Preparation method of natural polysaccharide-based organic / inorganic hybrid photo-thermal antibacterial hydrogel

Abstract

The invention discloses a preparation method of natural polysaccharide-based organic / inorganic hybrid photo-thermal antibacterial hydrogel, which comprises the following steps: 1) mixing and dissolving methacrylated gelatin, hydroformylated sodium alginate, tannic acid and a photoinitiator in a phosphate buffer solution to obtain a solution A; 2) dissolving carboxymethyl chitosan in a phosphate buffer solution to obtain a solution B; 3) adding a gold nanorod aqueous dispersion into the solution B to obtain a solution C; the volume ratio of the gold nanorod aqueous dispersion to the solution B is (1.5: 1)-(2: 1), the length of the gold nanorod is 30-70nm, and the width of the gold nanorod is 5-10nm; 4) blending the solution A and the solution C, and injecting to obtain a network hydrogel I; and 5) carrying out ultraviolet irradiation crosslinking on the hydrogel I to obtain the dual-network hydrogel II. The antibacterial hydrogel can be formed in a mold with a complex shape, in-situ injection photo-crosslinking gel forming can be carried out, a photo-thermal heating effect under near-infrared light irradiation can be achieved, and the range required by photo-thermal antibacterial can be reached.

Description

Technical Field

[0001] This invention relates to the field of wound dressing technology for repairing oral fistula tissue defects, specifically a method for preparing a natural polysaccharide-based organic / inorganic hybrid photothermal antibacterial hydrogel. Background Technology

[0002] Oropharyngeal fistulas are a common form of tissue defect in the human body. A typical complication is penetrating oral (pharyngeal) fistulas (POFs), a serious complication following unsuccessful oral or oropharyngeal reconstruction. Due to the lack of supporting tissue, contamination from saliva and chewed food, and the dynamic oral environment, it remains a complex clinical challenge. This disease also carries a serious risk of infection; invasion by bacteria, fungi, viruses, etc., can lead to irreversible tissue necrosis, partial or complete organ dysfunction, and other severe consequences. The variable physiological environment, complex anatomical structure, and continuous tissue movement of POFs make fistula repair extremely difficult. Currently, commonly used wound filling dressings for POF treatment include medical gelatin materials, iodoform gauze, and medical sponges. While these provide some physical barrier and absorbency, they generally suffer from poor mechanical properties, limited antibacterial capabilities, inability to dynamically respond to the wound environment, and a tendency to adhere to the wound surface, leading to secondary damage. Therefore, there is an urgent need to develop oral-oropharyngeal fistula repair scaffold materials with good antibacterial properties, good biocompatibility, shape matching to complex defect sites, and the ability to promote tissue repair.

[0003] While domestic patents related to antibacterial hydrogels for tissue defect repair dressings have been reported, several issues remain to be addressed. Chinese invention patent publication CN1 19838049A discloses a nano-silver composite hydrogel and its preparation method for promoting wound healing and repair. In this antibacterial hydrogel, black phosphorus nanosheets are mixed with hyaluronic acid and quaternized chitosan to enhance adhesion. Under near-infrared light irradiation, the silver nanoparticles loaded on the black phosphorus nanosheets release silver ions, exerting a highly efficient antibacterial effect. Combined with the photothermal effect of the black phosphorus nanosheets, this synergistic antibacterial effect promotes rapid wound healing and repair. However, this method has drawbacks. The antibacterial strategy of this antibacterial hydrogel involves a metal ion and silver ion release bactericidal step. The duration and range of action may be limited by the actual shape of the wound. Metal ions may also be toxic to normal human cells and inhibit the growth of fibroblasts and keratinocytes, which is not conducive to tissue growth and wound healing. In addition, this antibacterial hydrogel is mainly used for simple superficial skin defect models. The hydrogel can only be prepared into a gel in an in vitro mold and then demolded for use. It cannot be injected in situ to form a gel, and its shape adaptability is poor. Therefore, it is difficult to achieve the best effect on oral fistula defects with complex shapes.

[0004] Chinese invention patent publication CN1 15894985A discloses a bio-inducible tissue repair hydrogel, its preparation method, and its application. Using silkworm silk as raw material, a methacrylated silk fibroin solution is obtained by adding glycidyl methacrylate, followed by fullerol solution. The solution undergoes photocrosslinking and enzymatic crosslinking reactions sequentially to obtain a silk fibroin hydrogel composited with fullerol. However, this hydrogel lacks antibacterial components. In cases of oral fistula defects with a high risk of infection, this could lead to bacterial infection of the defect site, resulting in severe inflammatory reactions or even further serious tissue necrosis, significantly affecting repair time and effectiveness.

[0005] Chinese invention patent publication CN118304464A discloses a photothermal antibacterial hydrogel wound dressing and its preparation method, with raw materials including methacrylic acid-modified sericin and copper sulfide nanoparticles. However, the hydrogel matrix structure only contains methacrylic acid-modified sericin. Since it is a natural polymer derivative, using it as a single gelling component makes it difficult to match the mechanical properties of the gel with the actual situation of oral fistulas. At the same time, the photothermal conversion efficiency of the copper sulfide nanoparticle photothermal antibacterial strategy still needs to be improved, and it is easily affected by various biomolecules in blood and skin, resulting in poor antibacterial performance. Therefore, there is still room for improvement. Summary of the Invention

[0006] In view of this, the present invention provides a method for preparing a natural polysaccharide-based organic / inorganic hybrid photothermal antibacterial hydrogel. Specifically, the present invention provides the following technical solution:

[0007] 1. A method for preparing a natural polysaccharide-based organic / inorganic hybrid photothermal antibacterial hydrogel, comprising the following steps:

[0008] 1) Mix methacrylamide gelatin, sodium alginate aldehyde, tannic acid, and photoinitiator and dissolve them in phosphate buffer to obtain solution A;

[0009] 2) Dissolve carboxymethyl chitosan in phosphate buffer to obtain solution B;

[0010] 3) Add gold nanorod aqueous dispersion to solution B to obtain solution C; the volume ratio of gold nanorod aqueous dispersion to solution B is 1.5:1 to 2:1, and the gold nanorods are 30 to 70 nm long and 5 to 10 nm wide.

[0011] 4) Mix solutions A and C and inject them to obtain a network hydrogel I;

[0012] 5) Crosslink hydrogel I by ultraviolet light irradiation to obtain double-network hydrogel II.

[0013] Furthermore, based on mass-volume concentration, the concentration of methacrylamide gelatin in step 1) is 20-25%, the concentration of sodium alginate aldehyde is 5-8%, the concentration of photoinitiator is 0.1%-0.3%, and the concentration of tannic acid is 1%-2%.

[0014] Furthermore, the concentration of the carboxymethyl chitosan solution in step 2) is 5% to 8% by mass-volume concentration.

[0015] Furthermore, in step 4), the volume ratio of solution A to solution C is 2:1 to 2.5:1.

[0016] Furthermore, the methacrylated gelatin in step 1) is a reaction product of gelatin and methacrylic anhydride, with a degree of methacrylation of 70-95%.

[0017] Furthermore, the sodium alginate in step 1) is a reaction product of sodium periodate and sodium alginate, with a degree of aldehyde conversion of 50-90%.

[0018] Furthermore, the gold nanorod aqueous dispersion in step 3) is prepared using a seed-mediated method, and its concentration is 20–30 mmol / L, calculated based on the concentration of tetrachloroauric acid in the seed solution used in the seed-mediated method. -1 .

[0019] Furthermore, the preparation steps of the seed-mediated method are as follows:

[0020] 1) Preparation of seed solution: Hexadecyltrimethylammonium bromide and deionized water are added to the sample bottle. Tetrachloroauric acid is added to the sample bottle under low-speed stirring. The stirring speed is adjusted to high-speed stirring. Sodium borohydride is quickly added to reduce tetrachloroauric acid. Stir and let stand in a water bath at 27°C for 1 hour.

[0021] 2) Preparation of growth solution: Add hexadecyltrimethylammonium bromide and deionized water to a round-bottom flask, heat until the solution is completely clear, seal, keep warm at 27°C for 1 hour, add silver nitrate to the solution in sequence, then add tetrachloroauric acid, shake, add ascorbic acid, and observe the solution gradually turn colorless after shaking.

[0022] 3) Preparation and purification of gold nanorods: Add the seed solution from step 1) to the growth solution from step 2), seal, and let stand at 27°C for 24 hours to allow the gold seeds to grow fully into nanorods. Centrifuge the dispersion of gold nanorods, remove the supernatant, add deionized water to redisperse the gold nanorods, centrifuge again, remove the supernatant, repeat several times until the supernatant is colorless to obtain an aqueous dispersion of gold nanorods.

[0023] Furthermore, the photoinitiator in step 1) is a phenyl (2,4,6-trimethylbenzoyl) lithium phosphate salt.

[0024] Furthermore, in step 5), the ultraviolet light crosslinking process uses an ultraviolet light wavelength of 340–400 nm and an irradiation time of 30–60 s.

[0025] The beneficial effects of this invention are as follows: This invention constructs a first hydrogel network by reacting carboxymethyl chitosan with aldehyde-modified sodium alginate under a weakly alkaline environment via a Schiff base reaction to generate imine bonds. Tannic acid is added to enhance the network's mechanical strength through multiple hydrogen bonds and to prepare for a synergistic antibacterial strategy using a mild photothermal method. In the second step, methacrylamide gelatin is crosslinked under ultraviolet light in the presence of a photoinitiator to generate a second hydrogel crosslinked network. During this process, inorganic gold nanorods are incorporated to prepare a polysaccharide-based organic / inorganic hybrid antibacterial hydrogel material. The gold nanorod mixing ratio was subsequently controlled during preparation, thereby characterizing the material's near-infrared photothermal heating ability and mild photothermal antibacterial ability. This invention, by controlling the ratio of components forming the first and second networks and the initiator ratio, enables the hydrogel to gel during the first-step injection, maintaining its morphology, filling complex wound shapes, and preparing for the second-stage ultraviolet photocrosslinking. After the second-stage ultraviolet photocrosslinking, the mechanical properties increase, providing a certain degree of mechanical support. After screening, the following parameters were determined to meet the requirements: 1. Gold nanorod particle mixing amount; 2. Volume ratio and mutual proportion of aldehyde-modified sodium alginate and carboxymethyl chitosan, as well as the amount of tannic acid added; 3. Volume ratio and mutual proportion of methacrylamide gelatin and photoinitiator. Furthermore, this antibacterial hydrogel can be molded in complex morphologies and can even be photocrosslinked in situ, depending on the situation, to repair irregular defects. In addition, the mild photothermal antibacterial strategy avoids the risks of antibiotic resistance caused by antibiotic overuse, and the hydrogel has broad-spectrum antibacterial capabilities; simultaneously, the raw materials for preparation are readily available, the preparation process is relatively simple, and the preparation cost is relatively low. Attached Figure Description

[0026] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided:

[0027] Figure 1 This is a flowchart illustrating the actual process of hydrogel formation.

[0028] Figure 2 The image shows the FT-IR infrared spectroscopy test results.

[0029] Figure 3 The image shows the test results from the X-ray photoelectron spectroscopy instrument.

[0030] Figure 4 The figure shows the results of the hydrogel strain rheology test.

[0031] Figure 5 A graph showing the statistical results of the compression modulus test;

[0032] Figure 6 Photothermal heating curve of hydrogel;

[0033] Figure 7 Image taken by an infrared camera;

[0034] Figure 8 The graph shows the test results of the antibacterial properties of the hydrogel.

[0035] Figure 9 The image shows the results of the hydrogel biotoxicity test. Detailed Implementation

[0036] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0037] Example 1

[0038] 1. Preparation of methacrylamide gelatin

[0039] 1) Add 10g of gelatin to 100mL of PBS buffer and stir to dissolve at 60℃. Add 9mL of methacrylic anhydride and react at 60℃ for 4h. Then add 400mL of PBS buffer to terminate the reaction. Dialyze the solution at 40℃ for 7 days, and lyophilize to obtain methacrylated gelatin with a degree of methacrylation of 86.96%.

[0040] 2. Preparation of sodium alginate aldehyde

[0041] 1) Dissolve 4g of sodium alginate in 400mL of deionized water, add 8g of sodium periodate, stir and react in the dark for 24h, then add 2mL of ethylene glycol to terminate the reaction. The solution is then rotary evaporated, dialyzed for 3 days, and lyophilized to obtain aldehyde-modified sodium alginate with a degree of aldehyde modification of 83.2%.

[0042] 3. Preparation of gold nanorods using a seed-mediated method

[0043] (1) Preparation of seed solution: 128.8 mg cetyltrimethylammonium bromide (CTAB) and 3.545 mL deionized water were added to the sample vial. 74 μL of 24.28 mmol / L solution was added under low-speed stirring. -1 Tetrachloroauric acid (HAuCl4) solution was added to the sample vial. The mixture was then stirred at high speed, and 500 μL of NaBH4 solution was quickly added to reduce HAuCl4. The mixture was stirred for 2 min and then incubated in a 27°C water bath for 1 h.

[0044] (2) Preparation of growth medium: Add 1.288 g CTAB and 45.95 mL deionized water to a round-bottom flask, heat gently until the solution is completely clear, seal, and incubate in a 27°C water bath for 1 hour. Add 1.7 mL of 4 mmol·L⁻¹ silver nitrate (AgNO₃) to the solution, followed by 1.545 mL of HAuCl₄. After shaking, add 620 μL of 78.8 mmol·L⁻¹ growth medium. -1 Ascorbic acid (AA) solution, after shaking, was observed to gradually turn colorless.

[0045] (3) Formal preparation and purification: Add 500 μL of the seed solution from step (1) to the growth solution from step (2), seal, and let stand in a constant temperature water bath at 27°C for 24 h to allow the gold seeds to fully grow into nanorods, obtaining gold nanorods. Centrifuge the gold nanorod dispersion for 20 min using a centrifuge (12000 rpm) and remove the supernatant. To thoroughly remove impurities such as CTAB and AgNO3, add deionized water again to redisperse the gold nanorods, centrifuge at 12000 rpm for 20 min, and remove the supernatant. Repeat this step twice until the supernatant is colorless to obtain an aqueous dispersion of gold nanorods.

[0046] This gold nanorod aqueous dispersion, with a concentration of 24.28 mmol / L, was used in all the following examples and comparative examples. -1 Each gold nanorod is approximately 25 nm long and 6 nm wide.

[0047] Example 2

[0048] 1) Add 270 mg of sodium alginate, 50 mg of tannic acid, 1 g of methacrylamide gelatin, and 10 mg of photoinitiator to 5 mL of phosphate buffer. Shake and stir at 40 °C until completely dissolved to obtain solution A.

[0049] 2) Add 270 mg of carboxymethyl chitosan to 5 mL of phosphate buffer and stir at 25 °C until completely dissolved to obtain solution B. Then mix in the gold nanorod aqueous dispersion, with a volume ratio of gold nanorod aqueous dispersion to solution B of 2:1, and mix well to obtain solution C.

[0050] 3) Take 1 mL of solution A and 500 μL of solution C and place them into the two injection chambers of a double syringe. Mix and extrude the mixture at 25°C into a suitable mold to obtain hydrogel I-A1;

[0051] 4) Irradiate the hydrogel with a UV lamp with a wavelength of 365nm for 30s. Hydrogel II-A1 is then obtained.

[0052] Example 3

[0053] 1) Add 270 mg of sodium alginate, 50 mg of tannic acid, 1 g of methacrylamide gelatin, and 10 mg of photoinitiator to 5 mL of phosphate buffer. Shake and stir at 40 °C until completely dissolved to obtain solution A.

[0054] 2) Add 270 mg of carboxymethyl chitosan to 5 mL of phosphate buffer and stir at 25 °C until completely dissolved to obtain solution B. Then mix in the gold nanorod aqueous dispersion, with a volume ratio of gold nanorod aqueous dispersion to solution B of 1.5:1, and mix well to obtain solution C.

[0055] 3) Take 1 mL of solution A and 500 μL of solution C and place them into the two injection chambers of a double syringe. Mix and extrude the mixture at 25°C into a suitable mold to obtain hydrogel I-A2.

[0056] 4) Irradiate the hydrogel with a UV lamp with a wavelength of 365nm for 30s. Hydrogel II-A2 is then obtained.

[0057] Comparative Example 1

[0058] 1) Add 270 mg of sodium alginate, 50 mg of tannic acid, 1 g of methacrylamide gelatin, and 10 mg of photoinitiator to 5 mL of phosphate buffer. Shake and stir at 40 °C until completely dissolved to obtain solution A.

[0059] 2) Add 270 mg of carboxymethyl chitosan to 5 mL of phosphate buffer and stir at 25 °C until completely dissolved to obtain solution B. Then mix in the gold nanorod aqueous dispersion, with a volume ratio of 1:1 between the gold nanorod aqueous dispersion and solution B, and mix well to obtain solution C.

[0060] 3) Take 1 mL of solution A and 500 μL of solution C and place them into the two injection chambers of a double syringe. Mix and extrude the mixture at 25°C into a suitable mold to obtain hydrogel I-A3;

[0061] 4) Irradiate the hydrogel with a UV lamp with a wavelength of 365nm for 30s. Hydrogel II-A3 is then obtained.

[0062] The amount of gold nanorods added in this comparative example is less than the scope claimed in the claims.

[0063] Comparative Example 2

[0064] 1) Add 270 mg of sodium alginate, 50 mg of tannic acid, 1 g of methacrylamide gelatin, and 10 mg of photoinitiator to 5 mL of phosphate buffer. Shake and stir at 40 °C until completely dissolved to obtain solution A.

[0065] 2) Add 270 mg of carboxymethyl chitosan to 5 mL of phosphate buffer and stir at 25 °C until completely dissolved to obtain solution B. Then mix in the gold nanorod aqueous dispersion, with a volume ratio of gold nanorod aqueous dispersion to solution B of 0.5:1, and mix well to obtain solution C.

[0066] 3) Take 1 mL of solution A and 500 μL of solution C and place them into the two injection chambers of a double syringe. Mix and extrude the mixture at 25°C into a suitable mold to obtain hydrogel I-A4;

[0067] 4) Irradiate the hydrogel with a UV lamp with a wavelength of 365nm for 30s. Hydrogel II-A4 is then obtained.

[0068] The amount of gold nanorods added in this comparative example is less than the scope claimed in the claims.

[0069] Comparative Example 3

[0070] 1) Add 270 mg of sodium alginate, 50 mg of tannic acid, 1 g of methacrylamide gelatin, and 10 mg of photoinitiator to 5 mL of phosphate buffer. Shake and stir at 40 °C until completely dissolved to obtain solution A.

[0071] 2) Add 270 mg of carboxymethyl chitosan to 5 mL of phosphate buffer and stir at 25 °C until completely dissolved, i.e., solution B.

[0072] 3) Take 1 mL of solution A and 500 μL of solution B and place them into the two injection chambers of a double syringe. Mix and extrude the mixture at 25°C into a suitable mold to obtain hydrogel I-A5.

[0073] 4) Irradiate the hydrogel with a UV lamp with a wavelength of 365nm for 30s. Hydrogel II-A5 is then obtained.

[0074] This comparative example does not include gold nanorods.

[0075] Comparative Example 4

[0076] 1) Add 270 mg of sodium alginate, 50 mg of tannic acid, 1 g of methacrylamide gelatin, and 10 mg of photoinitiator to 5 mL of phosphate buffer. Shake and stir at 40 °C until completely dissolved to obtain solution A.

[0077] 2) Add 270 mg of carboxymethyl chitosan to 5 mL of phosphate buffer and stir at 25 °C until completely dissolved to obtain solution B. Then mix in the gold nanorod aqueous dispersion, with a volume ratio of gold nanorod aqueous dispersion to solution B of 2:1, and mix well to obtain solution C.

[0078] 3) Take 1 mL of solution A and 500 μL of solution C and place them into the two injection chambers of a double syringe. Mix and extrude the mixture at 25°C into a suitable mold to obtain hydrogel I-B1.

[0079] This comparative example added gold nanorods, but did not undergo UV cross-linking, and did not form a double network.

[0080] Comparative Example 5

[0081] 1) Add 270 mg of sodium alginate, 50 mg of tannic acid, 1 g of methacrylamide gelatin, and 10 mg of photoinitiator to 5 mL of phosphate buffer. Shake and stir at 40 °C until completely dissolved to obtain solution A.

[0082] 2) Add 270 mg of carboxymethyl chitosan to 5 mL of phosphate buffer and stir at 25 °C until completely dissolved, i.e., solution B. Mix with deionized water (the volume ratio of deionized water to solution B is 2:1).

[0083] 3) Take 1 mL of solution A and 500 μL of solution (2) and put them into the two injection chambers of a double syringe respectively. Mix them at 25°C and extrude them into a suitable mold to obtain hydrogel I-B2;

[0084] 4) Irradiate the hydrogel with a UV lamp with a wavelength of 365nm for 30s. Hydrogel II-B2 is then obtained.

[0085] This comparative example of gold nanorod aqueous dispersion was replaced with deionized water.

[0086] Test Example 1: Characterization Tests of Hydrogels

[0087] Figure 1 This document presents the actual synthesis process of hydrogel II-A1 as an example. It demonstrates the overall steps involved in the hydrogel synthesis. The vial inversion method verified the successful synthesis of the hydrogel.

[0088] Figure 2 FT-IR infrared spectroscopy was performed on the hydrogels of Examples II-A1 and I-A1. The two hydrogels were measured at 1640 cm⁻¹. -1 The characteristic peaks of the -C=N- bond stretching vibration of the Schiff base structure were observed at all locations, proving that Schiff base structures are generated in both single-network and double-network hydrogels.

[0089] Figure 3 For X-ray photoelectron spectroscopy testing. Figure 3 X-ray photoelectron spectroscopy (XPS) was used to analyze the hydrogel of Example II-A1. Characteristic peaks of gold (88.0 eV, 84.5 eV) were observed in the spectrum, indicating the presence of gold in the hydrogel and demonstrating the successful introduction of gold nanorod particles.

[0090] Test Example 2: Strain Rheology Test and Compression Modulus Test

[0091] This test was conducted using three representative hydrogels as examples: Example 1 (double-network hydrogel II-A1 with added gold nanorods), Comparative Example 5 (double-network hydrogel II-B2 without added gold nanorods), and Comparative Example 4 (single-network hydrogel I-B1 without added gold nanorods).

[0092] 1. Hydrogel strain rheology test: Cut a regular disc-shaped sample, calibrate the rheometer and set the test environment such as temperature and humidity. Then fix the sample between the upper and lower clamps to ensure that there are no air bubbles and the force is uniform. Then determine the linear viscoelastic region through small amplitude oscillation shear test. Then carry out strain scanning according to the set strain rate or range and record data such as storage modulus and loss modulus.

[0093] Figure 4 The results are from hydrogel strain rheological tests. Figure 4 As can be seen, the storage modulus of hydrogels II-A1, II-B2, and I-B1 are all about one order of magnitude higher than the loss modulus. This proves that the gelation was successful and verifies the feasibility of the dual-network gelation method. On the other hand, it demonstrates the excellent damping properties of the hydrogel, which are suitable for the complex situation of continuous tissue movement in the actual situation of oral fistula defects.

[0094] 2. Hydrogel compressive strength test: Cut hydrogel samples into regular cylindrical shapes, calibrate the testing machine and set the test environment, place the sample between the upper and lower pressure plates to ensure alignment without deviation, then set the compression rate, start the machine to apply axial pressure, and record stress-strain data in real time; stop the test when the strain reaches the set value and remove the sample.

[0095] Figure 5 This is a graph showing the statistical results of the compressive strength test. From... Figure 5 As can be seen, the maximum compressive strength of hydrogel II-A1 is about 300 kPa, the maximum compressive strength of II-B2 is about 170 kPa, and the maximum compressive strength of I-B1 is about 50 kPa.

[0096] Therefore, it can be seen that:

[0097] 1) The compressive modulus of hydrogel II-A1 (hydrogel with added gold nanorods) of the present invention is significantly higher than that of hydrogel II-B2 without added gold nanorods. This is because the gold nanoparticles play a role in the hydrogel system as physical cross-linking points, enhancing the mechanical properties of the hydrogel, increasing the network density, and improving the strength and toughness of the hydrogel.

[0098] 2) The compressive modulus of the dual-network hydrogels II-A1 and II-B2 is significantly higher than that of the single-network hydrogel I-B1. This is due to the synergistic effect between the two networks. On the one hand, the dual-network strategy allows the hydrogel to absorb and dissipate more energy, thus enabling it to withstand greater pressure. On the other hand, the dual-network structure significantly enhances intermolecular forces, making it less prone to relative sliding and separation of the molecular networks under external forces, thereby improving strength.

[0099] In summary, the dual-network hydrogel with added gold nanorods in this invention has better mechanical properties and is more effective in supporting wounds.

[0100] Test Example 6: Photothermal Heating Capacity Test

[0101] The purpose of the test is to determine whether the photothermal heating effect under near-infrared light irradiation can be achieved, and whether the final heating temperature can reach the range required for photothermal antibacterial activity. This is the most important research standard indicator of this invention.

[0102] Test Method: The photothermal heating capacity of the hydrogels from Examples 1-2 and Comparative Examples 1-3 was tested. The test procedure is as follows: Near-infrared light with a wavelength of 808 nm was used to test the hydrogels from the examples and the comparative examples (the results were tested at light powers of 1W, 1.5W, and 2W, respectively. The light power density was set to 350mW / cm²). 2 Irradiate for 10 minutes and measure its temperature rise curve. Figure 6 The final heating temperatures of four hydrogels with added gold nanorods were monitored using an infrared camera, and photographs were taken to record the results. Figure 7 ).

[0103] Figure 6 Photothermal temperature rise curves of the hydrogels. Under near-infrared light irradiation at three different power densities, the temperature stabilizes after approximately 10 minutes. All hydrogel groups with added gold nanorods exhibited a certain temperature rise. However, only the II-A1 and II-A2 hydrogels of this invention achieved a mild photothermal antibacterial effect (around 46°C to 50°C). In the control examples, the II-A3 and II-A4 hydrogels also showed a temperature rise, but their stable temperatures were slightly lower than this temperature range.

[0104] Figure 7 The image was taken with an infrared camera. The two hydrogels, II-A1 and II-A2, of this invention achieve a mild photothermal antibacterial effect (around 46℃ to 50℃). In the comparative examples, the hydrogels II-A3 and II-A4 also showed an increase in temperature, but their stable temperatures were slightly lower than this temperature range.

[0105] Because too few gold nanorods were added during the preparation of solution C, although there was some photothermal conversion effect, it failed to meet the application requirements. However, no significant heating effect was observed in Comparative Example 3 (II-A5 without added gold nanorods). This indicates that the hydrogel of the present invention can achieve a photothermal heating effect under near-infrared light irradiation, reaching the range required for photothermal antibacterial properties.

[0106] Test Example 7: Photothermal Antibacterial Ability Test

[0107] The photothermal antibacterial activity was tested using the hydrogels of Examples 1-2 and Comparative Examples 1-3. The test procedure is as follows: Hydrogels with a concentration of 1×10⁻⁶ were used respectively. 6 CFU / mL of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli were inoculated onto hydrogels of Examples II-A1-2 and Comparative Examples II-A3-5. Near-infrared light at a wavelength of 808 nm (test light power 1.5 W, light power density set to 350 mW / cm²) was used. 2 Irradiate for 15 minutes. Then, irradiate the antibacterial bacterial suspension for 10 minutes. 3 After a 1:1 dilution, 50 μL was spread onto an agar plate and incubated at 37°C for 24 hours. Colony counting was then performed and the plate was photographed. Figure 8 a) Counting and statistics ( Figure 8 b).

[0108] Figure 8 This is a graph showing the test results for the antibacterial properties of the hydrogel. From... Figure 8 As can be seen, no obvious antibacterial effect was observed in control examples II-A5, II-A4, and II-A3, while the hydrogels of examples II-A2 and II-A1 had an antibacterial rate of greater than 99% against both bacteria.

[0109] The main reason why control examples II-A5, II-A4, and II-A3 failed to achieve the required antibacterial effect is that mild photothermal antibacterial requires a specific temperature to destroy the structure and function of microorganisms. When the temperature is not up to standard, the added tannins cannot be used to achieve the purpose of rapidly and effectively damaging microorganisms within 15 minutes. Because the number of added nano gold rods in the control examples was insufficient, the temperature could not reach the required level, and therefore the antibacterial effect could not be demonstrated.

[0110] In summary, the hydrogel of this invention, through the combined action of a mild photothermal strategy and the antibacterial agent tannic acid, exhibits highly effective killing effects against Gram-positive bacteria *S. aureus* and Gram-negative bacteria *E. coli*. This demonstrates that the hydrogel of this invention can effectively kill common pathogenic bacteria and holds promise for use in severely infected environments such as oral fistula defects.

[0111] Test Example 8: Biocompatibility Test

[0112] Good biocompatibility is essential for wound dressings that are applied directly to the body. Therefore, the biocompatibility of the hydrogel was evaluated using two indicators: cytotoxicity and blood compatibility. Representative samples were selected from II-A1 of Example 1, II-A2 of Example 2, and II-B2 of Comparative Example 5 to evaluate the in vitro biocompatibility of the antibacterial hydrogel material of this invention.

[0113] First, the cytotoxicity of three hydrogels against mouse fibroblasts (L929) was detected using the MTT assay. The procedure was as follows: 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin were added to the cell culture medium. Cells were cultured in a CO2 humidified incubator at 37°C, with the culture medium changed every two days. 200 μL of culture medium was added to each container containing antibacterial hydrogel sheets (II-A1, II-A2, and II-B2) with a diameter of 4 mm and a thickness of 3 mm for extraction at 37°C for 24 h. 10,000 cells (L929) were seeded into each well of a 96-well plate and cultured for 12 h. The extracted culture medium was then replaced with the extracted medium, and the cells were cultured for another 24 h. Finally, cell viability was determined using the MTT assay. Figure 9 The image shows the results of the hydrogel biotoxicity test.

[0114] Figure 9 a represents the cytotoxicity test results. It can be seen that the viability of II-A1, II-A2, and II-B2 relative to L929 cells is approximately 75%, 80%, and 110%, respectively, meeting the standard of cell compatibility greater than 70%. This indicates that the material itself has good biocompatibility, and the potential dissolution process of the gold nanorods did not produce significant toxicity to the cells.

[0115] Next, the blood compatibility of four representative hydrogels (II-A1, II-A2, II-A3, and II-A5) and the gold nanorods themselves was evaluated. The test procedure was as follows: Blood from BALB / c mice was collected, mixed with physiological saline, and gently shaken to wash. Centrifugation was performed at 3000 rpm for 5 minutes each time, discarding the supernatant and aspirating the residual liquid each time to remove fragmented blood cells and obtain intact blood cells. This operation was repeated three times. The centrifuged blood cells were then dissolved in physiological saline to prepare a 4% blood cell solution. 300 μL of the above blood cell solution was mixed with 300 μL of the corresponding material's physiological saline extract (or gold nanorod aqueous dispersion) and co-cultured at 37°C for 3 hours. After the co-culture, a centrifugation at 3000 rpm for 5 minutes was performed to centrifuge the blood cells to the bottom. Images were then taken, and the absorbance of the supernatant at 545 nm was measured to evaluate the degree of hemolysis. An additional negative and positive control group were included.

[0116] Figure 9 Figure b shows the results of the blood compatibility test. Gold nanorods themselves exhibit significant hemolysis, but when loaded into hydrogels, neither the II-A1~3 materials containing gold nanorods nor the II-A5 materials without gold nanorods showed hemolysis exceeding 5%, indicating good blood compatibility of the materials.

[0117] The two tests above together demonstrate that the hydrogel of this invention possesses excellent biocompatibility. This invention uses biocompatible natural polysaccharides and natural polyphenols as the hydrogel matrix; the raw material molecules are all derived from natural organisms, and their original molecules and degradation products do not exhibit corresponding biotoxicity. While gold nanorods themselves possess some biotoxicity, after being loaded into the hydrogel, Au-S bond interactions are generated using the existing sulfur element in GelMA, anchoring the gold nanorods within the hydrogel without leakage, effectively avoiding potential biotoxicity hazards.

[0118] In summary, this invention modifies two naturally derived polysaccharides with gelatin to obtain an injectable dual-network hydrogel with good mechanical properties and biocompatibility. Furthermore, by physically blending inorganic gold nanorods with the antibacterial component tannic acid, a mild photothermal antibacterial effect with near-infrared light response is achieved, exhibiting an inhibition rate greater than 99% against two typical pathogenic bacteria (Staphylococcus aureus and Escherichia coli). This was verified through mechanical property testing, photothermal heating testing, antibacterial testing, and biocompatibility testing.

[0119] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing a natural polysaccharide-based organic / inorganic hybrid photothermal antibacterial hydrogel, characterized in that, The preparation steps are as follows: 1) Mix methacrylamide gelatin, sodium alginate aldehyde, tannic acid, and photoinitiator and dissolve them in phosphate buffer to obtain solution A; 2) Dissolve carboxymethyl chitosan in phosphate buffer to obtain solution B; 3) Add gold nanorod aqueous dispersion to solution B to obtain solution C; the volume ratio of gold nanorod aqueous dispersion to solution B is 1.5:1 to 2:1, and the gold nanorods are 30 to 70 nm long and 5 to 10 nm wide. 4) Mix solutions A and C and inject them to obtain a network hydrogel I; 5) Crosslink hydrogel I by ultraviolet light irradiation to obtain double-network hydrogel II.

2. The method for preparing a natural polysaccharide-based organic / inorganic hybrid photothermal antibacterial hydrogel according to claim 1, characterized in that, Based on mass-volume concentration, the concentration of methacrylamide gelatin in step 1) is 20-25%, the concentration of sodium alginate is 5-8%, the concentration of photoinitiator is 0.1%-0.3%, and the concentration of tannic acid is 1%-2%.

3. The method for preparing a natural polysaccharide-based organic / inorganic hybrid photothermal antibacterial hydrogel according to claim 1, characterized in that, The concentration of the carboxymethyl chitosan solution in step 2) is 5% to 8% by mass-volume concentration.

4. The method for preparing a natural polysaccharide-based organic / inorganic hybrid photothermal antibacterial hydrogel according to claim 1, characterized in that, In step 4), the volume ratio of solution A to solution C is 2:1 to 2.5:

1.

5. The method for preparing a natural polysaccharide-based organic / inorganic hybrid photothermal antibacterial hydrogel according to claim 1, characterized in that, The methacrylated gelatin in step 1) is a product of the reaction between gelatin and methacrylic anhydride, with a degree of methacrylation of 70-95%.

6. The method for preparing a natural polysaccharide-based organic / inorganic hybrid photothermal antibacterial hydrogel according to claim 1, characterized in that, The sodium alginate in step 1) is a reaction product of sodium periodate and sodium alginate, with a degree of aldehyde conversion of 50-90%.

7. The method for preparing a natural polysaccharide-based organic / inorganic hybrid photothermal antibacterial hydrogel according to claim 1, characterized in that, The gold nanorod aqueous dispersion in step 3) was prepared using a seed-mediated method, with a concentration of 20–30 mmol / L, calculated based on the concentration of tetrachloroauric acid in the seed solution. -1 .

8. The method for preparing a natural polysaccharide-based organic / inorganic hybrid photothermal antibacterial hydrogel according to claim 7, characterized in that, The preparation steps of the seed-mediated method are as follows: 1) Preparation of seed solution: Hexadecyltrimethylammonium bromide and deionized water are added to the sample bottle. Tetrachloroauric acid is added to the sample bottle under low-speed stirring. The stirring speed is adjusted to high-speed stirring. Sodium borohydride is quickly added to reduce tetrachloroauric acid. Stir and let stand in a water bath at 27°C for 1 hour. 2) Preparation of growth solution: Add hexadecyltrimethylammonium bromide and deionized water to a round-bottom flask, heat until the solution is completely clear, seal, keep warm at 27°C for 1 hour, add silver nitrate to the solution in sequence, then add tetrachloroauric acid, shake, add ascorbic acid, and observe the solution gradually turn colorless after shaking. 3) Preparation and purification of gold nanorods: Add the seed solution from step 1) to the growth solution from step 2), seal, and let stand at 27°C for 24 hours to allow the gold seeds to grow fully into nanorods. Centrifuge the dispersion of gold nanorods, remove the supernatant, add deionized water to redisperse the gold nanorods, centrifuge again, remove the supernatant, repeat several times until the supernatant is colorless to obtain an aqueous dispersion of gold nanorods.

9. The method for preparing a natural polysaccharide-based organic / inorganic hybrid photothermal antibacterial hydrogel according to claim 1, characterized in that, The photoinitiator in step 1) is a phenyl (2,4,6-trimethylbenzoyl) lithium phosphate salt.

10. The method for preparing a natural polysaccharide-based organic / inorganic hybrid photothermal antibacterial hydrogel according to claim 1, characterized in that, In step 5), the ultraviolet light crosslinking process uses an ultraviolet light wavelength of 340–400 nm and an irradiation time of 30–60 s.

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