A hydrogel material that promotes diabetic wound repair and degrades on demand, and its preparation method and application

Abstract

The present invention is a hydrogel material that promotes diabetic wound repair and is degraded on demand, and its preparation method and application, and discloses a preparation method of a polyethylene glycol hydrogel material that is degraded on demand, comprising the following steps: S1. reacting a multi-arm polyethylene glycol amine solution with a cross-linking agent to obtain a modified polyethylene glycol amine; S2. mixing the modified polyethylene glycol amine, polydopamine and Prussian blue nanoparticles, and stirring to react; S3. causing the mixture of step S2 to undergo an oxidation reaction with an oxidant to obtain the polyethylene glycol hydrogel material; wherein the cross-linking agent is a selenolactone compound having the following structure: n is selected from any integer of 1 to 3; X is selenium, sulfur or tellurium; and the multi-arm polyethylene glycol amine is a three-arm polyethylene glycol amine, a four-arm polyethylene glycol amine or a six-arm polyethylene glycol amine. The polyethylene glycol hydrogel material prepared by the present invention has excellent anti-inflammatory, antioxidant and antibacterial properties, can be degraded on demand, and can significantly accelerate the healing of diabetic wounds.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials and regenerative medicine, and in particular to an on-demand degradable hydrogel material for promoting diabetic wound repair, and a preparation method and application thereof. Background Art

[0002] Diabetic wounds are one of the most common complications of diabetes, which can lead to serious morbidity, mortality and amputation. Its susceptibility to infection and immune dysfunction are one of the key factors that seriously hinder the healing of diabetic wounds. Infection often leads to serious consequences, and patients with diabetic wound infections have a greatly increased risk of hospitalization compared with non-infected patients. In addition to infection, the oxidative microenvironment and persistent inflammation also hinder the recovery of diabetic wounds. Under diabetic conditions, immune cells produce more reactive oxygen species (ROS), which cause damage to body organs and hinder the healing of diabetic wounds. The inflammatory regulation function is impaired in diabetic wounds, and persistent inflammation also hinders the healing of diabetic wounds. Therefore, there is an urgent need to develop a treatment method that can resist infection, antioxidant and regulate immune function to effectively treat diabetic wounds.

[0003] Hydrogels have been widely used in the repair of diabetic wounds due to their unique advantages. The continuous porous structure of hydrogels can provide a long-lasting moist environment for wound healing, allowing oxygen penetration and inhibiting bacterial proliferation. Traditionally, research on hydrogel wound dressings has focused on delivering drugs, growth factors or cytokines to accelerate wound closure and the regeneration process of skin tissue. However, in recent years, various inorganic nanomaterials and nanotechnology-driven therapies have received more extensive attention in skin wound healing.

[0004] Prussian blue (PB) is an iron-based nanozyme. It has been reported that PB nanozymes not only exhibit hydrogen peroxide (H2O2) degradation activity, but also exhibit strong superoxide scavenging ability, ROS scavenging ability and significant anti-inflammatory activity. In addition, under the irradiation of near-infrared light, PB nanozymes also exhibit good photothermal properties and can kill bacteria efficiently and quickly. Summary of the invention

[0005] The purpose of the present invention is to provide a hydrogel dressing for diabetic wound repair, which has excellent anti-inflammatory, antioxidant and antibacterial properties, can be degraded on demand, and can significantly accelerate the healing of diabetic wounds.

[0006] To solve the above technical problems, the first aspect of the present invention provides a polyethylene glycol hydrogel material loaded with Prussian blue nanoparticles. The hydrogel material is prepared by a one-pot process using commercial multi-arm polyethylene glycol amine (n-PEG-NH2), selenolactone compounds, polydopamine nanoparticles (PDANPs) and Prussian blue nanoparticles (PBNPs) as raw materials. The preparation method is simple, without organic solvent residue, mild reaction conditions, simple operation and low raw material cost.

[0007] Specifically, the preparation method of the polyethylene glycol hydrogel material comprises the following steps:

[0008] S1. reacting a polyethylene glycol solution having an amino group terminal with a cross-linking agent to obtain a modified polyethylene glycol;

[0009] S2. mixing the modified polyethylene glycol and polydopamine nanoparticles and Prussian blue nanoparticles of step S1, and stirring to react;

[0010] S3. allowing the mixture of step S2 to undergo an oxidation reaction with an oxidant to obtain the polyethylene glycol hydrogel material;

[0011] Wherein, the cross-linking agent is a lactone compound having a structure shown in formula (I):

[0012]

[0013] n is selected from any integer of 1 to 3; X is sulfur, selenium or tellurium;

[0014] The polyethylene glycol is three-arm polyethylene glycol, four-arm polyethylene glycol or six-arm polyethylene glycol.

[0015] In the present invention, firstly, modified polyethylene glycol is generated through the amino ring-opening lactone of multi-arm polyethylene glycol, and then the modified polyethylene glycol and polydopamine nanoparticles undergo Michael addition reaction; finally, the polymer is oxidized to obtain a cross-linked polyethylene glycol hydrogel material, and Prussian blue nanoparticles are loaded therein.

[0016] Furthermore, in step S1, the polyethylene glycol is four-arm polyethylene glycol with a molecular weight of 500-50000; preferably, the molecular weight of the polyethylene glycol is 2000-20000; more preferably, the molecular weight of the polyethylene glycol is 10000.

[0017] Furthermore, the concentration of the polyethylene glycol solution is 1 to 80 wt %; preferably, the concentration of the polyethylene glycol solution is 5 to 30 wt %; more preferably, the concentration of the polyethylene glycol solution is 10 wt %.

[0018] Furthermore, in step S1, the solvent in the polyethylene glycol solution includes water or a phosphate buffer solution; and the pH value of the phosphate buffer solution is 7.0 to 8.0.

[0019] Preferably, the pH value of the phosphate buffer solution is 7.4.

[0020] Furthermore, in step S1, the reaction temperature is 0-100°C, and the reaction time is 1-12 hours. Preferably, the reaction temperature is 15-30°C, and the reaction time is 6-12 hours.

[0021] Furthermore, in step S1, the molar ratio of the polyethylene glycol to the cross-linking agent is 1:2 to 4, preferably 1:4.

[0022] Furthermore, in step S2, polydopamine nanoparticles are added to the modified polyethylene glycol and stirred for 5-10 minutes.

[0023] Furthermore, in steps S1 and S2, the reaction is carried out under a nitrogen atmosphere or an argon atmosphere.

[0024] Furthermore, in step S2, the mass ratio of the modified polyethylene glycol to the polydopamine nanoparticles is 10000:1-1000, and the mass ratio of the modified polyethylene glycol to the Prussian blue nanoparticles is 10000:1-80;

[0025] Preferably, the mass ratio of the modified polyethylene glycol to the polydopamine nanoparticles is 1000:1-100;

[0026] Preferably, the mass ratio of the modified polyethylene glycol to polydopamine nanoparticles is 100:1;

[0027] Preferably, the mass ratio of the modified polyethylene glycol to the Prussian blue nanoparticles is 10000:1-8, for example, 10000:1, 10000:2, 10000:5, 10000:8, etc.

[0028] Further, in step S3, the oxidant includes any one of air, oxygen, sodium hypochlorite, and hydrogen peroxide;

[0029] Preferably, the oxidant is selected from air and / or oxygen.

[0030] Furthermore, in the oxidation reaction, air and / or oxygen are used in excess.

[0031] Furthermore, in step S3, the reaction temperature is 0-80°C, and the reaction time is 4-24h.

[0032] The second aspect of the present invention provides a polyethylene glycol hydrogel material that can be degraded on demand and is prepared by the method.

[0033] The third aspect of the present invention provides the use of the polyethylene glycol hydrogel material as a medical dressing, especially as a diabetic wound repair dressing.

[0034] Diabetic wounds can cause persistent inflammation, and the PB nanoparticles released slowly in the polyethylene glycol hydrogel dressing can reduce the burden of macrophages at the wound, inhibit the secretion of inflammatory factors, reduce inflammatory reactions, and are conducive to diabetic wound healing. Diabetic wounds usually have an unbalanced wound microenvironment, resulting in the production of reactive oxygen species (ROS) such as excessive hydrogen peroxide, hydroxyl radicals and superoxide anions. The PB nanoparticles released slowly in the hydrogel dressing reduce the generation of ROS by decomposing hydrogen peroxide and removing superoxide anion radicals, thereby achieving an antioxidant effect and promoting the repair and regeneration of diabetic wounds. Therefore, the polyethylene glycol hydrogel material loaded with Prussian blue nanoparticles of the present invention is particularly suitable for use as a dressing for diabetic wound repair.

[0035] Furthermore, when the polyethylene glycol hydrogel material loaded with Prussian blue nanoparticles is used, the degradation rate of the hydrogel material is adjusted by light irradiation to achieve controlled sustained release of the Prussian blue nanoparticles.

[0036] The polyethylene glycol hydrogel material loaded with Prussian blue nanoparticles of the present invention, PB nanoparticles can be well loaded in the hydrogel, and degraded and slowly released on the wound by means of illumination within a certain period of time, ensuring that the wound has been supplemented with sufficient PB nanoparticles for a period of time, thereby achieving continuous regulation of the diabetic wound microenvironment, preventing infection, and promoting repair, which provides a longer healing time for the wound, and avoids the pain caused to the patient by continuous replacement of dressings. At the same time, as a material with a high water content, the hydrogel provides a moist repair and healing environment for chronic wound ulcers of diabetes, reducing the itching, pain and other problems caused by dryness of the wound in the recovery stage. In addition, since diabetic wounds are susceptible to infection, the PB nanoparticles released slowly in the hydrogel dressing are irradiated with 730nm red light, and through photothermal properties, a bactericidal effect is achieved, infection is prevented, and it is beneficial to diabetic wound healing.

[0037] The above technical solution of the present invention has the following advantages compared with the prior art:

[0038] 1. The present invention uses commercial multi-arm polyethylene glycol amine (n-PEG-NH2), selenolactone compounds, polydopamine nanoparticles (PDANPs) and Prussian blue nanoparticles (PBNPs) as raw materials and is prepared by one-pot cooking. The preparation method is simple, no organic solvent residue, mild reaction conditions, simple operation, and low raw material cost.

[0039] 2. The polyethylene glycol hydrogel material prepared in the present invention has a high water content and exhibits the behavior of releasing PB nanoparticles on demand over a long period of time. Therefore, when used for the treatment of difficult-to-heal diabetic wounds, it exhibits a strong ability to promote wound healing, regulate the microenvironment at the wound (anti-oxidation and anti-inflammatory), prevent bacterial infection and tissue repair, and can effectively relieve the patient's pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the reaction equation of DSeP@PB hydrogel;

[0041] Figure 2 The state diagram of the hydrogel before and after gelation prepared by the inverted bottle method;

[0042] Figure 3 This is the rheological test diagram of the hydrogel;

[0043] Figure 4 is the swelling ratio diagram of the hydrogel;

[0044] Figure 5 Schematic diagram and data of the photothermal controlled release of Prussian blue from hydrogel: the left figure shows the change of temperature over time: 5 minutes of light exposure + 10 minutes without light exposure, 4 cycles; the right figure is the kinetic diagram of the photo-thermal effect, and the temperature reaches equilibrium after 15 minutes;

[0045] Figure 6 This is the process of light-controlled drug release. When the light is on all the time, the drug release is the fastest. When the light is off, the black line is the slowest. When the light is on and off, the blue line cycles every 2 minutes.

[0046] Figure 7 The bactericidal effect of hydrogels: (A) Bacteria and hydrogels were co-cultured with colony-forming units after illumination; (B) Quantitative analysis results; (C) Representative transmission electron microscopy images of bacteria after contact with hydrogels;

[0047] Figure 8 The biocompatibility test diagram of the hydrogel: (A) Live / dead cell staining diagram of NIH-3T3 cells after co-culture with hydrogel samples for 24 hours; (B) Cell viability of NIH-3T3 cells after co-culture with Prussian blue nanoparticles and hydrogel samples for 24 hours;

[0048] Fig. 9 The antioxidant effect of the hydrogel is shown in Figure 1: (A) ROS fluorescence image of cells after treatment with different samples under hydrogen peroxide conditions; (B) Viability of NIH-3T3 fibroblasts treated with different samples under hydrogen peroxide conditions;

[0049] Fig.10Diagram of the wound repair process in mice with diabetes: (A) Representative images of the wound healing process in mice treated with control group, DSeP, DSeP+RL, PB, PB+RL, DSeP@PB, and DSeP@PB+RL; (B) Wound closure rate at different time points;

[0050] Fig.11 Pathological examination of wound tissue: (A) H&E staining of wound; (B) Masson staining of wound tissue sections; (C) quantitative analysis of epidermal thickness; (D) semi-quantitative analysis of collagen deposition;

[0051] Fig.12 Capillary and vascular growth in wound tissue: (A) Fluorescent staining of wound tissue sections using CD31 antibody and a-SMA antibody; (B) Semi-quantitative analysis results of CD31 antibody; (C) Semi-quantitative analysis results of a-SMA antibody;

[0052] Fig.13 Inflammatory conditions of wound tissue: (A) Fluorescent staining of wound tissue sections using TNF-a antibody and F4 / 80 antibody; (B) Semi-quantitative analysis results of TNF-a; (C) Semi-quantitative analysis results of F4 / 80. DETAILED DESCRIPTION

[0053] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0055] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

[0056] The fibroblast NIH-3T3 of the present invention was purchased from Shanghai Meiyan Biotechnology Co., Ltd. Hydrogen peroxide (H2O2) was purchased from Shanghai Maokang Biotechnology Co., Ltd.; and the ROS detection kit was purchased from Shanghai Biyuntian Co., Ltd.

[0057] The equipment used in the present invention, the field emission scanning electron microscope, was purchased from Hitachi, Japan; the rheometer was purchased from HAAKE, Germany; the microplate reader was purchased from Molecular Devices, USA; the living cell workstation was purchased from Leica, Germany; the confocal microscope and the inverted microscope were purchased from Olympus, Japan.

[0058] This study was approved by the Animal Ethics Committee of Mudanjiang Medical College, and all animal care, husbandry, and sacrifice procedures were carried out in accordance with the national animal experiment regulations, the Regulations on the Administration of Laboratory Animals (revised in 2017).

[0059] All statistical significance measurements of the present invention were performed using analysis of variance (ANOVA) followed by Dunnett's test. A P value < 0.05 was considered statistically significant.

[0060] Example 1: Synthesis of DSeP@PB(0.1) hydrogel

[0061] Under argon protection, selenolactone (60 mg) was added dropwise to a polyethylene glycol aqueous solution (10 g, 10 wt%), and the mixture was reacted at 25° C. for 10 h to obtain selenol-modified polyethylene glycol.

[0062] Selenol-modified polyethylene glycol and polydopamine nanoparticles were reacted at a mass ratio of [polyethylene glycol] 0: [polydopamine nanoparticles] 0: [Prussian blue nanoparticles] 0 = 1000:10:1 to obtain polyethylene glycol hydrogel (DSeP@PB (0.1)).

[0063] Example 2: Synthesis of DSeP@PB(1) hydrogel

[0064] In this embodiment, the mass ratio of [polyethylene glycol] 0: [polydopamine nanoparticles] 0: [Prussian blue nanoparticles] 0 = 1000:10:10, and the other reaction steps and reaction conditions are the same as those in Example 1 to obtain polyethylene glycol hydrogel DSeP@PB (1).

[0065] Example 3: Synthesis of DSeP@PB(8) hydrogel

[0066] In this example, the mass ratio of [polyethylene glycol] 0: [polydopamine nanoparticles] 0: [Prussian blue nanoparticles] 0 = 1000:10:80, and other reaction steps and reaction conditions were the same as in Example 1 to obtain polyethylene glycol hydrogel DSeP@PB (8).

[0067] Comparative Example 1: Synthesis of DSeP hydrogel

[0068] Under argon protection, selenolactone (60 mg) was added dropwise to a polyethylene glycol aqueous solution (10 g, 10 wt%), and the mixture was reacted at 25° C. for 10 h to obtain selenol-modified polyethylene glycol.

[0069] Selenol-modified polyethylene glycol and polydopamine were reacted at a mass ratio of [polyethylene glycol] 0:[polydopamine nanoparticles] 0 = 1000:10 to obtain polyethylene glycol hydrogel (DSeP).

[0070] Performance Testing and Characterization

[0071] The relevant properties of the DSeP@PB hydrogel prepared in Example 1 and the DSeP hydrogel prepared in Comparative Example 1 were tested as follows.

[0072] 1. Light-controlled drug release

[0073] like Figure 6 As shown in the figure, the drug release rate of DSeP@PB hydrogel is the fastest when it is always illuminated, and the slowest when it is always dark. When the illumination and no illumination cycle is every 2 minutes, the drug release rate is in the middle. This shows that the degradation rate of the hydrogel can be controlled by illumination, thereby achieving controlled sustained release of the drug.

[0074] (II) Antibacterial performance test

[0075] 0.3 g of DSeP, PB, and DSeP@PB were added to 1 mL of bacterial solution (Escherichia coli and Staphylococcus aureus) and exposed to light (730 nm, 1 W / cm 2 ). Then 40 μL of the solution was extracted, diluted 1000 times, and smeared on the plate. After 16 hours of incubation, the plate was imaged and counted. These experiments were repeated more than three times. Transmission electron microscopy was used to capture the morphological changes of bacteria after different sample treatments.

[0076] like Figure 7 As shown in A, the number of colonies in the DSeP@PB+RL group was the lowest, followed by PB+RL, and there were no significant differences among the other groups. Figure 7 The semi-quantitative analysis of B also illustrates this point. Figure 7 As shown in Figure C, the structure of the bacterial samples in the DSeP@PB+RL group has been severely damaged, followed by the PB+RL group, while the colonies in the control group have a smooth appearance and good morphology.

[0077] This shows that the hydrogel without PB nanoparticles and the samples without light treatment have no antibacterial function, while the PB nanoparticles and DSeP@PB hydrogel with light treatment have significant antibacterial effect.

[0078] (III) Biocompatibility of hydrogels

[0079] Live and dead cell staining:

[0080] The biocompatibility of the prepared DSeP@PB hydrogel dressings was tested, and each prepared gel was sterilized by ultraviolet irradiation for 30 min before the cell experiment.

[0081] The dual fluorescent dye method was used to image bacterial viability. In particular, the DSeP, PB, and DSeP@PB treatments were used, and PBS treatment was used as the control group. The RL treatment group was imaged with red light (1 W / cm 2 After 24 h of co-culture, the cells were co-stained with green fluorescent dye (calcein-am) and red fluorescent dye (PI) at 37°C for 20 min according to the instructions of the live-dead cell staining kit. NIH-3T3 cells treated with different methods were imaged under a live cell workstation. Figure 8 A.

[0082] MTT:

[0083] Mouse fibroblasts (NIH-3T3) were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin and incubated in a CO2 incubator at 37°C for 16 h. To determine the in vitro cytotoxicity of PB nanoparticles, NIH-3T3 cells were seeded in 96-well tissue culture plates (5000 cells / well). After cell attachment, different concentrations of PB nanoparticles, DSeP hydrogel, and DSeP@PB hydrogel were added to the cells, respectively. PBS treatment was used as the control group, and the RL treatment group was illuminated with red light (1 W / cm 2 , 5min) irradiation. After 24h of culture, the cells were washed with fresh medium containing MTT reagent at 37°C and incubated for 4h. Finally, the absorbance of formazan crystals dissolved in DMSO was measured at a wavelength of 490nm using an ELISA reader. The cell survival rate was then calculated based on the absorbance. The calculation results are shown in Figure 8 B.

[0084] The biocompatibility of the dressing was evaluated based on the results of co-culture of the hydrogel with HIN3T3 cells. Figure 8 A is a confocal image of live and dead cell staining, in which many live cells but no dead cells can be seen in the field of view. Figure 8 B is the result of MTT method to detect cell survival rate. There is no significant difference between the cell survival rate after different treatments and different concentrations and the control group. It can be seen that DSeP@PB hydrogel has good biocompatibility and no cytotoxicity.

[0085] (IV) Intracellular ROS determination

[0086] In order to verify the ability of DSeP@PB hydrogel to remove ROS and reduce oxidative stress, the present invention used DCFH-DA as a fluorescent probe to detect the level of ROS in cells. NIH-3T3 cells attached to six-well plates were exposed to 1mM hydrogen peroxide, and DSeP, PB nanoparticles, DSeP@PB, PB were added. PBS was used as a control treatment, and the RL group was irradiated with red light (730nm, 1W / cm 2 After 24 h of treatment, the cells were washed and incubated with 10 μM DCFH-DA for 30 min. Finally, the fluorescence signal in the cells was observed using a living cell workstation.

[0087] like Fig. 9 As shown in A, after hydrogen peroxide treatment, the intracellular fluorescence signal is higher, corresponding to the increase in intracellular ROS levels. In the presence of PB and DSeP@PB, the fluorescence signal is significantly reduced, indicating that the sustained-release PB nanoparticles in the hydrogel of the present invention have effective ROS scavenging activity.

[0088] (V) Cytoprotective Effect

[0089] To verify the cytoprotective effect of PB nanoparticles, NIH-3T3 cells were incubated with hydrogen peroxide to simulate a ROS environment. NIH-3T3 cells were grown in a 96-well plate and treated with DSeP, PB, and DSeP@PB hydrogels, respectively. The RL group was exposed to light and then treated with 1 mM hydrogen peroxide. The PBS treatment was the control group. After incubation for 24 h, the cells were washed and the cell viability was determined by the MTT method.

[0090] like Fig. 9 As shown in Figure B, with the increase of hydrogen peroxide concentration, cell viability decreased significantly. Treatment with PB+RL and DSeP@PB+RL increased cell viability.

[0091] (VI) In vivo experiments in diabetic mice

[0092] Eight-week-old male ICR mice were intraperitoneally injected with 100 mg / kg streptozotocin (STZ) after fasting, followed by continuous injections of 50 mg / kg for 3 consecutive days to induce a type I diabetes model. Mice with blood glucose levels exceeding 16.7 mM were considered diabetic. A total of 63 diabetic mice were used. Two circular full-thickness skin wounds (φ6 mm) were formed on the back of each mouse. The mice were then randomly divided into 7 groups, namely PBS control group, DSeP, DSeP+RL, PB, PB+RL, DSeP@PB, and DSeP@PB+RL.

[0093] After the wounds were treated as described above, they were bandaged with medical transparent tape. The wound size was photographed at fixed time intervals, and the wound size was measured and analyzed using a standard ruler and image processing software (Imagej). Fig.10 As shown in A, Fig.10 B is the wound healing rate at different time points.

[0094] First, from Fig.10 It can be seen from the control group that the mice have a certain self-healing ability; secondly, the wound area of ​​the mice in other groups at different times is smaller than that of the control group, indicating that the DSeP@PB hydrogel dressing has a significant effect on promoting the healing of diabetic wounds in mice, and light assistance has the best effect on promoting the healing of diabetic wounds.

[0095] 7. Histological Detection of Wound Healing Sites in Mice with Diabetes

[0096] The specific operation was as follows: on the 7th, 14th, and 21st days after wound induction, three diabetic mice in each group were killed by cervical dislocation, the wounds were excised, and then subsequent experiments were performed. The excised wound tissues were randomly divided into two groups, one group was fixed with formaldehyde for 24 hours and then embedded in paraffin, and the other group was fixed with OTC glue and quickly frozen in liquid nitrogen and stored at -80°C.

[0097] Paraffin sections were cut into 5 μm thin slices and then subjected to histopathological studies: (1) epidermis was observed by hematoxylin and eosin staining (H&E); (2) collagen deposition was observed by Masson staining;

[0098] The frozen sections were cut into 5 μm thin layers and then subjected to histopathological studies: (1) CD31 and a-SMA fluorescent staining of endothelial cells were used to observe angiogenesis; (2) TNF-a fluorescent staining of inflammatory factors and F4 / 80 fluorescent labeling of macrophages were used for inflammation diagnosis.

[0099] The stained tissue samples were observed and photographed under an optical microscope and a confocal microscope, and the cell number was counted using image processing software (ImageJ) for semi-quantitative analysis. (The above detection methods are all standard detection methods)

[0100] In wound healing, the wound healing process can be divided into the inflammatory response phase, the granulation phase and the epithelial formation phase, and each phase overlaps with each other. Fig.11 As shown in A, after 3 weeks of treatment, the wounds in each group were significantly healed and covered with new epidermis. However, the thickness and differentiation status of the new epidermis were different among the groups. Compared with the control group, the epidermal thickness in the DSeP group was significantly reduced, which was due to the higher maturity of the regenerated tissue at the wound edge ( Fig.11C), indicating that wound regeneration was enhanced compared with the control group, especially the DSeP@PB group. In addition, there were a large number of skin appendages in the tissues of the DSeP@PB+RL group, while no or a small number of skin appendages were found in the other groups. The H&E staining results suggest that the wounds treated with DSeP@PB+RL can achieve a relatively satisfactory healing effect, not only with a fast healing speed, but also with the formation of skin appendages.

[0101] During the wound healing process, collagen synthesized by fibroblasts is the main component of the extracellular matrix. The presence of collagen in the wound site is crucial to the final repair of the wound. Fig.11 Masson staining in B showed that collagen deposition was observed in the wounds treated with different methods. On day 21, the newly formed collagen increased in all groups. Nevertheless, the collagen deposition in the DSeP@PB group and the DSeP@PB+RL group was greater than that in the other groups ( Fig.11 D). In addition, a large number of skin appendages can also be clearly seen in the DSeP@PB+RL group, which confirms the results of the above H&E staining.

[0102] In summary, the histological results provided by H&E and Masson staining indicate that the DSeP@PB hydrogel dressing is very suitable for diabetic wounds. After exposure to light, it can better promote epidermal formation and differentiation, promote collagen formation, produce skin appendages, and ultimately accelerate the healing process of diabetes.

[0103] 8. Angiogenesis in diabetic wounds of mice

[0104] Due to wound hypoxia, blood vessel formation is a prerequisite for fibroblasts to form granulation tissue to fill the wound. The present invention uses antibodies CD31 and a-SMA for immunofluorescence staining to evaluate the neovascularization of 7 groups of wound tissues for 7 days, and observes the mode and degree of neovascularization in different treatment groups. The present invention uses CD31 antibody staining to detect capillaries in the wound area. Fig.12 As shown in A, compared with the control group, the expression of CD31 in the DSeP@PB group and the DSeP@PB+RL group was significantly higher than that in the other groups, and the expression in the DSeP@PB+RL group was the highest. Fig.12 This was also confirmed by the semi-quantitative results of B.

[0105] In addition, if Fig.12 As shown in A, compared with the control group, the number of arterioles in the DSeP group increased significantly, with the largest number in the DSeP@PB+RL group. Fig.12 This was also confirmed by the semi-quantitative results of C.

[0106] These results suggest that DSeP@PB+RL can induce high levels of angiogenesis and neovascularization, thereby promoting successful wound healing through proper re-epithelialization, collagen deposition, and maturation of the skin layer.

[0107] (IX) Analysis of inflammation on diabetic wounds in mice

[0108] Diabetic wounds can cause persistent inflammation, and excessive inflammatory responses may delay wound healing and produce scar tissue after healing. The present invention stains TNF-α, a pro-inflammatory cytokine secreted by activated macrophages that participates in the acute phase of inflammation. Fig.13 As shown in A, after treatment with DSeP, the expression of TNF-α in the wound area was significantly reduced, and the expression in the DSeP@PB+RL group was the lowest ( Fig.13 B).

[0109] To analyze the extent of macrophage recruitment, the present invention used immunostaining of the F4 / 80 glycoprotein, which is expressed on the surface of macrophages and is commonly used to identify tissue macrophages. Fig.13 As shown in C, in the wound area of ​​the DSeP@PB+RL group, the percentage of F4 / 80 positive area was significantly lower than that of the other groups.

[0110] Therefore, the present invention confirms the effective anti-inflammatory effect of DSeP@PB+RL in vivo, which can reduce the macrophage burden in the infected wound model and prevent the secretion of pro-inflammatory cytokines. In summary, the anti-inflammatory activity of DSeP@PB hydrogels with the assistance of light may help prevent prolonged inflammation and promote effective wound healing.

[0111] Obviously, the above embodiments are merely examples for the purpose of clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A method for preparing a hydrogel material that can be degraded on demand, characterized in that: The following steps are involved: S1. reacting an amino-terminated polyethylene glycol solution with a cross-linking agent to obtain a modified polyethylene glycol; S2. The modified polyethylene glycol and polydopamine nanoparticles of step S1 are mixed with Prussian blue nanoparticles and stirred to react; S3. causing the mixture of step S2 to undergo an oxidation reaction with an oxidant to obtain the hydrogel material; Wherein, the cross-linking agent is a lactone compound having a structure shown in formula (I): ， n is selected from any integer between 1 and 3; X is selenium, sulfur or tellurium; The polyethylene glycol is three-arm polyethylene glycol, four-arm polyethylene glycol or six-arm polyethylene glycol.

2. The method for preparing a hydrogel material that can be degraded on demand according to claim 1, characterized in that: In step S1, the polyethylene glycol is four-arm polyethylene glycol with a molecular weight of 500-50000; the concentration of the polyethylene glycol solution is 1-80wt%.

3. The method for preparing a hydrogel material that can be degraded on demand according to claim 2, characterized in that: The molecular weight of the polyethylene glycol is 10,000; the concentration of the polyethylene glycol solution is 5-30 wt%.

4. The method for preparing a hydrogel material that can be degraded on demand according to claim 1, characterized in that: In step S1, the solvent in the polyethylene glycol solution is water or a phosphate buffer solution; The pH value of the phosphate buffer solution is 7.0-8.

0.

5. The method for preparing a hydrogel material that can be degraded on demand according to claim 1, characterized in that: In step S1, the molar ratio of polyethylene glycol to the cross-linking agent is 1:2-4.

6. The method for preparing a hydrogel material that can be degraded on demand according to claim 1, characterized in that: In steps S1 and S2, the reaction is carried out under a nitrogen atmosphere or an argon atmosphere.

7. The method for preparing a hydrogel material that can be degraded on demand according to claim 1, characterized in that: In step S2, the mass ratio of the modified polyethylene glycol and polydopamine nanoparticles to the Prussian blue nanoparticles is 10000:1-1000:1-80.

8. The method for preparing a hydrogel material that can be degraded on demand according to claim 7, characterized in that: The mass ratio of the modified polyethylene glycol and polydopamine nanoparticles to the Prussian blue nanoparticles is 10000:10-1000:1-8.

9. The method for preparing a hydrogel material that can be degraded on demand according to claim 1, characterized in that: In step S3, the oxidant includes any one of air, oxygen, sodium hypochlorite, and hydrogen peroxide.

10. The method for preparing a hydrogel material that can be degraded on demand according to claim 9, characterized in that: The oxidant is selected from air or oxygen.

11. A hydrogel material degradable on demand prepared according to the method according to any one of claims 1 to 10.

12. Use of the on-demand degradable hydrogel material according to claim 11 as a diabetic wound repair dressing.

13. The use according to claim 12, characterized in that: When the hydrogel material is in use, the degradation rate of the hydrogel material is adjusted by light irradiation to achieve controlled sustained release of Prussian blue nanoparticles.

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