Photosensitive and thermosensitive hybrid rapid hemostatic hydrogel

Abstract

This invention belongs to the field of biomedical polymer hydrogel technology, and specifically relates to a photosensitive and thermosensitive hybrid rapid hemostatic hydrogel. The hydrogel components of this invention include poly(N-isopropylacrylamide) grafted modified gelatin, poly(N-isopropylacrylamide) modified hyaluronic acid, quaternized gelatin, photoresponsive molecule-modified hyaluronic acid, and photoinitiator LAP. Due to the presence of modified gelatin and hyaluronic acid, the hydrogel can effectively improve its adhesion to bleeding wounds and achieve effective hemostasis; the modified molecules used have photosensitive or thermosensitive environmental responsiveness, and by applying specific conditions, the curing speed of the hemostatic hydrogel can be accelerated, preventing the hydrogel components from being washed away by blood, thus enabling rapid wound hemostasis.

Description

[0001] This application is a divisional application of application number 202211167578.7 (filed on 2022-09-23, entitled "A High-Strength Hydrogel with Photosensitive and Thermosensitive Properties"). Technical Field

[0002] This invention belongs to the field of biomedical polymer hydrogel technology, specifically relating to a photosensitizing and thermosensitive hybrid rapid hemostatic hydrogel. Background Technology

[0003] Collagen is an important structural protein in animal connective tissue, playing a vital role in maintaining the normal function of cells, tissues, and organs, as well as in repairing damage. Collagen possesses excellent biological properties such as low antigenicity, good biocompatibility, biodegradability, and sol-gel properties, making it widely used in research fields such as tissue repair materials, hemostatic materials, drug sustained-release materials, and biomimetic scaffolds.

[0004] Clinically used hemostatic materials can be classified according to their hemostatic mechanism. The first category includes hemostatic gauze containing high-molecular-weight polysaccharides, inorganic zeolites, etc. This type requires physical compression of the bleeding wound during hemostasis, resulting in a slow hemostatic process, suitable for small wounds with minimal bleeding. The second category contains hemostatic materials containing thrombin and coagulation factors, commonly used for surgical wounds, but prone to the problem of thrombin protein molecules leaking into normal blood vessels, posing a risk of inducing thrombosis. The third category consists of occlusive wound materials with extremely strong tissue adhesion, such as adhesive bandages. All three categories of hemostatic materials struggle to address common clinical problems during wound healing, such as exudation, infection, and pain.

[0005] To address the significant risk of death due to blood loss in emergency situations and hospital settings, new hemostasis methods and strategies have been continuously researched. In recent years, Johnson & Johnson's Su product—an injectable gel-based hemostatic agent—has gained widespread attention in clinical applications. This material, primarily composed of gelatin, rapidly forms a foam-like gel upon dissolving in water. It can be injected into the wound to stop bleeding, protect exposed nerve endings, promote wound healing, and inhibit scarring. This novel in-situ gel-forming hemostasis method effectively alleviates the aforementioned clinical problems.

[0006] Currently, injectable, photoresponsive, and thermosensitive hemostatic gels have all been reported. Synthetic polymer-based hemostatic gels have high mechanical strength, but they solidify immediately upon contact with water and exhibit poor adhesion to wet tissue surfaces, lack elasticity, or produce toxic degradation products, limiting their clinical application. Natural biopolymers possess excellent biological properties, making them more suitable for hemostatic gels; however, these materials generally suffer from poor mechanical strength and slow curing speed, making them unsuitable for handling acute bleeding, large-volume bleeding, and tissue bleeding under high vascular pressure. Summary of the Invention

[0007] To address the technical problems of poor adhesion to wet tissue, lack of elasticity, or biotoxicity of synthetic hemostatic gels, and the poor mechanical properties, slow curing speed, and inability of natural biopolymers to cope with acute bleeding, large bleeding volume, and high vascular pressure tissue bleeding, this invention provides a high-strength hydrogel with a combination of photosensitivity and temperature sensitivity.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] One object of the present invention is to provide a high-strength hydrogel composition of mixed photosensitivity and thermosensitivity, characterized in that the hydrogel composition comprises the following components in mass fractions:

[0010] Poly(N-isopropylacrylamide) grafted modified gelatin: 14-16%, poly(N-isopropylacrylamide) modified hyaluronic acid: 0.5-1.5%, quaternized gelatin: 0.5-1.5%, photoresponsive molecule modified hyaluronic acid: 0.1-0.8%, photoinitiator: 0.05-1%; the balance is water or phosphate buffer solution.

[0011] Preferably, the hydrogel composition comprises the following components in mass fractions:

[0012] Poly(N-isopropylacrylamide) grafted modified gelatin (PG): 14.5-15.5%; poly(N-isopropylacrylamide) modified hyaluronic acid (P-HA): 0.8-1.2%; quaternized gelatin: 0.8-1.2%; photoresponsive molecularly modified hyaluronic acid (HA-NB): 0.2-0.6%; photoinitiator: 0.05-0.15%; the balance is water or phosphate buffer solution. The preferred content of photoresponsive molecularly modified hyaluronic acid (HA-NB) is 0.3-0.5%.

[0013] The present invention also provides a photosensitive and temperature-sensitive hybrid high-strength hydrogel, which is obtained by irradiating the above hydrogel composition with ultraviolet light at 20-25°C and then placing it in an environment of 30-40°C.

[0014] The resulting hydrogel has a swelling ratio of 800–900%, a fracture stress of 0.13–0.15 MPa, a storage modulus of 30,000–40,000 Pa, an adhesion capacity of 200–230 kPa, and a rupture pressure of 250–300 mmHg.

[0015] This hydrogel has strong adhesion and can withstand stronger burst pressure, making it suitable for treating bleeding from irregularly shaped organs and arteries during surgery, or for stopping bleeding from rapidly bleeding wounds on the battlefield or from external injuries.

[0016] This invention provides the use of the above-mentioned hydrogel composition or hydrogel as a rapid hemostatic material for bleeding wounds of irregularly shaped organs, incompressible internal organs, and high-pressure arteries during surgery. After irradiating the above-mentioned hydrogel composition with ultraviolet light, it is injected into the desired site, and hemostasis is achieved within 6-10 seconds.

[0017] Another object of the present invention is to provide a photosensitizing and thermosensitive hybrid rapid hemostatic hydrogel composition, characterized in that the hydrogel composition comprises the following components in mass fractions:

[0018] Poly(N-isopropylacrylamide) grafted modified gelatin: 14-16%; Poly(N-isopropylacrylamide) modified hyaluronic acid: 0.5-1.5%; Quaternized gelatin: 2.5-3.5%; Photoresponsive molecule modified hyaluronic acid: 0.1-0.8%; Photoinitiator: 0.05-1%; Balance is water or phosphate buffer solution.

[0019] Preferably, the hydrogel composition comprises the following components in mass fractions:

[0020] Poly(N-isopropylacrylamide) grafted modified gelatin (PG): 14.5-15.5%; poly(N-isopropylacrylamide) modified hyaluronic acid (P-HA): 0.8-1.2%; quaternized gelatin: 2.8-3.2%; photoresponsive molecule modified hyaluronic acid (HA-NB): 0.2-0.6%; photoinitiator: 0.05-0.15%; the balance is water or phosphate buffer solution. Preferably, HA-NB is 0.35-0.45%.

[0021] The present invention also provides a photosensitive and thermosensitive hybrid rapid hemostatic hydrogel, which is obtained by irradiating the above hydrogel composition with ultraviolet light at 20-25°C and then placing it in an environment of 30-40°C.

[0022] The resulting hydrogel has a swelling ratio of 780–790%, a fracture stress of 0.11–0.125 MPa, a storage modulus of 5000–6500 Pa, and a rupture pressure of 170–180 mmHg.

[0023] This invention provides the use of the above-mentioned hydrogel as a hemostatic material for traumatic bleeding in the digestive tract and common skin.

[0024] Another object of the present invention is to provide a photosensitive and thermosensitive hybrid antibacterial hydrogel composition, characterized in that the hydrogel comprises the following components in mass fractions:

[0025] Poly(N-isopropylacrylamide) grafted modified gelatin: 14-16%; Poly(N-isopropylacrylamide) modified hyaluronic acid: 0.5-1.5%; Quaternized gelatin: 4.5-5.5%; Photoresponsive molecule modified hyaluronic acid: 0.1-0.8%; Photoinitiator: 0.05-1%; Balance is water or phosphate buffer solution.

[0026] Preferably, the hydrogel composition comprises: poly(N-isopropylacrylamide) grafted modified gelatin (PG): 14.5–15.5%; poly(N-isopropylacrylamide) modified hyaluronic acid (P-HA): 0.8–1.2%; quaternized gelatin: 4.8–5.2%; photoresponsive molecule modified hyaluronic acid (HA-NB): 0.2–0.6%; photoinitiator: 0.05–0.15%; the balance being water or phosphate buffer solution. Preferably, HA-NB comprises 0.35–0.45%.

[0027] The present invention also provides a photosensitive and thermosensitive hybrid rapid hemostatic hydrogel, which is obtained by irradiating the above hydrogel composition with ultraviolet light at 20-25°C and then placing it in an environment of 30-40°C.

[0028] The resulting hydrogel had a swelling ratio of 550–600%, a fracture stress of 0.16–0.2 MPa, a storage modulus of 3000–3500 Pa, and a rupture pressure of 150–160 mmHg. The inhibition zone diameter against *Escherichia coli* was 15–16 mm, and against *Staphylococcus aureus*, it was 10–10.5 mm.

[0029] This invention provides the use of the above-mentioned hydrogel as a hemostatic material for easily infected wounds or chronic wounds.

[0030] Preferably, the ultraviolet irradiation time is 2–8 minutes, and the ultraviolet energy density is 5–40 mW / cm². 2 .

[0031] Preferably, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphite (LAP).

[0032] Preferably, the mass ratio of poly-N-isopropylacrylamide (PNIPAM) to gelatin in the poly-N-isopropylacrylamide grafted modified gelatin (PG) is (1-50):1; more preferably (3-18):1, and even more preferably 10-15:1.

[0033] Preferably, the mass ratio of poly-N-isopropylacrylamide (PNIPAM) to hyaluronic acid in the poly-N-isopropylacrylamide modified hyaluronic acid (P-HA) is (0.5-10):1; more preferably (1-1.5):1.

[0034] The quaternized gelatin is preferably diepoxy quaternary ammonium salt modified gelatin (DG). The DG is prepared by reacting gelatin with diepoxy quaternary ammonium salt (DEQAS), wherein the molar ratio of primary amino groups in the gelatin to epoxy groups in the DEQAS is 1:(1-10); more preferably 1:(2-3).

[0035] Preferably, the photoresponsive molecule in the photoresponsive molecule-modified hyaluronic acid (HA-NB) is N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy)butyramide. Preferably, the molar ratio of the photoresponsive molecule to hyaluronic acid in the HA-NB is (100-500):1; more preferably, the molar ratio of the photoresponsive molecule to HA in the HA-NB is (135-200):1.

[0036] One or more technical solutions provided in the embodiments of the present invention have at least the following technical effects:

[0037] (1) The components of this invention contain modified natural polymer materials, which can effectively improve the adhesion of hydrogel to bleeding wound and achieve effective hemostasis; the natural polymer materials are chemically modified in this invention, and the modified molecules have photosensitive or temperature-sensitive environmental responsiveness. By applying specific conditions, the curing speed of the hemostatic hydrogel can be accelerated, the hydrogel components can be prevented from being washed away by blood, and the wound can be stopped quickly.

[0038] (2) Several hydrogel components of the present invention undergo chemical cross-linking under ultraviolet light. The hydroxyl groups in HA-NB are oxidized to aldehyde groups, and then cross-link with the primary amino groups of gelatin to form Schiff base covalent bonds, which improves the strength of the gel and can withstand blood pressure of up to 280-320 mmHg. It is suitable as a quick-release hemostatic material for bleeding wounds of irregularly shaped organs, incompressible internal organs, and high-pressure arteries during surgery.

[0039] (3) Photosensitive and thermosensitive mixed antibacterial hydrogels have high antibacterial properties and are suitable as hemostatic materials for easily infected wounds or chronic wounds. Attached Figure Description

[0040] Figure 1 These are SEM images of hydrogels: (a) PG hydrogel, (b) PG / P-HA hydrogel, (c) hydrogel-3, (d) hydrogel-6, and (e) hydrogel-9.

[0041] Figure 2 It is the hydrogel compressive stress-strain curve;

[0042] Figure 3 This is a hydrogel modulus scanning test graph;

[0043] Figure 4 These are the results of the hydrogel antibacterial test; a represents Escherichia coli, and b represents Staphylococcus aureus.

[0044] Figure 5 These are the results of a cytotoxicity test;

[0045] Figure 6 These are pictures showing the healing process on the mouse's back;

[0046] Figure 7 This is a picture showing the hemostasis of a mouse liver. Detailed Implementation

[0047] The present invention will be further described below with reference to specific embodiments, but is not limited thereto.

[0048] It should be noted that, unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; and the reagents, materials and equipment described are commercially available unless otherwise specified.

[0049] The hyaluronic acid used in this invention was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., and the gelatin and other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0050] The PG was prepared by the following method described in the prior art: Shoji Ohyaa, et al. Poly(N-isopropylacrylamide)(PNIPAM)-grafted gelatinhydrogel surfaces: interrelationship between microscopic structure and mechanical property of surface regions and cell adhesiveness. Biomaterials 26(2005)3105-3111.

[0051] The EG was prepared by the following method described in the prior art: Shilin Xu, et al., A multifunctional gelatine-quaternary ammonium copolymer: An efficient material for reducing dye emission in leather tanning process by superior anionic dye adsorption, Journal of Hazardous Materials 383(2020)121142.

[0052] The P-HA was prepared by the following method described in the prior art: Huaping Tan, et al. Thermosensitive injectable hyaluronic acid hydrogel for adipose tissue engineering. Biomaterials 30(2009)6844-6853.

[0053] The HA-NB was prepared by the following method described in the prior art: Yi Hong, et al. A strongly adhesive hemostatic hydrogel for the repair of arterial and heartbleeds. Nature communications, 2019 May 14; 10(1):2060.

[0054] The mass ratio of poly(N-isopropylacrylamide) (PNIPAM) to gelatin in PG is (12:1);

[0055] In P-HA, the mass ratio of PNIPAM to HA is (1:1);

[0056] The molar ratio of primary amino groups in gelatin in EG to epoxy groups in EPTAC is (1:2.5).

[0057] The photoresponsive molecule of HA-NB is N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy)butyramide.

[0058] The molar ratio of photoresponsive molecules to hyaluronic acid (HA) in HA-NB is (168:1).

[0059] Example 1

[0060] A method for preparing a photosensitizing and thermosensitive hybrid high-strength hydrogel involves mixing the previously prepared PG, P-HA, DG, LAP, and HA-NB with a phosphate buffer solution at room temperature below 30°C to obtain a hydrogel composition with a final concentration of 15% PG, 1% P-HA, 1% DG, 0.2% HA-NB, and 0.1% LAP, with the balance being a phosphate buffer solution (PBS buffer solution, 0.01M, pH 7.2–7.4). The hydrogel is then subjected to an energy density of 5 mW / cm². 2 Irradiated with 365nm ultraviolet light for 5 minutes and placed in an environment of 30-40℃, it is denoted as hydrogel-2.

[0061] Examples 2 and 3

[0062] Following the preparation method of Example 1, the final concentrations of HA-NB were changed to 0.4% and 0.6%, and the resulting hydrogels were designated as hydrogel-3 and hydrogel-4.

[0063] Example 4

[0064] A method for preparing a photosensitizing and thermosensitive hybrid high-strength hydrogel involves mixing PG, P-HA, DG, LAP, and HA-NB with a phosphate buffer solution at room temperature to obtain a hydrogel composition with a final concentration of 15% PG, 1% P-HA, 3% DG, 0.2% HA-NB, and 0.1% LAP, with the balance being a phosphate buffer solution (PBS buffer solution, 0.01M, pH 7.2–7.4). The hydrogel is then subjected to an energy density of 5 mW / cm². 2 Irradiated with (365nm) ultraviolet light for 5 minutes, and it is recorded as hydrogel-5.

[0065] Examples 5 and 6

[0066] Following the preparation method of Example 4, the final concentration of HA-NB was changed to 0.4% and 0.6%, and the resulting hydrogels were designated as hydrogel-6 and hydrogel-7.

[0067] Example 7

[0068] A method for preparing a photosensitizing and thermosensitive hybrid high-strength hydrogel involves mixing pre-prepared PG, P-HA, DG, LAP, and HA-NB with a phosphate buffer solution at room temperature to obtain a hydrogel composition with a final concentration of 15% PG, 1% P-HA, 5% DG, 0.2% HA-NB, and 0.1% LAP, with the balance being a phosphate buffer solution (PBS buffer, 0.01M, pH 7.2–7.4). The hydrogel is then subjected to an energy density of 5 mW / cm². 2 Irradiated with (365nm) ultraviolet light for 5 minutes, and it was named hydrogel-8.

[0069] Examples 8 and 9

[0070] Following the preparation method of Example 7, the final concentrations of HA-NB were changed to 0.4% and 0.6%, respectively, and the resulting hydrogels were designated as hydrogel-9 and hydrogel-10.

[0071] Comparative Example 1

[0072] A method for preparing a hydrogel involves mixing PG, P-HA and a phosphate buffer solution at room temperature below 30°C to obtain a hydrogel composition with a final concentration of 15% PG, 1% P-HA, and the remainder being a phosphate buffer solution. This composition is then placed in an environment of 30–40°C and is designated as hydrogel-1.

[0073] Comparative Example 2

[0074] A method for preparing a hydrogel, which differs from Example 1 in that a monoepoxy quaternary ammonium salt (EG) modified gelatin is used to replace DG in Example 1, while all other conditions are the same as in Example 1, is referred to as hydrogel-11.

[0075]

[0076] Comparative Example 3

[0077] A method for preparing a hydrogel, differing from Example 4 in that a monoepoxide quaternary ammonium salt (EG) modified gelatin is used instead of DG in Example 4, while all other conditions are the same as in Example 4. This is designated as Hydrogel-12.

[0078] Comparative Example 4

[0079] A method for preparing a hydrogel, which differs from Example 7 in that a monoepoxy quaternary ammonium salt (EG) modified gelatin is used to replace DG in Example 7, while all other conditions are the same as in Example 7, is referred to as hydrogel-13.

[0080] (1) Microstructure of hydrogels

[0081] After freeze-drying the hydrogel, the specimen was sliced ​​into thin sections using a scalpel, and the surface of each section was sprayed with gold. The cross-sectional microstructure was then observed using a scanning electron microscope.

[0082] The microstructure of hydrogels has a significant impact on their water retention and mechanical properties. Figure 1 The image shows the microstructure of the freeze-dried hydrogel. Compared to PG and PG / P-HA single-network hydrogels, the dual-network hydrogel of this invention has a denser microstructure and smaller pore size. Furthermore, scanning electron microscopy images reveal an interconnected porous structure within the hydrogel. This dense structure improves the hydrogel's mechanical properties. Simultaneously, the interconnected porous structure enables efficient nutrient and oxygen transport, increasing the hydrogel's potential application in wound dressings.

[0083] (2) Swelling rate

[0084] The prepared hydrogel was freeze-dried and weighed (Wd). It was then soaked in phosphate-buffered saline (PBS) solution (pH = 7.4) for 24 hours until swelling equilibrium was reached. After wiping away surface water, the sample was removed and weighed (Ws). The swelling ratio (Q0) was calculated as follows:

[0085] Q0(%)=(Ws-Wd) / W×100%.

[0086] The swelling ratio of the hydrogel is shown in Table 1. The hydrogel can absorb large amounts of blood more quickly and can also absorb the surrounding tissue fluid, preventing its accumulation in the wound and the resulting inflammation.

[0087] (3) Mechanical properties

[0088] Fracture stress:

[0089] The compressibility of hydrogels was evaluated using a general mechanical testing machine. Cylindrical hydrogels with a diameter of 10 mm × 4 mm were prepared and compressed at a strain rate of 3 mm / min until they ruptured. The stress-strain curves were then obtained.

[0090] During wound healing, hydrogel dressings are inevitably subjected to external forces. To prevent secondary damage to the wound tissue, hydrogel dressings must possess suitable mechanical properties. The compressive stress-strain curve of the prepared hydrogel is shown below. Figure 2 As shown in Table 1, the hydrogels of this invention exhibit higher fracture stress (0.14 MPa) compared to single-network hydrogels. Compression tests indicate that the dual-network hydrogels possess better mechanical properties, which may be attributed to the increased crosslinking density resulting from the introduction of the dual-network structure.

[0091] Table 1:

[0092]

[0093] Energy storage modulus:

[0094] Storage modulus and loss modulus: The sol-gel transition of the hydrogel composition was studied using a rotational rheometer. The heating rate was set to 2℃ / min. Before the test, the sample stage was set to 25℃ to prevent gelation. The strain % was set to 1%, and the frequency was 1Hz.

[0095] Storage modulus represents a material's ability to store elastic deformation energy. A higher storage modulus indicates greater material hardness and resistance to deformation. Loss modulus represents the energy lost by a material under irreversible deformation and represents its viscosity. When loss modulus < storage modulus, the material is a gel, with elasticity as its primary characteristic; when loss modulus > storage modulus, the material is a fluid, with viscosity as its primary characteristic; when loss modulus = storage modulus, the material is at the sol-gel transition point, where viscosity and elasticity are equal. During the scan, with increasing temperature, around 31℃, we observed that the storage modulus gradually exceeded the loss modulus, indicating a transition from sol to gel. The results are shown below. Figure 3 The storage modulus is shown in Table 1. The results indicate that the hydrogels are in a stable elastic state and are suitable for wound healing applications. Among them, hydrogel-3 has the highest storage modulus, at 35127 Pa, making it suitable as a rapid hemostatic material for bleeding wounds of irregularly shaped organs, incompressible internal organs, and high-pressure arteries during surgery.

[0096] Hydrogel-6, with a storage modulus of 6142 Pa, is suitable for hemostasis of gastrointestinal and general skin traumatic bleeding where the hemostatic pressure is not too high. Hydrogel-9, with a storage modulus of 3264 Pa, is suitable for hemostasis of easily infected wounds, ordinary wounds, or chronic wounds due to its strong antibacterial effect.

[0097] (4) In vitro adhesion, burst pressure, antibacterial properties and cytotoxicity of hydrogels

[0098] Adhesion ability: Take two pieces of pigskin of the same size (2cm×5cm), irradiate the hydrogel composition with ultraviolet light, apply the irradiated hydrogel composition to one of the pigskin pieces (application area 2cm×2cm, application amount 1.5mL), and then bond the two pieces of pigskin together. Apply uniaxial tension using a universal testing machine, keeping the loading rate constant at 2mm / min.

[0099] Hydrogel dressings with adhesive properties generally have good application prospects in the field of wound repair. A hydrogel with an adhesion capacity greater than 30 kPa can generally meet the application requirements in the field of wound repair. For irregularly shaped organs, incompressible internal organs, high-pressure arteries, etc., the greater the adhesion capacity, the better. For other parts, lower adhesion performance can also meet the requirements.

[0100] Hydrogel-3 showed a significantly higher viscosity (215 kPa) than other samples, while hydrogel-6 exhibited a viscosity of 67 kPa and hydrogel-9 an adhesion strength of 66 kPa, all meeting the requirements for adhesion performance. These results indicate that, under certain hydrogel compositions, the formation of Schiff base covalent bonds enhances the adhesion of the hydrogel. This is because, upon light exposure, the HA-NB component in the hydrogel generates aldehyde groups, increasing the number of binding sites and strengthening the binding of HA-NB to tissue proteins. The high adhesion strength is achieved because the grafted gelatin molecular chain contains a large number of carboxyl and amino groups, which can interact with skin tissue through hydrogen bonds. This material shows broad application prospects in wound healing tissue adhesion, demonstrating that hydrogels possess excellent adhesion capabilities, forming a long-term physical barrier at the wound site and promoting wound repair.

[0101] Burst pressure: Excess fat was removed from pigskin (4×4 cm). The pigskin was used to cover and seal the bottle opening. The bottle was connected to a pressure pump, allowing gas to be introduced into the bottle for pressurization. A 2 mm diameter circular hole was cut in the pigskin, and 500 μL of a hydrogel composition irradiated with ultraviolet light was filled into the cut. The bottle was then placed at 30–40 °C to allow hydrogel to form in situ at the puncture site. The hydrogel thickness was approximately 4.4 mm. Burst pressure was measured after gelation. Gas was introduced into the bottle, and the peak pressure before pressure loss was considered the burst pressure. All measurements were repeated three times. The results showed that hydrogel-3 achieved a burst pressure of up to 280 mmHg, significantly higher than other hydrogel groups, indicating a promising hemostatic sealant.

[0102] Antibacterial properties: The antibacterial activity of the hydrogel against Staphylococcus aureus and Escherichia coli was tested using the inhibition zone method. Bacterial solutions (1×10⁻⁶) were then subjected to the following tests: 6 CFU / mL was inoculated onto an agar plate, and 10 μL of the UV-irradiated hydrogel composition was injected onto a 6 mm diameter filter paper disc. The filter paper disc was placed on the agar plate and incubated at 37 °C for 24 hours. The diameter of the antibacterial region around each sample was measured to evaluate the antibacterial activity of the hydrogel.

[0103] See results Figure 4 Figures a and b show the inhibition results for *Escherichia coli* and *Staphylococcus aureus*, respectively. In the figure, A, B, C, and D represent hydrogel-1, hydrogel-3, hydrogel-6, and hydrogel-9, respectively. For *Escherichia coli*, the inhibition zone diameter of hydrogel-1 was 0 mm, that of hydrogel-3 was 8.3 mm, that of hydrogel-6 was 12.3 mm, and that of hydrogel-9 was 15.5 mm. For *Staphylococcus aureus*, the inhibition zone diameter of hydrogel-1 was 0 mm, that of hydrogel-3 was 9.1 mm, that of hydrogel-6 was 9.9 mm, and that of hydrogel-9 was 10.4 mm.

[0104] Hydrogel wound dressings can act as a barrier to isolate wounds from external bacterial infection. The hydrogel of this invention exhibits significant inhibitory effects against *Escherichia coli* and *Staphylococcus aureus* after 24 hours. It also shows moderate antibacterial activity against *Staphylococcus aureus* and *Escherichia coli*, thus promoting wound healing more effectively. The antibacterial effect against *Escherichia coli* and *Staphylococcus aureus* can be attributed to the compatibility of long-chain alkanes with the bacterial outer cell wall. The positively charged quaternary ammonium compounds attract the negatively charged bacterial cell membrane, disrupting it and causing cytoplasmic leakage, thereby killing the bacteria.

[0105] Cytotoxicity: To test the cytotoxicity of the hydrogel, cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrabromonitride (MTT) method (Sigma, USA). HFF-1 fibroblasts were seeded in 96-well plates (100 μL / well containing 1.0 × 10⁵ cells / mL suspension) and incubated for 18 h, followed by treatment with C hydrogel for 24 h. After treatment, 10 μL of 5 mg / mL MTT was added to each well, and the 96-well plates were incubated at 37 °C for 4 h. Cells in each well were then washed with phosphate-buffered saline (PBS) and dissolved in 100 μL of dimethyl sulfoxide (DMSO). The absorbance of the samples was measured using a microplate reader.

[0106] Detected using the MTT method Figure 5 The cell survival rate is above 80%, indicating that the hydrogel has good biocompatibility and low cytotoxicity.

[0107] (5) In vivo wound repair ability of hydrogel and hemostasis in mouse liver

[0108] The effect of hydrogel on wound healing was investigated using a mouse dorsal full-thickness incision model. Methods: Male ICR mice (18-20g) were anesthetized, their backs were shaved, and the wounds were disinfected with 75% ethanol cotton balls. Circular full-thickness skin defects with a diameter of 7mm and a depth of 1mm were created on the dorsal side of each mouse. A hydrogel precursor solution was injected into the skin defect, and gelation was achieved through UV treatment. Skin defects treated with physiological saline served as a control. The wounds were photographed at selected time points to observe the healing process.

[0109] A full-thickness skin defect repair experiment was conducted in mice using hydrogel-3. Figure 6 As shown, the wound area in each group decreased over time. On day 3, the wounds in all groups were dry with no obvious redness or swelling. On days 5 and 7, the hydrogel-treated wounds showed significant healing compared to those treated with normal saline (NS). By day 11 after hydrogel treatment, the wounds were basically healed, significantly better than the other groups. After 13 days of treatment, the wounds in the NS group were still not completely healed, covered with scabs, while the hydrogel group showed no obvious scarring or elevation, and the skin color was similar to the adjacent normal tissue. Numerous new hair growths were observed at the wound healing site. Therefore, hydrogel is more effective in promoting wound healing. This is because the in-situ formed hydrogel can adhere well to the wound and adhere tightly to the wound site, preventing microbial infection. Simultaneously, the antibacterial properties of the diepoxide quaternary ammonium salt give the hydrogel excellent antibacterial ability; on the other hand, the water-retaining capacity of hyaluronic acid maintains the moist environment required for wound healing, accelerating the wound healing process.

[0110] To further demonstrate the potential of hydrogels as clinical hemostatic materials, they were applied to repair hemorrhage in the liver and heart. The hemostatic properties of the hydrogels were evaluated using a rat liver model (a specific visceral organ with a rich blood supply). In mice, liver puncture was performed to observe bleeding amounts under different conditions. A control group was also included to observe bleeding amounts. It was observed that blood gushed from the needle puncture site during rapid puncture of the liver. In this liver puncture model, blood continued to flow even without treatment or gauze coverage, thus establishing a liver hemorrhage model. Figure 7 In the study, group a was the control group and group b was the experimental group. Applying hydrogel-3 to the wound showed faster and more significant hemostasis compared to the untreated group, with bleeding amounts of 0.2642g and 0.0773g respectively, representing a reduction of over 70%. The hydrogel swells and forms a sealant, transforming from a sol to a gel in 6-10 seconds, immediately reducing bleeding and allowing for direct observation of complete wound hemostasis.

Claims

1. A photosensitive and thermosensitive hybrid rapid hemostatic hydrogel composition, characterized in that, The hydrogel composition comprises the following components by mass fraction: Poly(N-isopropylacrylamide) grafted modified gelatin: 14~16%, Poly(N-isopropylacrylamide) modified hyaluronic acid: 0.5-1.5%, Quaternized gelatin: 2.5-3.5%, Photoresponsive molecularly modified hyaluronic acid: 0.1-0.8%, photoinitiator: 0.05-1%; the balance is water or phosphate buffer solution; the quaternized gelatin is biepoxide quaternary ammonium salt modified gelatin, and the photoresponsive molecule in the photoresponsive molecule modified hyaluronic acid is N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy)butyramide.

2. The hydrogel composition according to claim 1, characterized in that it comprises the following components in mass fractions: Poly(N-isopropylacrylamide) grafted modified gelatin: 14.5-15.5%; Poly(N-isopropylacrylamide) modified hyaluronic acid: 0.8-1.2%; Quaternized gelatin: 2.8-3.2%; Photoresponsive molecularly modified hyaluronic acid: 0.2-0.6%; Photoinitiator: 0.05-0.15%; Balance is water or phosphate buffer solution.

3. The hydrogel composition according to claim 1, characterized in that, The mass ratio of poly-N-isopropylacrylamide to gelatin in the poly-N-isopropylacrylamide grafted modified gelatin is (1-50):

1.

4. The hydrogel composition according to claim 3, characterized in that, The mass ratio of poly-N-isopropylacrylamide to gelatin in the poly-N-isopropylacrylamide grafted modified gelatin is (3-18):

1.

5. The hydrogel composition according to claim 4, characterized in that, The mass ratio of poly-N-isopropylacrylamide to gelatin in the poly-N-isopropylacrylamide grafted modified gelatin is (10-15):

1.

6. The hydrogel composition according to claim 1, characterized in that, The mass ratio of poly-N-isopropylacrylamide to hyaluronic acid in the poly-N-isopropylacrylamide modified hyaluronic acid is (0.5-10):

1.

7. The hydrogel composition according to claim 6, characterized in that, The mass ratio of poly-N-isopropylacrylamide to hyaluronic acid in the poly-N-isopropylacrylamide modified hyaluronic acid is (1-1.5):

1.

8. The hydrogel composition according to claim 1, characterized in that, The bisepoxy quaternary ammonium salt modified gelatin is prepared by reacting gelatin with bisepoxy quaternary ammonium salt, wherein the molar ratio of the primary amino group of the gelatin reactant to the epoxy group of the bisepoxy quaternary ammonium salt in the bisepoxy quaternary ammonium salt modified gelatin is 1:(1-10).

9. The hydrogel composition according to claim 8, characterized in that, In the biepoxide quaternary ammonium salt modified gelatin, the molar ratio of the primary amino group of the reactant gelatin to the epoxy group of the biepoxide quaternary ammonium salt is 1:(2-3).

10. The hydrogel composition according to claim 1, characterized in that, The molar ratio of photoresponsive molecules to hyaluronic acid in the photoresponsive molecule-modified hyaluronic acid is (100-500):

1.

11. The hydrogel composition according to claim 10, characterized in that, The molar ratio of photoresponsive molecules to hyaluronic acid in the photoresponsive molecule-modified hyaluronic acid is (135-200):

1.

12. A photosensitizing and thermosensitive hybrid rapid hemostatic hydrogel, characterized in that, The hydrogel composition of claim 1 or 2 is irradiated with ultraviolet light at 20-25°C and then placed at 30-40°C to obtain a hydrogel.

13. The hydrogel according to claim 12, characterized in that, The ultraviolet light irradiation time is 2-8 minutes, and the ultraviolet light energy density is 5-40 mw / cm2.

14. The hydrogel according to claim 12, characterized in that, The hydrogel has a swelling rate of 780-790%, a fracture stress of 0.11-0.125 MPa, a storage modulus of 5000-6500 Pa, and a rupture pressure of 170-180 mmHg.

15. Use of the hydrogel composition according to any one of claims 1 to 11 or the hydrogel according to any one of claims 12 to 14 in the preparation of hemostatic materials, wherein the hemostatic materials are applied to gastrointestinal or common skin traumatic bleeding.

Images