Nano-component gel for repairing and regenerating infectious diabetes skin defects

Abstract

According to the nano-component gel for repairing and regenerating the infectious diabetes skin defects, the hyaluronic acid modified by the o-nitrobenzyl alcohol derivative and the nano-zinc oxide are subjected to in-situ cross-linking to form the hydrogel, reactive oxygen free radicals at the skin defects can be removed, and the skin defects can be repaired and regenerated. The expression of TNF-alpha of skin defect tissues is reduced, the expression of VEGF is improved at the same time, and collagenous fiber deposition at the diabetic skin defect repair position is promoted, so that rapid repair of infectious diabetic skin defects is promoted, and the problem that the infectious diabetic skin defects are difficult to repair is effectively solved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials and skin defect repair, in particular to a nano-component gel for repairing and regenerating skin defects caused by infectious diabetes. Background Art

[0002] Infectious diabetic skin defects are a difficult problem to repair clinically. Due to abnormal blood sugar, once the skin is infected, it will be more difficult to heal than normal skin. It is necessary to solve the problems of bacterial infection and skin regeneration in diabetic conditions at the same time. Sometimes it is difficult to heal even for several months.

[0003] Hydrogels are three-dimensional hydrophilic polymer networks that are widely used in tissue engineering, regenerative medicine, and implantable devices due to their properties similar to those of biological tissue. Recently, adhesive hydrogels have become a powerful tool for tissue repair, wound dressing, and hemostasis. Adhesive hydrogels are compatible with wet surfaces and can act as a protective layer by adhering to biological tissues, polymers, and metal surfaces. Depending on the chemical properties of the surface, different functional chemical groups, such as catechol, carboxyl, and nitrobenzyl groups, are grafted onto the polymer chains to improve the adhesion of the hydrogel. For adhesion to biological tissues, aldehyde groups are often used because they can form dynamic covalent bonds with the amino groups of collagen through Schiff base reactions. However, for difficult-to-heal infected diabetic skin defects, traditional hydrogels lack the integrated functionalities of antibacterial properties, scavenging reactive oxygen species, promoting tissue regeneration, and tissue adhesion.

[0004] Therefore, there is an urgent need to develop a new type of hydrogel for repairing and regenerating infectious diabetic skin defects, so as to accelerate the healing of infectious diabetic skin defects. Summary of the Invention

[0005] To solve the above problems, the present invention provides a nano-component gel for the repair and regeneration of infectious diabetic skin defects. By using hyaluronic acid modified with an o-nitrobenzyl alcohol derivative and nano-zinc oxide, a hydrogel is formed through in situ cross-linking. The hydrogel can eliminate reactive oxygen free radicals at the skin defect site, downregulate the expression of TNF-α in the skin defect tissue and simultaneously increase the expression of VEGF, promote the deposition of collagen fibers at the diabetic skin defect repair site, thereby promoting the rapid repair of infectious diabetic skin defects and effectively solving the problem that infectious diabetic skin defects are difficult to repair.

[0006] In one aspect, the present invention provides a hydrogel for repairing skin defects caused by infectious diabetes. The hydrogel is composed of modified hyaluronic acid and zinc oxide, which are formed by in situ cross-linking. The modified hyaluronic acid and nano-zinc oxide are effectively combined through chemical coordination to form a stable nano-component hydrogel.

[0007] Infectious diabetic skin defects are a difficult problem to repair clinically. Due to abnormal blood sugar, once the skin is infected, it will be more difficult to heal than normal skin. It is necessary to solve both bacterial infection and skin regeneration in diabetic conditions at the same time.

[0008] To achieve skin regeneration, bioactive molecules such as hyaluronic acid, gelatin, sodium alginate, chondroitin sulfate, silk fibroin, chitosan, carboxymethyl cellulose, or collagen are required. Hyaluronic acid is one of the most commonly used biomacromolecules and is widely used in medical aesthetics. However, unmodified hyaluronic acid is water-soluble and easily lost during practical application. Chemical modification is required to enhance its gel-forming ability and the aforementioned tissue adhesion properties.

[0009] Existing hyaluronic acid modification technologies only give hyaluronic acid material properties and biological activity that promotes regeneration, but lack the ability to scavenge active oxygen free radicals and antibacterial properties when applied to the epidermis.

[0010] Antimicrobial properties can be achieved by specially formulated nanomaterials or antimicrobial peptides with antimicrobial activity. Among these, nanozinc oxide is a common nanomaterial, and zinc oxide is also commonly used in cosmetic skin products. However, its antimicrobial properties in the repair of diabetic skin have not been fully explored.

[0011] Most metal oxides have antibacterial properties, such as manganese dioxide, iron oxide, etc. However, since the Zn element has always been in the form of Zn in biological systems, 2+ It exists in a stable form and, unlike Cu and Fe ions, does not undergo redox reactions, which can involve reactive free radicals and destructive reactive oxygen species. In addition, Zn and Zn-containing proteins play an important role in every stage of skin repair; and Zn also plays an important role in EM modification, cell migration, protein synthesis, and inflammation reduction. Therefore, Zn is preferred. 2+ to provide antibacterial properties to the hydrogel.

[0012] Nano zinc oxide has good antibacterial properties, but whether its antibacterial properties can be fully utilized in hydrogel materials remains to be explored. After extensive research, the present invention has proved that the use of ZnO in a suitable ratio to prepare hydrogels not only has good biocompatibility and antibacterial properties, but also can provide Zn with both biological regulatory effects. 2+ , which helps to better repair infectious diabetic skin defects.

[0013] Furthermore, the modified hyaluronic acid is hyaluronic acid modified with an o-nitrobenzyl alcohol derivative; and the zinc oxide is nano zinc oxide.

[0014] Furthermore, the mass ratio of the o-nitrobenzyl alcohol derivative-modified hyaluronic acid to nano-zinc oxide is (10:1) to (40:1).

[0015] Furthermore, the mass ratio of the o-nitrobenzyl alcohol derivative-modified hyaluronic acid to nano-zinc oxide is preferably (15:1) to (25:1), and most preferably 20:1.

[0016] Furthermore, the crosslinking is light crosslinking. The crosslinking of the present invention only requires light crosslinking, without adding other crosslinking agents.

[0017] The o-nitrobenzyl alcohol derivative-modified hyaluronic acid and nano zinc oxide provided by the present invention can be directly in-situ cross-linked into a hydrogel upon irradiation without adding a cross-linking agent.

[0018] In another aspect, the present invention provides a method for preparing a hydrogel for repairing infectious diabetic skin defects, comprising the following steps:

[0019] (1) preparing hyaluronic acid modified with o-nitrobenzyl alcohol derivatives;

[0020] (2) Hyaluronic acid modified with o-nitrobenzyl alcohol derivatives is mixed with nano-zinc oxide and cross-linked under light to form a hydrogel.

[0021] In another aspect, the present invention provides a composition for preparing a hydrogel for removing free radicals from skin defects, wherein the composition comprises hyaluronic acid modified with an o-nitrobenzyl alcohol derivative and nano zinc oxide.

[0022] Furthermore, the skin defect is an infectious diabetic skin defect, the infectivity is infection with Staphylococcus aureus, and the free radical is a DPPH free radical and / or a HO free radical.

[0023] On the other hand, the present invention provides a composition for preparing a hydrogel for removing reactive oxygen free radicals from infectious diabetic skin defects, wherein the composition comprises hyaluronic acid modified with an o-nitrobenzyl alcohol derivative and nano-zinc oxide, wherein the skin defect is an infectious diabetic skin defect, and the infection is infection with Staphylococcus aureus.

[0024] On the other hand, the present invention provides a composition for preparing a hydrogel for repairing infectious diabetic skin defects, wherein the composition comprises hyaluronic acid modified with an o-nitrobenzyl alcohol derivative and nano-zinc oxide, wherein the skin defect is an infectious diabetic skin defect, and the infection is infection with Staphylococcus aureus.

[0025] In another aspect, the present invention provides a composition for preparing a hydrogel for downregulating the expression of TNF-α in skin defect tissue and simultaneously increasing the expression of VEGF, wherein the composition comprises hyaluronic acid modified with an o-nitrobenzyl alcohol derivative and nano zinc oxide.

[0026] Furthermore, the skin defect tissue is tissue from an infectious diabetic skin defect.

[0027] In another aspect, the present invention provides a use of a composition for preparing a hydrogel for promoting the repair of diabetic skin defects, wherein the composition comprises hyaluronic acid modified with an o-nitrobenzyl alcohol derivative and nano zinc oxide.

[0028] In another aspect, the present invention provides a use of a composition for preparing a hydrogel for improving the antibacterial effect of skin defects, wherein the composition comprises hyaluronic acid modified with an o-nitrobenzyl alcohol derivative and nano zinc oxide.

[0029] Furthermore, the skin defect is an infectious diabetic skin defect, and the infection is infection with Staphylococcus aureus.

[0030] In another aspect, the present invention provides a composition for preparing a hydrogel for promoting collagen fiber deposition at the repair site of diabetic skin defects, wherein the composition comprises hyaluronic acid modified with an o-nitrobenzyl alcohol derivative and nano zinc oxide.

[0031] The hydrogel for repairing infectious diabetic skin defects provided by the present invention has the following beneficial effects:

[0032] 1. By using hyaluronic acid modified with o-nitrobenzyl alcohol derivatives and nano-zinc oxide to form a hydrogel through in situ cross-linking, it can promote the rapid repair of infectious diabetic skin defects;

[0033] 2. Hyaluronic acid modified with o-nitrobenzyl alcohol derivatives can undergo photocrosslinking reaction and form hydrogel without the need for additional crosslinking agents;

[0034] 3. The prepared hydrogel has wet surface tissue adhesion and can effectively adhere to the surface of biological tissue;

[0035] 4. The prepared hydrogel can effectively resist Escherichia coli and Staphylococcus aureus and has good antibacterial properties;

[0036] 5. The prepared hydrogel can effectively remove reactive oxygen free radicals in skin defects caused by infectious diabetes and promote the accelerated repair of skin defects;

[0037] 6. The prepared hydrogel can downregulate the expression of TNF-α in skin defect tissue and simultaneously increase the expression of VEGF, promoting the accelerated repair of skin defects;

[0038] 7. The prepared hydrogel can effectively promote the deposition of collagen fibers in the repair site of diabetic skin defects and accelerate the repair of skin defects;

[0039] 8. It can basically repair the skin defects of mice with infectious diabetes within 14 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The preparation process and detection profile of the ZnO@HN hydrogel in Example 1, wherein (a) is a flow chart for preparing hyaluronic acid (HN) modified with an o-nitrobenzyl alcohol derivative, (b) is a photograph of HN and ZnO@HN before and after cross-linking, (c) is a TEM image of nano-ZnO particles, (d) is an XRD pattern of nano-ZnO particles, and (e) is a SEM image and EDS image of the ZnO@HN hydrogel;

[0041] Figure 2 Detection spectra of the ZnO@HN hydrogel in Example 1, where (a) is the H NMR spectrum of HA and the prepared HN, (b) is the full XPS spectrum analysis of ZnO@HN, and (c) to (f) are the XPS narrow spectrum analyses of Zn, C, O, and N elements, respectively;

[0042] Figure 3 Schematic diagram of the antibacterial ability test results of the ZnO@HN hydrogel in Example 2, where (a) shows the OD value of Escherichia coli inhibited by the compound for 3, 6, and 9 hours, (b) shows the OD value of Staphylococcus aureus inhibited by the compound for 3, 6, and 9 hours, and (c) is a photograph of the bacterial colonies after treatment with ZnO and ZnO@HN.

[0043] Figure 4 Analysis of the effect of ZnO@HN hydrogel in Example 3 on scavenging reactive oxygen species in skin defects, (a) is the result of the proliferation experiment based on BMSCs and CCK8, (b) is the Live / Dead detection image of BMSCs cultured in ZnO and ZnO@HN hydrogels, (c) is the test result of scavenging DPPH free radical ability, (d) is the test result of scavenging HO free radical ability, (e) is the test result of BMSCs cell viability cultured under H2O2 conditions, (f) is the fluorescence image of ROS scavenging ability at the cellular level (scale: 50m), (g) is the quantitative analysis of ROS in BMSCs (Mean±SD, n=3, *p<0.05, ***p<0.001, ****p<0.0001);

[0044] Figure 5 Schematic diagram of the analysis results of the effect of the hydrogels prepared by modifying hyaluronic acid with different methods in Example 4 on the scavenging ability of intracellular DPPH free radicals;

[0045] Figure 6Figure 5 shows the wound healing performance of the ZnO@HN hydrogel in Example 5 on full-thickness skin defects on the backs of diabetic rats infected with Staphylococcus aureus. (a) shows representative photographs of skin defects on days 0, 3, 7, and 14; (b) shows the quantitative analysis of wound area in each group; and (c) shows the histological evaluation of wound regeneration.

[0046] Figure 7 Analysis of collagen deposition in the repair tissue in Example 5, where (a) is a Masson staining image on day 14 (scale bar 100 μm), (b) shows the expression of HIF-1α (red arrow) and the observed Staphylococcus aureus (yellow arrow), and (c) the collagen volume fraction in the wound tissue on day 14 by Masson staining (Mean ± SD, n = 3, *p < 0.05, ***p < 0.001, ****p < 0.0001);

[0047] Figure 8 This is the immunohistochemical analysis of the repair tissue in Example 5, wherein (a) is the CD68 immunohistochemical staining image on day 7, (b) is the CD31 immunohistochemical staining image on day 14, (c) is the MMP-2 immunohistochemical staining image on day 7, (d) is the MMP-9 immunohistochemical staining image on day 7, (e) is the α-SMA immunohistochemical staining image on day 7, (f) is the CD68 immunological analysis result, (g) is the CD31 immunological analysis result, (h) is the MMP-2 immunological analysis result, (i) is the MMP-9 immunological analysis result, and (j) is the α-SMA immunological analysis result;

[0048] Figure 9 This is the immunofluorescence analysis of the repair tissue in Example 5, wherein (a) is a representative image of a skin tissue section 7 days after immunofluorescence staining with TNF-α marker, (b) is a representative image of a skin tissue section 7 days after immunofluorescence staining with VEGF marker, (c) is the quantitative analysis result of TNF-α, and (d) is the quantitative analysis result of VEGF. DETAILED DESCRIPTION

[0049] The preferred embodiments of the present invention are described in further detail below with reference to the accompanying drawings. It should be noted that the following embodiments are intended to facilitate understanding of the present invention and do not limit it in any way. The raw materials and equipment used in the specific embodiments of the present invention are all known products and were obtained by purchasing commercially available products.

[0050] Example 1: Hydrogel provided by the present invention and its preparation method

[0051] 1. Preparation of hyaluronic acid modified with o-nitrobenzyl alcohol derivatives

[0052] The hyaluronic acid (HA) used in this example (approximately 200-400 kDa, Bloomage Biotechnology Co., Ltd.) and the o-nitrobenzyl alcohol derivative was N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide, purchased from Haining Jurassic Biotechnology Co., Ltd.

[0053] The flow chart for preparing hyaluronic acid (HN) modified with o-nitrobenzyl alcohol derivatives is as follows: Figure 1 (a) is shown, and the specific process is as follows:

[0054] Dissolve 2g of HA in 100mL of 0.01mol / L MES buffer (pH approximately 5.3) and react at 35°C. Once HA is completely dissolved, add 60mg of NB to the reaction system in 10mL of DMSO (dimethyl sulfoxide). Add 1.2g of DMTMM (4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride) to the reaction system in three equal additions, with 0.5-1h intervals between additions. The reaction is terminated three hours after the final addition of DMTMM. The product is then dialyzed against 0.1M NaCl and lyophilized to yield approximately 1.8g of the final product, HN.

[0055] 2. Preparation of Nano-ZnO

[0056] Add 3.29g Zn(CH3COO)2·2H2O to 30mL ethanol and stir at 60℃. At the same time, add 2.24g triethanolamine (TEA) to the solution. 2+ The final concentrations of NH4Cl and TEA were both 0.5 M. The mixture was stirred at 60°C for 1 h and aged at room temperature for 1 h. The resulting homogeneous solution was poured into a 50 mL hydrothermal autoclave and heated in an oven at 200°C for 18 h. The white precipitate was washed with 30% ethanol and separated by centrifugation (3000 rpm, 5 min). The synthesized ZnO nanoparticles were collected and dried in a vacuum oven at 60°C overnight.

[0057] TEM (transmission electron microscope) images of nano ZnO particles are shown in Figure 2. Figure 1 As shown in (c), the XRD (X-ray diffraction analysis) of the nanoparticles is as follows Figure 1 (d) shown.

[0058] 3. Preparation of hydrogel

[0059] 5 mg of zinc oxide was ultrasonically dispersed in 5 mL of deionized water to form a 0.1% zinc oxide suspension. Then, 100 mg of HN and 25 mg of a photoinitiator (lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, LAP) were dissolved in the ZnO suspension in the dark at final concentrations of 2% and 0.5%. The composite hydrogel was photocured using 365 nm UV irradiation.

[0060] Photos of HN and ZnO@HN before and after cross-linking are shown in Figure 1 As shown in (b), the SEM (scanning electron microscopy) image and EDS (energy dispersive spectrometry) image of ZnO@HN hydrogel are as follows: Figure 1 As shown in (e), it can be seen from the element distribution that ZnO is uniformly dispersed in the gel system.

[0061] The H NMR spectra of HA and prepared HN are as follows Figure 2 As shown in (a), the two proton peaks in the spectrum a and b of HN belong to the NB benzene ring, which indicates that NB is successfully grafted onto HA.

[0062] XPS (X-ray photoelectron spectroscopy) of ZnO@HN Figure 2 Figures b, c, d, e, and f, among which the most important is that in Figure c, the peaks at 1045.6 and 1026.5 eV belong to the Zn-N peaks, proving that there is a coordination bond between ZnO and HN, rather than a simple physical mixture.

[0063] Example 2: Verification of the antibacterial ability of hydrogel

[0064] This example uses two methods to evaluate the antibacterial properties of the hydrogel prepared in Example 1. First, the hydrogel (Ø10 mm × 2 mm) was immersed in a Luria-Bertani (LB) solution of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) (initial OD = 0.03, 600 nm); the bacterial solution was shaken at 37°C, and the OD value at the desired time point was recorded. Second, the above bacterial solution was incubated for 9 hours and then diluted 10,000 times. 100 μL of the diluent was evenly spread on an agar plate (Ø10 cm). The colony was incubated at 37°C for 12 hours, and the antibacterial properties were evaluated. Direct addition of sterile water was used as a blank control.

[0065] The antibacterial ability test results of ZnO@HN hydrogel are as follows Figure 3 As shown, Figure 3 (a) is the detection result of the OD value of Escherichia coli being inhibited by the compound for 3, 6 and 9 hours, Figure 3 (b) is the detection result of the OD value of Staphylococcus aureus being inhibited by the compound for 3, 6 and 9 hours, Figure 3 (c) Photos of bacterial colonies in ZnO suspension and after treatment with ZnO@HN.

[0066] according to Figure 3 It can be seen that ZnO@HN hydrogel has good antibacterial ability.

[0067] Example 3: Study on the effect of hydrogel in removing reactive oxygen free radicals in skin defects

[0068] 1. Interaction between ZnO@HN hydrogel and cells

[0069] The hydrogel (Ф10mm×2mm) prepared according to the method provided in Example 1 was immersed in 10mL of cell culture medium (DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture) for 12 hours, and the hydrogel extract was obtained after sterilization with a 0.22μm filter. Rat bone marrow mesenchymal stem cells (BMSCs) were cultured with hydrogel extract as the experimental group, and those cultured with untreated cell culture medium as the control group, and compared with ZnO solution. The culture temperature was 37°C and 5% CO2. BMSCs were treated with CCK-8 method for the required time, and the OD value (450nm) was measured with a microplate reader. The cells were stained with Live / Dead staining method, and the images were observed under a fluorescence microscope. The results are shown in Figure 2. Figure 4 As shown in (a) and (b), Figure 4 (a) is the result of the proliferation experiment based on BMSCs and CCK8. It can be seen that ZnO@HN hydrogel can significantly improve cell viability; Figure 4 (b) Live / Dead detection images of BMSCs cultured in ZnO and ZnO@HN hydrogels. It can be seen that compared with ZnO, ZnO@HN hydrogel can significantly improve the viability of BMSCs.

[0070] 2. ROS scavenging ability of ZnO@HN hydrogel

[0071] 2.1 Extracellular DPPH free radical and H2O2 scavenging ability of ZnO@HN hydrogel

[0072] ZnO@HN hydrogel (Ø10mm×2mm) was immersed in 5mL DPPH solution (250μM, 95% ethanol) and stirred at 37℃ for 2h. The absorbance of DPPH at 520nm was measured using a microplate reader. The results of the hydrogel scavenging DPPH free radical test were compared with those of HN hydrogel without ZnO, ZnO alone, and ZnO@HN hydrogel. Figure 4 As shown in (c), it can be seen that compared with HN hydrogel alone and ZnO alone, ZnO@HN hydrogel can significantly improve the ability to scavenge DPPH free radicals. The reason may be that HN hydrogel and ZnO have synergistic effects in scavenging DPPH free radicals. The hydrogel was immersed in 5mL H2O2 solution (1mM) and stirred at 37℃ for 8h. The inhibitory effect of H2O2 was evaluated by hydrogen peroxide content determination kit. The test results of the hydrogel's ability to scavenge H2O2 are shown in Figure 2. Figure 4 As shown in (d), it can be seen that HN and ZnO@HN hydrogels effectively remove H2O2, and HN hydrogel and ZnO have synergistic effects in removing H2O2.

[0073] 2.2 Intracellular HO free radical scavenging ability of ZnO@HN hydrogel

[0074] First, we experimentally confirmed that the addition of 50μM and 80μM H2O2 to the cell culture medium caused moderate cellular oxidative damage (approximately 50-70%). BMSCs were seeded in 96-well plates and cultured with gel extract for 12 hours. The cells were then treated with the aforementioned H2O2 concentrations for 6 hours. The cells' ability to scavenge intracellular ROS was assessed using CCK-8 staining and a reactive oxygen species detection kit.

[0075] The results of the BMSCs cell viability test under H2O2 conditions are as follows Figure 4 As shown in (e), it can be seen that under the conditions of 50μM and 80μM H2O2, the BMSCs cells treated with gel can maintain a high cell viability.

[0076] Fluorescence images of ROS scavenging ability at the cellular level (scale bar: 50 μm) are shown in Figure 4 (f) Quantitative analysis of ROS in BMSCs (Mean±SD, n=3, *p<0.05, ***p<0.001, ****p<0.0001) Figure 4 (g), it can be seen that the hydrogel has good ROS scavenging ability.

[0077] Example 4: Study on the effect of hydrogels prepared by modifying hyaluronic acid with different methods on the scavenging ability of intracellular DPPH free radicals

[0078] Hyaluronic acid modified by different methods was used to prepare hydrogels. The hydrogels were prepared in the following groups: 1. A solution prepared from unmodified hyaluronic acid; 2. A hydrogel prepared from hyaluronic acid modified with methacrylic acid (HAMA) (HAMA was dissolved in a 0.1% ZnO solution (final HAMA concentration was 2%), and then 0.5% LAP was added and irradiated with 365nm UV for 20s to form a gel). 3. A hydrogel prepared according to the method of Example 1 (hyaluronic acid HN modified with an o-nitrobenzyl alcohol derivative); the intracellular DPPH free radical scavenging ability was tested according to the method provided in Example 3. The test results are shown in FIG. Figure 5 shown.

[0079] from Figure 5 It can be seen that compared with methacrylic acid modification, the ability of hyaluronic acid modified with o-nitrobenzyl alcohol derivatives in scavenging intracellular DPPH free radicals is significantly improved. Therefore, hyaluronic acid modified with o-nitrobenzyl alcohol derivatives is preferably used to prepare hydrogels.

[0080] Example 5: Repair effect of hydrogel on full-thickness skin defects infected by Staphylococcus aureus in diabetic rats

[0081] In this example, the wound healing properties of the hydrogel dressing were further investigated using full-thickness skin defects infected with Staphylococcus aureus on the backs of diabetic rats. Methods: All animal experiments were conducted in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals. C57BL / 6 male mice (6 weeks old, 20-25 g, purchased from Hangzhou Shengtian Biotechnology Co., Ltd.) were used as experimental subjects. A type 1 diabetic mouse model was established by injection with a pre-formulated streptozotocin (STZ) solution (60 mg / kg). A successful model was established when blood glucose levels were >16.7 mmol / L after one week. A full-thickness skin defect (Ø10 mm) was created on the upper back. All mice were randomly divided into five groups: wild-type control group (normal mice), DM group (diabetic mice), DM+SA group (diabetic mice infected with Staphylococcus aureus), DM+H group (diabetic mice treated with hydrogel), and DM+SA+H group (diabetic mice infected with Staphylococcus aureus treated with hydrogel). Wound conditions were recorded daily. The animals were sacrificed, and the skin was harvested for histological analysis.

[0082] 1. Analysis of wound healing performance

[0083] The skin defects were observed on days 0, 3, 7, and 14 to investigate the wound healing performance of ZnO@HN hydrogel on full-thickness skin defects infected with Staphylococcus aureus on the backs of diabetic mice. Representative photos of skin defects on days 0, 3, 7, and 14 (scale bar: 3 mm) are shown. Figure 6 As shown in (a), quantitative analysis of wound area in each group (n=3, *p<0.05, **p<0.01) Figure 6 (b) shown.

[0084] according to Figure 6 As shown in (a) and (b), compared to the wild-type group, skin defects in diabetic mice were more difficult to heal, especially those infected with Staphylococcus aureus. However, after hydrogel treatment, the wound area was significantly reduced and essentially healed within 14 days, a rate even faster than that of the wild-type group. On day 14, the wounds in the DM and DM+SA groups still had noticeable scarring, while the wounds in the DM+H and DM+SA+H groups had almost completely recovered.

[0085] In order to further explore the healing process of diabetic wounds, HE staining of skin tissue was performed in this example, and wound regeneration histological evaluation (scale bar: 100 μm) was performed. Figure 6 (c) As shown. Figure 6As shown in (c), on day 3, the wound surface was primarily characterized by inflammatory exudate and inflammatory cell infiltration. On day 7, the epidermis gradually re-epithelialized, with a complete epithelial lamina visible in the DM+H and DM+SA+H groups, whereas no epithelial lamina was observed in the Wild, DM, and DM+SA groups. Granulation tissue and fibroblast proliferation in the dermis reached a peak. On day 14, inflammation essentially subsided, the epidermis was covered, and newly formed capillaries transformed into mature small vessels, with collagen fiber deposition. New hair follicles and collagen fiber deposition were observed in the DM+H and DM+SA+H groups, whereas granulation tissue remained the primary component, with less collagen fiber deposition in the DM and DM+SA groups. Hematoxylin and eosin (H&E) staining revealed a higher degree of re-epithelialization, better hair follicle repair, more angiogenesis, and more collagen fiber formation in the hydrogel dressing group.

[0086] 2. Analysis of repair tissue

[0087] Tissue samples collected on days 3, 7, and 14 were fixed in 4% (v / v) paraformaldehyde solution for 1 day. Samples were then embedded in paraffin and sectioned into 4-μm-thick slices. Sections were stained with hematoxylin and eosin (HE). All sections were scanned using a digital slide scanner (Ningbo KFBIO). Masson staining was performed on days 3 and 14 to evaluate collagen fiber deposition. Wound healing was assessed on days 7 or 14 using immunohistochemical staining for CD68, CD31, MMP-2, MMP-9, and α-SMA, and immunofluorescent staining for TNF-α and VEGF.

[0088] 2.1 Analysis of collagen deposition

[0089] Masson staining images on day 14 are shown in Figure 7 (a) (Scale bar 100 μm); HIF-1α expression (red arrow) and Staphylococcus aureus (yellow arrow) were observed on day 3. Figure 7 (b) Masson-stained collagen volume fraction in wound tissue on day 14 (Mean±SD, n=3, *p<0.05, ***p<0.001, ****p<0.0001) Figure 7 (c).

[0090] Masson staining was used to evaluate collagen fiber deposition. Figure 7 (a) Compared with the Wild group, DM group, and DM+SA group, the collagen fibers in the DM+H group and DM+SA+H group were more abundant and more neatly arranged on the 14th day. Quantitative analysis of collagen fibers in the regenerated dermis was performed. The collagen volume fractions in the DM+H group and DM+SA+H group were 63.4% and 61.6%, respectively, which were significantly higher than those in the DM group and DM+SA group ( Figure 7(c)). The results showed that ZnO@HN promoted the formation of collagen fibers during the healing process of bacterial-infected diabetic wounds. Immunohistochemical staining was used to detect the expression of hypoxia-inducible factor-1α (HIF-1α) ( Figure 7 (b)). On the third day, a small number of HIF-1α positive cells were observed in the tissues surrounding the wounds in the Wild, DM+H, and DM+SA+H groups, while almost no positive cells were found in the DM and DM+SA groups. Staphylococcus aureus was observed in the scabs of the wounds in the DM+SA and DM+SA+H groups ( Figure 7 Yellow arrows in (b). The DM+SA+H group showed a significant decrease in bacterial colonies and was surrounded by inflammatory cells. These results suggest that ZnO@HN hydrogels have potential applications in diabetic wound repair.

[0091] 2.2 Immunohistochemical analysis

[0092] This example also evaluated the expression of macrophages in the inflammatory-proliferative transition phase by CD68 immunohistochemical staining ( Figure 8 On day 7, the expression of CD68 in the DM and DM+SA groups was significantly higher than that in the hydrogel dressing-treated group, and was associated with tissue inflammatory infiltration. However, after ZnO@HN hydrogel treatment, the expression of CD68 was significantly reduced, indicating that the hydrogel treatment can eliminate inflammation and accelerate wound repair.

[0093] CD31 was used to mark mature blood vessels in wound tissue ( Figure 8 (b) Figure 8 (g) Compared with the DM and DM+SA groups, the DM+H and DM+SA+H groups showed increased numbers of new blood vessels and increased CD31 expression. Compared with the untreated group, the DM+H and DM+SA+H groups showed more granulation tissue formation at the wound surface, with the latter group showing denser granulation tissue, suggesting that it promotes matrix reorganization and repair during wound healing.

[0094] The therapeutic effect of ZnO@HN was further explored by detecting the expression of MMP-2 and MMP-9. After treatment with the composite hydrogel, the expression levels of MMP-2 and MMP-9 decreased significantly, indicating that ZnO@HN hydrogel can consume MMPs ( Figure 8 (c), 8(d), 8(h) and 8(i)), thereby accelerating wound healing.

[0095] Myofibroblasts are mainly distributed at the wound edge, and their contractile function is crucial for wound healing. The formation of myofibroblasts was assessed by α-smooth muscle actin (α-SMA) staining. Figure 8As shown in Figures e and 8j, the number of α-SMA-positive cells increased significantly after hydrogel treatment, indicating that ZnO@HN can promote the proliferation of myofibroblasts in vivo, thereby improving the therapeutic effect on diabetic skin bacterial infection wounds.

[0096] 2.3 Immunofluorescence analysis

[0097] Moderate tumor necrosis factor (TNF) can enhance immune cell recruitment and promote proliferation, thereby accelerating skin regeneration. This example evaluates the infection prevention effect of ZnO@HN by studying tumor necrosis factor-α (TNF-α). Figure 9 As shown in (a) and 9(c), the expression of TNF-α (green dots) was significantly higher in the group without hydrogel treatment, which was attributed to the antibacterial property of ZnO@HN.

[0098] VEGF is a factor related to collagen synthesis, angiogenesis and re-epithelialization. The expression of VEGFA in the DM+H group and the DM+SA+H group was significantly higher than that in the DM group and the DM+SA group ( Figure 9 (b) Figure 9 (d)). These results indicate that ZnO@HN accelerates skin regeneration by downregulating the expression of TNF-α and simultaneously increasing the expression of VEGF.

[0099] Although the present invention is disclosed as above, the present invention is not limited thereto. For example, the present invention can be expanded according to its medical application range. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the scope defined by the claims.

Claims

1. A hydrogel for repairing skin defects caused by infectious diabetes, characterized in that: It is composed of modified hyaluronic acid and zinc oxide, which form a hydrogel through in situ cross-linking.

2. The hydrogel according to claim 1, wherein The modified hyaluronic acid is hyaluronic acid modified with an o-nitrobenzyl alcohol derivative; and the zinc oxide is nano zinc oxide.

3. The hydrogel according to claim 2, wherein The mass ratio of the o-nitrobenzyl alcohol derivative-modified hyaluronic acid and nano zinc oxide is (10:1) to (40:1).

4. The hydrogel according to claim 3, wherein The crosslinking is photocrosslinking.

5. The method for preparing a hydrogel according to any one of claims 1 to 4, wherein: The following steps are involved: (1) Preparation of o-nitrobenzyl alcohol derivative-modified hyaluronic acid and nano-zinc oxide; (2) Hyaluronic acid modified with o-nitrobenzyl alcohol derivatives is mixed with nano-zinc oxide and cross-linked under light to form a hydrogel.

6. A composition for preparing a hydrogel for removing free radicals from skin defects caused by infectious diabetes, characterized in that: The composition comprises hyaluronic acid modified with an o-nitrobenzyl alcohol derivative and nano zinc oxide; the skin defect is an infectious diabetic skin defect, the infection is infection with Staphylococcus aureus, and the free radicals are active oxygen free radicals.

7. A composition for preparing a hydrogel for repairing skin defects caused by infectious diabetes, characterized in that: The composition comprises hyaluronic acid modified with an o-nitrobenzyl alcohol derivative and nano zinc oxide. The skin defect is an infectious diabetic skin defect, and the infection is infection with Staphylococcus aureus.

8. A composition for preparing a hydrogel for down-regulating the expression of TNF-α in skin defect tissue and simultaneously increasing the expression of VEGF, characterized in that: The composition comprises hyaluronic acid modified with an o-nitrobenzyl alcohol derivative and nano zinc oxide.

9. A composition for preparing a hydrogel that promotes the repair of diabetic skin defects, characterized in that: The composition comprises hyaluronic acid modified with an o-nitrobenzyl alcohol derivative and nano zinc oxide.

10. A composition for preparing a hydrogel for promoting collagen fiber deposition in the repair of diabetic skin defects, characterized in that: The composition comprises hyaluronic acid modified with an o-nitrobenzyl alcohol derivative and nano zinc oxide.

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