A gel material for hemostasis and promoting healing of gastrointestinal mucosal layering and its preparation method.

Abstract

This invention provides a gel material for hemostasis and promoting healing of the digestive tract mucosa, and its preparation method, belonging to the field of biomedical materials technology. The material consists of two separately packaged solutions: Solution A contains sodium alginate with an amination degree of 0.5-2.0 mmol / g, RGD peptide, quaternized chitosan, growth factors, and indigo carmine; Solution B consists of liposome microspheres encapsulated with pre-chelated calcium ions. This invention achieves controllable cross-linking through a "dual regulation mechanism": modified sodium alginate adjusts the cation capture sensitivity, and liposomes control the ion release rate, allowing the gel formation time to be precisely controlled within 30-60 seconds, solving the needle blockage problem caused by the instantaneous solidification of traditional gels. This gel combines physical support, immediate hemostasis, and long-term healing promotion, and has good biocompatibility, making it particularly suitable for mucosal elevation and postoperative repair in endoscopic submucosal dissection (ESD).

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a gel material for hemostasis and healing promotion of gastrointestinal mucosal layering and its preparation method. Background Technology

[0002] Endoscopic submucosal dissection (ESD) is a standard minimally invasive surgical procedure for treating early-stage gastrointestinal tumors. Its key step involves injecting fluid into the submucosal layer beneath the lesion to form a sufficiently high and rigid cushion layer to separate the mucosal layer from the muscularis propria, thereby ensuring complete resection of the lesion and reducing the risk of perforation. An ideal submucosal injection solution should not only have a long cushion maintenance time but also good tissue adhesion, immediate hemostasis, and the ability to promote postoperative wound healing.

[0003] Existing submucosal injection solutions, such as normal saline and sodium hyaluronate solution, although clinically mature, generally have significant drawbacks such as short duration of cushioning, easy diffusion into surrounding tissues, and inability to provide hemostasis and promote healing. These often lead to unclear surgical field, the need for repeated injections, and increased operation time and patient suffering.

[0004] In recent years, sodium alginate-based in-situ cross-linked gels have shown application potential due to their good biocompatibility and biodegradability. However, such materials face serious challenges in practical applications: the cross-linking speed of traditional sodium alginate mixed with calcium ion solution is too fast (usually <5 seconds), which easily leads to premature coagulation inside the needle, causing injection failure. This forces doctors to complete the injection in a very short time, making it difficult to control the spread and extent of the gel under the mucosa, increasing the difficulty of the operation and the risk of perforation. In addition, existing gel materials have limited functions, only acting as a physical cushioning layer, and cannot effectively control intraoperative bleeding, prevent delayed postoperative bleeding and stenosis, and lack proactive intervention for the postoperative healing process.

[0005] More importantly, existing technologies struggle to organically integrate functions such as cross-linking rate regulation, hemostatic drug loading, and tissue activity repair. For instance, attempts to simply add growth factors or hemostatic drugs often result in drug inactivation or burst release due to the rapid cross-linking process, failing to achieve the desired synergistic therapeutic effect.

[0006] Therefore, developing an integrated gel material with precise and controllable cross-linking speed and both efficient hemostasis and active repair promotion functions is of great clinical significance and urgent need for improving the safety of ESD surgery, reducing the incidence of complications, and promoting rapid postoperative recovery of patients. Summary of the Invention

[0007] In view of the above, the purpose of this invention is to provide a gel material for hemostasis and promoting healing of the digestive tract mucosa and its preparation method, belonging to the field of biomedical materials technology. The material consists of two separately packaged solutions: Solution A contains sodium alginate, RGD peptide, quaternized chitosan, growth factors, and indigo carmine with an amination degree of 0.5-2.0 mmol / g; Solution B is liposome microspheres encapsulated with pre-chelated calcium ions.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A gel material for hemostasis and promoting healing of gastrointestinal mucosal layering, comprising separately packaged solution A and solution B;

[0010] Solution A contains amination-modified sodium alginate, positively charged hemostatic material, active pharmaceutical ingredient, and chromogenic agent; solution B is a suspension of cross-linked ion-source microspheres encapsulated in liposomes.

[0011] The color developer is indigo carmine, and its final concentration in solution A is 0.05%-0.2% (w / v), preferably 0.1% (w / v).

[0012] In this invention, the degree of amination of the aminated sodium alginate is 0.5-2.0 mmol / g; the solution A also contains RGD peptide grafted onto the sodium alginate chain through an EDC / NHS activation system, and the grafting density of the RGD peptide is 0.5-3 μmol / mg.

[0013] In this invention, the degree of amination of the aminated sodium alginate is 1.2 mmol / g; and the grafting density of the RGD peptide is 1.8 μmol / mg.

[0014] In this invention, the positively charged hemostatic material is quaternized chitosan; the active pharmaceutical ingredient includes a hemostatic drug and a healing-promoting drug; the hemostatic drug is selected from at least one of thrombin and tranexamic acid; the healing-promoting drug is selected from growth factors and natural antioxidants, the growth factors include FGF and EGF (preferably bFGF), and the natural antioxidants include tea polyphenols, resveratrol, and curcumin.

[0015] In this invention, further, the cross-linked ion source is encapsulated in liposomes in the form of a pre-chelate; the cross-linked ion source is Ca... 2+ Zn 2+ or Sr 2+ At least one of the following, the prechelate is formed by prechelating a cross-linked ion source with a chelating agent; the chelating agent is selected from DTPA, EDTA, glucono-δ-lactone or gluconic acid.

[0016] In this invention, the liposomes have a particle size of 100-1000 nm; and the encapsulation efficiency of the cross-linked ion source is greater than 80%.

[0017] In this invention, the volume ratio of solution A to solution B is further 5:3.

[0018] The present invention also provides the use of the gel material in the preparation of mucosal pad products for endoscopic submucosal dissection (ESD).

[0019] The present invention also provides a method for preparing the gel material described above, comprising the following steps:

[0020] Step 1, Preparation of Solution A: Sodium alginate is aminated and RGD peptide is grafted onto the sodium alginate chain through an EDC / NHS activation system. Then, it is mixed with positively charged hemostatic material, active pharmaceutical ingredient, and colorimetric agent to prepare Solution A.

[0021] Step 2, Preparation of Solution B: The cross-linked ion source is pre-chelated with a chelating agent to form a pre-chelate, and then the pre-chelate is encapsulated in liposomes by a thin-film hydration method to obtain a liposome-encapsulated cross-linked ion source microsphere suspension, which is Solution B;

[0022] Step 3, Independent Packaging: Separately package solution A and solution B into separate containers.

[0023] The present invention has at least the following beneficial effects:

[0024] 1. This invention achieves precise control of gel formation time through a "dual regulation mechanism": on the one hand, sodium alginate is aminated to adjust its sensitivity to crosslinking ions (locking tightness); on the other hand, a crosslinking ion source (such as Ca) is used... 2+ The ion release rate is controlled by encapsulating the liposomes in a pre-chelate form within liposome microspheres, utilizing the physical barrier effect of the liposome phospholipid bilayer. The synergistic effect of these two technologies extends the gel formation time from less than 5 seconds in the traditional sodium alginate-calcium ion system to a controllable range of 30-60 seconds. Comparative experiments show that when using liposome sustained-release technology alone without modified sodium alginate, the gel formation time is only 12±5 seconds, which is still insufficient to meet the injection and spreading requirements in endoscopic surgery; only by combining the two can an ideal operating window of 45±3 seconds be achieved. This controllable cross-linking property avoids the risk of premature coagulation within the needle, providing doctors with ample operating time while ensuring uniform spreading of the gel in the submucosal layer.

[0025] 2. This invention integrates two mechanisms of hemostasis: physical and pharmacological hemostasis. For physical hemostasis, the aminated sodium alginate carries a positive charge, which can rapidly capture negatively charged blood cells through electrostatic adsorption and promote platelet activation and adhesion, forming a physical hemostatic barrier on the wound surface. For pharmacological hemostasis, thrombin and tranexamic acid in the gel sustained-release system are gradually released, exerting their enzymatic coagulation and antifibrinolytic pharmacological effects. The two mechanisms work synergistically at different time scales: physical hemostasis takes effect immediately, while pharmacological hemostasis is maintained continuously. Experimental data show that the whole blood clotting time of the gel in this invention is 85±12 seconds, significantly shorter than that of the unmodified sodium alginate gel group (180±15 seconds) and the saline control group (320±25 seconds); the number of platelets adhering is 2.3 times that of the unmodified sodium alginate gel; in the pig liver incision model, the hemostasis time is 125±18 seconds, superior to the positive control group (185±22 seconds). This dual hemostatic design controls intraoperative bleeding and reduces the risk of delayed postoperative bleeding.

[0026] 3. This invention constructs a microenvironment conducive to tissue repair through the synergistic effect of RGD peptide grafting and a drug sustained-release system. RGD peptides are covalently grafted onto sodium alginate chains via succinimide ester method. These peptides can form hydrogen bonds and hydrophobic interactions with fibronectin receptors in wound tissue, stably anchoring the gel to the exposed tissue surface of the wound with an adhesion strength of 1.8 ± 0.3 kPa (0.5 ± 0.1 kPa for unmodified sodium alginate gel), providing stable matrix support for the subsequent repair process. Based on this, the three-dimensional network structure of the gel enables the sustained release of growth factors (FGF, EGF) and natural antioxidants (tea polyphenols): the release rate is less than 22% in the first 24 hours, and the cumulative release rate exceeds 80% after 7 days. The sustained-released growth factors continuously promote cell proliferation and migration, while the sustained-released antioxidants continuously scavenge free radicals and protect newly formed tissue. Cellular experiments showed that after 3 days of treatment with the gel extract of this invention, the proliferation rate of GES-1 cells reached 145.3%, significantly higher than that of the gel group without growth factors (112.5%) and the blank control group (100%); the scratch healing rate reached 78.5%, significantly higher than that of the blank control group (45.2%); in animal experiments, the wound healing rate reached 85.3% after 7 days, significantly better than that of the unmodified gel group (62.1%) and the blank control group (48.7%). Furthermore, all components are biocompatible materials, and the cell viability (92.5%), hemolysis rate (2.3%), and acute systemic toxicity test results all meet the safety standards for medical materials; endoscopic visualization was achieved through indigo carmine staining, facilitating precise intraoperative positioning. Attached Figure Description

[0027] Figure 1 This is a transmission electron microscope (TEM) image of the solution B (slow-release calcium ion liposome microspheres) prepared in Example 1 of the present invention.

[0028] Figure 2 This is a scanning electron microscope (SEM) image of the internal structure of the gel material formed by crosslinking in Example 1 of the present invention, showing its three-dimensional porous network structure.

[0029] Figure 3 This is an endoscopic photograph of the gel material of Embodiment 1 of the present invention after injection into the submucosa of an animal (miniature pig) to form a gel pad, showing that the mucosal layer was effectively lifted.

[0030] Figure 4 The image shows the gel material of Embodiment 1 of the present invention in an isolated porcine stomach ESD simulation surgery, demonstrating that the gel pad does not leak under the incision and can continuously maintain mucosal elevation.

[0031] Figure 5 The image shows the wound healing effect after treatment with the gel material in Example 1 of this invention, indicating that the wound has fully recovered after the operation. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in further detail below with reference to the accompanying drawings and 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 this invention.

[0033] In the embodiments and comparative experiments described in this section, the crosslinking ion source was Ca. 2+ .

[0034] Example 1

[0035] This embodiment illustrates the optimal preparation parameters and performance verification of the gel material described in this invention. The crosslinking ion source used is Ca2+. 2+ The volume ratio of solution A to solution B is 5:3, and the injection interval is 12 seconds. The specific preparation steps are as follows.

[0036] I. Preparation of solution A.

[0037] 1. Modification of sodium alginate and RGD grafting.

[0038] Weigh 1.2 g of sodium alginate and dissolve it in 80 mL of deionized water to prepare a 1.5% (w / v) solution. Add hexamethylenediamine (molar ratio to sodium alginate carboxyl groups of 2:1), EDC (molar ratio to sodium alginate carboxyl groups of 1.5:1), and NHS (molar ratio to sodium alginate carboxyl groups of 1:1). Stir the reaction mixture at pH 6.0 and room temperature for 12 hours. After dialyzing and lyophilization, modified sodium alginate with an amination degree of 1.2 mmol / g is obtained.

[0039] The above-mentioned aminated sodium alginate was redissolved in deionized water, and 0.25 g of EDC and 0.15 g of NHS were added. The mixture was activated by stirring at 4°C for 2 hours. Then, 0.05 g of RGD peptide (sequence: arginine-glycine-aspartic acid, purity >95%) was added, and the reaction was carried out at room temperature (25°C) and in the dark, with stirring at 200 rpm for 24 hours. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 8000-14000 Da and dialyzed against deionized water for 48 hours, with the deionized water replaced every 6 hours. After dialysis, the product was lyophilized to obtain RGD-grafted sodium alginate powder. The grafting density of the RGD peptide was determined to be 1.8 μmol / mg.

[0040] 2. Drug loading and preparation.

[0041] Redissolve the lyophilized powder in 50 mL of deionized water and stir until completely dissolved. Add the following components in sequence.

[0042] 0.3 g quaternized chitosan (a preferred form of the positively charged hemostatic material of this invention, which refers to a natural or synthetic polymeric material with a positive charge that can capture negatively charged blood cells through electrostatic adsorption and promote platelet adhesion, with a degree of substitution >90%); 1000 IU thrombin; 5 mg tranexamic acid; 50 µg bFGF (basic fibroblast growth factor, bFGF is a preferred form of FGF of this invention); 30 µg EGF (epidermal growth factor); 10 mg tea polyphenols (purity >98%); appropriate amount of indigo carmine to make the solution appear clear blue (final concentration 0.1% w / v).

[0043] Adjust the pH to 7.0 using 1M NaOH solution, and finally bring the volume to 100 mL with deionized water. Stir at 300 rpm for 30 minutes at 25°C to ensure thorough mixing of all components, obtaining solution A. Aliquot solution A into sterile syringes and store at 4°C protected from light for later use.

[0044] II. Preparation of Solution B (Sustained-release calcium ion liposome microspheres).

[0045] 1. Calcium ion prechelation.

[0046] 0.2 g of anhydrous calcium chloride (CaCl2, analytical grade) was mixed with 0.1 g of DTPA (diethylenetriaminepentaacetic acid), and 10 mL of deionized water was added. The mixture was stirred at 200 rpm for 1 hour in a 37°C water bath to form CaCl2. 2+ -DTPA complex solution. This pre-chelation step prevents Ca from forming during subsequent liposome preparation. 2+ It interacts adversely with phospholipids.

[0047] 2. Liposome preparation (thin-film hydration method).

[0048] Weigh 1.0 g of soybean lecithin (purity >98%) and 0.2 g of cholesterol, dissolve them in 20 mL of anhydrous ethanol, and sonicate until completely dissolved. Transfer the lipid ethanol solution to a 250 mL round-bottom flask and evaporate it under reduced pressure using a rotary evaporator at 45°C water bath and -0.1 MPa vacuum to remove the ethanol until a uniform, transparent lipid film forms on the inner wall of the flask.

[0049] Add 10 mL of the Ca prepared in the first step to the flask 2+ The DTPA complex solution was hydrated in a 45°C water bath for 30 minutes, during which the flask was slowly rotated to completely detach and disperse the lipid film.

[0050] The hydrated suspension was transferred to a high-pressure homogenizer and homogenized five times at 1000 bar. Subsequently, the homogenized suspension was extruded ten times through a polycarbonate membrane extruder with a 200 nm pore size to obtain liposome microspheres of uniform size. The resulting microsphere suspension was stored at 4°C, which is solution B.

[0051] 3. Characterization of solution B.

[0052] Take an appropriate amount of solution B, dilute it with deionized water, and observe the morphology of the microspheres using transmission electron microscopy (TEM). The results are as follows: Figure 1 As shown: Liposomes are spherical or near-spherical in shape, with uniform particle size and narrow distribution, and an average particle size of approximately 200 nm. The complete liposome bilayer structure is clearly visible, and the internally encapsulated Ca2+ microspheres are clearly visible. 2+ The -DTPA complex exists in the form of an electron-dense core, confirming that the cross-linked ion source was successfully encapsulated within the liposomes. This microstructure is key to achieving sustained release of cross-linked ions and thus precisely controlling gel formation time.

[0053] The zeta potential of the microspheres was determined to be -15 mV using a zeta potential analyzer. Free Ca was determined using inductively coupled plasma mass spectrometry (ICP-MS). 2+ Concentration, calculated to obtain Ca 2+ The encapsulation rate is greater than 80%.

[0054] III. Formation and Properties of Gel Materials

[0055] The prepared solutions A and B were respectively filled into the two chambers of a double-barrel syringe (A:B volume ratio = 5:3). Under simulated usage conditions, solution A was injected into the target location first, and after a 12-second interval, solution B was injected into the same location. The Ca released slowly from solution B... 2+It undergoes an ionic cross-linking reaction with the modified sodium alginate in solution A, forming a gel pad in situ.

[0056] The properties of this gel material were tested and are as follows.

[0057] Gel formation time: 45±3 seconds (inverted test tube method).

[0058] Compression modulus: 8.5±1.2 kPa (universal testing machine).

[0059] Elastic modulus: 6.3±0.8 kPa.

[0060] Adhesion strength to porcine gastric mucosa: 1.8 ± 0.3 kPa (peel test).

[0061] The cross-linked gel sample was observed using a scanning electron microscope, such as... Figure 2 As shown, the gel exhibits a typical three-dimensional porous network structure, which provides space for loading and sustained release of the drug's active ingredients.

[0062] Example 2: Amination degree of 0.5 mmol / g

[0063] The preparation method is the same as in Example 1, except that the amination reaction conditions of sodium alginate are adjusted to control the degree of amination at 0.5 mmol / g. The remaining steps and parameters remain unchanged.

[0064] Testing revealed that this method achieved a gel formation time of 55±4 seconds, a compressive modulus of 7.2±1.0 kPa, an adhesive strength of 1.4±0.2 kPa, and a padding duration of 52±5 minutes. Compared to Example 1, the reduced degree of amination resulted in fewer active sites for crosslinking on the sodium alginate molecular chain, leading to a slight decrease in gel crosslinking density and consequently, slightly lower compressive modulus and adhesive strength. Simultaneously, the reduced number of crosslinking sites also resulted in a slightly slower gel network formation, extending the gel formation time to approximately 55 seconds, but still within a controllable window of 30-60 seconds. The padding duration exceeding 50 minutes still meets the clinical requirements for mucosal elevation duration in ESD surgery (typically not less than 30 minutes). This method is suitable for surgical scenarios where the mechanical strength requirements of the gel pad are relatively low, but a more flexible operation time is desired.

[0065] Example 3: Liposome particle size of 500 nm

[0066] The preparation method is the same as in Example 1, except that a 500 nm pore size polycarbonate membrane extruder is used instead of a 200 nm membrane to control the liposome particle size to 500 ± 50 nm. The remaining steps and parameters remain unchanged.

[0067] The tested results showed that the gel formation time of this method was 38±4 seconds, the compressive modulus was 8.2±1.1 kPa, the adhesion strength was 1.7±0.3 kPa, and the padding maintenance time was 55±4 minutes. Compared with Example 1, the increased liposome particle size led to a decrease in their specific surface area, and the phospholipid bilayer had a greater impact on the internal Ca2+. 2+ The physical barrier effect is relatively weakened, Ca 2+ The release rate was slightly accelerated, thus shortening the gel formation time to 38 seconds, but still within the controllable range of 30-60 seconds. The compressive modulus and adhesive strength were similar to those of Example 1, and the padding duration still exceeded 50 minutes, fully meeting the clinical requirements of ESD surgery. This approach is suitable for surgical scenarios where a slightly faster gel formation speed and shorter surgical waiting time are desired.

[0068] Performance testing methods.

[0069] 1. Gel formation time: The inverted test tube method was used. Solution A and solution B were mixed in a test tube at a volume ratio of 5:3. The time from the end of mixing to the point where the gel stopped flowing after the test tube was inverted was recorded. Each group was repeated 5 times, and the mean ± standard deviation was taken.

[0070] 2. Compression modulus and elastic modulus: Tested using a universal testing machine (Instron 3343). The gel was prepared into cylindrical samples with a diameter of 10 mm and a height of 5 mm, and compressed to 30% of the strain at a rate of 1 mm / min. The stress-strain curves were recorded, and the compression modulus and elastic modulus were calculated.

[0071] 3. Adhesion strength: The peel test method was used. The gel was applied to the surface of isolated porcine gastric mucosa (area 2 cm × 2 cm), and a universal testing machine was used to peel it 180° at a rate of 5 mm / min. The peel force was recorded and divided by the area to obtain the adhesion strength.

[0072] 4. In vitro drug release assay: The gel sample was placed in a centrifuge tube containing 10 mL of PBS buffer (pH 7.4, with 0.02% NaN3 preservative) and shaken at 37°C and 100 rpm. At predetermined time points (0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, and 168 hours), all release media were collected and replenished with an equal volume of fresh PBS. The concentrations of bFGF and EGF in the release solution were determined using an ELISA kit, and the concentration of tea polyphenols was determined using ultraviolet spectrophotometry (wavelength 280 nm).

[0073] 5. Whole blood clotting time (WBCT): Fresh rabbit heart blood was collected, mixed with the gel sample, and placed in a glass test tube. The time required for complete clotting was recorded. Each group was repeated 5 times.

[0074] 6. Platelet adhesion assay: After co-incubating the gel sample with platelet-rich plasma (PRP) for 30 minutes, the sample was gently washed with PBS, fixed with 2.5% glutaraldehyde, dehydrated in a gradient manner, sputter-coated with gold, and the number of platelets adhering was observed and counted by scanning electron microscopy (SEM).

[0075] 7. Hemostasis time in a simulated bleeding model: A standardized incision (1 cm long, 0.5 cm deep) was made on the surface of fresh pig liver. Solution A and solution B were injected sequentially to form a gel cover, and the time for complete cessation of bleeding was recorded. A commercially available absorbable gelatin hemostatic material (Surgiflo®, Johnson & Johnson) was used as a positive control.

[0076] 8. Cell proliferation assay (CCK-8 assay): GES-1 gastric epithelial cells were seeded in 96-well plates (density 1×10⁶). 4 After culturing for 24 hours, the culture medium was replaced with gel extraction medium (gel to medium ratio 1:10, extraction at 37℃ for 24 hours). After culturing for 1, 2, and 3 more days, 10 µL of CCK-8 reagent was added to each well, and the cells were incubated for 2 hours. The absorbance at 450 nm was measured, and the cell proliferation rate was calculated.

[0077] 9. Cell scratch assay: GES-1 cells were cultured in 6-well plates to a confluence of approximately 90%. Straight scratches were made using a 200 µL pipette tip. After washing with PBS, the cells were replaced with culture medium containing gel extraction solution. Images were taken at 0 and 24 hours, and the scratch healing rate was calculated using ImageJ software.

[0078] 10. Animal Model Wound Healing Rate: A gastric mucosal injury model was established using miniature pigs (weighing 20-25 kg). Under endoscopic guidance, a mucosal defect approximately 1 cm in diameter was created in the gastric antrum, and the resulting gel (Example 1), unmodified sodium alginate gel, and physiological saline (blank control) were injected, respectively. Animals were sacrificed on postoperative day 7, and gastric tissue was harvested. The unhealed area was measured, and the wound healing rate was calculated as (initial defect area - unhealed area) / initial defect area × 100%. Another miniature pig was used to establish a gastric mucosal injury model using the same method. These pigs were fed normally postoperatively, and wound healing was observed endoscopically periodically for 3 consecutive months. Figure 5 As shown, the wound healed completely after the operation, with no obvious scarring or tissue stenosis.

[0079] 11. Cytotoxicity test (MTT method): Refer to ISO 10993-5 standard. L929 mouse fibroblasts were co-cultured with gel extract for 72 hours, and the absorbance at 570 nm was measured after adding MTT reagent to calculate cell viability.

[0080] 12. Hemolysis rate test: Fresh rabbit anticoagulated blood was incubated with the gel sample at 37°C for 1 hour. After centrifugation, the absorbance of the supernatant at 545 nm was measured, and the hemolysis rate was calculated. Deionized water was used as a positive control (100% hemolysis), and physiological saline was used as a negative control (0% hemolysis).

[0081] 13. Acute systemic toxicity test: Kunming mice (weighing 18-22 g, 10 mice per group, half male and half female) were injected with gel extract (50 mg / kg) via the tail vein and observed for 7 consecutive days. Changes in body weight, activity, food intake, and death were recorded.

[0082] Test results.

[0083] 1. Verification of the controllability of crosslinking speed.

[0084] To verify the effect of the "dual regulation mechanism" of this invention on the gel formation time, the gel formation times of ordinary sodium alginate + free CaCl2 solution (control group), ordinary sodium alginate + sustained-release microspheres (comparative group), and Example 1 (modified sodium alginate + sustained-release microspheres) were tested respectively. The results are shown in Table 1. The gel formation time of the control group (ordinary sodium alginate + free CaCl2 solution) was <5 seconds, solidifying instantly and impossible to inject; the formation time of the comparative group (ordinary sodium alginate + sustained-release microspheres) was 12±5 seconds, which was still too fast even with the sustained release of solution B alone, indicating that it must be modified with solution A; while the formation time of Example 1 (modified sodium alginate + sustained-release microspheres) was 45±3 seconds, with good rheological properties and an ideal operating window.

[0085] Table 1: Verification of Crosslinking Rate Controllability

[0086]

[0087] 2. Evaluation of hemostatic effect.

[0088] To evaluate the hemostatic performance of the gel of the present invention, the whole blood clotting time and platelet adhesion count were tested in Example 1, the unmodified sodium alginate gel group, and the saline control group. The results are shown in Table 2. It can be seen that the whole blood clotting time of Example 1 (85±12 seconds) was significantly shorter than that of the unmodified sodium alginate gel group (180±15 seconds) and the saline control group (320±25 seconds); the platelet adhesion count was 2.3 times that of the unmodified sodium alginate gel group.

[0089] Table 2: Comparison and Evaluation of Hemostatic Effects

[0090]

[0091] Note: "—" indicates that the test was not performed.

[0092] Furthermore, the in vivo hemostatic properties of the gel of this invention were tested in a pig liver incision model. The hemostasis time in Example 1 was 125±18 seconds, significantly better than that in the positive control group (185±22 seconds), further verifying its rapid hemostatic effect.

[0093] 3. In vitro sustained-release experiment.

[0094] To evaluate the sustained-release behavior of the gel of the present invention, the cumulative release amounts of bFGF, EGF, and tea polyphenols in Example 1 were measured at different time points. The results are shown in Table 3. It can be seen that bFGF, EGF, and tea polyphenols in Example 1 all exhibited good sustained-release characteristics: the release amount in the first 24 hours was less than 22%, and the cumulative release amount in 7 days was more than 80%.

[0095] Table 3: Drug sustained-release effect (Example 1)

[0096]

[0097] 4. Wound healing capacity assessment.

[0098] To verify the synergistic effect of the gel of this invention in promoting tissue repair, the following control group was set up for parallel comparison:

[0099] Gel group without growth factors: The preparation method of Example 1 is the same as that of Example 1, except that bFGF, EGF and tea polyphenols are not added, while the other components and preparation conditions remain unchanged.

[0100] Unmodified sodium alginate gel group: The preparation method is the same as in Example 1, except that unmodified ordinary sodium alginate is used instead of aminated modified sodium alginate, and RGD peptide grafting is not performed. The other components and preparation conditions remain unchanged.

[0101] Blank control group: physiological saline.

[0102] The test results are shown in Tables 4-1 to 4-3. The results show that the cell proliferation rate, scratch healing rate, and wound healing rate of the animal model in Example 1 (the present invention) were significantly higher than those of the control groups, indicating that the present invention achieves a significant healing-promoting effect through a multiple synergistic mechanism of "modified carrier + RGD anchoring + drug sustained release".

[0103] Table 4-1: Cell proliferation rate (day 3)

[0104]

[0105] Table 4-2: Cell scratch healing rate (24h)

[0106]

[0107] Table 4-3: Wound healing rate in animal models (7 days)

[0108]

[0109] 5. Biosafety assessment.

[0110] To assess the biocompatibility of the gel of this invention, cytotoxicity, hemolysis rate, and acute systemic toxicity tests were conducted according to ISO 10993-5. The results are shown in Table 5. The cell viability (92.5±3.8%) in Example 1 met the ISO 10993-5 standard (>80%); the hemolysis rate (2.3±0.5%) was far below the safety threshold (5%); mice were observed for 7 days after tail vein injection, and there were no deaths, no weight loss, and no other abnormal symptoms.

[0111] Table 5: Biosafety Assessment

[0112]

[0113] Application example: Simulation of endoscopic submucosal dissection (ESD).

[0114] Solution A and solution B prepared in Example 1 were respectively filled into the two chambers of a double-barrel syringe (A:B volume ratio = 5:3). ESD procedure was simulated on an isolated porcine stomach model: under direct endoscopic visualization, approximately 5 mL of solution A was injected into the submucosa using a needle. After a 12-second interval, approximately 3 mL of solution B was injected at the same location. A raised, stable blue gel pad was observed to form rapidly in the local area, as shown... Figure 3 As shown, the gel pad effectively separated the mucosal lesions from the muscle layer, providing a good lifting effect. Measurements showed that the pad maintained its position for over 60 minutes, significantly longer than the saline control group (<10 minutes). Figure 4 As shown, during the simulated dissection procedure, the gel pad remained intact below the incision without leakage or loss outside the incision. It continuously maintained mucosal elevation, providing a clear dissection plane for lesion resection, and no significant bleeding was observed during the cutting process. Postoperatively, the excised tissue and gel pad were sent for examination. Histological examination showed that the gel and tissue interface were tightly bonded, with no acute inflammatory reaction. The pad's maintenance time and elevation effect fully meet the clinical requirements for support and stability in ESD surgery.

[0115] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A gel material for hemostasis and promoting healing of the digestive tract mucosa, characterized in that, This includes separately packaged solutions A and B; Solution A contains amination-modified sodium alginate, positively charged hemostatic material, active pharmaceutical ingredient, and chromogenic agent; solution B is a suspension of cross-linked ion-source microspheres encapsulated in liposomes.

2. The gel material according to claim 1, characterized in that, The amination degree of the aminated sodium alginate is 0.5-2.0 mmol / g; the solution A also contains RGD peptide grafted onto the sodium alginate chain through an EDC / NHS activation system, and the grafting density of the RGD peptide is 0.5-3 μmol / mg.

3. The gel material according to claim 2, characterized in that, The amination degree of the aminated sodium alginate is 1.2 mmol / g; the grafting density of the RGD peptide is 1.8 μmol / mg.

4. The gel material according to claim 1, characterized in that, The positively charged hemostatic material is quaternized chitosan; the active pharmaceutical ingredient includes a hemostatic drug and a healing-promoting drug; the hemostatic drug is selected from at least one of thrombin and tranexamic acid; the healing-promoting drug is selected from growth factors and natural antioxidants, the growth factors include FGF and EGF, and the natural antioxidants include tea polyphenols, resveratrol, and curcumin.

5. The gel material according to claim 1, characterized in that, The cross-linked ion source is encapsulated in liposomes in the form of a prechelate; the cross-linked ion source is Ca. 2+ Zn 2+ or Sr 2+ At least one of the following, the prechelate is formed by prechelating a cross-linked ion source with a chelating agent; the chelating agent is selected from DTPA, EDTA, glucono-δ-lactone or gluconic acid.

6. The gel material according to claim 1, characterized in that, The liposomes have a particle size of 100-1000 nm; the encapsulation efficiency of the cross-linked ion source is greater than 80%.

7. The gel material according to claim 1, characterized in that, The volume ratio of solution A to solution B is 5:

3.

8. A method for preparing the gel material according to any one of claims 1-7, characterized in that, Includes the following steps: Step 1, Preparation of Solution A: Sodium alginate is aminated and RGD peptide is grafted onto the sodium alginate chain through an EDC / NHS activation system. Then, it is mixed with positively charged hemostatic material, active pharmaceutical ingredient, and colorimetric agent to prepare Solution A. Step 2, Preparation of Solution B: The cross-linked ion source is pre-chelated with a chelating agent to form a pre-chelate, and then the pre-chelate is encapsulated in liposomes by a thin-film hydration method to obtain a liposome-encapsulated cross-linked ion source microsphere suspension, which is Solution B; Step 3, Independent Packaging: Separately package solution A and solution B into separate containers.

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