Hydrogel dressing for chronic wound surface of diabetes mellitus as well as preparation method and application of hydrogel dressing

Abstract

The invention belongs to the technical field of medical products, and provides a hydrogel dressing for diabetic chronic wounds and a preparation method and application of the hydrogel dressing. The antibacterial effect of the hydrogel dressing for the chronic wound surface of diabetes mellitus is improved by increasing the ZIF-8 coated AgNPs; in the hydrogel matrix part, the pH response is dominated by adjusting the component proportion of the temperature-sensitive agent N-isopropylacrylamide and the pH response 1-vinyl imidazole; the N-isopropylacrylamide assists the extension of a macromolecular chain segment in the composite hydrogel dressing, so that the dressing can fully absorb wound exudate and is easy to peel off, and secondary damage can be avoided.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical products, and particularly relates to a hydrogel dressing for chronic wounds of diabetes, a preparation method and application thereof. BACKGROUND

[0002] Human skin can resist bacterial and viral invasion, and after the formation of a wound caused by external force, the action will be disturbed. Normal wound healing needs to go through four stages of hemostatic inflammatory response, proliferation and reconstruction. Bacterial infection is one of the main reasons for delayed wound healing, which makes the healing process stagnate at the inflammatory response stage, and further becomes a chronic wound. Diabetic patients are prone to chronic wounds, and the most typical one is diabetic foot ulcer. The amputation rate of diabetic foot ulcer patients is 7-20%, and the mortality rate within 5 years after the occurrence of ulcer is nearly 50%. Chronic wounds seriously affect the quality of life and health of patients, and significantly increase the social medical expenses. In summary, the effective treatment of chronic wounds is an urgent and challenging public health and clinical problem.

[0003] In clinical practice, there are many kinds of dressings for chronic wounds, such as foam dressings, which can cause water loss of dry wounds and some products can be thick and not flexible. Transparent film dressing is also one of the common dressings, but it cannot absorb wound exudate, resulting in surrounding skin maceration. In addition, the secondary damage caused by peeling of most dressings is also a big problem in clinical practice. In recent years, hydrogel has attracted widespread attention in the field of chronic wounds. It is rich in water and can provide moisture for dry wounds; its porous structure allows gas exchange and can also absorb wound exudate; it can load bioactive molecules. In addition, different monomers of synthetic hydrogel also have their own characteristics. Such as antibacterial, temperature response, pH response, etc. These are the properties that an ideal dressing should have.

[0004] Antibacterial hydrogel is a material that combines the antibacterial properties and the physical properties of hydrogel. According to the classification of hydrogel matrix and antibacterial agent, antibacterial hydrogel is divided into three types: antibiotic-containing hydrogel, hydrogel with inherent antibacterial ability and nanoparticle-containing hydrogel. The team of Ding Xin of Sun Yat-Sen University developed a double cross-linked interpenetrating polymer network hydrogel with gentamicin as a dynamic cross-linking agent. Hydroxyethyl methacrylate (HEMA) and acrylic acid (AA) are polymerized by free radicals to form a primary chemical cross-linking network; a secondary physical cross-linking network of the hydrogel is constructed through the electrostatic interaction between the carboxyl group of polyacrylic acid and the amino group of gentamicin. Gentamicin is chosen as the main antibacterial material, but it is also easy to produce bacterial resistance. The team of Liang Shengnan of Ocean University of China synthesized a gel with chitosan as an antibacterial material. Chitosan relies on the amino group in its structure for antibacterial properties. However, it also relies on the interaction between the amino group protonated and the negative charge groups in the system to form a gel, which means that the antibacterial effect of chitosan polymer gel is greatly weakened. Therefore, 2-chloroethylamine hydrochloride (ACH) is introduced at C6−OH of chitin in this scheme, hoping to increase the amino group in the structure to increase the antibacterial effect of the gel. However, since the gel is only composed of soft polymer chains, its mechanical properties are not ideal. The team of Chen Qian of Soochow University used tannic acid to coordinate with silver ions (Ag + ) to form TA-Ag NPs, which can act as a catalyst for the polymerization reaction of N-isopropyl acrylamide (NIPAM) to form PNIPAM / TA-Ag hydrogel. The antibacterial effect of this gel mainly relies on TA-Ag NPs, and the gel itself does not have antibacterial effect, so the antibacterial effect of a single antibacterial mechanism dressing is not ideal. The team of Deng Yi of Sichuan University encapsulated Er-doped carbon dots (Er:CDs) inside ZIF-8, and then loaded the synthesized C@Z on a hydrogel made of methacrylated gelatin (Gel-MA) and N-(2-hydroxyethyl) acrylamide (HEAA) polymerization to obtain the final gel dressing: GH-C@Z. The hypothetical antibacterial mechanism: GH-C@Z is applied to the wound surface of an infected wound, partially destroying the bacterial biofilm matrix, C@Z contacts the weak acid environment, and the ZIF-8 shell is broken to release Er:CDs and Zn 2+Then, near-infrared light is used to realize the synergistic antibacterial treatment of photothermal, photodynamic and ion interference. After the bacterial biofilm is eliminated, the near-infrared is turned off, and the antioxidant and anti-inflammatory effects of the Er:CDs are used to promote wound healing. The dressing avoids the possibility of bacterial resistance, but it can be found that the antibacterial effect of the dressing mainly depends on the triggering of near-infrared light irradiation, so the antibacterial effect after the near-infrared light is turned off is very weak. And when the near-infrared is irradiated, •OH is generated, and there is a possibility that the wound will be worsened if the •OH enters the wound due to the untimely turning off. From the above analysis, it can be seen that the existing dressing has high drug resistance, poor antibacterial property, poor mechanical property and dependence on external factors, which further makes the effect of wound healing not ideal. SUMMARY

[0005] To solve the above technical problems, the present application provides a hydrogel dressing for diabetic chronic wounds and a preparation method thereof.

[0006] The first aspect of the present application provides a hydrogel dressing for diabetic chronic wounds, which is obtained by mixing a pH-responsive agent, a temperature-sensitive agent, acrylamide and a crosslinking agent, and then irradiating with ultraviolet light to obtain a polymer chain segment; adding a ZIF-8@Ag NPs dispersion and a photoinitiator, and connecting the ZIF-8@Ag NPs and the polymer chain segment through hydrogen bonds and electrostatic interactions to obtain a pre-gel suspension; and solidifying the pre-gel suspension to obtain the hydrogel dressing. The mass ratio of the temperature-sensitive agent, the pH-responsive agent and the acrylamide is 1:2-10:2-8; and the mass ratio of the crosslinking agent, the photoinitiator and the acrylamide is 0.001-0.1:0.001-0.1:2-8. The concentration of the ZIF-8@Ag NPs in the pre-gel suspension is 0.001g / mL-0.08g / mL. The pH-responsive agent is 1-vinylimidazole, and the temperature-sensitive agent is N-isopropyl acrylamide.

[0007] In the present application, the temperature-sensitive agent, the pH-responsive agent and the acrylamide are mixed in a mass ratio of 1:2-10:2-8, and according to the response principle of the pH-responsive agent and the temperature-sensitive agent, when the pH-responsive agent is dominant, i.e. the amount of the pH-responsive agent is higher than that of the temperature-sensitive agent, the hydrogel dressing will show a swelling effect, and the swollen hydrogel dressing can absorb wound exudate to achieve the purpose of promoting wound healing. The added ZIF-8@Ag NPs nanoparticles can easily form hydrogen bonds and electrostatic interactions with the surrounding water and polymer chain segments to increase the mechanical properties of the hydrogel. In addition, the mechanical properties, adhesion and biocompatibility of the hydrogel system meet the requirements, and it is easy to peel off, which can avoid secondary damage.

[0008] In another preferred embodiment, the ZIF-8@Ag NPs dispersion liquid is obtained by dispersing the ZIF-8@Ag NPs in water. The ZIF-8@Ag NPs are prepared by solution chemistry method from ZIF-8 and silver salt solution under the reduction of a reducing agent.

[0009] In another preferred embodiment, the crosslinking agent is N,N'-methylenebisacrylamide, and the photoinitiator is 2-hydroxy-2-methylpropiophenone.

[0010] The second aspect of the present application provides a preparation method of the hydrogel dressing, comprising the following steps: In a one-pot method, N-isopropylacrylamide, 1-vinylimidazole and acrylamide are added into water in a mass ratio of 1:2-10:2-8, and N,N-methylenebisacrylamide, 2-hydroxy-2-methylpropiophenone and acrylamide are added into water in a mass ratio of 0.001-0.1:0.001-0.1:2-8, and stirring is performed until complete dissolution; ZIF-8@Ag NPs are added and uniformly dispersed by ultrasonic, to obtain a pre-gel suspension; The pre-gel suspension is cured to obtain the hydrogel dressing.

[0011] In another preferred embodiment, the curing is performed by ultraviolet light.

[0012] In another preferred embodiment, the mass-volume ratio of the 1-vinylimidazole and water is 1:5-20.

[0013] In another preferred embodiment, the ZIF-8@Ag NPs are obtained by the following process: ZIF-8 is added into an AgNO3 solution, stirring is performed for 12-16 hours to obtain a suspension, centrifugation is performed, the supernatant is taken, alcohol washing is performed, and the primary product is obtained after drying; wherein, the concentration of the AgNO3 solution is 0.02-0.2 mol / L, and the mass ratio of ZIF-8 to AgNO3 is 1:4-10. A sodium borohydride solution is mixed with the primary product as a reducing agent, stirring is performed for 30 minutes, centrifugation is performed, the precipitate is taken, washing is performed, and drying is performed to obtain the ZIF-8@Ag NPs; wherein, the concentration of the sodium borohydride solution is 0.001-0.01 g / mL.

[0014] In another preferred embodiment, the alcohol washing agent is methanol.

[0015] The third aspect of the present application provides an application of the hydrogel dressing in preparing a wound healing auxiliary material.

[0016] In another preferred embodiment, the wound is a wound caused by diabetes.

[0017] Compared with the prior art, the present application has the following beneficial effects: The present application uses N-isopropyl acrylamide with a temperature-sensitive effect and 1-vinylimidazole with pH response effect and antibacterial effect as monomers, obtains a gel under the action of a crosslinking agent, loads ZIF-8@Ag NPs on the gel, and enhances the mechanical strength of the gel by adding acrylamide. According to the response principle of 1-vinylimidazole and the temperature-sensitive agent, when 1-vinylimidazole is dominant, that is, the amount of 1-vinylimidazole is higher than that of the temperature-sensitive agent, the hydrogel dressing will show a swelling effect, and the swollen hydrogel dressing can absorb wound exudate, so as to promote wound healing and effectively reduce the dependence on the outside world. And Ag NPs is selected as the main antibacterial material to realize antibiosis together with 1-vinylimidazole, which effectively improves the antibacterial effect. The Ag NPs are encapsulated in ZIF-8 to obtain ZIF-8@Ag NPs, which forms hydrogen bonds and electrostatic interaction forces with the surrounding water and polymer chains, thereby further increasing the mechanical properties of the hydrogel. The gel in the present application not only has good antibacterial effect, but also is easy to peel off and does not easily cause secondary damage after use. In addition, the hydrogel in the present application has good adhesion and biocompatibility, and is easy to peel off, so that secondary damage can be avoided. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a preparation process schematic diagram of the hydrogel dressing for chronic wounds of diabetes in the present application; wherein A is a ZIF-8@Ag NPs preparation process schematic diagram, and B is a preparation flowchart of the hydrogel dressing, wherein b is a high molecular chain segment formation schematic diagram.

[0019] Figure 2 It is TEM and SEM results of ZIF-8 and ZIF-8@Ag NPs; (a) is an SEM graph of ZIF-8, (b) is an enlarged SEM graph of ZIF-8, (c) is a TEM graph of ZIF-8, (d) is an SEM graph of ZIF-8@Ag NPs, (e) is an enlarged SEM graph of ZIF-8@Ag NPs, and (f) is a TEM graph of ZIF-8@Ag NPs.

[0020] Figure 3 It is element analysis and TEM-EDS result graph of ZIF-8@Ag NPs; wherein (a) is a high-angle annular dark field image, (b) is a TEM-EDS result graph of all elements distribution, (c) is a TEM-EDS result graph of Zn element, (d) is a TEM-EDS result graph of Ag element, and (e) is a TEM-EDS energy spectrum graph.

[0021] Figure 4 XRD results of ZIF-8, ZIF-8@Ag NPs and standard ZIF-8.

[0022] Figure 5 FIR results of ZIF-8 and ZIF-8@Ag NPs.

[0023] Figure 6 Stress-strain curves of composite hydrogels of each group.

[0024] Figure 7 Swelling ratio-temperature curves of composite hydrogels of each group.

[0025] Figure 8 Swelling ratio-time curves of composite hydrogels of each group in different pH environments, wherein (a) is the swelling ratio-time curve in the environment with pH value of 5, and (b) is the swelling ratio-time curve in the environment with pH value of 8.

[0026] Figure 9 Cell survival rate diagrams of L929 cells treated by PVNA and PVNA-Z@Ag NPs for different times, wherein (a) is the cell survival rate after treating L929 cells for 24 h, and (b) is the cell survival rate diagram after treating L929 cells for 48 h.

[0027] Figure 10 Antibacterial effect diagrams of PVNA and PVNA-Z@Ag NPs of G5, G6 and G9 groups, wherein (a) is the antibacterial effect diagram of treating Escherichia coli, and (b) is the antibacterial effect diagram of treating Staphylococcus aureus.

[0028] Figure 11 Adhesion effect diagrams of 2G9 group PVNA-Z@Ag NPs composite hydrogel on different articles, wherein (a) is the adhesion effect diagram on plastic, (b) is the adhesion effect diagram on glass, (c) is the adhesion effect diagram on wood, (d) is the adhesion effect diagram on metal, and (e) is the adhesion effect diagram at the joint.

[0029] Figure 12 Normal mouse wound healing situation diagram, wherein (a) is the real scene diagram of wound closure process of the control group and the experimental group, (b) is the wound overlapping diagram, and (c) is the statistical result diagram of wound closure rate. In the diagram, Control represents the control group, and Experimental represents the experimental group.

[0030] Figure 13 Diabetic mouse wound healing situation diagram, wherein (a) is the real scene diagram of wound closure process of the control group and the experimental group, (b) is the wound overlapping diagram, and (c) is the statistical result diagram of wound closure rate.

[0031] Figure 14 HE and masson staining results of the skin of the acute wound model of normal mice.

[0032] Figure 15 HE and masson staining results of the skin of the acute wound model of diabetic mice.

[0033] Figure 16 HE staining results of the heart, liver, spleen, lung and kidney of the acute wound model of normal mice, in which, Heart represents the heart, Liver represents the liver, Spleen represents the spleen, Lung represents the lung, and Kidney represents the kidney.

[0034] Figure 17 HE staining results of the heart, liver, spleen, lung and kidney of the acute wound model of diabetic mice. DETAILED DESCRIPTION

[0035] The technical solutions in the present application will be clearly and completely described in combination with specific embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application. The methods described in the embodiments of the present application are all conventional methods unless otherwise specified. The materials, reagents and the like used are all available from commercial channels unless otherwise specified.

[0036] 1. Synthesis of ZIF-8 and synthesis of ZIF-8@Ag NPs 2-Methylimidazole and zinc nitrate hexahydrate were dissolved in methanol, and the molar ratio of the two was 6:1. The mixture was stirred at room temperature for 12 h. The resulting suspension was transferred to a 10-ml centrifuge tube, and after being balanced, it was centrifuged at 8000 rpm for 5 min to remove the supernatant. Then, the mixture was washed with methanol three times, and dried at 60°C for 12 h. The obtained nanoparticles were ZIF-8.

[0037] Synthesis of ZIF-8@Ag NPs 0.423 g of ZIF-8 prepared was added to an AgNO3 solution with a concentration of 0.025 mol / L, and stirred at 23°C for 12 h. The resulting suspension was transferred to a 10-ml centrifuge tube, and after being balanced, it was centrifuged at 10000 rpm for 5 min to remove the supernatant. Then, the mixture was washed with methanol three times, and dried at 60°C for 12 h. 0.2 g of sodium borohydride was dissolved in 16 mL of water, and mixed with the dried 0.423 g of the initial product. After stirring, the suspension was transferred to a 10-ml centrifuge tube, and centrifuged at 10000 rpm for 5 min to remove the supernatant. Then, the mixture was washed with methanol and deionized water alternately three times, and dried at 60°C for 12 h. Finally, ZIF-8@Ag NPs were obtained, as shown in FIG. 1. Figure 1As shown in A in the diagram.

[0038] The ZIF-8 and ZIF-8@Ag NPs were characterized as follows.

[0039] 1) Scanning electron microscopy characterization and transmission electron microscopy characterization The morphology of ZIF-8 and ZIF-8@AgNPs was observed using a scanning electron microscope (FEI LLC, Teneo VS, USA). 2 mg of the prepared ZIF-8 and ZIF-8@Ag@NPs were weighed and added to 1 ml of anhydrous ethanol. The mixture was ultrasonically dispersed to ensure uniform distribution of the ZIF-8 and ZIF-8@Ag NPs powders in the solvent. The suspension was resuspended using a 200 μl pipette tip and placed on a glass slide. After the anhydrous ethanol evaporated, the samples were sputter-coated with gold and then placed on a stage for observation of their microstructure under a scanning electron microscope.

[0040] The morphology of ZIF-8 and ZIF-8@Ag NPs and the elemental distribution in ZIF-8@Ag NPs were observed using a transmission electron microscope (FEI LLC, Talos F200X, USA). A suspension of 2 mg / ml ZIF-8 and ZIF-8@Ag NPs was prepared according to the above method. The suspension was then dropped onto a copper sheet, and after the anhydrous ethanol evaporated, morphological and elemental analyses were performed. The results are as follows: Figure 2 As shown.

[0041] from Figure 2 The TEM and SEM results show that ZIF-8 is a cubic nanoparticle with a diameter of 400-600 nm. Comparing the two nanoparticles, the radius of ZIF-8@Ag NPs is generally smaller than that of ZIF-8. This may be because during the synthesis of ZIF-8@Ag NPs, prolonged stirring in water caused slight surface wear on the nanoparticles. However, it is still evident that ZIF-8@Ag NPs has a cubic crystal structure.

[0042] Figure 3 This is the TEM-EDS result of ZIF-8@Ag NPs, which shows the distribution of each element in the ZIF-8@Ag NPs within the nanoparticles. The distribution of red dots represents the distribution of Ag NPs throughout the nanoparticles, and the varying shades of color indicate the different degrees to which Ag NPs have penetrated the nanoparticles.

[0043] 2) X-ray diffraction detection The phase structure of ZIF-8 and ZIF-8@Ag NPs was studied by powder X-ray diffraction (Bruker, D8 Advance, Germany). The data were collected using Cu-kα x-rays, 2θ scanning method, and the scanning range was 5°-80°.

[0044] Figure 4 For the XRD results, it can be seen from the observation results that the synthesized ZIF-8 has characteristic peaks: 2θ = 7.4°, 10.45°, 12.78°, 14.71°; in addition, the peak intensity is also similar to the standard XRD spectrum. In summary, it can be proved that ZIF-8 is successfully synthesized.

[0045] The Ag NPs@ZIF-8 spectrum has more characteristic peaks than ZIF-8: 2θ = 38.2° (111), 44.4° (200), 64.5° (220), 77.9° (310), which are consistent with the characteristic peaks of pure Ag NPs in the literature, which can further prove that Ag NPs@ZIF-8 is successfully synthesized. In addition, the characteristic peak intensity of ZIF-8 in the Ag NPs@ZIF-8 spectrum is significantly reduced, which may be caused by the wear of the crystal form during stirring.

[0046] 3) Fourier infrared detection The functional group structure of ZIF-8 and ZIF-8@Ag NPs was studied by Fourier infrared spectrometer (Shimadzu, 8400s, Japan). The sample was treated by KBr pressing method, the spectral range was 500-40000 cm -1 , the scanning rate was 2 cm -1 , and the results are shown in Figure 5 . It can be found from the observation of the Fourier infrared spectrum that the characteristic peak of ZIF-8 is 3636 cm −1 , the characteristic peak of 3128 cm −1 can be attributed to the stretching vibration of aromatic C-H, the characteristic peak of 2926 cm −1 is caused by the stretching vibration of methyl C-H, the characteristic peak of 1587 cm −1 can be attributed to the stretching vibration of imidazole ring C=N, the characteristic peak of 1350 cm −1 -1500 cm −1 is related to the stretching vibration of the entire imidazole ring, the characteristic peak of 950-1350 cm −1 is related to the in-plane bending, the characteristic peak of 600-950 cm −1 is related to the out-of-plane bending, and the characteristic peak of 535 cm −1 is the characteristic peak of Zn-O stretching vibration, and the characteristic peak of 430 cm −1Should be the characteristic peak of Zn-N stretching vibration. Comparing the two kinds of nanoparticles Fourier infrared spectrum, it is found that the spectrum of Ag NPs@ZIF-8 does not appear new characteristic peak, indicating that the combination of Ag NPs and ZIF-8 is not connected by chemical covalent bond. But compared with pure ZIF-8, the characteristic peak position of Ag NPs@ZIF-8 spectrum obviously shifts. It may be that the addition of Ag NPs affects the interaction of Zn 2+ and imidazole, thereby affecting the vibration frequency; it may also be that the surface plasmon resonance effect of Ag NPs affects the electronic distribution. In addition, the signal intensity of some characteristic peaks increases significantly, which may be caused by the surface enhanced Raman scattering effect of Ag NPs increasing the vibration signal of adjacent substances.

[0047] 2. Synthesis and characterization of PVNA-Z@Ag NPs hydrogel dressing 1) Synthesis of PVNA-Z@Ag NPs hydrogel dressing A one-pot method was used to prepare a composite hydrogel containing nanoparticles.

[0048] The pH-responsive agent 1-vinylimidazole (VI, Shanghai Aladdin Biochemical Technology Co., Ltd.), the temperature-sensitive agent N-isopropyl acrylamide (NIPAM, Anhui Zesheng Technology Co., Ltd.), the acrylamide (AM, Shanghai Aladdin Biochemical Technology Co., Ltd.) to increase the mechanical properties, and the crosslinking agent N,N-methylene bisacrylamide (MBA, Anhui Zesheng Technology Co., Ltd.) were added to water in a certain proportion and stirred until completely dissolved; then ZIF-8@Ag NPs was added and uniformly dispersed by ultrasonic; finally, the photoinitiator 2-hydroxy-2-methylpropiophenone was added, and a pre-gel suspension was obtained. Finally, the pre-gel suspension was poured into a mold, and the target hydrogel was obtained by ultraviolet curing, which was denoted as PVNA-Z@Ag NPs. To simplify the operation, three appropriate levels of NIPAM, VI, and ZIF-8@Ag NPs were selected for orthogonal experiment, and finally 9 groups of tests were obtained (the specific data are shown in Table 1), as shown in Table 1; in addition, the mass of AM, the mass of MBA, and the mass of photoinitiator added in all groups were controlled to be the same, which were 0.35 g, 0.01 g, and 100 ul, respectively.

[0049] Table 1 Composition and ratio of each group of hydrogel dressing 2) Characterization of PVNA-Z@Ag NPs hydrogel dressing Tensile property detection of PVNA-Z@Ag NPs hydrogel dressing The tensile properties of the PVNA-ZIF-8@Ag NPs hydrogel were detected by using a microcomputer-controlled electronic universal testing machine (China Matest Industrial System Co., Ltd., CMT6103). The total amount of substance (except ZIF-8) added was controlled to be 1:10 of the amount of substance of water added, and then the pre-prepared glue suspension was poured into a mold with a width of 20 mm, a length of 45 mm, and a thickness of 10 mm. After ultraviolet curing, the composite gel required for detection was obtained. The standard to be executed is GB 13022-1991.

[0050] Table 2 Elongation at break of each group of gels Figure 6 Table 2 is the tensile property detection result of nine groups of hydrogel dressings. The normal human skin elongation at break is about 60%-75%. Using the universal testing machine detection, it can be seen that the elongation at break of the nine groups of composite hydrogels obtained by the above orthogonal experiment is superior to that of human skin.

[0051] 2) Temperature response effect detection of PVNA-Z@Ag NPs hydrogel dressing Each group of composite hydrogels was soaked in a deionized water solution, placed at room temperature until swelling equilibrium, weighed and recorded as W1; then it was placed in a 55°C oven, weighed after standing for 16h, then placed at room temperature until the weight of the hydrogel returned to W1; repeat the above operation, put the hydrogel into 37°C, 25°C, 4°C environment in turn, take out after standing for 16h and weigh. After the above operation, the hydrogel was vacuum dried and weighed, recorded as Wd. Parallel experiment three times. Calculate the swelling rate (SR): SR=(WT-Wd) / Wd*100%; WT represents the weight of the hydrogel at T temperature.

[0052] Figure 7 is the temperature responsiveness result of each group of composite gels with the same component ratio. NIPAM has both hydrophobic and hydrophilic groups, so it has temperature responsiveness. From the experiment, it can be seen that the swelling rate change of the composite gel dressing of other groups except G1 and G3 is most obvious at 25°C-37°C, so the lowest critical solution temperature of other groups of composite gels is between 25°C-37°C. Control pH=7, i.e. exclude the pH response effect caused by VI. When the composite dressing is attached to the human skin, the hydrogen bond between the hydrophilic group inside the composite gel and the water molecules breaks, the hydrophobic group of isopropyl dominates, causing the molecular chain to cohere and exclude water, and the whole composite gel shrinks. In addition, the swelling rate change of G2 and G4 groups in the whole temperature change process is relatively weak compared with other groups, which means that its temperature response is not good; the swelling rate change of G5-G9 groups at 25°C-37°C is obvious, and the difference between groups is not very obvious, which means that G5-G9 all have good similar temperature responsiveness.

[0053] 3) pH response effect detection of PVNA-Z@Ag NPs hydrogel dressing Each group of composite hydrogel was immersed in a deionized water solution with pH = 7, and placed at room temperature until swelling equilibrium, and weighed. Then each group of gel was divided into two groups, and placed in phosphate buffer with pH = 5 and pH = 8, respectively, and weighed at regular intervals. Finally, the hydrogel was vacuum dried, weighed as Wd, and the surrounding environment was controlled at 37°C, and the experiment was repeated three times. The swelling ratio (SR) was calculated as (Wt-Wd) / Wd*100%; Wt represents the weight of the hydrogel at time t

[0054] Figure 8 The pH response results of each group of composite gels with the same composition ratio are shown in the table above. It can be found that under the conditions of 37°C, weak acid, and sufficient surrounding moisture, the pH response effect of the above 9 groups of composite gels with different proportions is stronger than the temperature response effect, and the overall swelling rate changes significantly, with G2, G3, G5, G6, and G9 showing more obvious changes in swelling rate at pH = 5, indicating that they have good pH response.

[0055] Based on the temperature response results and the pH response results, G5, G6, and G9 were selected for the following experiments.

[0056] 4) Cytotoxicity evaluation of PVNA-Z@Ag NPs hydrogel dressing Cell culture: The whole process was sterile, and the cells were cultured according to the growth conditions of mouse fibroblasts (Jiangsu Kaikai Biotechnology Co., Ltd., L929 cells). The cells in the cryopreserved tube taken out from the -80°C freezer were recovered. Then every 2 days, the cells were subcultured at a ratio of 1:3.

[0057] Cytotoxicity evaluation: The cell counting kit-8 (CCK8) was used to evaluate whether the synthetic dressing had toxicity to L929 fibroblasts. Based on the temperature response and pH response results, G5, G6, and G9 were selected for cytotoxicity evaluation. Before evaluation, the monomers were removed by soaking in PBS buffer; then the composite gel extraction solution was prepared, with an extraction ratio of 0.1 g / mL, and additional absorption of the absorption medium of the hydrogel. Extraction medium: DMEM medium; extraction conditions: 37±1°C for 24±2 hours; finally, the extracted sample was filtered with a bacteria filter.

[0058] Mouse L929 cells were inoculated on DMEM medium containing 10% fetal bovine serum at a concentration of 1×10 5The cell density of L929 cells was inoculated on a 96-well plate. The obtained hydrogel extract was added to the well plate and cultured at 37°C, 5% CO2 for 24h and 48h respectively. Then, 10uL CCK-8 was added to each well, and after 2h of incubation, the absorbance of the sample was detected at 450nm using a microplate reader and recorded. Blank control and negative control groups were set up, the blank control group only contained DMEM medium; the negative control group contained DMEM medium and cells without sample leachate, and five replicate wells were set up. The cell survival rate (CV) was calculated: CV = (As-Ab) / (Ac-Ad)*100%; where As is the absorbance of the experimental group: 100ul gel leaching liquid + cell well; Ab is the absorbance of the corresponding 100ul gel leaching liquid well; Ac is the absorbance of the control group: 100ul medium + cell well; Ad is the absorbance of the 100ul medium well, and the experimental results are shown in Figure 9 .

[0059] The temperature response results and pH response results were combined to select G5, G6 and G9 for cell toxicity evaluation. From the cell survival rate results of L929 cells treated with PVNA and PVNA-Z@Ag NPs dressings for 24h and 48h, it can be seen that the survival rates of L929 cells treated with the two gel dressings were both greater than 80% after 24h and 48h, so the synthesized composite gel dressing had no cytotoxicity; in addition, from a statistical point of view, there was no significant difference in cytotoxicity between the NPs-containing hydrogel dressing groups incubated for 24h and 48h.

[0060] 0) Evaluation of the antibacterial effect of the PVNA-Z@Ag NPs hydrogel dressing Bacterial culture: sterile operation throughout, according to the survival conditions of Staphylococcus aureus (BNCC186335) and Escherichia coli (BNCC133264) for culture. After opening the ampoule containing the bacterial freeze-dried powder, sterile water was slowly injected into the upper layer of the freeze-dried powder with a sterile Pasteur pipette. After the freeze-dried powder was dissolved by absorbing water, it was mixed gently with the pipette. The bacterial suspension was prepared. The bacterial solution was dropped on the solid culture medium and evenly coated, and then cultured at 37°C for 24h. After the bacteria recovered, a single colony was taken for streak inoculation, and subcultured twice to achieve the rejuvenation effect.

[0061] Bacteriostatic experiment: the composite gel dressing was first incubated with bacteria, and then the bacterial liquid was coated to observe the antibacterial effect of the dressing.

[0062] Preparation of bacterial solution: a single rejuvenated colony was picked up in a 12ml shaking tube containing 5ml liquid medium, shaken at 150rpm for 5h, and the OD600 was measured to be 0.8~1.2, i.e. the bacterial solution in the logarithmic growth phase was prepared.

[0063] The prepared logarithmic growth phase bacterial solution was diluted to 10 6CFU / ml were then inoculated in a shaker tube with 5 ml of liquid medium, and the composite hydrogel dressing was added. The shaking frequency was selected as 200 rpm according to the oxygen solubility efficiency of the human body, and the OD600 was measured after 12 h. The reason for selecting 12 h of shaking is that this time period includes the logarithmic growth phase of E. coli and S. aureus, ensuring the activity of the bacteria. In addition, according to the pH response performance experiment, it can also be confirmed that the pH response effect of the composite dressing is best in this time period, so setting 12 h as the co-incubation time can well evaluate the antibacterial performance of the dressing.

[0064] The composite gel dressing mainly relies on ZIF-8@Ag NPs and VI for antibacterial activity. In a weak acid environment, ZIF-8@Ag NPs are cleaved to release positively charged imidazole groups, Zn 2+ and Ag NPs. The composite gel dressing has positively charged imidazole groups, and Z@Ag NPs release positively charged imidazole groups and Zn 2+ By electrostatic adsorption, the bacterial membrane permeability is increased, making the bacterial contents more easily excreted, and similarly, Zn 2+ and Ag NPs are also more easily introduced into the bacterial interior. Ag + released by Ag NPs entering the bacterial interior interferes with bacterial metabolism together with Zn 2+ , accelerating bacterial death. Figure 10 The antibacterial effect of PVNA and PVNA-Z@Ag NPs in G5, G6 and G9 groups is shown in FIG. 6. Overall, PVNA also has antibacterial effect, and the antibacterial effect is better with more VI content. In addition, PVNA-Z@Ag NPs are better than PVNA alone in antibacterial effect, which shows the contribution of ZIF-8@Ag NPs to antibacterial activity.

[0065] Based on the antibacterial effect of PVNA and PVNA-Z@Ag NPs in G5, G6 and G9 groups on E. coli and S. aureus, we selected G9 group PVNA-Z@Ag NPs for subsequent experiments.

[0066] Evaluation of the adhesion effect of PVNA-Z@Ag NPs hydrogel dressing Human skin was used as the adhesion substrate for G9 group PVNA-Z@Ag NPs composite hydrogel, and the adhesion of the gel dressing to plastic, glass, wood and metal was observed. In addition, the hydrogel was placed on the finger joints, and the hydrogel dressing was observed to see if it was always attached to the skin by moving the finger joints.

[0067] Figure 11The adhesion effect diagram of the composite hydrogel dressing on human skin as the base can be clearly seen that the composite hydrogel containing nanoparticles can make plastic, glass, wood and metal with a certain weight adhere to the skin surface and hang in the air. In addition, the composite hydrogel is applied to the joint, and when the joint is bent, the gel dressing still adheres to the skin surface. In summary, the PVNA-Z@Ag NPs composite hydrogel dressing of G9 group has good adhesion, which basically meets the adhesion requirements of wound dressings.

[0068] 1. Animal experiment According to the results of the tensile properties, temperature and pH response effect, cytotoxicity, adhesion and antibacterial properties of the composite hydrogel, we selected the PVNA-Z@Ag NPs composite hydrogel dressing of G9 group for the following animal experiment.

[0069] 1) Construction of diabetic mouse model Male SPF BALB / c mice were selected and raised in a barrier environment with normal feeding and drinking water. After 1 week, the diabetic model was established, and the mice were fed with high-fat diet (D12492) for 7 days, and continuously fed for 5 weeks. Then, streptozotocin citric acid buffer (STZ, pH=4.5) was injected to induce diabetes. On the first day, after fasting for 12 h, 50 mg / kg STZ was injected intraperitoneally, and the injection was continued for 5 days, and only the first day was fasting. On the 3rd, 5th and 7th day after STZ injection, the body weight and blood glucose of the mice were measured. When the mice were observed to have polyuria and the blood glucose level of the mice was continuously higher than 11.1 mmol / L, it was considered that the induction of type II diabetes was successful. If not successful, STZ can be supplemented.

[0070] Wound characterization and results: Closure rate: The experiment was divided into two parts, the first part was to observe the healing process of normal mice, and the second part was to observe the wound healing process of diabetic mice. The mice were all established as full-thickness skin defect wound models. The mice were anesthetized with 2% isoflurane, and the concentration of isoflurane was maintained at 1.5% during the operation. After removing the back hair with a hair clipper, the skin was disinfected with 75% ethanol and iodophor solution, and a sterile skin puncher was used to generate two circular full-thickness defect areas with a diameter of 5 mm on the epidermis of the mouse back, one as a control group and the other as an experimental group. The control group was washed with normal saline, and the experimental group was treated with the PVNA-Z@Ag NPs composite hydrogel of G9 group, and the dressing was changed every 2 days. The wound healing was observed and recorded, and the results are shown in Figure 12 and Figure 13 .

[0071] The results of the normal mouse wound model closure rate: From the qualitative and quantitative results of the acute wound closure rate of normal mice, it can be found that the closure speed of the wound treated with G9 group PVNA-Z@Ag NPs composite hydrogel dressing is significantly faster than that of the control group. And from the statistical point of view, until the wound is completely closed, the closure rate results of the two have significant differences.

[0072] The results of the diabetic mouse wound model closure rate: On the first day, due to the large wound area, more tissue exudate, and other reasons, the interaction force between the dressing and the wound is larger, in order to avoid the possibility of secondary damage and considering that there is no cytotoxicity within 48 hours, therefore, the dressing is not removed on the first day. The corresponding naked eye cannot directly observe the real situation of wound closure. The actual situation is shown in the first day of normal mice and diabetic mice. Two days later, the wound area and exudate are reduced, and the water is wetted to remove it smoothly. After that, the first day situation will not occur again. According to the above situation, therefore, the first day situation is not analyzed in the chart analysis.

[0073] From the qualitative and quantitative results of the acute wound closure rate of diabetic mice, it can be found that the closure speed of the wound treated with G9 group PVNA-Z@Ag NPs composite hydrogel dressing is significantly faster than that of the control group. And from the statistical point of view, until the wound is completely closed, the closure rate results of the two have significant differences.

[0074] 3) Tissue section staining When the wound surface treated with PBS and G9 group PVNA-Z@Ag NPs composite hydrogel dressing has been completely closed, the internal situation of the tissue is analyzed. The removed skin is fixed with a 4% polyformaldehyde solution, and then embedded with paraffin. After staining the tissue sections with hematoxylin and eosin (H&E) and Masson staining, histological analysis and photographing are performed.

[0075] The results of H&E and Masson staining of the normal mouse acute wound model are shown in Figure 14 , as follows: The H&E staining results show that the skin of the experimental group completely recovered, the epidermis thickness was normal, the dermal collagen fibers were arranged closely and orderly, the number of accessory structures was large, and no obvious pathological changes were observed. In contrast, although the skin damage of the control group was obviously recovered, the regenerated epidermis was thicker, the dermal collagen fibers were arranged more loosely, the skin accessory structures such as hair follicles and sebaceous glands also had obvious regeneration, and more inflammatory cells were observed in the dermis and subcutaneous tissue. In addition, Masson staining can stain collagen fibers blue, which can further prove that the collagen fibers of the experimental group are more abundant and arranged in order than those of the control group. In summary, the 9 group PVNA-Z@Ag NPs composite hydrogel dressing has obvious help for the internal tissue repair of the normal mouse wound.

[0076] H&E and Masson staining results of acute wound model of diabetic mice are shown as follows: Figure 15 When the epidermis of the skin damage of the control group is completely repaired, the newly generated epidermis is thicker, and the dermis layer is filled with a large amount of granulation tissue, which is mainly composed of collagen fibers and newly generated blood vessels. The collagen fibers are arranged in disorder, accompanied by a small amount of inflammatory cell infiltration, and a small amount of regenerated hair follicles. In contrast, when the epidermis of the skin damage of the experimental group is completely repaired, the newly generated epidermis is not different from the normal epidermis, the collagen fibers of the dermis are arranged in order and closely, and a large amount of newly generated hair follicles and sebaceous glands can be seen, which can prove that the recovery is better than that of the control group.

[0077] In vivo biocompatibility: heart, liver, spleen, lung and kidney of normal mice, untreated diabetic mice and diabetic mice treated with G9 PVNA-Z@Ag NPs composite hydrogel dressing were taken for tissue section and H&E staining for in vivo toxicity evaluation. All mice were sacrificed by anesthesia and cervical dislocation, and then the heart, liver, spleen, lung and kidney were taken out, fixed with 4% polyformaldehyde solution, and then embedded with paraffin. After staining the tissue sections with hematoxylin and eosin (H&E), histological analysis and photographing were performed.

[0078] In vivo biocompatibility of acute wound model of normal mice is shown as follows: Figure 16 The heart, liver, spleen, lung and kidney of the control group and the experimental group mice are shown in the above figure. The internal organs of the two groups of mice are not obviously abnormal, so it can be determined that the 9 PVNA-Z@Ag NPs composite hydrogel dressing has no in vivo toxicity to normal mice

[0079] In vivo biocompatibility of acute wound model of diabetic mice is shown as follows: Figure 17 The heart, liver, spleen, lung and kidney of the control group and the experimental group are basically the same: the structure of the heart and the spleen is completely normal; there is a small amount of inflammatory cell infiltration in the lung; interstitial inflammatory cell infiltration can be seen in the kidney; hepatocellular ballooning and inflammatory cell infiltration can be seen in the liver. Since the lung, kidney and liver of the two groups are consistent in pathological changes, these pathological changes can be inferred to be caused by diabetes in mice, which can be considered as background pathological changes. In addition to the above background pathological changes, the experimental group does not have other pathological changes compared with the control group, so it can be basically determined that the 9 PVNA-Z@Ag NPs composite hydrogel dressing has no in vivo toxicity.

[0080] ​​​The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A hydrogel dressing for chronic wounds in diabetes, characterized in that, is that the pH responsive agent, the temperature-sensitive agent, the acrylamide and the crosslinking agent are mixed, and then the polymer chain segment is obtained under ultraviolet irradiation; the ZIF-8@Ag NPs dispersion liquid and the photoinitiator are added, and the ZIF-8@Ag NPs and the polymer chain segment are connected through hydrogen bond and electrostatic interaction to obtain a pre-gel suspension; and the pre-gel suspension is solidified to obtain the hydrogel dressing; The mass ratio of the temperature-sensitive agent, the pH responsive agent and the acrylamide is 1:2-10:2-8; and the mass ratio of the crosslinking agent, the photoinitiator and the acrylamide is 0.001-0.1:0.001-0.1:2-8. The concentration of the ZIF-8@Ag NPs in the pre-gel suspension is 0.001g / mL-0.08g / mL. The pH responsive agent is 1-vinylimidazole, and the temperature-sensitive agent is N-isopropyl acrylamide.

2. The hydrogel dressing of claim 1, wherein, The ZIF-8@Ag NPs dispersion liquid is obtained by dispersing ZIF-8@Ag NPs in water. The ZIF-8@Ag NPs are prepared by a solution chemical method under the reduction of a reducing agent.

3. The hydrogel dressing of claim 1, wherein, The crosslinking agent is N,N-methylenebisacrylamide, and the photoinitiator is 2-hydroxy-2-methylpropiophenone.

4. A method of preparing a hydrogel dressing according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: A one-pot method is adopted, and N-isopropyl acrylamide, 1-vinylimidazole and acrylamide are added into water in a mass ratio of 1:2-10:2-8, and N,N-methylenebisacrylamide, 2-hydroxy-2-methylpropiophenone and acrylamide are added into water in a mass ratio of 0.001-0.1:0.001-0.1:2-8, and then stirring is performed until complete dissolution; ZIF-8@Ag NPs are added and uniformly dispersed to obtain a pre-gel suspension; The pre-gel suspension is solidified to obtain the hydrogel dressing.

5. The preparation method according to claim 4, characterized in that, The solidification is performed by ultraviolet curing.

6. The preparation method according to claim 4, characterized in that, The mass-volume ratio of 1-vinylimidazole to water is 1:5-20.

7. The preparation method according to claim 4, characterized in that, The specific obtaining process of the ZIF-8@Ag NPs is as follows: ZIF-8 is added into an AgNO3 solution, stirring is performed for 6h-24h to obtain a suspension, centrifugation is performed, a supernatant is taken, alcohol washing is performed, and then an initial product is obtained after drying; wherein the concentration of the AgNO3 solution is 0.02mol / L-0.2mol / L, and the mass ratio of AgNO3 to ZIF-8 is 1:4-10; A sodium borohydride solution is mixed with the initial product as a reducing agent, and stirring is performed for 0.5h-2h, centrifugation is performed, a precipitate is taken, washing is performed, and then drying is performed to obtain the ZIF-8@Ag NPs; wherein the concentration of the sodium borohydride solution is 0.001g / mL-0.01g / mL.

8. The preparation method according to claim 7, characterized in that, The alcohol washing agent is methanol.

9. Use of the hydrogel dressing according to any one of claims 1-3 in the preparation of a wound healing auxiliary material.

10. Use according to claim 9, characterized in that, The wound is a wound caused by diabetes.

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