CN117752850A - Photosensitive and temperature-sensitive mixed antibacterial hydrogel - Google Patents

Abstract

The invention belongs to the technical field of biomedical polymer hydrogels, and particularly relates to a photosensitive and temperature-sensitive hybrid antibacterial hydrogel. The hydrogel component of the present invention includes poly-N-isopropylacrylamide graft-modified gelatin, poly-N-isopropylacrylamide-modified hyaluronic acid, quaternary aminated gelatin and light-responsive molecule-modified hyaluronic acid. Photoinitiator LAP. Because it contains modified gelatin and hyaluronic acid, the hydrogel can effectively improve the adhesion to the bleeding wound and effectively stop bleeding; the modified molecules used have photosensitive or temperature-sensitive environmental responsiveness, and can accelerate the process by applying specific conditions. The curing speed of the hemostatic hydrogel prevents the hydrogel components from being washed away by blood and can quickly stop the wound from bleeding.

Description

A kind of photosensitive and temperature-sensitive hybrid antibacterial hydrogel

This application is a divisional application based on the application number 202211167578.7 (the application date is 2022-09-23, the invention name is a photosensitive and temperature-sensitive hybrid high-strength hydrogel)

Technical field

The invention belongs to the technical field of biomedical polymer hydrogels, and specifically relates to a photosensitive and temperature-sensitive hybrid antibacterial hydrogel.

Background technique

Collagen is an important structural protein in animal connective tissue and plays an important role in maintaining the normal function and damage repair of cells, tissues, and organs. Collagen has excellent biological properties such as low antigenicity, good biocompatibility, degradability in the body, and sol-gel properties, making it widely used as tissue repair materials, hemostatic materials, drug sustained-release materials, bionic scaffolds, etc. in the research field.

Commonly used clinical hemostatic materials can be classified according to their hemostatic mechanisms. The first type is hemostatic gauze containing polymer polysaccharides, inorganic zeolites and other substances. During the hemostasis process, physical compression of the bleeding wound is required, and the hemostasis speed is slow. It is suitable for hemostasis of wounds with small wounds and low bleeding; Type 1 is a hemostatic material containing thrombin and coagulation factors. It is often used to stop bleeding in wounds such as surgery. However, it is prone to the problem of spillage of thrombin protein molecules into normal blood vessels, and there is a risk of thrombosis induced by coagulation; the third type is Alpha cyanoacrylate and other closed wound materials that have strong adhesion to tissues, such as band-aids, etc. These three types of hemostatic materials are difficult to solve common clinical problems in the wound healing process such as exudation, infection, and pain.

In response to the significant risk of mortality due to blood loss in emergency situations and hospital settings, new hemostatic methods and strategies are constantly being investigated. In recent years, Johnson & Johnson's Su product - injectable gel hemostatic products have entered clinical application and have received widespread attention. The material is mainly composed of gelatin. After being dissolved in water, it quickly forms a foamy gel that can be injected into the wound to stop bleeding, protect exposed peripheral nerves, promote wound healing, and inhibit scarring. This new method of hemostasis that forms gel in situ effectively alleviates the above clinical problems.

At present, injectable, light-responsive, and temperature-sensitive hemostatic gels have been reported. Synthetic polymer-type hemostatic gels have high mechanical strength, but they solidify immediately upon contact with water. They also have poor adhesion to wet tissue surfaces, lack of elasticity, or toxic degradation products, which limits their clinical application. The excellent biological properties of natural biopolymers make them more suitable for hemostatic gels. However, this type of hemostatic material generally has the disadvantages of poor mechanical strength and slow curing speed. It cannot cope with acute bleeding, large bleeding volume, and high vascular pressure in tissues. Bleeding.

Contents of the invention

The present invention aims to solve the problem that synthetic hemostatic gel has poor adhesion to wet tissue, lacks elasticity or has biological toxicity. Natural biopolymers have poor mechanical properties, slow curing speed and cannot cope with acute bleeding, large bleeding volume and high vascular pressure tissue bleeding. The technical problem is to provide a photosensitive and temperature-sensitive hybrid high-strength hydrogel.

In order to achieve the above objects, the present invention adopts the following technical solutions:

One object of the present invention is to provide a photosensitive and temperature-sensitive hybrid high-strength hydrogel composition, which is characterized in that the hydrogel composition contains the following components in mass fraction:

Poly-N-isopropylacrylamide graft-modified gelatin: 14-16%, poly-N-isopropylacrylamide-modified hyaluronic acid: 0.5-1.5%, quaternary aminated gelatin: 0.5-1.5%, light response Molecularly modified hyaluronic acid: 0.1-0.8%, photoinitiator: 0.05-1%; the balance is water or phosphate buffer solution.

Preferably, the hydrogel composition contains the following components in mass fraction:

Poly-N-isopropylacrylamide graft-modified gelatin (P-G): 14.5-15.5%; poly-N-isopropylacrylamide-modified hyaluronic acid (P-HA): 0.8-1.2%; quaternary aminated gelatin : 0.8-1.2%; light-responsive molecule 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 light-responsive molecule modified hyaluronic acid (HA-NB) is 0.3-0.5%.

The invention also provides a photosensitive and temperature-sensitive hybrid high-strength hydrogel. After the above hydrogel composition is irradiated with ultraviolet light at 20-25°C, it is placed in an environment of 30-40°C to obtain a hydrogel. .

The swelling rate of the obtained hydrogel is 800-900%, the fracture stress is 0.13-0.15MPa, the storage modulus is 30000-40000Pa, the adhesion capacity is 200-230kpa, and the rupture pressure is 250-300mmHg.

The hydrogel has strong adhesion and can withstand stronger burst pressure, and is suitable for hemostasis of irregularly shaped organs and arterial bleeding during surgery, or for hemostasis of rapidly bleeding wounds on the battlefield or trauma.

The present invention provides the use of the above-mentioned hydrogel composition or hydrogel as a quick-out hemostatic material for bleeding wounds of irregularly shaped organs, incompressible internal organs, and high-pressure arteries during surgery. After the above-mentioned hydrogel composition is irradiated with ultraviolet light, it is injected into the desired site, and the hemostatic effect can be achieved in 6 to 10 seconds.

Another object of the present invention is to provide a photosensitive and temperature-sensitive hybrid rapid hemostatic hydrogel composition, which is characterized in that the hydrogel composition contains the following components in mass fraction:

Poly-N-isopropylacrylamide graft-modified gelatin: 14-16%; poly-N-isopropylacrylamide-modified hyaluronic acid: 0.5-1.5%; quaternary aminated gelatin: 2.5-3.5%; light response Molecularly modified hyaluronic acid: 0.1-0.8%; photoinitiator: 0.05-1%; the balance is water or phosphate buffer solution.

Preferably, the hydrogel composition contains the following components in mass fraction:

Poly-N-isopropylacrylamide graft-modified gelatin (P-G): 14.5-15.5%; poly-N-isopropylacrylamide-modified hyaluronic acid (P-HA): 0.8-1.2%; quaternary aminated gelatin : 2.8-3.2%; light-responsive molecule modified hyaluronic acid (HA-NB): 0.2-0.6%, photoinitiator: 0.05-0.15%; the balance is water or phosphate buffer solution. Preferably, the HA-NB content is 0.35 to 0.45%.

The present invention also provides a photosensitive and temperature-sensitive hybrid rapid hemostatic hydrogel. After the above hydrogel composition is irradiated with ultraviolet light at 20-25°C, the hydrogel is obtained by placing it in an environment of 30-40°C. . The swelling rate of the obtained hydrogel is 780-790%, the rupture stress is 0.11-0.125MPa, the storage modulus is 5000-6500Pa, and the rupture pressure is 170-180mmHg.

The present invention provides the use of the above-mentioned hydrogel as a hemostatic material for traumatic bleeding of the digestive tract and ordinary skin.

Another object of the present invention is to provide a photosensitive and temperature-sensitive hybrid antibacterial hydrogel composition, which is characterized in that the hydrogel contains the following components in mass fraction:

Poly-N-isopropylacrylamide graft-modified gelatin: 14-16%; poly-N-isopropylacrylamide-modified hyaluronic acid: 0.5-1.5%; quaternary aminated gelatin: 4.5-5.5%; light response Molecularly modified hyaluronic acid: 0.1-0.8%; photoinitiator: 0.05-1%; the balance is water or phosphate buffer solution.

Preferably, the poly-N-isopropylacrylamide graft-modified gelatin (P-G) in the hydrogel composition: 14.5-15.5%; poly-N-isopropylacrylamide-modified hyaluronic acid (P-HA) ): 0.8~1.2%; quaternary aminated gelatin: 4.8~5.2%; 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, the HA-NB content is 0.35 to 0.45%.

The invention also provides a photosensitive and temperature-sensitive mixed antibacterial hydrogel. After the above hydrogel composition is irradiated with ultraviolet light at 20-25°C, it is placed in an environment of 30-40°C to obtain a hydrogel. The swelling rate of the obtained hydrogel is 550-600%, the rupture stress is 0.16-0.2MPa, the storage modulus is 3000-3500Pa, and the rupture pressure is 150-160mmHg. The diameter of the inhibition zone of the hydrogel against E. coli is 15 to 16 mm. The diameter of the inhibition zone against Staphylococcus aureus is 10 to 10.5 mm.

The present invention provides the use of the above-mentioned hydrogel as a hemostatic material for easily infected wounds or chronic wounds.

Preferably, the ultraviolet light irradiation time is 2 to 8 minutes, and the ultraviolet light energy density is 5 to 40 mw/cm ² .

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

Preferably, the mass ratio of polyN-isopropylacrylamide (PNIPAM) and gelatin in the polyN-isopropylacrylamide graft-modified gelatin (P-G) is (1-50):1; further preferably (3-18): 1, more preferably 10-15:1.

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.

The quaternized gelatin is preferably diepoxy quaternary ammonium salt modified gelatin (D-G). The D-G is prepared by reacting gelatin and diepoxy quaternary ammonium salt (DEQAS). The molar ratio of the primary amino group in the gelatin to the epoxy group in DEQAS is 1:(1-10); further preferably, it is 1:( 2-3).

Preferably, the photoresponsive molecule in the photoresponsive molecule modified hyaluronic acid (HA-NB) is N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy -5-Nitrosophenoxy)butanamide. Preferably, the ratio of the amount of light-responsive molecules to hyaluronic acid in the HA-NB is (100-500):1; further preferably, the amount of light-responsive molecules to HA in the HA-NB is The ratio is (135-200):1.

One or more technical solutions provided by embodiments of the present invention have at least the following technical effects:

(1) The components of the present invention contain modified natural polymer materials, which can effectively improve the adhesion between the hydrogel and the bleeding wound and effectively stop bleeding; the present invention chemically modifies the natural polymer materials, and uses modified The chemical molecules have photosensitive or temperature-sensitive environmental responsiveness. By applying specific conditions, they can accelerate the curing speed of the hemostatic hydrogel, prevent the hydrogel components from being washed away by blood, and quickly stop the wound from bleeding.

(2) Several hydrogel components of the present invention are chemically cross-linked under ultraviolet light. The hydroxyl groups in HA-NB are oxidized into aldehyde groups, and then cross-linked with the primary amino groups of gelatin to form Schiff base covalent bonds. , improves the strength of the gel and can withstand blood pressure up to 280-320 mmHg; it is suitable as a quick-out hemostatic material for bleeding wounds of irregular-shaped organs, incompressible internal organs, and high-pressure arteries during surgery.

(3) The photosensitive and temperature-sensitive hybrid antibacterial hydrogel has high antibacterial properties and is suitable as a hemostatic material for easily infected wounds or chronic wounds.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the SEM image of hydrogel, (a) P-G hydrogel, (b) P-G/P-HA hydrogel, (c) Hydrogel-3, (d) Hydrogel-6, (e )Hydrogel-9;

Figure 2 is the hydrogel compressive stress-strain curve;

Figure 3 is a scanning test chart of hydrogel modulus;

Figure 4 shows the results of the hydrogel antibacterial test. a is Escherichia coli and b is Staphylococcus aureus;

Figure 5 shows the cytotoxicity test results;

Figure 6 is a picture of the healing condition of the mouse back;

Figure 7 is a picture of mouse liver hemostasis.

Detailed ways

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

It should be noted that the experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents, materials and equipment described can be obtained from commercial sources unless otherwise specified.

The hyaluronic acid mentioned in the present invention was purchased from Shanghai McLean Biochemical Technology Co., Ltd., and the gelatin and other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

The P-G is prepared by the method recorded in the following 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.

The E-G is prepared by the method recorded in the following prior art: Shilin Xu, et al, Amultifunctional gelatine-quaternary ammonium copolymer: An efficient material for reducing dye emission in leather tanning process by superior anionic dyeadsorption, Journal of Hazardous Materials 383 (2020) 121142.

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

The HA-NB is prepared by the method recorded in the following prior art: Yi Hong, et al. A stronglyadhesive hemostatic hydrogel for the repair of arterial and heartbleeds. Nature communications, 2019May 14; 10(1): 2060.

Among them, the mass ratio of poly-N-isopropylacrylamide (PNIPAM) and gelatin in P-G is (12:1);

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

The ratio of the amount of gelatin primary amino groups in E-G to the epoxy groups in EPTAC is (1:2.5);

The photoresponsive molecule of HA-NB is N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy)butanamide.

The ratio of the amount of light-responsive molecules to hyaluronic acid (HA) in HA-NB is (168:1).

Example 1

A method for preparing a photosensitive and temperature-sensitive hybrid high-strength hydrogel. The above-prepared PG, P-HA, DG, LAP and HA-NB and phosphate buffer solution are mixed at room temperature below 30°C to obtain The final concentration is 15% PG, 1% P-HA, 1% DG, 0.2% HA-NB and 0.1% LAP, and the balance is water in phosphate buffer solution (PBS buffer solution, 0.01M, pH 7.2~7.4) Gel composition. Then, it was irradiated with ultraviolet light with an energy density of 5mW/cm ² (365nm) for 5 minutes, placed in an environment of 30 to 40°C, and was recorded as hydrogel-2.

Examples 2 and 3

According to the preparation method of Example 1, the final concentrations of HA-NB were changed to 0.4 and 0.6% respectively, and the obtained hydrogels were designated as Hydrogel-3 and Hydrogel-4.

Example 4

A method for preparing a photosensitive and temperature-sensitive hybrid high-strength hydrogel. The prepared PG, P-HA, DG, LAP, HA-NB and phosphate buffer solution are mixed at room temperature to obtain a final concentration of 15% PG. , 1% P-HA, 3% DG, 0.2% HA-NB and 0.1% LAP, and the balance is a hydrogel composition of phosphate buffer solution (PBS buffer solution, 0.01M, pH 7.2 to 7.4). Then, it was irradiated with ultraviolet light with an energy density of 5mW/cm ² (365nm) for 5 minutes, which was recorded as hydrogel-5.

Examples 5 and 6

According to the preparation method of Example 4, the final concentrations of HA-NB were changed to 0.4 and 0.6% respectively, and the obtained hydrogels were designated as Hydrogel-6 and Hydrogel-7.

Example 7

A method for preparing a photosensitive and temperature-sensitive hybrid high-strength hydrogel. The prepared PG, P-HA, DG, LAP, HA-NB and phosphate buffer solution are mixed at room temperature to obtain a final concentration of 15% PG. , 1% P-HA, 5% DG, 0.2% HA-NB and 0.1% LAP, and the balance is a hydrogel composition of phosphate buffer solution (PBS buffer solution, 0.01M, pH 7.2 to 7.4). Then, it was irradiated with ultraviolet light with an energy density of 5mW/cm ² (365nm) for 5 minutes, and was recorded as hydrogel-8.

Examples 8 and 9

According to the preparation method of Example 7, the final concentrations of HA-NB were changed to 0.4 and 0.6% respectively, and the obtained hydrogels were recorded as Hydrogel-9 and Hydrogel-10.

Comparative example 1

A method for preparing hydrogels. The prepared P-G, P-HA and phosphate buffer solutions are mixed at room temperature below 30°C to obtain a final concentration of 15% P-G, 1% P-HA, and the balance is phosphoric acid. The hydrogel composition of salt buffer solution was placed in an environment of 30 to 40°C and was recorded as hydrogel-1.

Comparative example 2

A method for preparing a hydrogel. The difference from Example 1 is that monoepoxy quaternary ammonium salt (E-G) modified gelatin is used to replace D-G in Example 1. Other conditions are the same as Example 1, recorded as water. Gel-11.

The chemical formula of monoepoxy quaternary ammonium salt is:

Comparative example 3

A method for preparing a hydrogel is different from Example 4 in that monoepoxy quaternary ammonium salt (E-G) modified gelatin is used to replace D-G in Example 4, and other conditions are the same as Example 4. Marked as hydrogel-12.

Comparative example 4

A method for preparing a hydrogel. The difference from Example 7 is that monoepoxy quaternary ammonium salt (E-G) modified gelatin is used to replace D-G in Example 7. Other conditions are the same as Example 7, recorded as water. Gel-13.

(1) Microstructure of hydrogel

After the hydrogel was freeze-dried, the specimen was cut into thin slices with a scalpel and its surface was spray-coated with gold. The cross-sectional microstructure was observed using a scanning electron microscope.

The microstructure of hydrogel has a great influence on its water retention and mechanical properties. Figure 1 shows the micromorphology of freeze-dried hydrogel. Compared with P-G and P-G/P-HA single network hydrogels, the double network hydrogel of the present invention has a denser microstructure and smaller pore size. Furthermore, SEM images revealed that the hydrogel had an interconnected porous structure. The dense structure can improve the mechanical properties of hydrogels. At the same time, the interconnected porous structure enables efficient transfer of nutrients and oxygen, increasing the application potential of hydrogels in wound dressings.

(2) Swelling rate

The prepared hydrogel was freeze-dried, weighed (Wd), soaked in phosphate buffered saline (PBS) solution (pH=7.4) for 24 hours, and after reaching swelling equilibrium, the surface water was wiped and the specimen was taken out and weighed (Ws). , the swelling ratio (Q ₀ ) is calculated as:

Q ₀ (%)=(Ws-Wd)/W×100%.

The swelling ratio of the hydrogel is shown in Table 1. Hydrogels can absorb large amounts of blood more quickly and absorb adjacent tissue fluid, preventing it from accumulating in the wound and causing inflammation.

(3)Mechanical properties

Breaking stress:

A general mechanical testing machine was used to evaluate the compressive properties of the hydrogel. Cylindrical hydrogels with a diameter (10 mm × 4 mm) were prepared and compressed at a strain rate of 3 mm/min until rupture, and then the stress-strain curve was obtained.

During the wound healing process, hydrogel dressings will inevitably be affected by external forces. In order to prevent secondary damage to wound tissue caused by external forces, hydrogel dressings are required to have appropriate mechanical properties. The compressive stress-strain curve of the prepared hydrogel is shown in Figure 2, and the results are shown in Table 1. Compared with single network hydrogels, the hydrogels of the present invention all have higher fracture stress, reaching 0.14MPa. Compression tests show that the double network hydrogel has better mechanical properties, which may be due to the introduction of the double network structure increasing the cross-linking density.

Table 1:

Storage modulus:

Storage modulus and loss modulus: A rotational rheometer was used to study the sol-gel transition of the hydrogel composition. The heating rate was set to 2°C/min. Before the test, the temperature was set to 25°C on the sample stage to prevent condensation. For gelling, strain % was set to 1% and frequency to 1Hz.

The storage modulus represents the material's ability to store elastic deformation energy. The greater the storage modulus, the harder the material is and is difficult to deform. The loss modulus represents the energy lost when a material is subjected to irreversible deformation and represents the viscosity. When the loss modulus < storage modulus, the material is a gel, and elasticity is the main characteristic; when the loss modulus > storage modulus, the material is a fluid, and viscosity is the main characteristic; loss modulus = storage modulus, the material At the sol-gel transition point, viscosity and elasticity are equal. In the scan, as the temperature increases, around 31°C we see that the storage modulus gradually becomes larger than the loss modulus, transitioning from sol to gel. The results are shown in Figure 3, and the storage modulus is shown in Table 1. The results show that the hydrogel is in a stable elastic state and is suitable for wound healing applications. Among them, Hydrogel-3 has the highest storage modulus of 35127 Pa, and is suitable as a quick-release hemostatic material for irregular-shaped organs, incompressible internal organs, and high-pressure arterial bleeding wounds during surgery.

The storage modulus of Hydrogel-6 is 6142Pa, which is suitable for hemostasis that requires less strong hemostatic pressure and traumatic bleeding of the digestive tract and ordinary skin. The storage modulus of Hydrogel-9 is 3264Pa. It relies on its strong antibacterial effect and is suitable for hemostasis of easily infected wounds, ordinary wounds or chronic wounds.

(4) In vitro adhesion, bursting pressure, antibacterial properties and cytotoxicity of hydrogels

Adhesion ability: Take two pieces of pigskin of the same size (2cm×5cm), irradiate the hydrogel composition with ultraviolet light, and apply the hydrogel composition after irradiation with ultraviolet light on one piece of pigskin (application area 2cm ×2cm, the amount of application is 1.5mL), glue the two pieces of pigskin together. A universal testing machine was used to apply unidirectional tension, and the loading rate was kept constant at 2 mm/min.

Hydrogel dressings with adhesive ability usually have good application prospects in the field of wound repair. Hydrogel dressings with an adhesive ability greater than 30kpa can generally meet the application in the field of wound repair. However, for irregularly shaped organs, incompressible viscera, For high-pressure arteries, etc., the greater the adhesion capacity, the better; for other parts, lower adhesion performance can also meet the usage requirements.

Among them, Hydrogel-3 is much higher than other samples at 215kpa, Hydrogel-6 has a viscosity capacity of 67kpa, and Hydrogel-9 has an adhesion capacity of 66Kpa, all of which can meet the requirements for adhesion performance. These results indicate that under a certain hydrogel composition, the formation of Schiff base covalent bonds improves the adhesion of hydrogels. This is because the HA-NB component in the hydrogel produces aldehyde groups after illumination, which increases the number of binding sites and strengthens the binding of HA-NB to tissue proteins. The reason why high adhesion strength can be achieved is that the grafted gelatin molecular chain contains a large number of carboxyl groups and amino groups, which can interact with skin tissue in the form of hydrogen bonds. This material has broad application prospects in the adhesion of healing wound tissue. It can be seen that the hydrogel has good adhesion ability and can form a long-term physical barrier on the wound site to promote wound repair.

Burst pressure: Clean and remove excess fat from pigskin (4×4 cm), cover the mouth of the bottle with pigskin and seal it. The bottle is connected to a pressure pump, which can pressurize the bottle with gas. Cut a circular hole with a diameter of 2 mm in the pig skin, fill the incision with 500 μL of the hydrogel composition irradiated by ultraviolet light, and then place it in an environment of 30 to 40°C to form hydrogel in situ at the puncture site. glue. The thickness of the hydrogel is about 4.4mm, and the explosion pressure is measured after gel formation. Gas is introduced into the bottle, and the peak pressure before pressure loss is considered the burst pressure. All measurements were repeated three times. As a result, the rupture pressure of hydrogel-3 reached a maximum of 280mmHg. Much higher than other groups of hydrogels and is a promising hemostatic sealant.

Antibacterial properties: The antibacterial properties of the hydrogels were tested against Staphylococcus aureus and Escherichia coli by the zone of inhibition method. The bacterial solution (1×10 ⁶ CFU/mL) was inoculated on the agar plate, and 10 μL of the UV-irradiated hydrogel composition was injected onto a filter paper piece with a diameter of 6 mm. The filter paper piece was placed on the agar plate and incubated at 37 Incubate at ℃ for 24 hours. The diameter of the antimicrobial area around each sample was measured to evaluate the antimicrobial activity of the hydrogel.

The results are shown in Figure 4. a and b are the antibacterial results of Escherichia coli and Staphylococcus aureus respectively. In the figure, A, B, C, and D are hydrogel-1, hydrogel-3, hydrogel-6, and hydrogel-9, respectively. For E. coli, the diameter of the inhibition zone of hydrogel-1 is 0, the diameter of the inhibition zone of hydrogel-3 is 8.3mm, the diameter of the inhibition zone of hydrogel-6 is 12.3mm, and the diameter of the inhibition zone of hydrogel-9 is 12.3mm. The diameter of the inhibition zone is 15.5mm. For Staphylococcus aureus, the diameter of the inhibition zone of Hydrogel-1 is 0, the diameter of the inhibition zone of Hydrogel-3 is 9.1mm, the diameter of the inhibition zone of Hydrogel-6 is 9.9mm, and the diameter of the inhibition zone of Hydrogel-6 is 9.9mm. The diameter of the inhibition zone of -9 is 10.4mm.

Hydrogel wound dressings can act as a barrier to isolate the wound from external bacterial infection. The hydrogel of the present invention shows obvious inhibitory effect on Escherichia coli and Staphylococcus aureus at 24 hours. The hydrogel also shows moderate antibacterial activity against Staphylococcus aureus and Escherichia coli, which will more effectively promote wound healing. . The antibacterial effect against Escherichia coli and Staphylococcus aureus can be attributed to the long-chain alkanes being compatible with the bacterial outer cell wall. The positively charged quaternary ammonium can attract the negatively charged bacterial cell membrane, destroy the cell membrane, and cause the cytoplasm to leak, thereby killing the bacteria. .

Cytotoxicity: To test the cytotoxicity of hydrogels, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrabromide (MTT) (Sigma, USA) method was used Perform cell viability assay. Fibroblast HFF-1 was seeded in a 96-well plate (100 μL/well containing a suspension of 1.0×105 cells/mL), incubated for 18 hours, and then treated with C hydrogel for 24 hours. After treatment, 10 μL of 5 mg/mL MTT was added to each well, and the 96-well plate was incubated at 37°C for 4 h. Then the cells in each well were washed with phosphate-buffered saline (PBS) and dissolved with 100 μL of dimethyl sulfoxide (DMSO). The absorbance of the sample was measured using a microplate reader.

Through the MTT method, Figure 5 shows that the cell survival rate is above 80%, indicating that the hydrogel has good biocompatibility and low cytotoxicity.

(5) In vivo wound repair ability of hydrogel and hemostasis of mouse liver

A mouse dorsal full-thickness incision model was used to study the effect of hydrogel on wound healing. Methods: Male ICR mice (18-20g) were anesthetized, shaved on the back during surgery, and disinfected with 75% ethanol cotton balls. A circular full-thickness skin defect with a diameter of 7 mm and a depth of 1 mm was made on the dorsal side of the mouse. The hydrogel precursor solution is injected into the skin defect and gelled by UV treatment. Skin defects treated with normal saline were used as controls. At selected time points, take photos of the wound surface to observe the healing status.

Hydrogel-3 was used to conduct full-thickness skin defect repair experiments on mice. As shown in Figure 6, the wound area in each group decreased with time. On the third day, the wounds in each group were dry with no obvious redness or swelling. On the 5th and 7th day, the wounds treated with hydrogel healed significantly compared with normal saline (NS). The wound was basically healed on the 11th day after hydrogel treatment, which was significantly better than that of other groups. After 13 days of treatment, the wound in the NS group had not yet completely healed and was covered with scabs. In the hydrogel group, no obvious scars or bumps were seen, and the skin color was similar to that of adjacent normal tissue. Extensive new hair growth was observed at the wound healing site. Therefore, hydrogels are more effective in promoting wound healing. This is because the hydrogel formed in situ can fit the wound well and adhere closely to the wound site, thus avoiding microbial infection. At the same time, the antibacterial properties of diepoxy quaternary ammonium salts give the hydrogel excellent antibacterial capabilities. On the other hand, the water retention ability of hyaluronic acid preserves the moist environment required for wound healing and accelerates the wound healing process.

To continue to demonstrate the potential of hydrogels as clinical hemostatic materials, they were used to repair bleeding in the liver and heart. A rat liver model (a specific internal organ with a rich blood supply) was used to evaluate the hemostatic performance of the hydrogel. Liver puncture was performed on mice to observe the amount of bleeding under different conditions. In the same way as above, a control group was set up to observe the amount of bleeding. It was observed that when a quick puncture was made on the liver, blood poured out of the needle hole. In this liver puncture model, if no treatment is performed or covered with gauze, blood is still flowing, establishing a liver bleeding model. In Figure 7, a is the control group and b is the experimental group. Hydrogel-3 was applied to the wound surface. Compared with the no treatment group, the bleeding was stopped quickly and the effect was obvious. The bleeding volume was 0.2642g and 0.0773g respectively, and the bleeding volume was reduced by 70%. above. The hydrogel swells and forms a sealant, and changes from sol to gel in 6-10 seconds, immediately reducing bleeding, and complete hemostasis of the wound can be visually observed.

Claims (10)

1. A photosensitive and temperature-sensitive hybrid antibacterial hydrogel composition, characterized in that the hydrogel composition contains the following components in mass fraction: Poly-N-isopropylacrylamide graft-modified gelatin: 14-16%, poly-N-isopropylacrylamide-modified hyaluronic acid: 0.5-1.5%, quaternary aminated gelatin: 4.5-5.5%, light response Molecularly modified hyaluronic acid: 0.1-0.8%, photoinitiator: 0.05-1%; the balance is water or phosphate buffer solution; the quaternized gelatin is diepoxy quaternary ammonium salt modified gelatin, the The photoresponsive molecule in the photoresponsive molecule modified hyaluronic acid is N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy) butylamide. 2. The hydrogel composition according to claim 1, characterized in that it contains the following components in mass fraction: Poly-N-isopropylacrylamide graft-modified gelatin: 14.5-15.5%; poly-N-isopropylacrylamide-modified hyaluronic acid: 0.8-1.2%; quaternary aminated gelatin: 4.8-5.2%; light response Molecularly modified hyaluronic acid: 0.2-0.6%, photoinitiator: 0.05-0.15%; the balance is water or phosphate buffer solution. 3. The hydrogel composition according to claim 1, wherein the mass ratio of polyN-isopropylacrylamide to gelatin in the polyN-isopropylacrylamide graft-modified gelatin is ( 1-50):1; preferably (3-18):1, more preferably 10-15:1. 4. 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; preferably (1-1.5):1. 5. The hydrogel composition according to claim 1, wherein the diepoxy quaternary ammonium salt modified gelatin is prepared by reacting gelatin and diepoxy quaternary ammonium salt, and the diepoxy quaternary ammonium salt is In the salt-modified gelatin, the molar ratio of the primary amino group of the reactant gelatin and the epoxy group in the diepoxy quaternary ammonium salt is 1: (1-10); more preferably, it is 1: (2-3). 6. The hydrogel composition according to claim 1, wherein the ratio of the amount of light-responsive molecules to hyaluronic acid in the light-responsive molecule-modified hyaluronic acid is (100-500): 1; Preferably, the ratio of the amount of light-responsive molecules to hyaluronic acid in the light-responsive molecule-modified hyaluronic acid is (135-200):1. 7. A photosensitive and temperature-sensitive hybrid antibacterial hydrogel, characterized in that, after the hydrogel composition according to claim 1 or 2 is irradiated with ultraviolet light at 20-25°C, it is placed at 30-40°C. Hydrogel was obtained under ℃ environment. 8. The hydrogel according to claim 7, wherein the ultraviolet light irradiation time is 2 to 8 minutes, and the ultraviolet light energy density is 5 to 40 mw/cm ² . 9. The hydrogel according to claim 7, characterized in that the hydrogel has a swelling rate of 550-600%, a fracture stress of 0.16-0.2MPa, a storage modulus of 3000-3500Pa, and a rupture pressure of 150-160mmHg. . 10. The use of the hydrogel composition according to any one of claims 1 to 6 or the hydrogel according to any one of claims 7 to 9 in the preparation of hemostatic materials, which are applied to wounds susceptible to infection, or It's a chronic wound.