An adherable hydrogel material and methods of making and using the same

Abstract

The application belongs to the field of biochemistry, and particularly relates to an adherable hydrogel material and a preparation method and use thereof. The application provides an adherable hydrogel material, wherein silver nanoparticles are prepared by a GA functionalized PLG mineralization reduction method, and then the silver nanoparticles are incorporated into a hydrogel network to obtain the adherable hydrogel material. The adherable hydrogel material provided by the application has excellent adhesion, and can be simply de-adhered after being treated by glutathione or sodium bicarbonate solution. The hydrogel material provided by the application also has antibacterial property, cell compatibility and low hemolysis rate, and can be used as a wound dressing to promote tissue repair.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biochemistry, and particularly relates to an adherable hydrogel material and a preparation method and use thereof. BACKGROUND

[0002] Skin is the largest organ of the human body (about 15% of the body mass) and the first line of defense, playing an important role in maintaining the internal environment and protecting internal organs from external environmental interference. However, due to its soft elasticity, once damaged and wounded (referred to as a wound), the protective function of the skin tissue against the invasion of harmful substances and microorganisms is destroyed, seriously endangering the health condition and even the life safety of human beings.

[0003] Traditional wound dressings, including non-degradable gauze, cotton wool, polyurethane foam and bandages, are difficult to adapt to the wound and maintain a moist environment. Worse still, the dressing needs to be updated constantly during the healing process, which will inevitably cause secondary damage to the wound. Recently, hydrogels with three-dimensional porous structures have shown comparable flexibility to the extracellular matrix and have become potential wound dressings. In order to promote the healing of chronic wounds, researchers have developed a series of hydrogels with multifunctional properties, including adhesion, injectability, antibacterial, antioxidant and pro-angiogenic properties. Benefiting from the rapid development of nanotechnology, various nanomaterials, including gold nanoparticles, silver nanoparticles and molybdenum disulfide nanosheets, have been used as antibacterial additives. Among them, silver nanoparticles prepared by a biomimetic method have attracted much attention due to their excellent antibacterial, biocompatibility, biodegradable and antioxidant properties.

[0004] Gallic acid (GA) is a polyphenol widely present in various plants and fruits. The o-phenol group is rich in GA, which has been found to be a mineralizer for the reduction preparation of silver nanoparticles, improving the dense crosslinking points of the system and excellent antibacterial properties.

[0005] In the prior art, a variety of hydrogels have been used as wound dressings (e.g., chitosan, gelatin, etc.), but generally have weak adhesion and lack antibacterial and hemostatic properties. The existing dressings are difficult to separate from the wound when they need to be replaced, which may damage the regenerating new skin tissue, leading to bleeding and new infection. In addition, some residual dressings may hinder wound repair. SUMMARY

[0006] In order to solve the problems of the lack of antibacterial and hemostatic properties of the existing hydrogel materials and the difficulty in separating from the wound, the present application provides an adherable hydrogel material. The present application first prepares silver nanoparticles by a GA (gallic acid) functionalized PL (polylysine) mineralization reduction method; then, the silver nanoparticles are incorporated into the hydrogel network to prepare the adherable hydrogel material.

[0007] The preparation method of the above-mentioned adhesive hydrogel material is as follows:

[0008] a. Preparation of silver nanoparticles (Ag-PLG): a solution of GA-functionalized polylysine (PLG) is prepared by EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and NHS (N-hydroxysuccinimide) reaction, and then freeze-dried to obtain GA-functionalized PLG powder; then silver nitrate and GA-functionalized PLG powder are mixed in water, the pH value is adjusted to 10-12, and after heating and stirring for 30 min, the solution is cooled to 0-5℃ to obtain Ag-PLG solution;

[0009] b. Preparation of hydrogel material: AA (acrylic acid), AANHS (acrylic acid-N-succinimidyl ester), OHA (aldehyde-hydroxylated hyaluronic acid), Ag-PLG solution, BAC (N,N'-bis(acryloyl)cystamine) are added to PBS solution (phosphate buffered saline) and stirred to dissolve, and then a photoinitiator LAP (phenyl-2,4,6-trimethylbenzoyl lithium phosphite) is added, and an adhesive hydrogel material (PAN / Ag-PLG) is obtained under 405 nm blue light irradiation for 5 min.

[0010] In the above-mentioned preparation method of the adhesive hydrogel material, the specific preparation steps of the GA-functionalized PLG powder in step a are as follows: PLG and GA are dissolved in MES (morpholine ethanesulfonic acid) buffer, and an EDC / NHS mixture is added dropwise to the above-mentioned solution, and the reaction is carried out at room temperature under nitrogen protection; after 8-16 h of reaction, the mixture solution is dialyzed in ultrapure water for 3 days, and then freeze-dried. The MES buffer is 0.05M. The molar ratio of the EDC / NHS mixture is EDC:NHS=3:1. The EDC / NHS mixture accounts for about 20%-25% of the total volume of the reaction solution. The molar ratio of the hydroxyl group in the NHS to the carboxyl group in the GA is 1:1.

[0011] In the above-mentioned preparation method of the adhesive hydrogel material, the mass content ratio of silver nitrate to PLG in step a is 1:10.

[0012] In the above-mentioned preparation method of the adhesive hydrogel material, the heating temperature in step a is 50-80℃.

[0013] In the above-mentioned preparation method of the adhesive hydrogel material, the mass content ratio of AANHS to AA in step b is 10-30wt%; the mass content ratio of OHA to AA is 5-10wt%; the mass content ratio of BAC to AA is 4-6wt%; and the mass content ratio of LAP to AA is 3-6wt%.

[0014] The application also provides use of the above-mentioned adherable hydrogel material in preparation of a wound dressing.

[0015] The hydrogel material provided by the application has excellent adhesion and can be simply de-adhered after treatment by glutathione (GSH) or sodium bicarbonate (SBC) solution. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Synthetic route of gallic acid functionalized polylysine.

[0017] Figure 2 Transmission electron microscope images and particle size distribution of Ag-PLG, (a) is a transmission electron microscope image of Ag-PLG nanoparticles, (b) is a particle size distribution diagram of Ag-PLG.

[0018] Figure 3 The hydrogel material provided by the application can adhere to biological tissues and engineering materials of different volumes.

[0019] Figure 4 Adhesion strength of the hydrogel material provided by the application, (a) is a real object diagram of shear adhesion test of the hydrogel to pig skin, (b) is shear adhesion strength of the hydrogel after different treatments.

[0020] Figure 5 Antibacterial performance of the hydrogel material provided by the application, (a) is antibacterial performance of different treatment groups to methicillin-resistant Staphylococcus aureus, (b) is survival rate of methicillin-resistant Staphylococcus aureus of different treatment groups, (c) is SEM of methicillin-resistant Staphylococcus aureus of different treatment groups, (d) is antibacterial performance of different treatment groups to Escherichia coli, (e) is SEM of Escherichia coli of different treatment groups, (f) is survival rate of Escherichia coli of different treatment groups.

[0021] Figure 6 Cell compatibility of the hydrogel material provided by the application, (a-c) are cell viability values of L929 cells at 24h, 48h and 72h respectively.

[0022] Figure 7 Hemostatic performance of the hydrogel material provided by the application, (a) is a real object diagram of bleeding of mouse liver of different treatment groups, (b) is amount of blood loss of mouse liver of different treatment groups, (c) is hemostatic time of bleeding of mouse liver of different treatment groups.

[0023] Figure 8The hydrogel material provided by the application has the ability to promote healing of mouse wounds. (a) Wound healing of mice in each group, (b) wound healing trajectory of each group, (c) wound closure rate of mice in each group. DETAILED DESCRIPTION

[0024] The preparation method of the adhesive hydrogel material includes the following specific operation steps:

[0025] a. Preparation of silver nanoparticles (Ag-PLG): PLG and GA are dissolved in MES (morpholine ethanesulfonic acid) buffer solution, and an EDC / NHS mixture is added dropwise to the above solution, and the reaction is carried out at room temperature under nitrogen protection; after 8-16 hours of reaction, the mixture solution is dialyzed in ultrapure water for 3 days, and then freeze-dried to obtain GA functionalized PLG powder; then silver nitrate and GA functionalized PLG powder are added to water and mixed, the pH value is adjusted to 10-12, and after heating and stirring for 30 min, it is cooled to 0-5℃ to obtain an Ag-PLG solution;

[0026] b. Preparation of hydrogel material: AA, AANHS, OHA, Ag-PLG solution, BAC are added to PBS solution and stirred to dissolve, then a photoinitiator LAP is added, and an adhesive hydrogel material (PAN / Ag-PLG) is obtained under 405 nm blue light irradiation for 5 min.

[0027] In the preparation method of the adhesive hydrogel material, the MES buffer solution in step a is 0.05M. The molar ratio of the EDC / NHS mixture is EDC:NHS=3:1. The EDC / NHS mixture is about 20%-25% of the total volume of the reaction solution. The molar ratio of hydroxyl in NHS to carboxyl in GA is 1:1.

[0028] In the preparation method of the adhesive hydrogel material, the mass content ratio of silver nitrate to PLG in step a is 1:10.

[0029] In the preparation method of the adhesive hydrogel material, the heating temperature in step a is 50-80℃.

[0030] In the preparation method of the adhesive hydrogel material, the mass content ratio of AANHS to AA in step b is 10-30wt%; the mass content ratio of OHA to AA is 5-10wt%; the mass content ratio of BAC to AA is 4-6wt%; and the mass content ratio of LAP to AA is 3-6wt%.

[0031] Methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli were purchased from Shanghai Luwei Technology Co., Ltd. Luria Bertani agar was prepared by the laboratory itself; fetal bovine serum was purchased from Cellgo Biological Products (Hong Kong) Trading Co., Ltd.; penicillin-streptomycin and DMEM cell culture medium were purchased from Jiangsu Kaikai Biotechnology Co., Ltd.; L929 fibroblasts were provided by the State Key Laboratory of Biotechnology, Sichuan University; Cell Counting Kit-8 cell counting reagent was purchased from Shanghai Taoxue Biotechnology Co., Ltd.; mice were purchased from Chengdu Dasuo Co., Ltd.

[0032] Example 1 Synthesis of hydrogel material

[0033] Preparation of Ag-PLG: 2 g of polylysine and 2 g of GA were dissolved in 200 mL of 0.05 M MES (morpholine ethanesulfonic acid) buffer, and EDC / NHS (molar ratio of EDC:NHS = 3:1, EDC / NHS mixed solution volume about 20%-25% of the total volume of the reaction solution) was added to the above solution, wherein the molar ratio of hydroxyl in NHS to carboxyl in GA was 1:1. The reaction was carried out at room temperature under nitrogen protection to remove oxygen. After 12 h of reaction, the mixture solution was dialyzed in ultrapure water for 3 days, and then freeze-dried to obtain Ag-PLG solution.

[0034] Preparation of hydrogel material: AA, AANHS, OHA, Ag-PLG solution, BAC were added to PBS solution (total volume 2 mL) and stirred to dissolve, then light initiator LAP was added, and hydrogel material (PAN / Ag-PLG) was formed under 405 nm blue light irradiation for 5 min.

[0035] Table 1 Preparation of different hydrogel materials

[0036]

[0037] Example 2 Transmission electron microscope photograph and particle size distribution of Ag-PLG

[0038] As Figure 2 The transmission electron microscope test of the prepared Ag-PLG is shown, and the Ag-PLG nanoparticles of dispersed single black particles are presented in the figure, and no agglomeration phenomenon occurs, indicating that it has good dispersibility. In addition, the particle size test of the obtained Ag-PLG is carried out, and the particle size distribution graph is an independent sharp peak, and the size is about 40 nm.

[0039] Example 3 Adhesion ability experiment of hydrogel

[0040] PAN / Ag 20-PLG hydrogel can adhere to biological tissues (heart, liver, spleen, lung) and engineering materials (polymethyl methacrylate, glass, copper sheet, weight). The picture shows PAN / Ag 20 -PLG hydrogel can adhere to different objects, indicating that it has good adhesion performance.

[0041] Rats were euthanized, and the heart, liver, spleen, and lung were immediately removed and rinsed with PBS, and then PAN / Ag 20 -PLG hydrogel material adhered to the heart, liver, spleen, and lung, and the adhesion performance of the hydrogel to biological tissues was observed. Similarly, the hydrogel was adhered to the surface of polymethyl methacrylate (5g), glass (37g), copper sheet (21g), and weight (80g), respectively.

[0042] Example 4: Debonding ability of hydrogel

[0043] A pig skin lap shear model was used to investigate the debonding ability of gel after glutathione (GSH) or sodium bicarbonate (SBC) treatment.

[0044] Freshly obtained pig skin was simply washed, and GSH or SBC soaked PAN / Ag 20 -PLG hydrogel material was adhered to the surface of pig skin, and then the adhesion performance of the hydrogel was tested using a universal tensile testing machine.

[0045] As Figure 4 shown, the adhesion strength of the control group (PAN / Ag 20 -PLG hydrogel adhered to the surface of pig skin) hydrogel was 20.97 kPa, the adhesion strength after GSH treatment was 8.59 kPa, the adhesion strength after SBC treatment was 4.7 kPa, and the adhesion strength after GSH and SBC treatment was 3.44 kPa. It can be seen that the adhesion ability of the hydrogel after GSH or / and SBC treatment is significantly reduced, indicating that the hydrogel stimulated by GSH and SBC has excellent debonding ability.

[0046] Example 5: Antibacterial performance of hydrogel

[0047] Methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) were used as model bacteria for testing. The bacterial suspension (100 μL, 10 8 CFU mL -1Different hydrogel materials were added to the surface, respectively. The control group (the control group, the cultured bacteria were not treated in any way, and directly coated on the agar plate). After the bacterial suspension was added dropwise on the surface of different hydrogels and incubated at 37°C for 2 hours, the bacteria on the surface of the hydrogel were resuspended with 500 μL of sterile PBS buffer, diluted to the appropriate concentration, and added to Luria Bertani agar plates and incubated for 24 hours. The bacterial suspension in PBS was used as a negative control. The colonies produced were photographed and recorded. The killing rate was calculated using the following formula: Bacterial survival rate (%) = (the number of viable bacteria on the agar plate after hydrogel treatment / the number of viable bacteria on the agar plate in the control group) x 100%.

[0048] Figure 5 The results show that the survival rate of bacteria treated with hydrogels (a-c: MRSA; d-f: E. coli) is significantly reduced, indicating that the hydrogel material provided by the present application has excellent antibacterial performance.

[0049] Example 6 Cytotoxicity of hydrogels

[0050] Hydrogel extracts were collected for in vitro biocompatibility studies. After 75% alcohol disinfection and PBS washing, different hydrogels (0.1 g mL -1 ) were immersed in complete high glucose DMEM cell culture medium containing fetal bovine serum (10%) and penicillin-streptomycin (1%) and extracted for 24 hours. The control group (control group, the complete high glucose DMEM cell culture medium containing fetal bovine serum (10%) and penicillin-streptomycin (1%) was used to culture cells, and the cells were not treated in any way). L929 fibroblasts (3000 cells / well) were inoculated into 96-well plates containing high glucose DMEM medium containing 10% fetal bovine serum and incubated at 37°C for 24 hours to adhere cells. Then, the medium was changed to the medium extract of the hydrogel (100 μL), and incubated for 24, 48 and 72 hours. At the predetermined time point, Cell Counting Kit-8 cell counting reagent (10 μL) was added to each well and incubated at 37°C for 1 hour, and then the absorbance at 450 nm was read with a microplate reader.

[0051] The results are shown in Figure 6 (a: 24h; b: 48h; c: 72h), the cell viability at 24, 48 and 72 hours was higher than 90%, indicating that the hydrogel material provided by the present application has excellent cell safety.

[0052] Example 7 Hemostatic performance of hydrogels in liver

[0053] The hemostatic performance of the hydrogels in vivo was evaluated using a Balb / c mouse liver hemorrhage model (male, 18-20 g). Intraperitoneal injection of sodium pentobarbital anesthesia (0.5%, 75 mg mL -1 ) was used, then the abdomen was incised to expose the liver. A 16G needle was used to puncture the liver to form a hemorrhage, and weighed filter paper was placed under the liver. The experimental group immediately adhered different hydrogel materials to the hemorrhage site after the needle formed a hemorrhage. The control group (control group, a 16G needle was used to puncture the liver of the mouse to form a hemorrhage, and no treatment was performed). The clotting time and bleeding amount of the wound were recorded.

[0054] Figure 7 The results show that the blood loss of the control group is about 145 mg, and the clotting time is about 196 s; the blood loss of the PAN / Ag0-PLG group is about 38 mg, and the clotting time is 69 s; the blood loss of the PAN / Ag 10 -PLG group is about 28 mg, and the clotting time is 49 s; the blood loss of the PAN / Ag 20 -PLG group is about 23 mg, and the clotting time is 40 s; the blood loss of the PAN / Ag 40 -PLG group is about 28 mg, and the clotting time is 38 s. Compared with the control group, the hydrogel material treatment group provided by the present application has low blood loss and fast clotting time.

[0055] Example 8 Hydrogel promotes wound repair

[0056] To evaluate the wound repair effect of the hydrogel, a methoxylated Staphylococcus aureus infected full-thickness wound model was used in diabetic mice. All animal experiments were performed according to the approved protocol of the Animal Experiment Ethics Committee of the State Key Laboratory of Biotherapy, Sichuan University. The diabetic mice were randomly divided into four groups: Control (control group), 3M, PAN / Ag0-PLG, PAN / Ag 20 -PLG. After anesthesia with isoflurane and removal of the back hair, a full-thickness skin circular wound with a diameter of 8 mm was formed, and immediately a Staphylococcus aureus suspension (100 μL, 1 × 10 7 CFU mL -1 ) was added to the wound to establish a full-thickness Staphylococcus aureus infected wound model. Treatment was performed 24 h later. At the predetermined time, the wound healing process of the mouse was recorded using a smart phone. The wound area was calculated by using ImageJ software.

[0057] As Figure 8 The results show that on the 9th day, the wound healing rate of the control group is 55.1%, the wound healing rate of the 3M group is 57.7%, the wound healing rate of the PAN / Ag0-PLG group is 64.6%, and the wound healing rate of the PAN / Ag 20- The wound healing rate of the PLG group was 70.2%. On day 15, the wound healing rate of the control group was 78.1%, the wound healing rate of the 3M group was 83.4%, the wound healing rate of the PAN / Ag0-PLG group was 87.9%, and the wound healing rate of the PAN / Ag 20 - The wound healing rate of the PLG group was 91.2%. On day 21, the wound healing rate of the control group was 91.2%, the wound healing rate of the 3M group was 94.8%, the wound healing rate of the PAN / Ag0-PLG group was 96.9%, and the wound healing rate of the PAN / Ag 20 - The wound healing rate of the PLG group was 99.6%. It can be seen from this that the PAN / Ag 20 - The PLG treatment group had the best effect on promoting wound healing.

Claims

1. An adhesive hydrogel material, characterized by: Silver nanoparticles are prepared by a reduction method of polylysine functionalized with gallic acid mineralization; then, the silver nanoparticles are incorporated into the hydrogel network to prepare the adhesive hydrogel material; the preparation method is as follows: a. Preparation of Ag-PLG: a solution of polylysine functionalized with gallic acid is prepared by reaction of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, and then freeze-dried to obtain polylysine functionalized with gallic acid powder; then, silver nitrate and polylysine functionalized with gallic acid powder are mixed in water, the pH value is adjusted to 10-12, heated and stirred for 30 min, and then cooled to 0-5℃ to obtain Ag-PLG solution; b. Preparation of the hydrogel material: acrylic acid, acrydite, aldehyde-hydroxylated hyaluronic acid, Ag-PLG solution, N,N'-bis(acryloyl)cystamine are added to a phosphate buffer solution and stirred to dissolve, then a photoinitiator, lithium phenyl-2,4,6-trimethylbenzoyl phosphinate, is added, and an adhesive hydrogel material, PAN / Ag-PLG, is obtained under 405 nm blue light irradiation for 5 min.

2. The adhesive hydrogel material of claim 1, wherein: The specific preparation steps of the polylysine functionalized with gallic acid in step a are as follows: polylysine and gallic acid are dissolved in a morpholine ethanesulfonic acid buffer solution, and a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysuccinimide mixture is added dropwise to the above solution, and the reaction is carried out at room temperature under nitrogen protection; after 8-16 h of reaction, the mixture solution is dialyzed in ultrapure water for 3 days, and then freeze-dried to obtain polylysine functionalized with gallic acid powder.

3. The adhesive hydrogel material of claim 2, wherein: The morpholine ethanesulfonic acid buffer solution is 0.05M; in the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysuccinimide mixture, the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to N-hydroxysuccinimide is 3:1; the volume of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysuccinimide mixture is 20%-25% of the total volume of the reaction solution; and the molar ratio of hydroxyl groups in N-hydroxysuccinimide to carboxyl groups in gallic acid is 1:

1.

4. The adhesive hydrogel material of claim 1, wherein: The mass content ratio of silver nitrate to polylysine in step a is 1:

10.

5. The adhesive hydrogel material of claim 1, wherein: The heating temperature in step a is 50-80℃.

6. The adhesive hydrogel material of claim 1, wherein: In step b, the mass content ratio of acrydite to acrylic acid is 10-30wt%; the mass content ratio of aldehyde-hydroxylated hyaluronic acid to acrylic acid is 5-10wt%; the mass content ratio of N,N'-bis(acryloyl)cystamine to acrylic acid is 4-6wt%; and the mass content ratio of lithium phenyl-2,4,6-trimethylbenzoyl phosphinate to acrylic acid is 3-6wt%.

7. Use of the adhesive hydrogel material according to any one of claims 1-6 in the preparation of a wound dressing.

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