Hydrogel with anti-infection and drug-loaded slow-release functions as well as preparation method and application of hydrogel

Abstract

The invention belongs to the field of hydrogel wound dressings, and particularly relates to hydrogel with anti-infection and drug-loaded slow-release functions, a preparation method and application. Based on HA-PBA (hyaluronic acid-phenylboronic acid) and four-arm PEG dopamine as a skeleton, the hydrogel dressing is formed through boric acid ester bond crosslinking; antibacterial peptides and drug-loaded micelles are doped in the hydrogel dressing, and the micelles load quercetin, so that the hydrogel dressing has a slow release function. The antibacterial peptide is combined with cell membranes of bacteria through the charge effect, the powerful anti-infection function is achieved, and breeding and colonization of pathogenic bacteria can be effectively inhibited. Quercetin plays an anti-inflammatory role through an Akt / STAT6 pathway, inflammatory response can be remarkably reduced, and tissue repair is promoted. In addition, the hydrogel has good biocompatibility and mechanical properties, and can provide a suitable moist environment for wounds and promote healing.

Description

Technical Field

[0001] The invention belongs to the field of hydrogel wound dressings, and in particular relates to a hydrogel with both anti-infection and drug-carrying sustained-release functions, a preparation method and application thereof. Background Art

[0002] With the development of social economy and the change of lifestyle, diabetes has become one of the chronic diseases with high morbidity and mortality rates worldwide. According to the report of the International Diabetes Federation, there are more than 400 million people with diabetes worldwide, and this number is still rising. Due to the long-term high blood sugar state, the functions of multiple systems of diabetic patients are impaired, especially the lesions of the nervous system and circulatory system are the most significant. In this case, the wound healing ability of diabetic patients is significantly reduced, and chronic wounds that are difficult to heal are easily formed.

[0003] The main reasons why diabetic wounds are difficult to heal include the following aspects: First, in a hyperglycemic environment, the functions of fibroblasts and keratinocytes are impaired, and the ability of cell proliferation and migration decreases, affecting wound healing. Secondly, diabetic patients often have peripheral vascular disease, and reduced blood flow leads to insufficient nutrition and oxygen supply in the wound area, hindering tissue repair. In addition, high blood sugar also inhibits the function of macrophages, affects the immune response, and increases the risk of infection. Finally, metabolic abnormalities and the accumulation of advanced glycation end products (AGEs) in diabetic patients will aggravate tissue damage and lead to persistent inflammatory responses.

[0004] Inflammation and infection are the two main obstacles to diabetic wound healing. Chronic inflammation can lead to persistent tissue damage and cell apoptosis, affecting the formation of new tissue. Infection not only aggravates the inflammatory response, but may also lead to more serious complications such as sepsis. Therefore, the development of wound dressings with anti-inflammatory and anti-infection functions is of great significance for the treatment of diabetic wounds.

[0005] At present, the treatment methods for chronic wounds in diabetes mainly include controlling blood sugar, local debridement, anti-infection, and promoting tissue regeneration. However, traditional wound dressings such as gauze and cellulose, although they can protect wounds and absorb exudates, have limited effects in anti-inflammation and anti-infection. In recent years, with the progress of materials science and biomedicine, various new wound dressings have been developed. Among them, dressings containing antibacterial drugs, antimicrobial peptides and anti-inflammatory drugs have attracted much attention. For example, silver ion dressings and dressings containing antibiotics can effectively kill bacteria, but they are prone to drug resistance and may be toxic to human cells. On the other hand, biomaterials such as hyaluronic acid (HA) and polyethylene glycol (PEG) have gradually become a research hotspot for wound dressings due to their good biocompatibility and adjustable physical and chemical properties. However, the functions of a single material are limited, and it is difficult to have the comprehensive properties of anti-inflammation, anti-infection and promotion of wound healing at the same time. Therefore, the development of multifunctional composite wound dressings has become an important direction of current research.

[0006] The centralized treatment methods in the current existing technologies have many defects. For example, when suturing after surgical resection, it cannot improve local microcirculation, is highly traumatic, and has a high recurrence rate. Traditional dressings also have the problems of single function, inability to self-degrade, limited efficacy, and the burden of frequent dressing changes. In addition, there are limitations of plant-derived drugs, including poor water solubility, short action time, and inability to achieve sustained-release effects. To address these problems, there is an urgent need for a hydrogel with both anti-infection and drug-carrying sustained-release functions, a preparation method, and its application. Summary of the invention

[0007] In view of the problems and shortcomings of the prior art, the present invention provides a method for preparing a hydrogel having both anti-infection and drug-carrying sustained-release functions, comprising the following steps:

[0008] S1: Dissolve HA in a solvent, adjust the pH, add EDC and NHS respectively, then add 3-APBA dissolved in dimethyl sulfoxide, stir to react, and dialyze to obtain HA-PBA;

[0009] S2: Dissolve 4A-PEG-COOH in a solvent, then drop the EDC solution into 4A-PEG-COOH, adjust the pH and stir to obtain a mixed solution, add NHS to the mixed solution, then add dopamine to the mixed solution for reaction, and obtain 4A-PEG-Dopa after dialysis and freeze-drying;

[0010] S3: Dissolve the HA-PBA obtained in S1 in a solvent, mix the HA-PBA solution with 4A-PEG-DA synthesized from 4A-PEG-COOH at a mass ratio of 5:1, and adjust the pH to obtain a hydrogel.

[0011] Furthermore, the mass ratio of HA to EDC to NHS described in S1 is 2.94:1.68:1:1.

[0012] Furthermore, the reaction time described in S1 is 48 h, the dialysis time is 5 days, and the pH is adjusted to 6.5.

[0013] Furthermore, the mass ratio of 4A-PEG-DA to EDC, NHS, and dopamine described in S2 is 1:1:0.001:0.002, and the pH is 4.75.

[0014] Furthermore, the reaction time of adding dopamine in S2 is 4 hours.

[0015] Furthermore, the pH described in S3 is 7.

[0016] A hydrogel with both anti-infection and drug-carrying sustained-release functions.

[0017] The invention discloses an application of a hydrogel having both anti-infection and drug-carrying sustained-release functions, wherein the hydrogel is applied in the preparation of wound dressings.

[0018] Technical Effects

[0019] (1) The hydrogel provided by the present invention has both anti-infection and drug-loaded sustained-release functions, and provides a step-by-step response multifunctional drug-loaded hydrogel drug delivery platform based on reversible boronate bonds; the platform can effectively carry QC (quercetin) through the intelligently designed hydrogel structure and accurately control the release order of the drug, thereby achieving multiple biological activities such as antibacterial, antioxidant, and anti-inflammatory; this platform provides a powerful solution in terms of structure and function, and provides new ideas and application prospects for the design of multifunctional hydrogel spatiotemporal controlled-release drug delivery systems.

[0020] (2) Through the preparation method of a hydrogel with both anti-infection and drug-carrying sustained-release functions provided by the present invention, the hydrogel formed by HA-PBA and four-arm PEG dopamine cross-linked by boronate bonds has good mechanical properties and adhesion. The introduction of antimicrobial peptides gives the hydrogel strong antibacterial properties; in the hydrogel, quercetin is encapsulated in drug-carrying micelles and gradually released through the sustained-release system to continuously exert anti-inflammatory effects, avoiding the disadvantages of frequent use of traditional anti-inflammatory drugs and reducing side effects.

[0021] (3) Through the application of a hydrogel with both anti-infection and drug-carrying sustained-release functions provided by the present invention, the hydrogel is of great significance for the treatment of chronic diabetic wounds. First, the synergistic effect of antimicrobial peptides and quercetin can effectively control inflammation and infection, break the vicious cycle of inflammation-infection, and significantly shorten the wound healing time. Secondly, the moist environment and suitable mechanical support provided by the hydrogel are conducive to cell migration and tissue reconstruction, and promote wound healing. Finally, the multifunctional properties of the composite dressing reduce the frequency of dressing changes during treatment, reduce the patient's pain and treatment costs. In addition, the invention also has broad application prospects. It is not only suitable for the treatment of chronic diabetic wounds, but also can be applied to other types of difficult-to-heal wounds, such as burns, bedsores and wound care after surgical operations. Through further research and clinical trials, it is expected that this multifunctional composite wound dressing will be brought to the market, providing a safe, effective and convenient treatment plan for the majority of patients, improving the success rate of wound healing and the quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without creative work. In the drawings:

[0023] Figure 1 The preparation route of HA-PBA and four-arm PEG dopamine of the present invention is shown;

[0024] Figure 2 The magnetic resonance hydrogen spectrum and synthesis schematic diagram of Example 1 of the present invention;

[0025] Figure 3 This is a physical and chemical characterization diagram of the hydrogel of Example 1 of the present invention;

[0026] Figure 4 This is a graph showing the antioxidant properties and biocompatibility of the hydrogel of Example 1 of the present invention;

[0027] Figure 5 This is a diagram of the antibacterial effect of the hydrogel in Example 1 of the present invention;

[0028] Figure 6 This is a schematic diagram of the hydrogel promoting angiogenesis in Example 1 of the present invention;

[0029] Figure 7 The immunomodulatory and anti-inflammatory mechanism diagram of Gel@AMP&MQ of the present invention;

[0030] Figure 8The in vivo experiment and pathological analysis diagram of the hydrogel of Example 1 of the present invention;

[0031] Fig. 9 This is a graph analyzing the cytokine levels in rat regenerated skin after treatment with the hydrogel of Example 1 of the present invention. DETAILED DESCRIPTION

[0032] The following will be combined with Example 1 of the present invention and the attached Figures 1 to 9 , the technical solution of the present invention is clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] In view of the problem that traditional antibiotics in the prior art are prone to drug resistance in the treatment of diabetic chronic wounds, the present invention introduces antimicrobial peptides. Antimicrobial peptides interact with the negative charges on the bacterial cell membrane, causing the rupture of the bacterial cell membrane and the leakage of cell contents, thereby killing bacteria;

[0034] The treatment of diabetic chronic wounds requires continuous drug action, but traditional drugs are difficult to provide long-term therapeutic effects due to their short action time. The present invention achieves the sustained release function of the drug by doping drug-loaded micelles in the hydrogel and loading quercetin inside the micelles. The drug-loaded micelles can gradually release quercetin locally in the wound, prolong the action time of the drug, ensure that the drug continues to exert anti-inflammatory and antioxidant effects for a long time, avoid frequent dressing changes, and improve the compliance and effect of treatment.

[0035] Therefore, the present invention provides a hydrogel having antibacterial, anti-inflammatory, antioxidant and tissue regeneration promoting properties, and the preparation method thereof comprises the following steps:

[0036] S1: HA was dissolved in deionized water, stirred and dissolved, pH was adjusted, EDC and NHS were added for activation, 3-APBA dissolved in dimethyl sulfoxide was added after 6 hours, stirred at room temperature, reacted for 48 hours, dialyzed with deionized water for 5 days to remove the reactants that were not fully reacted, and then freeze-dried to obtain HA-PBA;

[0037] S2: Dissolve 4A-PEG-COOH in deionized water, then dissolve EDC in deionized water, slowly drip into 4A-PEG-COOH to obtain a mixed system, adjust the pH of the system and stir, dissolve NHS in deionized water and add to the mixed system after adjusting the pH, react as a whole and adjust the pH to neutral; then add dopamine to the reaction system, react for 4 hours, dialyze with deionized water for 5 days to remove the reactants that have not reacted fully, and then freeze-dry to obtain 4A-PEG-Dopa;

[0038] S3: Dissolve the HA-PBA obtained in S1 into a phosphate buffer solution and stir thoroughly to dissolve, mix the HA-PBA solution with 4A-PEG-DA synthesized from 4A-PEG-COOH at a mass ratio of 5:1, and obtain a hydrogel when the pH is 7.0.

[0039] Example 1

[0040] S1: 2 g of HA was dissolved in 200 ml of deionized water, stirred and dissolved at 37 ° C, the pH of the solution was adjusted to 6.5, 1.14 g of EDC and 0.68 g of NHS were added to activate the carboxyl groups in HA, 3-APBA dissolved in dimethyl sulfoxide (DMSO) was added after 6 hours of activation, 3-APBA was 0.68 g, stirred at room temperature, fully reacted for 48 hours, and the reactant was dialyzed with deionized water for 5 days to remove the reactant that was not fully reacted, and then freeze-dried to obtain HA-PBA;

[0041] S2: Dissolve 0.1g of 4A-PEG-DA in 50ml of deionized water, dissolve 0.1g of EDC in 10ml of deionized water, and then slowly drip into 4A-PEG-COOH to obtain a mixed system, adjust the pH to 4.75 and stir, dissolve 0.1mg of NHS in 10ml of deionized water, add to the mixed system, react for 30min as a whole and adjust the pH to neutral to obtain a reaction system, at which point the carboxyl group of 4A-PEG-COOH is successfully activated; take 0.2mg of dopamine and add it to the reaction system, react for 4h, dialyze with deionized water for 5 days to remove the reactants that have not reacted fully, and then freeze-dry to obtain 4A-PEG-Dopa;

[0042] S3: Dissolve the HA-PBA of S1 in phosphate buffer solution (PBS, pH 7.2-7.4) and stir thoroughly to dissolve; mix HA-PBA and 4A-PEG-DA in a mass ratio of 5:1; when the pH is 7.0, a hydrogel is obtained.

[0043] Combination Figure 1 It can be obtained that the preparation routes of HA-PBA and four-arm PEG dopamine are combined Figure 2 Can get, Figure 2 In a, in the magnetic resonance hydrogen spectrum, a peak appears at 7.0-8.0 ppm, which represents that HA carries the protons on the benzene ring; Figure 2 In b, in the FTIR spectra of HA-PBA and PBA, 1583 cm -1 and 1350cm -1 The peaks are tentatively attributed to the stretching vibration of C=C and the bending vibration of BO; Figure 2 In c, 4A-PEG-Dopa showed a peak similar to Dopa at 6.0-7.0 ppm, proving that dopamine was grafted onto the four-arm PEG; Figure 2 In d, FTIR shows that 4A-PEG-Dopa -1 Typical NH vibrations appeared on the Figure 2 In e, the UV absorption curve shows that 4A-PEG-Dopa has a characteristic absorption peak similar to Dopa at 285 nm; Figure 2 f is a schematic diagram of the synthesis of HA-PBA;

[0044] Figure 2 In g, the UV-visible absorption curve shows that MIC@QC and QC form a significant peak at 368 nm, which indicates that QC is successfully encapsulated into the interior of MIC to form MIC@QC.

[0045] The preparation method of MIC@QC is as follows: QC and MIC are mixed in a mass ratio of 1:10, added into 1 ml of DMSO to fully dissolve, and then 19 ml of deionized water is added, ultrasonically shaken for 10 minutes to obtain the product, and the product is dialyzed with deionized water for 3 days, and the unreacted QC and DMSO are removed and concentrated using a 0.22 um ultrafiltration tube to obtain MIC@QC.

[0046] Figure 2 h, the particle size and ζ potential test showed that both MIC and MIC@QC were negatively charged, which was caused by the negative charge of mPEG and PLGA in the micelles; the average particle size of MIC without QC encapsulation was 34.69±0.20, and the average particle size of MIC@QC after encapsulation was 40.17±0.98, the particle size was slightly increased, and their PDI was less than 0.3, which showed that the distribution of nanomicelle MIC was highly consistent; Figure 2 i is a schematic diagram of the synthesis of four-arm PEG dopamine; Figure 2 In j, the size distribution of MIC@QC treated with H2O2 changed from unimodal to multimodal, the particle size distribution increased significantly, and the PDI increased to 0.7; Figure 2 In k, TEM shows that individual MICs are distributed in a spherical shape under TEM, with a darker center; Figure 2 l is TEM showing that the shape of MIC@QC encapsulating QC has not changed, but its center is brighter; Figure 2 m, after MIC@QC was treated with H2O2, obvious structural collapse could be observed under TEM, and the spherical appearance was obviously broken, which was caused by the breaking of TK bonds.

[0047] Four-arm PEG dopamine was added to the HA-PBA solution before gelation. The microstructure of the hydrogel was observed by SEM and EDS. Its injectability, self-healing, swelling and degradability were verified by in vitro experiments. The rheometer was used to dynamically observe its gelation time and rheological properties such as elastic modulus and loss modulus. The hydrogel was placed in an environment with different pH and H2O2 to verify its pH / ROS multi-responsiveness and drug release performance. The physical and chemical characterization of the hydrogel is shown in Figure 3 . Figure 3 In the figure, Gel@AMP&MQ is formed by carrying AMP&MQ on hydrogel.

[0048] Figure 3 In a, after adding four-arm PEG dopamine to HA-PBA, rapid gelation can be seen, and it has good injectability; Figure 3 In b-3c, the hydrogel was freeze-dried and then quenched with liquid nitrogen. SEM observation revealed an obvious pore structure and sulfur was visible in the element distribution, proving that AMP was loaded. Figure 3 d shows the self-healing property of the hydrogel; Figure 3 In e, the frequency sweep G′ exceeds G″, and the hydrogel exhibits frequency dependence, indicating the formation of an elastic network; Figure 3 In f-3g, after applying 1000% strain, the G' and G" of the hydrogel decreased significantly, and G' was higher than G". After applying 1% strain, it recovered quickly after two cycles, indicating that the hydrogel has extraordinary self-healing ability; Figure 3 h is a schematic diagram of the dynamic bonding of hydrogel; Figure 3 In i, the hydrogels showed different degrees of disintegration in acidic and ROS-rich environments; Figure 3 k, the in vitro drug release curves showed that the release of QC exhibited a dual response to pH and redox, because the hydrogel first dissociated and released QC at a significantly faster rate under acidic and oxidative conditions. Figure 3 j, the hydrogel has excellent swelling properties, and its volume swells to about 40 times within 2 h.

[0049] The hydrogel (Gel@AMP&MQ) was biologically characterized. First, the experimental groups were divided into groups: different drugs were loaded into the hydrogel drug delivery system to verify the antibacterial activity, antioxidant activity and biocompatibility of different hydrogels. The groups included: blank group, Gel group, Gel@AMP group, Gel@MQ group and Gel@AMP&MQ group. Then the antibacterial activity was verified: L929 cells were used as the research object, and the hydrogel extract was co-cultured with the cells. The live and dead cell staining method and CCK-8 were used to detect the biocompatibility of different hydrogels. Finally, L929 was used as the experimental object for scratch tests and oxygen free radical scavenging experiments.

[0050] Figure 4In a, live and dead cell staining was performed after Gel@AMP&MQ was co-cultured with L929, and no obvious cell death was observed; Figure 4 In b, after Gel@AMP&MQ was co-cultured with L929 cells, a CCK-8 experiment was performed, and no decrease in cell survival rate was observed in the Gel@AMP&MQ group; Figure 4 In c-4e, scratch test was performed after Gel@AMP&MQ was co-cultured with L929, and the scratch healing in the Gel@AMP&MQ group was significantly accelerated; Figure 4 In f~4g, Gel@AMP&MQ was co-cultured with L929 cells, and the oxygen free radical scavenging rate of the Gel@AMP&MQ group was the highest.

[0051] For the antibacterial effect of Gel@AMP&MQ, we first conducted experimental grouping: different drugs were loaded into the hydrogel drug delivery system to verify the antibacterial activity, antioxidant activity and biocompatibility of different hydrogels. The groups included blank group, Gel group, Gel@AMP group, Gel@AMP group and Gel@AMP&MQ group. As for the antibacterial activity: S. aureus was used as the representative of Gram-positive bacteria and P. aeruginosa was used as the representative of Gram-negative bacteria. After the hydrogel was co-cultured with bacteria, the OD600 method, bacterial plating method and bacterial live-dead fluorescence staining method were used to detect the broad-spectrum antibacterial activity of different hydrogels. See the antibacterial activity. Figure 5 .

[0052] Figure 5 In a, the antibacterial activity of the hydrogel was further studied by the agar plate culture method, and it was found that a small number of colonies were formed on the agar plate after co-culture with Gel@AMP&MQ hydrogel; Figure 5 In b, Gel@AMP&MQ hydrogel exhibited good broad-spectrum antibacterial activity against Staphylococcus aureus (93.2%); Figure 5 In c, Gel@AMP&MQ hydrogel showed good broad-spectrum antibacterial activity against Pseudomonas aeruginosa (87.7%); Figure 5 In d, a large number of dead bacteria were observed in the Gel@AMP&MQ group under a fluorescence microscope; Figure 5 In e, after Staphylococcus aureus and Pseudomonas aeruginosa were incubated with Gel@AMP&MQ hydrogel, the normal physiological morphology of the bacteria changed. These results show that Gel@AMP&MQ hydrogel has good antibacterial activity; Figure 5 f is a schematic diagram of the antibacterial principle of hydrogel.

[0053] Based on RAW 264.7, we explored the mechanism of hydrogel promoting anti-inflammatory effect. Taking RAW 264.7 as a representative, we explored the mechanism of Gel@AMP&MQ exerting anti-inflammatory effect based on Akt / STAT6 signaling pathway. After co-culturing hydrogel and RAW 264.7, we performed bioinformatics analysis by transcriptome sequencing, and detected its effect on the expression of downstream marker proteins and genes of Akt / STAT6 signaling pathway by Western-blot. Its anti-inflammatory mechanism was verified by Western-blot, ELISA, immunofluorescence and bioinformatics methods. Figure 6 , Figure 7 .

[0054] Figure 6 In a, ELISA test showed that the expression of IL-10 in the Gel@AMP&MQ group was significantly higher than that in the control group. Figure 6 In b, ELISA test showed that the expression of TNF-α in the Gel@AMP&MQ group was significantly lower than that in the control group; Figure 6 In c, Q-PCR detection indicated that the expression of IL-10 in the Gel@AMP&MQ group was significantly higher than that in the control group; Figure 6 In d, Q-PCR indicated that the expression of TNF-α in the Gel@AMP&MQ group was significantly lower than that in the control group; Figure 6 e, immunofluorescence showed that the IL-10 level in the Gel@AMP&MQ group was significantly higher than that in the control group, and the TNF-α level was significantly lower than that in the control group; Figure 6 In f, flow cytometry examination indicated that M2 macrophages showed obvious differentiation after Gel@AMP&MQ treatment. Figure 6 g is the schematic diagram of Gel@AMP&MQ immune regulation, Figure 6 h, WB detection indicated that the Gel@AMP&MQ group showed high expression of IL-10 and significantly low expression of TNF-α.

[0055] Figure 7 In a, the GO enrichment results after Gel@AMP&MQ treated RAW264.7 cells showed that a large number of genes were enriched in reducing oxidative stress and promoting cytokines; Figure 7 b is the heat map of RAW264.7 cells treated with Gel@AMP&MQ. Figure 7 The volcano plot in c shows 155 up-regulated genes and 318 down-regulated genes in the mRNA-seq analysis; Figure 7 KEGG enrichment analysis in d showed that the PI3K-Akt signaling pathway was significantly upregulated; Figure 7 e is a schematic diagram of the anti-inflammatory effect of QC; Figure 7f The expressions of STAT6, p-STAT6, Akt, and p-Akt in different groups were detected by WB, among which AKt and STAT6 showed obvious phosphorylation in the Gel@AMP&MQ group; Figure 7 The molecular docking results of QC and Akt protein in g~h suggest that the two molecules have strong binding force.

[0056] An animal model of chronic infectious full-thickness skin defect was established based on diabetic rats to verify the in vivo chronic wound repair effect of the Gel@AMP&MQ oxygen-generating drug delivery system. First, an animal model was established. A rat diabetes model was established by injecting streptozotocin into the tail vein. On this basis, a skin punch was used to create a full-thickness skin wound with a diameter of 10 mm on the back of the rat, and 100 μL of S. aureus (1.0×10 8 CFU / mL) to establish a chronic infection wound model; then the chronic wound repair effect was verified. After the model was successfully established, different hydrogels were implanted in the wound site for intervention treatment, and the wound healing of the rats was observed and recorded within 14 days. On the third day, the rats were euthanized by inhalation of an overdose of isoflurane and the wound tissue was obtained. After the tissue was cooled by liquid nitrogen, the antioxidant activity in vivo was evaluated by DHE staining of frozen sections.

[0057] The rats were euthanized on the 7th and 14th days, and the wound tissues were taken for HE and Masson staining to evaluate the degree of wound healing, angiogenesis and collagen deposition. Immunofluorescence staining was used to detect the expression of angiogenesis-related cytokines (VEGF) and inflammatory-related cytokines (IL-10, TNF-α) in the wound tissues to comprehensively evaluate the in vivo wound repair efficacy. Figure 8 and Fig. 9 .

[0058] Figure 8 a is a schematic diagram of the animal experiment process. Figure 8 b is the contraction of wound edge. Figure 8 c is the number of new hair follicles, Figure 8 d is a photo of wound healing and a schematic diagram of wound contraction. Figure 8 e shows that DHE staining shows that the hydrogel has obvious oxygen free radical scavenging ability. Figure 8 f is the collagen deposition. Figure 8 g is the wound contraction area statistics, Figure 8 h shows that HE staining showed that the infiltration of inflammatory cells in Gel@AMP&MQ was significantly reduced, the number of hair follicles was significantly increased, and the wound was significantly healed; Figure 8 i shows that Masson staining showed that the Gel@AMP&MQ group had more collagen deposition.

[0059] Fig. 9In a, compared with the control group, on the 14th day after application of Gel@AMP&MQ hydrogel, the expressions of CD206, VEGF, and CD31 were significantly increased, and iNOS was significantly decreased; Fig. 9 In b, immunohistochemical analysis showed that the expression of proinflammatory factor TNF-α in the Gel@AMP&MQ hydrogel group was significantly lower than that in the control group, and the expression of IL-10 was significantly higher than that in the control group. Sirius red staining showed obvious deposition of type III collagen; Fig. 9 In c, the expression of CD31 in the Gel@AMP&MQ hydrogel group was significantly higher than that in the control group on day 14; Fig. 9 d shows that on the 14th day, the expression of VEGF in the Gel@AMP&MQ hydrogel group was significantly higher than that in the control group; Fig. 9 e, the expression of CD206 in the Gel@AMP&MQ hydrogel group was significantly higher than that in the control group on day 14; Fig. 9 f, the expression of iNOS in the Gel@AMP&MQ hydrogel group was significantly lower than that in the control group on day 14.

[0060] The present invention provides a hydrogel dressing formed by cross-linking boronate bonds based on HA-PBA (hyaluronic acid-phenylboronic acid) and four-arm PEG dopamine as a skeleton. Antimicrobial peptides and drug-loaded micelles are doped inside the hydrogel, and the micelles are loaded with quercetin, which has a sustained release function. Antimicrobial peptides bind to the cell membrane of bacteria through charge action, exert a powerful anti-infection function, and can effectively inhibit the reproduction and colonization of pathogens. Quercetin exerts an anti-inflammatory effect through the Akt / STAT6 pathway, can significantly reduce inflammatory reactions, and promote tissue repair. In addition, the hydrogel has good biocompatibility and mechanical properties, can provide a suitable moist environment for wounds, and promote healing.

[0061] The present invention provides a hydrogel with both anti-infection and drug-loaded sustained-release functions, which has excellent mechanical properties and adhesion, and also has anti-infection and anti-inflammatory functions. Since a moist environment is one of the important conditions for wound healing, and the hydrogel has good moisturizing properties, it can provide a suitable moist environment for the wound to prevent the wound from drying and scabbing. The moist environment not only helps cell migration and the formation of new tissue, but also reduces pain and itching, and improves the patient's comfort. In addition, the hydrogel can absorb wound exudate, prevent the occurrence of infection, and further promote wound healing; the hydrogel can also promote tissue regeneration; the hydrogel, as a multifunctional composite dressing, reduces the frequency of dressing changes during treatment, reduces the patient's pain and treatment costs.

[0062] The present invention provides an application of a hydrogel with both anti-infection and drug-carrying sustained-release functions. The hydrogel can be used not only in the treatment of chronic wounds of diabetes, but also in wound care after surgical operations such as burns, thereby broadening the application prospects of the hydrogel and improving the success rate of wound healing.

Claims

1. A method for preparing a hydrogel having both anti-infection and drug-carrying sustained-release functions, characterized in that: The following steps are involved: S1: Dissolve HA in a solvent, adjust the pH, add EDC and NHS respectively, then add 3-APBA dissolved in dimethyl sulfoxide, stir to react, and dialyze to obtain HA-PBA; S2: Dissolve 4A-PEG-COOH in a solvent, then drop the EDC solution into 4A-PEG-COOH, adjust the pH and stir to obtain a mixed solution, add NHS to the mixed solution, then add dopamine to the mixed solution for reaction, and obtain 4A-PEG-Dopa after dialysis and freeze-drying; S3: Dissolve the HA-PBA obtained in S1 in a solvent, mix the HA-PBA solution with 4A-PEG-DA synthesized from 4A-PEG-COOH at a mass ratio of 5:1, and adjust the pH to obtain a hydrogel.

2. The method for preparing a hydrogel having both anti-infection and drug-carrying sustained-release functions according to claim 1, characterized in that: The mass ratio of HA to EDC to NHS described in S1 is 2.94:1.68:1:

1.

3. The method for preparing a hydrogel having both anti-infection and drug-carrying sustained-release functions according to claim 1, characterized in that: The reaction time described in S1 was 48 h, the dialysis time was 5 days, and the pH was adjusted to 6.

5.

4. The method for preparing a hydrogel having both anti-infection and drug-carrying sustained-release functions according to claim 1, characterized in that: The mass ratio of 4A-PEG-DA to EDC, NHS, and dopamine described in S2 is 1:1:0.001:0.002, and the pH is 4.

75.

5. The method for preparing a hydrogel having both anti-infection and drug-carrying sustained-release functions according to claim 1, characterized in that: The reaction time of adding dopamine as described in S2 is 4 hours.

6. The method for preparing a hydrogel having both anti-infection and drug-carrying sustained-release functions according to claim 1, characterized in that: The pH described in S3 is 7.

7. A hydrogel having both anti-infection and drug-carrying sustained-release functions obtained by the preparation method according to any one of claims 1 to 6.

8. The use of a hydrogel having both anti-infection and drug-carrying sustained-release functions according to claim 7, characterized in that: The hydrogel is used in preparing wound dressing.

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