Composite hydrogel, preparation method and application of composite hydrogel in preparation of medicine for treating diabetic foot ulcer infection

Abstract

The invention discloses composite hydrogel, a preparation method and application of the composite hydrogel in preparation of a medicine for treating diabetic foot ulcer infection. The composite hydrogel comprises a methacrylated gelatin hydrogel and a cyclocarya paliurus extract product or cyclocarya paliurus leaf powder compounded in the methacrylated gelatin hydrogel. The dressing prepared by the invention realizes drug sustained release, which indicates that the hydrogel can promote long-term release of drugs, further reduce loss of liquid drugs and improve the utilization rate of the drugs, is used for inhibiting healing of diabetic foot ulcer infected wounds, and has wide clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a composite hydrogel, a preparation method and application of the composite hydrogel in preparing a medicine for treating diabetic foot ulcer infection. Background Art

[0002] Diabetic foot ulcer is one of the most serious and costly complications of diabetes. About a quarter of diabetic patients will develop foot ulcers in their lifetime. Among them, a large number of patients with diabetic foot ulcers will suffer from foot infections. Moderate or severe diabetic foot infections can lead to minor or major amputations and are one of the main causes of disability or premature death.

[0003] When diabetic foot ulcers occur, the normal barrier function is lost, leading to an increased risk of foot infection. Staphylococcus aureus and Streptococcus agalactiae are the most commonly isolated pathogens. Microorganisms gather in specific areas within diabetic foot ulcers, where they grow and multiply and wrap themselves with extracellular polymers containing polysaccharides and lipids. Polymeric substances secreted by cells embedded in the ulcers include proteins, lipids, nucleic acids, polysaccharides and other components, which aggregate with microorganisms to form biofilms. These membranes enable bacteria to attach to both biotic and inbiotic surfaces. Because biofilms are resistant to antimicrobial agents, the immune system and chemicals, they can delay wound healing and cause chronic inflammation and recurrent infections. Hyperglycemia reduces leukocyte function, most notably that of neutrophils, which is reflected in reduced production of chemokines, increased production of reactive oxygen species, and reduced cell phagocytosis and migration caused by the complement system. At the same time, keratinocyte migration is impaired in the infected wound surface of diabetic foot ulcers, which is also one of the reasons for slow wound healing.

[0004] Dressings are an integral part of the treatment of diabetic foot ulcers. High-quality dressings should help relieve symptoms, protect diabetic foot wounds, and promote wound healing. Currently accepted wound dressings should also have the following characteristics: (1) promote tissue reconstruction by providing insulation, gas exchange, increased drainage, and debris removal; (2) be biocompatible and not cause allergic or immune reactions; (3) be antimicrobial and be able to treat / prevent secondary wound infections; and (4) be easy to remove without causing trauma.

[0005] Diabetic foot ulcers have a special pathogenesis different from ordinary skin ulcers. Different types of wounds and different stages of wound healing have different characteristics. Whether traditional or modern dressings, they do not provide targeted treatment for the particularity of diabetic foot ulcers and cannot be used throughout the whole process. Therefore, they are always unsatisfactory in promoting wound healing. Dressings loaded with traditional Chinese medicine have unique advantages in application. Traditional Chinese medicine has a variety of active substances and can achieve the purpose of treatment through multiple targets. It is suitable for all stages of clinical treatment, and has significant efficacy and fewer side effects.

[0006] Therefore, it is of great significance to develop a drug or dressing loaded with important active ingredients that can be used to treat diabetic foot ulcer infection. Summary of the invention

[0007] The purpose of the present invention is to provide a composite hydrogel, a method for preparing the composite hydrogel and application of the composite hydrogel in preparing a medicament for treating diabetic foot ulcer infection.

[0008] The present invention is achieved in that:

[0009] In a first aspect, the present invention provides a composite hydrogel, comprising a methacrylated gelatin hydrogel and a Cyclocarya paliurus extract product or a Cyclocarya paliurus leaf powder composited in the methacrylated gelatin hydrogel.

[0010] In an optional embodiment, the Cyclocarya paliurus extract product includes at least one of Cyclocarya paliurus squeezed juice, Cyclocarya paliurus extract, a Cyclocarya paliurus squeezed juice freeze-dried product, and a Cyclocarya paliurus extract freeze-dried product.

[0011] In an optional embodiment, all components except water in the Cyclocarya paliurus extract preparation are used as active ingredients, and the concentration of the active ingredients in the composite hydrogel is 5%-30% (w / v).

[0012] In an optional embodiment, the Cyclocarya paliurus juice is prepared from fresh, green, smallest, fully expanded leaves picked from March to June as raw materials;

[0013] And / or, the preparation method of the Cyclocarya paliurus extract comprises: mixing crushed Cyclocarya paliurus leaves and an extractant, obtaining an extract after reflux extraction, concentrating and drying the extract under reduced pressure to obtain a powder extract, and diluting and filtering the powder extract to obtain the Cyclocarya paliurus extract.

[0014] In an optional embodiment, the crushed Cyclocarya paliurus leaves have a particle size of 10-50 mesh;

[0015] and / or, the volume-to-mass ratio of the extractant to the Cyclocarya paliurus leaves in the reflux extraction step is 15-25 mL / g;

[0016] And / or, the number of reflux extractions is one or more than two times. When the number of reflux extractions is more than two times, solid-liquid separation is performed after each reflux extraction to obtain a filtrate, and the filtrates obtained from each reflux extraction are combined to obtain the extract;

[0017] and / or, the reflux extraction temperature is 110-130°C, and the single reflux extraction time is 0.5-2h;

[0018] And / or, the mass fraction of the powder extract in the diluted liquid is 5%-20%.

[0019] In a second aspect, the present invention provides a method for preparing a composite hydrogel as described in any one of the aforementioned embodiments, comprising: using ultraviolet light to cross-link and solidify a composite hydrogel precursor containing methacrylated gelatin, acylphosphonate lithium salt and Cyclocarya paliurus extract to obtain the composite hydrogel.

[0020] In an optional embodiment, the preparation of the composite hydrogel precursor includes: mixing an aqueous solution containing methacryloylated gelatin and a photoinitiator with the Cyclocarya paliurus extract product, wherein the concentration of methacryloylated gelatin in the aqueous solution is 8wt%-12wt%, and the concentration of the photoinitiator in the aqueous solution is 0.23wt%-0.27wt%;

[0021] Preferably, after the aqueous solution is prepared, it is filtered and sterilized before being mixed with the Cyclocarya paliurus extract product.

[0022] In an optional embodiment, the method further includes filtering and sterilizing the Cyclocarya paliurus extract or Cyclocarya paliurus juice, and then using the extract to prepare a composite hydrogel precursor;

[0023] Or, it also includes filtering, sterilizing and freeze-drying the Cyclocarya paliurus extract or Cyclocarya paliurus juice to obtain a freeze-dried product of the Cyclocarya paliurus extract or a freeze-dried product of the Cyclocarya paliurus juice, which is then used to prepare a composite hydrogel precursor.

[0024] In an optional embodiment, the wavelength of the ultraviolet light is 400-410 nm, and the cross-linking time is 15-30 s.

[0025] In a third aspect, the present invention provides a use of the composite hydrogel described in any one of the aforementioned embodiments in the preparation of a medicament for diabetic foot ulcer infection.

[0026] The present invention has the following beneficial effects:

[0027] The dressing prepared by the present invention realizes sustained drug release, which indicates that the hydrogel can promote the long-term release of the drug, thereby reducing the loss of liquid drugs, improving the utilization rate of the drug, and is used to inhibit the healing of diabetic foot ulcer infection wounds, and has broad clinical application prospects. Rat experiments show that the composite hydrogel prepared by the present invention can better improve and promote the healing of diabetic infection wounds, and the healing effect is better than that of the Cyclocarya paliurus extract product. The composite hydrogel can significantly reduce the levels of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 and IL-1β in the wound tissue, and the activity of superoxide dismutase (SOD) and malondialdehyde (MDA) levels are significantly improved, and it can promote the secretion of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF). BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 SEM images of GelMA and GelMA-h-CP hydrogels;

[0030] Figure 2 is the swelling properties of GelMA and GelMA-h-CP hydrogels;

[0031] Figure 3 The stretching curves of hydrogels; (a) GelMA; (b) GelMA-h-CP;

[0032] Figure 4 is the standard curve of chlorogenic acid (CA) solution with different concentrations;

[0033] Figure 5 is the drug release curve of GelMA-h-CP hydrogel;

[0034] Figure 6 Wound surface changes and healing rates in different treatment groups; (a) Wound surface changes; (b) Wound surface healing rate;

[0035] Figure 7The expressions of IL-6, TNF-α and IL-1β in the granulation tissue of the wound surface of rats in each group; (a) IL-6; (b) TNF-α; (c) IL-1β;

[0036] Figure 8 The expressions of SOD and MDA in wound tissues of rats in each group; (a) SOD; (b) MDA;

[0037] Fig. 9 Expression of VEGF and EGF in wound tissues of rats in each group; (a) VEGF; (b) EGF. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional ones that can be purchased commercially.

[0039] An embodiment of the present invention provides a composite hydrogel, comprising a methacrylated gelatin hydrogel and a Cyclocarya paliurus extract product or a Cyclocarya paliurus leaf powder composited in the methacrylated gelatin hydrogel.

[0040] The composite hydrogel of the present invention contains a Cyclocarya paliurus extract or Cyclocarya paliurus leaf powder, which has the effects of anti-infection, anti-inflammatory, anti-oxidation and promoting wound healing. The dressing can keep the wound moist, absorb wound exudate and release effective ingredients for a long time during the period of adhering to the diabetic ulcer wound, effectively exert antibacterial, anti-inflammatory and anti-oxidation effects, improve the infected ulcer wound environment, reduce inflammatory reactions, promote the regeneration of granulation tissue, and promote wound healing.

[0041] The hydrogel matrix of the present invention is a gelatin derivative, which contains rich arginine-glycine-aspartic acid and matrix metalloproteinase sensitive sequences, and has excellent cell attachment and remodeling capabilities. Its unique in situ photocrosslinking properties give it a significant advantage in treating irregular wounds of diabetic foot ulcers.

[0042] By physically blending the hydrogel with a Cyclocarya paliurus extract or Cyclocarya paliurus leaf powder, it was found that the prepared hydrogel exhibited a porous three-dimensional morphology, certain moisturizing properties, and anti-drying properties, which helped to maintain a moist environment for the wound, promote the production of endogenous enzymes, and effectively remove necrotic tissue; at the same time, the dressing prepared by the present invention had certain permeability, swelling capacity and mechanical properties, could absorb wound exudate, resist local stress during application and maintain the structural integrity of the wound dressing.

[0043] In addition, the dressing prepared by the present invention achieves sustained drug release, which indicates that the hydrogel can promote the long-term release of drugs, thereby reducing the loss of liquid drugs and improving drug utilization. It is used to inhibit the healing of infected wounds of diabetic foot ulcers and has broad clinical application prospects.

[0044] In an optional embodiment, the Cyclocarya paliurus extract product includes at least one of Cyclocarya paliurus squeezed juice, Cyclocarya paliurus extract, a Cyclocarya paliurus squeezed juice freeze-dried product, and a Cyclocarya paliurus extract freeze-dried product.

[0045] The extract product of Cyclocarya paliurus can be a water extract of Cyclocarya paliurus, an alcohol extract of Cyclocarya paliurus, or a squeezed juice of young leaves of Cyclocarya paliurus, or a powder or paste obtained by drying the water extract of Cyclocarya paliurus, the alcohol extract of Cyclocarya paliurus, or the squeezed juice of young leaves of Cyclocarya paliurus.

[0046] In an optional embodiment, all components except water in the Cyclocarya paliurus extract preparation are used as active ingredients, and the concentration of the active ingredients in the composite hydrogel is 5%-30% (w / v).

[0047] In an optional embodiment, the Cyclocarya paliurus juice is prepared using fresh, green, smallest fully expanded leaves picked from March to June as raw materials.

[0048] Fresh, green, smallest, fully expanded young leaves of Cyclocarya paliurus picked from March to June are selected, washed and immediately squeezed with a juicer, or stored in a refrigerated vacuum state, and squeezed within 48 hours to obtain a coarse squeezed juice. The coarse squeezed juice is filtered through four layers of gauze and a disposable sterile syringe filter to obtain the Cyclocarya paliurus young leaf juice extract, which is then used to prepare a drug for diabetic foot ulcer infection.

[0049] The juice squeezing temperature may be room temperature, preferably 22±3° C., and the sterile syringe filter may be a disposable sterile syringe filter of 0.45 μm.

[0050] In an optional embodiment, the preparation method of the Cyclocarya paliurus extract includes: mixing crushed Cyclocarya paliurus leaves and an extractant, obtaining an extract after reflux extraction, concentrating and drying the extract under reduced pressure to obtain a powder extract, diluting and filtering the powder extract to obtain the Cyclocarya paliurus extract.

[0051] In an optional embodiment, the crushed Cyclocarya paliurus leaves have a particle size of 10-50 mesh;

[0052] and / or, in the reflux extraction step, the volume mass ratio of the extractant to the Cyclocarya paliurus leaf is 15-25 mL / g, preferably 20 mL / g;

[0053] And / or, the number of reflux extractions is one or more than two times. When the number of reflux extractions is more than two times, solid-liquid separation is performed after each reflux extraction to obtain a filtrate, and the filtrates obtained from each reflux extraction are combined to form the extract. Preferably, the number of reflux extractions is two times.

[0054] And / or, the reflux extraction temperature is 110-130°C, and the single reflux extraction time is 0.5-2h, preferably the temperature is 120°C, and the single reflux extraction time is 1h;

[0055] And / or, the mass fraction of the powder extract in the diluted liquid is 5%-20%, preferably 10%.

[0056] An embodiment of the present invention also provides a method for preparing the composite hydrogel described in any one of the aforementioned embodiments, comprising: using ultraviolet light to cross-link and solidify a composite hydrogel precursor containing methacrylated gelatin, acylphosphonic acid lithium salt and Cyclocarya paliurus extract to obtain the composite hydrogel.

[0057] In an optional embodiment, the preparation of the composite hydrogel precursor includes: mixing an aqueous solution containing methacryloylated gelatin and a photoinitiator with the Cyclocarya paliurus extract product, wherein the concentration of methacryloylated gelatin in the aqueous solution is 8wt%-12wt%, and the concentration of the photoinitiator in the aqueous solution is 0.23wt%-0.27wt%;

[0058] Preferably, after the aqueous solution is prepared, it is filtered and sterilized before being mixed with the Cyclocarya paliurus extract product.

[0059] The photoinitiator may be selected from acylphosphonate lithium salts, such as phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt and the like.

[0060] In some embodiments, the preparation method of the composite hydrogel comprises:

[0061] S1: The freeze-dried GelMA was dissolved in deionized water containing acylphosphonate lithium salt at 45° C. in the dark to obtain a GelMA solution, and the GelMA solution was immediately filtered using a 0.22 μm sterile syringe filter.

[0062] S2: Mix the GelMA solution of step 1 with the Cyclocarya paliurus extract product, and stir evenly at 45° C. in the dark to prepare a composite hydrogel precursor.

[0063] S3: Use ultraviolet light to cross-link and solidify the composite hydrogel precursor obtained in step 2 into a hydrogel.

[0064] In an optional embodiment, the method further includes filtering and sterilizing the Cyclocarya paliurus extract or Cyclocarya paliurus juice, and then using the extract to prepare a composite hydrogel precursor;

[0065] Or, it also includes filtering, sterilizing and freeze-drying the Cyclocarya paliurus extract or Cyclocarya paliurus juice to obtain a freeze-dried product of the Cyclocarya paliurus extract or a freeze-dried product of the Cyclocarya paliurus juice, which is then used to prepare a composite hydrogel precursor.

[0066] In an optional embodiment, the wavelength of the ultraviolet light is 400-410 nm, and the cross-linking time is 15-30 s.

[0067] An embodiment of the present invention further provides a use of the composite hydrogel described in any one of the aforementioned embodiments in the preparation of a medicament for treating diabetic foot ulcer infection.

[0068] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0069] Example 1 - Preparation of a hydrogel system loaded with Cyclocarya paliurus extract

[0070] Fresh, green, smallest, fully expanded young leaves of Cyclocarya paliurus picked from March to June are picked, cleaned (dust and impurities are removed and selected), and then a certain mass of Cyclocarya paliurus young leaves are squeezed to extract juice with a juicer, the coarse squeezed juice is filtered with 4 layers of gauze, and the supernatant is filtered through a 0.45 μm microporous filter membrane to obtain the Cyclocarya paliurus young leaf squeezed juice extract (CPJ), and the Cyclocarya paliurus young leaf squeezed juice extract is further freeze-dried at -25°C until the weight no longer changes, to obtain a squeezed juice extract freeze-dried powder (CP).

[0071] The detailed process of preparing gelatin methacryloyl (GelMA) hydrogel is as follows:

[0072] Preparation of 0.25% (w / v) initiator standard solution: Add 20 mL of PBS buffer solution to a brown bottle containing 0.05 g of phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt initiator, then place it in a 45°C water bath to heat and dissolve in the dark, oscillate several times during the process to ensure that the initiator is fully dissolved, seal it and store it at 0°C in the dark.

[0073] Preparation of 10% methacryloyl gelatin solution: 1g of methacryloyl gelatin (GelMA) was placed in a centrifuge tube wrapped with tin foil, 10mL of 0.25% initiator standard solution was added to fully soak the GelMA, and then transferred to a water bath and heated at 65°C in the dark for 25min, during which it was shaken several times to completely dissolve it, and the GelMA solution was filtered. To ensure the sterility of the gel and low-temperature gelation, the GelMA solution was immediately filtered with a 0.22μm sterile syringe filter.

[0074] The GelMA solution was uniformly mixed with CP to prepare a 13.7% (w / v) gel mixed solution, which was sterilized by filtering the gel mixed solution with a 0.22 μm sterile syringe filter and then cured by ultraviolet light for 30 seconds to obtain a GelMA-h-CP hydrogel.

[0075] Example 2 - Preparation of a hydrogel system loaded with CA (chlorogenic acid, the main component of CPJ) products

[0076] Determination of CA concentration: According to the liquid chromatography analysis of the CPJ stock solution, the CA content was about 2.8%, and it was calculated that 13.7g of Cyclocarya paliurus stock solution contained 0.3836g of CA. In order to prove the main effect of CA in the Cyclocarya paliurus stock solution, the concentration of CA was appropriately increased to prepare a gel solution with a concentration of 0.5% (w / v) for subsequent experiments.

[0077] The detailed process of preparing gelatin methacryloyl (GelMA) hydrogel is as follows:

[0078] Preparation of 0.25% (w / v) initiator standard solution: Add 20 mL of PBS buffer solution to a brown bottle containing 0.05 g of phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt initiator, then place it in a 45°C water bath to heat and dissolve in the dark, oscillate several times during the process to ensure that the initiator is fully dissolved, seal it and store it at 0°C in the dark.

[0079] Preparation of 10% methacryloyl gelatin solution: 1g of methacryloyl gelatin (GelMA) was placed in a centrifuge tube wrapped with tin foil, 10mL of 0.25% initiator standard solution was added to fully soak the GelMA, and then transferred to a water bath and heated at 65°C in the dark for 25min, during which it was shaken several times to completely dissolve it, and the GelMA solution was filtered. To ensure the sterility of the gel and low-temperature gelation, the GelMA solution was immediately filtered with a 0.22μm sterile syringe filter.

[0080] The GelMA solution and CA powder were uniformly mixed to prepare a 0.5% gel mixed solution, which was sterilized by filtering the gel mixed solution with a 0.22 μm sterile syringe filter and then cured by ultraviolet light for 30 seconds to obtain a GelMA-h-CA hydrogel.

[0081] Comparative Example 1-Preparation of a hydrogel system without loading of Cyclocarya paliurus extract

[0082] The detailed process of preparing gelatin methacryloyl (GelMA) hydrogel is as follows:

[0083] Preparation of 0.25% (w / v) initiator standard solution: Add 20 mL of PBS buffer solution to a brown bottle containing 0.05 g of phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt initiator, then place it in a 45°C water bath to heat and dissolve in the dark, oscillate several times during the process to ensure that the initiator is fully dissolved, seal it and store it at 0°C in the dark.

[0084] Preparation of 10% methacryloyl gelatin solution: 1g of methacryloyl gelatin (GelMA) was placed in a centrifuge tube wrapped with tin foil, 10mL of 0.25% initiator standard solution was added to fully soak the GelMA, and then transferred to a water bath and heated at 65°C in the dark for 25min, during which it was shaken several times to completely dissolve it, and the GelMA solution was filtered. To ensure the sterility of the gel and low-temperature gelation, the GelMA solution was immediately filtered with a 0.22μm sterile syringe filter and cured with ultraviolet light for 30s to obtain a GelMA hydrogel.

[0085] Comparative Example 2-Preparation of extract from young leaves of Cyclocarya paliurus

[0086] Fresh, green, smallest, fully expanded young leaves of Cyclocarya paliurus picked from March to June are picked, cleaned (dust and impurities are removed and selected), and then a certain mass of Cyclocarya paliurus young leaves are squeezed to extract juice with a juicer, the coarsely squeezed juice is filtered with 4 layers of gauze, and the supernatant is filtered through a 0.45 μm microporous filter membrane to obtain the Cyclocarya paliurus young leaf squeezed juice extract (CPJ) with a water content of 66.3%.

[0087] Comparative Example 3 - Preparation of Cyclocarya paliurus aqueous extract

[0088] Take dry leaves of Cyclocarya paliurus, grind and sieve them to obtain dry powder for use; mix pure water and dry powder in a volume-to-mass ratio of 20, reflux extract at 120°C for 1 hour, extract twice in total, combine the filtrates, concentrate by rotary evaporation at 750°C to a volume concentration of 0.4 times, and then freeze-dry at -25°C until the weight no longer changes, to obtain a water extract dry powder extract.

[0089] Comparative Example 4-Preparation of Cyclocarya paliurus ethanol extract

[0090] Take the dry leaves of Cyclocarya paliurus, grind and sieve them to obtain dry powder for use; accurately weigh 15.0 g of the powder and place it in a 500 mL round-bottom flask, reflux extract with 80% ethanol solution, the solid-liquid ratio is 1:20 (mass / volume), reflux extraction at 120°C for 1 hour, extract twice in total, combine the filtrate, rotary evaporate and concentrate at 75°C to 0.4 times the volume, then freeze-dry at -25°C until the weight no longer changes, and then further freeze-dry the extract to obtain an alcohol extract powder extract.

[0091] Experimental Example 1 Characterization and physicochemical properties of GelMA-h-CP hydrogel and GelMA hydrogel

[0092] 1. Materials and reagents

[0093] 1.1 Main Materials

[0094] The main experimental materials are shown in Table 1.

[0095] Table 1 Main experimental materials

[0096]

[0097] 1.2 Main experimental instruments

[0098] The main experimental instruments are shown in Table 2

[0099] Table 2 Main experimental instruments

[0100]

[0101] 2 Internal morphology and structure of hydrogel

[0102] The morphology of the composite hydrogel obtained in Example 1 was studied using a scanning electron microscope. Figure 1 The hydrogels were shown to have a porous structure, indicating the successful preparation of supported hydrogels.

[0103] 3. Hydrogel equilibrium swelling rate experiment

[0104] Take a gel of size 10×10×5 (length×width×height) mm, freeze-dry it to constant weight, record the mass as M0, then transfer it to a beaker and completely immerse it in a PBS solution with pH=7.4, let it stand at room temperature in the dark, take out the sample after 0.5, 1, 2, 3, 6, 9, 12, 24, 48, and 72 hours, and calculate the swelling rate. Figure 2 The swelling ratio of the CP-loaded GelMA gel at 72 h was 580.7%.

[0105] 4 Mechanical properties test of hydrogel

[0106] The mechanical properties of the gel were tested using a German KW compression (tensile) stage equipped with an in-situ microscope. The experiment was terminated after the gel was compressed until it broke, and the compression strain was recorded. Figure 3 The results show that the tensile strength of GelMA-h-CP gel is 43.59 kPa, and the corresponding elongation at break is 62.19%.

[0107] 5. Drug release experiment

[0108] Through preliminary experiments, it was found that chlorogenic acid is one of the main components of Cyclocarya paliurus. Figure 4As shown in FIG. 1 , after the standard curve is established, the GelMA-h-CP hydrogel is dried in a vacuum oven at 60° C. for 2 h, and then a certain amount of the gel is packaged into a dialysis bag (molecular weight: 300-500), as shown in FIG. Figure 5 As shown, 3 mL of the solution was taken after each interval to perform UV-visible absorption spectrum test, and it was found that the prepared loaded GelMA hydrogel had a certain drug sustained-release effect.

[0109] Experimental Example 2 Experimental evaluation of hydrogel loaded with Cyclocarya paliurus juice to promote wound healing in vivo

[0110] 1. Materials and reagents

[0111] 1.1 Main Materials

[0112] Streptozotocin (STZ) (MP Biomedicals, 0210055701), sodium citrate buffer (Beijing Solebow Technology Co., Ltd., C1013), Staphylococcus aureus (Qingdao Haibo Biotechnology Co., Ltd., ATCC25923), and Cyclocarya paliurus were purchased from Nanjiang County People's Hospital, Bazhong, Sichuan.

[0113] 1.3 Instruments

[0114] Blood glucose meter, blood glucose test strips (Sinocare Biosensor Co., Ltd., GA-3, measuring range: 1.1-33.3 mmol / L, display HI when higher than 33.3 mmol / L), enzyme labeled detector (BioTek, Epoch, USA).

[0115] 1.4 Animal grouping and modeling

[0116] 72 SPF healthy male 6-week-old SD rats, with a body weight of (200±10)g, were fed with SD rat basic feed and purified water for one week. The rats' grip, mental state, stool, fur color, etc. were observed, and rats with abnormal fur color, mental depression or excitement, diarrhea or constipation were removed in time. After the adaptive feeding of the remaining rats, they were randomly divided into two parts: control group: CON group (12 rats) and DM model group (60 rats). The CON group continued to be fed with basic maintenance feed, and the DM model group was fed with high-fat and high-sugar feed. After 2 weeks of feeding, they were fasted but not watered for 16h, and 1% STZ was injected intraperitoneally at a uniform speed at 50mg / kg to complete the preparation of DM rat model. After the modeling of the experimental rats, the SD rats were continued to be fed with high-fat and high-sugar feed and pure water. The blood glucose of the rats was measured by the rat tail vein blood sampling method after 72h, 7d, and 14d, and the rats with blood glucose ≥16.7mmol / L were selected as stable DM model rats. The selected model rats were randomly divided into 5 groups, 12 in each group, namely MOD group: model group; CPJ group-Cyclocarya paliurus juice group; GelMA-h-CP group-loaded juice gel group; CA group: chlorogenic acid group; GelMA-h-CA group: loaded chlorogenic acid gel group. On the day of grouping, the rats in each group were marked with numbers, weighed, and the subsequent DM full-thickness skin wound infection model was carried out.

[0117] On the premise that DM model was successfully established, the rats were anesthetized with 3% sodium pentobarbital, the back hair was removed, and a skin biopsy sampler with a diameter of 10 mm was used to create a full-thickness skin defect on the back of the rats. The wound depth reached the muscle fascia layer, and the wound edge was trimmed with ophthalmic scissors. After gauze was applied to stop bleeding, 50 μL (2×10 6 CFU / mL) Staphylococcus aureus (S. aureus) was suspended in PBS and applied to the wound surface, and a retention patch was applied to prevent contamination by other bacteria. A wound infection model was established, and rats were placed on a warming pad and put into a cage after they woke up naturally, and their physical signs were observed.

[0118] 2 Methods

[0119] 2.1 Intervention methods

[0120] After successful modeling, the GelMA-h-CP group and the GelMA-h-CA group were prepared with gel mixed solutions according to the methods of Examples 1 and 2, respectively. The gel mixed solutions were sterilized by filtering with a 0.22 μm sterile syringe filter, covered on the wound surface, cured with ultraviolet light for 30 s, covered with six layers of medical gauze (4 cm × 4 cm), and fixed with a medical elastic bandage for 2 weeks.

[0121] After successful modeling, the ulcer wounds of rats in the CPJ group and the CA group were given the above-mentioned Cyclocarya paliurus juice and chlorogenic acid solution dripped into the silicone gasket (about 0.2 mL) every day, and the wounds were covered with six layers of medical gauze (4 cm × 4 cm) and fixed with medical elastic bandages for 2 weeks.

[0122] The ulcer wounds of rats in the normal control group and the model group were wiped with an equal amount of normal saline once a day. After wiping, the wounds were covered with six layers of medical gauze (4cm×4cm) and fixed with medical elastic bandages for 2 weeks.

[0123] Six rats were randomly selected from each group and killed for relevant tests after 7 and 14 days of intervention.

[0124] 2.2 Observation indicators

[0125] 2.2.1 Blood glucose status of rats

[0126] After the experimental grouping and drug administration, blood sugar was measured at 8 am every other week. The measurement method adopted the rat tail vein blood sampling method. First, the rat's tail was cleaned with water, and the moisture was absorbed with a cotton ball. The rat's tail was slowly massaged from the proximal end to the distal end of the tail to make the tip of the rat's tail fully congested. The tip of the tail was wiped with a sterilized cotton swab soaked in 75% ethanol to fully disinfect the blood sampling area. The ethanol in the tail was then wiped dry with a dry, clean, sterile cotton swab, and a disposable blood collection needle was used to pierce the congested area of ​​the tail tip (one needle for one mouse, and the blood collection needle was not reused). After the first drop of blood flowed out, it was wiped off with a clean, dry, sterilized cotton swab, and the second drop of blood was drawn with a blood glucose meter equipped with a clean blood glucose test strip. The blood glucose value displayed on the blood glucose meter was read in real time and accurately recorded.

[0127] 2.2.2 Wound healing rate

[0128] After 2 days of bacterial infection (marked as day 0), the dressing was changed every morning, the retention patch was removed, and after treatment, another layer of retention patch was covered on the wound site to exclude external contamination. The wound area on day 0 was taken as 100%, and the wound area of ​​each group was quantified on days 1, 3, 5, 7, 11, and 14, and the size of the wound was calculated using Fiji Image J images.

[0129]

[0130] 2.2.3 Detection of IL-6, TNF-α, and IL-1β levels in granulation tissue of ulcer wounds

[0131] ELISA was used to detect the levels of IL-6, TNF-α, and IL-1β in the granulation tissue of the ulcer wound. After the experimental rats were killed and the ulcer surface tissue was wrapped in tin foil and frozen in liquid nitrogen, an appropriate amount of the cut skin tissue was weighed, added to a 9-fold sterile PBS solution, ground and mixed thoroughly, and then the skin granulation tissue homogenate was centrifuged at 3000-4000 rpm at 4°C for 10 min, the supernatant was collected, and the test was completed in strict accordance with the instructions of the kit.

[0132] 2.2.4 Detection of SOD and MDA levels in granulation tissue of ulcer wound

[0133] Biochemical method was used to detect the SOD activity and MDA level of granulation tissue of ulcer wound. The frozen tissue was taken out and thawed on ice. According to the instructions of the kit, SOD activity was calculated by colorimetric analysis of WST-1 product. MDA in lipid peroxidation degradation products can react with thiobarbituric acid under high temperature and acidic environment. The MDA content in the skin granulation tissue of rats in each group was detected by thiobarbituric acid method.

[0134] 2.2.5 Detection of VEGF and EGF levels in granulation tissue of ulcer wound

[0135] The expression of related healing factors VEGF and EGF in each group of rats was detected by ELISA. An appropriate amount of cut skin tissue was weighed and ground with 9-fold sterile PBS solution. Then the skin granulation tissue homogenate was centrifuged at 3000-4000rpm for 10 minutes at 4°C, and the supernatant of the homogenate sample was collected and stored at 4°C. The VEGF and EGF content in the skin granulation tissue of each group of rats was determined according to the instructions, and the final results were standardized according to the Bicinchoninic Acid Assay (BCA) method for the protein concentration detected.

[0136] 3 Results

[0137] 3.1 Blood sugar status

[0138] The blood glucose levels of rats in each group are shown in Table 3. Compared with the CON group, all DM rats were stable in a hyperglycemic state during the experiment (P<0.01); during the 14-day treatment, there was no statistical difference in blood glucose between the intervention group and the MOD group.

[0139] Table 3 Comparison of blood glucose levels in each group at different time points

[0140]

[0141] Note: Compared with the model group *P<0.05, **P<0.01

[0142] 3.2 Wound healing rate

[0143] The experimental results are shown in Figure 6 Table 4

[0144] Table 4 Comparison of wound healing rate in each group at different time points after treatment (%, )

[0145]

[0146] Note: Compared with the model group *P<0.05, **P<0.01

[0147] Depend on Figure 6 From Table 4, we can see that:

[0148] After S. aureus infection, white pus forms initially on the normal wound surface at about 24 hours, and obvious purulent secretions can be seen at 48 hours, and redness and swelling appear around the skin. However, the white pus appears relatively slowly in the wound surface of DM rats, and initially forms at 48 hours. After 3 days of intervention, the wound healing rate of the GelMA-h-CP group was significantly improved compared with the MOD group (P < 0.05). After 5 days of administration, the healing rate of the MOD group was significantly lower than that of the CON group and each intervention group (P < 0.05 or P < 0.01). There was light yellow thin exudate on the wound surface of the CON group and each intervention group, and there was no statistically significant difference between the intervention groups (P > 0.05). After 7 days of administration, bright red new granulation tissue could be clearly seen in each intervention group and the CON group. The healing rate of the MOD group was slightly better than before, but the healing rate was still significantly lower than that of the CON group and each intervention group. Compared with the CON group, the healing rates of each intervention group were lower than that of the CON group. However, except for the CA group (P<0.05), the differences were not statistically significant (P>0.05). After 11 days of administration, inflammatory reaction was still observed at the junction of the wound and normal tissue in the MOD group. The healing rates of the CON group and each intervention group were significantly increased compared with the MOD group, and the differences were statistically significant in each group (P<0.05 or P<0.01). Compared with the CON group, the healing rates of the CPJ, GelMA-h-CP, and GelMA-h-CA groups were not statistically significant. The healing rate of the CA group was lower than that of the CON group, and the difference was statistically significant (P<0.01). Compared with the intervention groups, the effect of the GelMA-h-CP group on promoting the healing of DM-infected wounds was better than that of the CPJ, CA, and GelMA-h-CA groups, and the difference was statistically significant (P<0.05 or P<0.01). After 14 days of administration, compared with the MOD group, the wounds in each group healed significantly (P<0.05), the scabs were absorbed and fell off, and all 4 rats in the GelMA-h-CP group were completely healed. Compared with the different intervention groups, the healing rate showed a trend of GelMA-h-CP>GelMA-h-CA>CPJ>CA, and the difference between GelMA-h-CP and CA groups was statistically significant.

[0149] 3.3 Levels of IL-6 and TNF-α in wound tissue

[0150] The experimental results are shown in Figure 7 Table 5

[0151] Table 5 Expression of TNF-α and IL-6 inflammatory factors in the wound surface of rats in each group at different time points

[0152]

[0153] Note: Compared with the model group *P<0.05, **P<0.01

[0154] Depend on Figure 7 From Table 5, we can see that:

[0155] Compared with the CON group, the levels of IL-6, TNF-α, and IL-1β in the wound of rats in the MOD group were significantly increased (P < 0.05 or P < 0.01), indicating that after bacterial induction, in the abnormal state of DM, the body showed an uncontrolled "overreaction" and released excessive inflammatory factors. Compared with the MOD group, the levels of IL-6 and TNF-α decreased significantly after CPJ, GelMA-h-CP, CA, and GelMA-h-CA intervention for 7d and 14d, improving the inflammatory state of the wound. There was no significant statistical difference in the expression of IL-6 and TNF-α between the intervention groups and the CON group, indicating that CPJ, GelMA-h-CP, CA, and GelMA-h-CA restored the control of the secretion of inflammatory factors in response to bacteria in the DM state. Compared with the CON group, the MOD group significantly increased the IL-1β protein expression level. After 7 days of intervention, CPJ, GelMA-h-CP, and GelMA-h-CA could significantly inhibit the overexpression of IL-1β in DM infected wounds (P<0.05 or P<0.01). The expression level in the CA group showed a downward trend, but there was no statistical significance compared with the MOD group. After 14 days, the IL-1β levels in each group showed a downward trend compared with 7 days before. The IL-1β level in the MOD group was significantly higher than that in the CON group (P<0.01). The IL-1β protein expression levels in the wounds of each intervention group and the CON group were still significantly lower than those in the MOD group (P<0.01). There was no statistical significance between the different intervention groups.

[0156] 3.4 SOD activity and MDA expression levels in wound tissue

[0157] The experimental results are shown in Figure 8 Table 6

[0158] Table 6 SOD activity and MDA expression levels in wound tissue

[0159]

[0160] Note: Compared with the model group *P<0.05, **P<0.01

[0161] Depend on Figure 8 From Table 6, we can see that:

[0162] On the 7th day after administration, compared with the MOD group, the SOD activity in the CON group, CPJ group, GelMA-h-CP group, CA group, and GelMA-h-CA group increased (P<0.05 or P<0.01). Compared with the CON group, there was no significant difference in the expression between the intervention groups and the CON group (P>0.05). Comparison between the intervention methods showed that the SOD expression in the GelMA-h-CP group was higher than that in the CA group and the GelMA-h-CA group, and the difference was statistically significant (P<0.01). In the wound tissue on the 14th day, the SOD expression level in each group increased to varying degrees compared with that on the 7th day. The SOD expression in the CON group, CPJ group, and GelMA-h-CP group was still significantly higher than that in the MOD group. The SOD expression in the CA group and the GelMA-h-CA group showed an increasing trend compared with the MOD group, but the difference was not statistically significant (P>0.05). The SOD expression levels of the CPJ group and the GelMA-h-CP group at 14 days were significantly higher than those of the CA group (P < 0.01, P < 0.05). Malondialdehyde (MDA) is one of the end products of lipid peroxidation and represents the degree of lipid peroxidation damage in tissues. At 7 days and 14 days after wound healing, the MDA level in the MOD group was significantly higher than that in the CON group (P < 0.01), reflecting that the degree of tissue peroxidation damage in DM infection was higher than that in the CON group. The MDA levels of each group at 14 days were lower than those at 7 days. The MDA levels of each intervention group were significantly lower than those of the MOD group at both 7 days and 14 days, but there was no significant statistical difference between the intervention methods.

[0163] 3.4 Expression levels of VEGF and EGF in wound tissue

[0164] The experimental results are shown in Fig. 9 Table 7

[0165] Table 7 Expression of VEGF and EGF in wounds of rats in each group at different time points

[0166]

[0167]

[0168] Note: Compared with the model group *P<0.05, **P<0.01

[0169] Depend on Fig. 9 , Table 7 shows that:

[0170] VEGF can mediate the proliferation, sprouting, migration and lumen formation of endothelial cells. On the 7th day after treatment, the expression of VEGF in the wound surface of the CON group, CPJ group and GelMA-h-CP group was higher than that of the MOD group (P<0.05), and there was no significant difference between each intervention group and the CON group (P>0.05). Compared with the 7th day, the expression of VEGF in the wound surface on the 14th day showed a downward trend. On the 14th day after treatment, the expression of VEGF in the CON group, CPJ group and GelMA-h-CP group was still higher than that of the MOD group (P<0.05), and the expression of each group showed a downward trend compared with the previous day, and there was no statistical difference compared with the CON group; the increased VEGF level in the GelMA-h-CP group was significantly higher than that in the CA group (P<0.01).

[0171] EGF can promote the growth and division of epidermal cells, keratinocytes, fibroblasts and other cells, accelerate metabolism and enhance the activity of epidermal cells. Compared with the CON group, the EGF secretion level of the 7d MOD group was lower (P<0.01), and the EGF levels of each intervention group were slightly higher than those of the MOD group, but the difference was not statistically significant (P>0.05). Compared with the CON group, the EGF expression of the GelMA-h-CP group had no significant difference (P>0.05), and the other intervention groups were lower than the CON group and the difference was statistically significant (P<0.05). After 14 days, the EGF expression of each group was significantly higher than before, and the EGF expression of the wound surface of the CPJ, GelMA-h-CP and CON groups was higher than that of the MOD group (P<0.05), and there was no difference between the CPJ, GelMA-h-CP and CON groups (P>0.05). Among them, the GelMA-h-CP group was significantly superior to the CA group and the GelMA-h-CA group in increasing the level of EGF. In summary, the GelMA hydrogel loaded with Cyclocarya paliurus can significantly reduce the secretion of inflammatory factors TNF-α, IL-6, and IL-1β, improve the oxidative stress state (SOD, MDA), and increase the expression of healing factors VEGF and EGF to promote the healing of DM-infected wounds. CA may be one of the main components of CPJ, which can better correct excessive inflammation and oxidative stress. The effect of GelMA hydrogel and the synergistic effect of CA and other plant components may be the factors that promote the better healing effect of CPJ and GelMA-h-CP. The above studies show that GelMA-h-CP loaded with CPJ can be used as an effective therapeutic drug for the treatment of chronic DM-infected wounds, and the effect is more significant than that of single-component medication, which provides new ideas for treatment strategies and promotes the application in this field.

[0172] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A composite hydrogel, characterized in that: The invention comprises a methacrylated gelatin hydrogel and a Cyclocarya paliurus extract product or a Cyclocarya paliurus leaf powder compounded in the methacrylated gelatin hydrogel.

2. The composite hydrogel according to claim 1, characterized in that The Cyclocarya paliurus extract product comprises at least one of Cyclocarya paliurus squeezed juice, Cyclocarya paliurus extract, Cyclocarya paliurus squeezed juice freeze-dried product and Cyclocarya paliurus extract freeze-dried product.

3. The composite hydrogel according to claim 1, characterized in that All components except water in the Cyclocarya paliurus extract product are used as active ingredients, and the concentration of the active ingredients in the composite hydrogel is 5%-30% (w / v).

4. The composite hydrogel according to claim 2, characterized in that The Cyclocarya paliurus juice is prepared from fresh, green, smallest, fully expanded leaves picked from March to June as raw materials; And / or, the preparation method of the Cyclocarya paliurus extract comprises: mixing crushed Cyclocarya paliurus leaves and an extractant, obtaining an extract after reflux extraction, concentrating and drying the extract under reduced pressure to obtain a powder extract, and diluting and filtering the powder extract to obtain the Cyclocarya paliurus extract.

5. The composite hydrogel according to claim 4, characterized in that The particle size of the crushed Cyclocarya paliurus leaves is 10-50 mesh; and / or, the volume-to-mass ratio of the extractant to the Cyclocarya paliurus leaves in the reflux extraction step is 15-25 mL / g; And / or, the number of reflux extractions is one or more than two times. When the number of reflux extractions is more than two times, solid-liquid separation is performed after each reflux extraction to obtain a filtrate, and the filtrates obtained from each reflux extraction are combined to obtain the extract; and / or, the reflux extraction temperature is 110-130° C., and the single reflux extraction time is 0.5-2 h; And / or, the mass fraction of the powder extract in the diluted liquid is 5%-20%.

6. A method for preparing the composite hydrogel according to any one of claims 1 to 5, characterized in that: include: The composite hydrogel precursor containing methacrylic gelatin, acylphosphonic acid lithium salt and Cyclocarya paliurus extract is cross-linked and cured by ultraviolet light to obtain the composite hydrogel.

7. The method for preparing the composite hydrogel according to claim 6, characterized in that: The preparation of the composite hydrogel precursor comprises: mixing an aqueous solution containing methacryloylated gelatin and a photoinitiator with the Cyclocarya paliurus extract product, wherein the concentration of methacryloylated gelatin in the aqueous solution is 8wt%-12wt%, and the concentration of the photoinitiator in the aqueous solution is 0.23wt%-0.27wt%; Preferably, after the aqueous solution is prepared, it is filtered and sterilized before being mixed with the Cyclocarya paliurus extract product.

8. The method for preparing the composite hydrogel according to claim 6, characterized in that: The method also includes filtering and sterilizing the Cyclocarya paliurus extract or Cyclocarya paliurus squeezed juice, and then using it to prepare a composite hydrogel precursor; Or, it also includes filtering, sterilizing and freeze-drying the Cyclocarya paliurus extract or Cyclocarya paliurus juice to obtain a freeze-dried product of the Cyclocarya paliurus extract or a freeze-dried product of the Cyclocarya paliurus juice, which is then used to prepare a composite hydrogel precursor.

9. The method for preparing the composite hydrogel according to claim 6, characterized in that: The wavelength of the ultraviolet light is 400-410 nm, and the cross-linking time is 15-30 s.

10. Use of the composite hydrogel according to any one of claims 1 to 5 in the preparation of a medicament for treating diabetic foot ulcer infection.

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