEPES buffer and degassed to obtain component B. Component A and Component B are mixed during use.

2. The method for preparing the two-stage composite gel according to claim 1, characterized in that, In S2, Span80 is added to the liquid paraffin as an emulsifier beforehand; calcium chloride aqueous solution is used as a crosslinking liquid for calcium ion crosslinking; and anhydrous ethanol is used for demulsification.

3. The method for preparing the two-stage composite gel according to claim 1, characterized in that, In S3, 3,4-dihydroxyphenylpropionic acid was activated in MES buffer using EDC and NHS; 4-aminophenylboronic acid was used as a phenylboronic acid group donor.

4. The method for preparing the two-stage composite gel according to claim 1, characterized in that, In S5, the preparation of the ROS-responsive polymer containing thioketal bonds specifically includes: 3-Mercapto-1,2-propanediol was added to anhydrous acetone, and trifluoroacetic acid was added as an acid catalyst. The reaction was carried out under nitrogen protection to form a polyhydroxy monomer containing a thioketal bond and retaining a hydroxyl group. After the reaction was completed, triethylamine was added to neutralize the trifluoroacetic acid and remove the acetone. The crude product was dissolved in ethyl acetate, washed, dried, filtered, and concentrated under reduced pressure to obtain the polyhydroxy monomer containing a thioketal bond. A polyhydroxy monomer containing thioketal bonds and hexamethylene diisocyanate were dissolved in anhydrous dimethyl sulfoxide. Under nitrogen protection, the diisocyanate solution was added dropwise to the polyhydroxy monomer solution containing thioketal bonds, and dibutyltin dilaurate was added as a catalyst to induce a polycondensation reaction, forming a polyurethane ROS-responsive polymer containing thioketal bonds. After the reaction was completed, the precipitate was collected to obtain the ROS-responsive polymer containing thioketal bonds.

5. The method for preparing the two-stage composite gel according to claim 1, characterized in that, In S5, the active substance promoting epithelialization is EGFR-affinity short peptide GE11, whose amino acid sequence is YHWYGYTPQNVI.

6. The method for preparing the two-stage composite gel according to claim 1, characterized in that, In S6, component A, by mass percentage, comprises 1%–2% aldehyde-modified dextran, 2%–5% sodium alginate-γ-polyglutamic acid composite microgel particles, 0.1%–0.2% medical layered silicate nanosheets, 0.005%–0.02% indigo carmine, and the balance being HEPES buffer.

7. The method for preparing the two-stage composite gel according to claim 1, characterized in that, In S7, the B component, by mass percentage, comprises: 0.5%–2.0% aminoglucan-masked catechol graft, 0.5%–1% carboxymethyl chitosan, 0.2%–1% cationic gelatin-tannic acid nanocomposite, 0.1%–0.3% ROS-responsive epithelialization nanocapsules, 0.2%–0.3% calcium gluconate, 0.01%–0.1% magnesium chloride, with the balance being HEPES buffer.

8. A two-stage composite gel, characterized in that, The two-stage composite gel is prepared by the preparation method of any one of claims 1 to 7, and is composed of component A and component B; Component A includes aldehyde-modified dextran, sodium alginate-γ-polyglutamic acid composite microgel particles, medical layered silicate nanosheets, and indigo carmine; Component B includes amino-glucan-masked catechol graft, carboxymethyl chitosan, cationic gelatin-tannic acid nanocomposite, ROS-responsive epithelialization nanocapsules, calcium gluconate and magnesium chloride. When used, components A and B are mixed and first form a first gel network through a Schiff base reaction. Then, the adhesive groups are activated and secondary crosslinking occurs to form a second gel network, thus exhibiting a two-stage characteristic.

9. The two-stage composite gel according to claim 8, characterized in that, Component A and Component B are respectively filled into two independent chambers of a dual-chamber pre-filled syringe, and a static mixer is installed at the front end of the dual-chamber pre-filled syringe.

10. The application of the two-stage composite gel according to claim 8 in the preparation of materials for ESD submucosal injection, characterized in that, The submucosal injection material forms a first gel network for support after being injected into the submucosal layer during surgery, and forms a second gel network for closing the wound and promoting re-epithelialization after the wound is exposed postoperatively.

Images