An antibacterial composite hydrogel loaded with nanoparticles, its preparation method and application

Abstract

The present invention relates to an antibacterial composite hydrogel loaded with nanoparticles, a preparation method and an application thereof. The main body of the hydrogel network is composed of phenylboronic acid grafted methacrylated type I collagen rCol1MA-PBA and methacrylated polylysine EPLMA, crosslinked by adding glucose oxidase GoX, and simultaneously loaded with core-shell nanozyme C@Z@T. The core-shell nanozyme C@Z@T consists of a spherical CeO2 nanoparticle core, a zeolitic imidazolate framework material TIF-8 shell, and tannic acid TA coated on the shell. The raw materials of this hydrogel are easily obtained, the preparation method is simple, the price is cheap, and the components are safe and reliable; moreover, it integrates multiple functions such as antibacterial, anti-inflammatory, antioxidant and promoting angiogenesis, providing a more comprehensive therapeutic effect and can be used for the healing of diabetic wounds.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical preparations, and particularly to an antibacterial composite hydrogel loaded with nanoparticles, a preparation method thereof, and an application thereof. Background Art

[0002] The continuous challenge of chronic wound non-healing has been a major burden on healthcare, and such wounds are common in diabetic patients. This has greatly increased the financial and medical costs of the global healthcare system, ranging from $9.9 billion to $35.8 billion annually. It has specific characteristics, including persistent inflammation, blocked cell proliferation, blocked angiogenesis, increased susceptibility to infection, and extracellular matrix alteration, etc., and about 20% of cases require amputation. Diabetic patients are particularly vulnerable to these problems during the healing process. Debridement is currently the most common method for treating diabetic wounds. Debridement involves removing dead, damaged, or infected tissue, thereby enhancing the healing potential of the remaining healthy tissue. Another important wound care method after surgery and systemic care is local treatment. Currently, there are many local treatment methods available for treating diabetic foot wounds, including gauze, hydrogel, hydrocolloid, alginate dressings, foam dressings, silver ion dressings, dressings containing growth factors, silicone-impregnated non-invasive dressings, hyperbaric oxygen therapy, etc. Among them, the method of treating with dressings has received extensive attention in recent years. A dressing actually refers to a class of materials applied to cover, fill, and heal a wound surface. As an effective treatment means, it can reduce the situation where the patient's wound is exposed to the outside and infected.

[0003] As a traditional dressing, gauze is still widely used clinically. However, with the research on wounds, it has been found that this dry dressing is extremely prone to losing water and adhering to the wound site, which is likely to cause discomfort to the patient during replacement, and the effect of gauze in preventing microbial invasion is poor. Transparent film dressings can promote wound healing, but due to their limited absorption capacity, they can only be used for shallow wounds with a small amount of exudate and cannot be applied to highly exudative wounds. Foam dressings also have the disadvantages of requiring a specific thickness to absorb exudate, relying on specific polymer materials, being difficult to remove from the wound, and having a relatively single function, which limits their application in wound healing. The hydrophilicity of hydrocolloid dressings enables them to handle a certain amount of wound exudate. However, high exudate will cause the dressing to separate from the wound surface; at the same time, the preparation of hydrocolloid dressings has great limitations on materials, and this disadvantage limits their use range to only chronic wounds with mild exudate. Therefore, there is an urgent need to develop wound dressings that are more suitable for chronic wounds.

[0004] An ideal wound dressing should have the following characteristics: (1) sterile, easy to use, and low cost; (2) able to maintain a moist wound healing environment; (3) able to absorb excess wound exudate; (4) non-adherent to the wound, non-toxic, and non-allergenic; (5) prevent foreign body contamination of the wound; (6) protect the wound from microbial invasion; (7) allow gas exchange between the inside of the wound and the outside; (8) provide heat insulation and a certain degree of mechanical protection.

[0005] Compared with other commonly used dressings, hydrogel dressings have the advantages of reducing wound exudate, providing a moist environment, ensuring gas exchange, having a modulus matching that of human soft tissue, and reducing the patient's pain perception during dressing changes. At the same time, they also reduce the limitations on materials. Summary of the Invention

[0006] In order to overcome at least one problem existing in the above-mentioned prior art, the present invention has developed a novel antibacterial composite hydrogel loaded with nanoparticles. Using phenylboronic acid-grafted methacrylated type I collagen (rCol1MA-PBA) and methacrylated polylysine (EPLMA) as the substrate, glucose oxidase (GOx) is added as a pH-regulating component, and a hydrogel carrier is prepared by photocuring crosslinking, and loaded with core-shell nanozyme (C@Z@T), which can spontaneously activate the generation and scavenging functions of ROS, balance the ROS homeostasis, and promote the healing of diabetic wounds.

[0007] One aspect of the present invention provides an antibacterial composite hydrogel loaded with nanoparticles. The main body of the hydrogel network is composed of phenylboronic acid-grafted methacrylated type I collagen rCol1MA-PBA and methacrylated polylysine EPLMA, crosslinked with the addition of glucose oxidase GOx, and simultaneously loaded with core-shell nanozyme C@Z@T. The core-shell nanozyme C@Z@T consists of a spherical CeO2 nanoparticle core, a zeolitic imidazolate framework material TIF-8 shell, and tannic acid TA coated on the shell.

[0008] Further, in the hydrogel, it contains 10wt%-20wt% rCol1MA-PBA, 7.5wt%-12.5wt% EPLMA, 10mg / mL GOx, and 50-200μg / mL C@Z@T.

[0009] Further, the rCol1MA-PBA is obtained by mixing and reacting 3-aminophenylboronic acid, EDC, NHS, and type I recombinant collagen at room temperature; the ratio of 3-aminophenylboronic acid: EDC: NHS: type I recombinant collagen = 1.0g: 1.0g: 0.5g: 2g.

[0010] Further, the EPLMA is prepared by the ring-opening reaction of nucleophilic amination of polylysine EPL and polymethyl methacrylate MA, and the molar ratio of MA to EPL is 11:1.

[0011] Further, the particle size of the core-shell nanozyme C@Z@T is 20 - 700 nm, and the ζ potential is -26.9 ± 0.721 mV.

[0012] Further, the preparation method of the core-shell nanozyme C@Z@T includes the steps:

[0013] S1 Preparation of CeO2@ZIF-8 nanoparticles;

[0014] Dissolve spherical CeO2 nanoparticles and PVP in an organic solvent respectively, mix them evenly, then centrifuge the product and immediately redisperse it in a 2-methylimidazole / organic solvent solution until completely mixed; subsequently, add a zinc salt and mix and react to obtain the CeO2@ZIF-8 nanoparticles;

[0015] S2 Preparation of CeO2@ZIF-8@TA nanozyme;

[0016] Disperse the prepared CeO2@ZIF-8 nanoparticles evenly in water, then add tannic acid and disperse evenly, then centrifuge, wash, and dry to obtain the core-shell nanozyme.

[0017] Further, in the step S1, the reaction temperature is 24 - 37 °C, and the ratio of spherical CeO2 nanoparticles to PVP is 30 - 40 mg: 0.5 - 1.5 g; in the step S2, the reaction temperature is 25 - 35 °C, and the ratio of CeO2@ZIF-8 nanoparticles to tannic acid is 0.1 - 0.5 g: 0.05 - 0.15 g.

[0018] On the other hand, the present invention also provides a preparation method of the antibacterial composite hydrogel, including the following steps:

[0019] Prepare a solution containing a photoinitiator, then dissolve rCol1MA-PBA and EPLMA in it. After complete dissolution, add GOx, vortex to make it completely dissolve and disperse evenly, then add the C@Z@T core-shell nanozyme and stir to disperse; then under vacuum conditions, irradiate with ultraviolet light for crosslinking to obtain the antibacterial composite hydrogel.

[0020] The photoinitiator can be a water-soluble photoinitiator commonly used in hydrogels, such as LAP, NAP, etc. The addition amount is determined according to the specific type of photoinitiator.

[0021] On yet another aspect, the present invention also provides the application of the antibacterial composite hydrogel in wound dressings.

[0022] Furthermore, since the glucose oxidase (GOx) is loaded in the hydrogel of the present invention, it can rapidly respond to the special microenvironment of diabetic wounds, exert cascade enzyme catalytic activity, consume blood glucose at the wound site to generate H2O2 and gluconic acid. Moreover, the core-shell nanozyme C@Z@T decomposes in an acidic microenvironment to generate O2, scavenge ROS, promote the generation of mature collagen and the deposition of fibroblasts, so it is particularly suitable for diabetic wound dressings.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1) The raw materials of the hydrogel of the present invention are easily obtained, the preparation method is simple, the price is cheap, and the components are safe and reliable;

[0025] 2) Compared with the traditional wound healing materials with single function, the hydrogel of the present invention integrates multiple functions such as antibacterial, anti-inflammatory, antioxidant and promoting angiogenesis, providing a more comprehensive treatment effect;

[0026] 3) The hydrogel of the present invention is loaded with glucose oxidase (GOx), which can rapidly respond to the special microenvironment of diabetic wounds, exert cascade enzyme catalytic activity, and consume blood glucose at the wound site to generate H2O2 and gluconic acid.

[0027] 4) The hydrogel of the present invention can generate O2 in a slightly acidic environment under pH response, scavenge ROS, promote the generation of mature collagen and the deposition of fibroblasts, thereby accelerating the healing of diabetic wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the nuclear magnetic resonance spectrum of synthesized rCol1MA-PBA;

[0029] Figure 2 is the nuclear magnetic resonance spectrum of synthesized EPLMA;

[0030] Figure 3 is the TEM image of the core-shell nanozyme C@Z@T;

[0031] Figure 4 is the particle size distribution diagram of the core-shell nanozyme C@Z@T;

[0032] Figure 5 is the Zeta potential of CeO2, ZIF-8, C@Z, C@Z@T nanoparticles;

[0033] Figure 6 is the gelation diagram of the composite hydrogel of the embodiment of the present invention;

[0034] Figure 7 is the SEM image of the composite hydrogel of the embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0035] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings and specific embodiments. Preferred embodiments of the present invention are shown in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0036] In the description of the present invention, unless otherwise clearly defined, words such as heating, cleaning, weighing, freezing, etc. should be understood in a broad sense, and those skilled in the art can reasonably determine the specific meanings of the above words in the present invention in combination with the specific content of the technical solution.

[0037] In the description of the present invention, the description referring to terms such as "some embodiments", "examples", etc. means that the specific methods and materials described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific methods and materials described can be combined in a suitable manner in any one or more embodiments or examples.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention in this specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0039] In the following examples and comparative examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial sources.

[0040] Examples 1 - 6

[0041] This example provides an antibacterial composite hydrogel loaded with nanoparticles. The main body of the hydrogel network is composed of phenylboronic acid grafted methacrylated type I collagen rCol1MA - PBA and methacrylated polylysine EPLMA, crosslinked by adding glucose oxidase GOx, and simultaneously loaded with core - shell nanozyme C@Z@T. The core - shell nanozyme C@Z@T consists of a spherical CeO2 nanoparticle core, a zeolitic imidazolate framework material ZIF - 8 shell, and tannic acid TA coated on the shell.

[0042] Specifically, its preparation method includes the steps:

[0043] 1) Dissolve LAP in PBS buffer or physiological saline to form an aqueous solution with a mass fraction of 0.2%.

[0044] 2) Then, dissolve rCol1MA-PBA and EPLMA in 0.2% LAP aqueous solution. After ultrasonic treatment to completely dissolve them, add GOx and vortex for 10 min to completely dissolve it and uniformly mix it in the solution. Subsequently, add C@Z@T nanozyme and stir magnetically at 600 revolutions per minute for 10 minutes to uniformly disperse the microspheres in the solution. Finally, pour it into a mold, remove air bubbles under a vacuum of -1 bar, and irradiate with ultraviolet light at a wavelength of 365 nm for 30 seconds to complete crosslinking. Demold to obtain the hydrogel.

[0045] The preparation methods of methacrylated type I collagen rCol1MA-PBA, methacrylated polylysine EPLMA, and core-shell nanozyme C@Z@T used in the examples are as follows:

[0046] (1) Preparation of phenylboronic acid grafted methacrylated type I collagen (rCol1MA-PBA)

[0047] First, dissolve 2 g of type I recombinant collagen in 100 mL of water and stir until completely dissolved. Dissolve 1.0 g of 3-aminophenylboronic acid, 1.0 g of EDC, and 0.5 g of NHS in 50 mL of water and 10 mL of DMSO solution, and then add this mixed solution to the type I recombinant collagen solution. React at room temperature for 3 days, dialyze with a dialysis bag of 3500 Da, and freeze-dry to obtain rCol1-PBA. Weigh 1.0 g of rCol1-PBA and dissolve it in 100 mL of deionized water. After complete dissolution, add 1 mL of methacrylic anhydride and react at room temperature for 4 h. Dialyze with a dialysis bag of 8000 Da and freeze-dry to obtain rCol1MA-PBA.

[0048] Figure 1 Figure 13 is the nuclear magnetic resonance spectrum of the synthesized rCol1MA-PBA. Characteristic peaks of the benzene ring on phenylboronic acid appear at 7.74, 7.53, and 7.41 ppm, indicating the successful synthesis of rCol1-PBA. After methacrylation modification, characteristic peaks of the vinyl group on methacryloyl appear at 5.68 and 5.40 ppm, indicating the successful synthesis of rCol1MA-PBA.

[0049] (2) Preparation of methacrylated polylysine (EPLMA)

[0050] EPLMA was prepared by the ring-opening reaction of EPL with MA through nucleophilic amination. Specifically, EPL was dissolved in deionized water at 25 °C with stirring for 2 h to obtain a 10 wt% solution. At 80 °C, MA was added dropwise to the prepared EPL solution under mechanical stirring, and the reaction was carried out for 6 h until the oily liquid disappeared. The molar ratio of MA to EPL was 11:1. After the reaction, the obtained solution was filtered and dialyzed for 4 days using a dialysis bag (1000 Da). Finally, the obtained EPLMA solution was freeze-dried for 48 h. The freeze-dried EPLMA powder was stored at -20 °C for subsequent use.

[0051] Figure 2 It is the nuclear magnetic resonance spectrum of the synthesized EPLMA. The results show that the characteristic peaks of methacrylate vinyl (δ = 5.62 and 5.40 ppm) and methyl (δ = 1.8 ppm) appear in EPLMA, indicating that lysine has been successfully modified.

[0052] (3) Preparation of core-shell nanozyme C@Z@T

[0053] 3.1 Preparation of spherical CeO2

[0054] 2 g of cerium nitrate hexahydrate was added to 80 mL of ethylene glycol, and 4 mL of ultrapure water was added dropwise. The mixture was vigorously stirred at room temperature for 30 min. The mixed solution was transferred to a polytetrafluoroethylene liner, and the stainless-steel autoclave was sealed with a lid. Hydrothermal synthesis was carried out at 180 °C for 8 h. After centrifugal separation, it was repeatedly washed with ultrapure water and then dried overnight in an oven at 110 °C. The solid was ground into powder and placed in a muffle furnace. It was heated to 500 °C in air at a heating rate of 2 °C / min and calcined for 6 h.

[0055] 3.2 Preparation of CeO2@ZIF-8 nanoparticles

[0056] CeO2 nanoparticles (35 mg) and PVP (1 g) were respectively dissolved in methanol (50 mL), and then mixed at 25 °C for 24 h. After centrifugation of the product, it was immediately redispersed in 50 ml of 25.6 mM 2-methylimidazole (methanol solvent), and stirred at 25 °C for 20 min until completely mixed. Subsequently, Zn(NO3)2·6H2O (50 mL of 25.2 mM methanol solvent) was added, and mixed for another 30 minutes until the product was completely reacted. The final product was centrifuged at 12,000 rpm, purified with methanol 3 times, and dried in vacuo to obtain CeO2@ZIF-8 nanoparticles, denoted as C@Z.

[0057] 3.3 Preparation of CeO2@ZIF-8@TA core-shell nanozyme

[0058] Add 0.3 g of the prepared CeO2@ZIF-8 to 100 mL of water and stir for 30 min to disperse it evenly. Add 0.1 g of tannic acid (TA) to the solution, stir at 30 °C for 10 min, centrifuge to obtain the precipitate, and wash it several times with deionized water to remove the residual TA. Then, dry the wet CeO2@ZIF-8@TA nanoparticles at 60 °C for 12 h to obtain the CeO2@ZIF-8@TA core-shell nanozyme, denoted as C@Z@T.

[0059] (4) Preparation of ZIF-8 nanoparticles

[0060] Dissolve 3.3 g of 2-methylimidazole in 70 mL of methanol solution to form solution A. Subsequently, dissolve 1.5 g of zinc nitrate hexahydrate in 70 mL of methanol solution to form solution B. Slowly add solution A to solution B to form a mixed solution, stir at room temperature for 24 h, and perform centrifugal separation. The obtained solid is washed 3 times with methanol solution and then dried overnight at 80 °C. The obtained sample is denoted as ZIF-8.

[0061] Figure 3 Figure 10 is the transmission electron microscope image of the prepared C@Z@T core-shell nanozyme. It can be seen that since the core cerium oxide is spherical, the obtained C@Z@T nanoparticles also show uniform spherical shapes and uniform sizes. Figure 4 Figure 11 shows the particle size distribution of the C@Z@T core-shell nanozyme. It can be seen that the particle size is between 20 - 700 nm, and the average particle size is 214 ± 8.246 nm.

[0062] Figure 5 Figure 12 shows the Zeta potential statistics of the individual CeO2 and ZIF-8 nanoparticles, C@Z, and C@Z@T nanoparticles. It can be seen that the potentials of the individual CeO2 and ZIF-8 nanoparticles are -7.56 ± 1.58 mV and 23.8 ± 1.65 mV, respectively. Due to the negative charge of tannic acid, the overall shows a negative charge. The Zeta potential of the synthesized C@Z@T is significantly increased to -26.9 ± 0.721 mV, indicating an increase in the electrostatic repulsion between particles and a significant improvement in stability, and the core-shell nanozyme has good dispersibility.

[0063] Comparative Example 1

[0064] This comparative example provides a hydrogel. The main body of the hydrogel network is composed of phenylboronic acid-grafted methacrylated type I collagen rCol1MA-PBA, crosslinked with glucose oxidase GOx, and simultaneously loaded with the core-shell nanozyme C@Z@T. The core-shell nanozyme C@Z@T consists of a spherical CeO2 nanoparticle core, a zeolitic imidazolate framework material shell, and tannic acid coated on the shell.

[0065] The difference from Example 1 is only that in step 2), rCol1MA-PBA is dissolved in 0.2% LAP aqueous solution, and the rest is the same as in Example 1.

[0066] Comparative Example 2

[0067] This comparative example provides a hydrogel. The main body of the hydrogel network is crosslinked by phenylboronic acid-grafted methacrylated type I collagen rCol1MA-PBA, and simultaneously loaded with core-shell nanozyme C@Z@T. The core-shell nanozyme C@Z@T consists of a spherical CeO2 nanoparticle core, a zeolitic imidazolate framework material shell, and tannic acid coated on the shell.

[0068] The difference from Example 3 is that in step 2), there is no step of adding GOx, and the rest is the same as in Example 3.

[0069] Comparative Example 3

[0070] This comparative example provides a hydrogel composed of phenylboronic acid-grafted methacrylated type I collagen rCol1MA-PBA and methacrylated polylysine EPLMA.

[0071] The difference from Example 3 is that there is no step 1). In step 2), rCol1MA-PBA and EPLMA are directly dissolved in water, and there is no step of adding GoX. The rest is the same as in Example 3.

[0072] Since the solution does not contain a photo-crosslinking agent, direct ultraviolet light irradiation cannot crosslink to form a gel network.

[0073] Comparative Example 4

[0074] This comparative example provides a hydrogel. The main body of the hydrogel network is crosslinked by methacrylated polylysine EPLMA with the addition of glucose oxidase GOx.

[0075] The difference from Example 3 is that in step 2), EPLMA is dissolved in 0.2% LAP aqueous solution, and there is no step of adding C@Z@T nanoparticles. The rest is the same as in Example 3.

[0076] Comparative Example 5

[0077] This example provides a hydrogel. The main body of the hydrogel network is crosslinked by phenylboronic acid-grafted methacrylated type I collagen rCol1MA-PBA and methacrylated polylysine EPLMA.

[0078] The difference from Example 3 is that in step 2), there is no step of adding C@Z@T nanoparticles and GOx. The rest is the same as in Example 3.

[0079] The hydrogel formulation tables for each example and comparative example are shown in Table 1.

[0080] Table 1 Hydrogel Formulation Tables for Examples and Comparative Examples

[0081]

[0082]

[0083] Implementation analysis of hydrogel effects:

[0084] 1. Analysis of hydrogel storage modulus

[0085] Table 2 Hydrogel Storage Modulus

[0086]

[0087] Note: Comparative Example 3 is a liquid and not tested.

[0088] Test method for storage modulus: Demold a cylindrical hydrogel sample with a liquid volume of 400 μL. Place it on a stainless steel parallel plate rotor with a diameter of 25 mm for rheological measurement. The test instrument is a rotational rheometer. The test temperature is 37 °C, and the scanning strain is 1%. G' characterizes the elastic modulus of the sample.

[0089] Result description: The storage modulus of the hydrogel is mainly affected by the concentrations of the hydrogel substrates rCol1MA-PBA and EPLMA. The higher the concentrations of rCol1MA-PBA and EPLMA, the higher the storage modulus. The addition of GOx and C@Z@T does not have much impact on its storage modulus. Comparative Example 3 cannot be photocured to form a hydrogel without adding LAP and thus cannot be tested for storage modulus.

[0090] 2. Analysis of hydrogel swelling properties

[0091] Table 3 Hydrogel Swelling Ratio and Volume Expansion Ratio

[0092]

[0093] Note: Comparative Example 3 is a liquid and not tested.

[0094] Swelling ratio test method: Place 400 μL of the hydrogel precursor solution in a 48-well plate and irradiate it with ultraviolet light to form a gel. Then, weigh the material using a balance and record it as M0. Place the sample material in PBS and after swelling for 6 h, quickly blot the surface moisture with filter paper and weigh the sample to record the mass as Mw. The water absorption rate (X) of the sample is calculated according to the following formula: X(%) = (Mw - M0) / M0 × 100%. Volume swelling ratio test method: Weigh the hydrogel sample using a balance and measure the diameter and height of the dressing using a vernier caliper. Place the measured sample material in distilled water and after swelling to equilibrium (6 h), quickly blot the surface moisture with filter paper and measure the diameter and height of the dressing after absorbing water. The water absorption swelling rate formula of the dressing is as follows: VR(%) = (D1 2 ×H1) / (D0 2 ×H0)×100%, where D0 and D1 are the diameters of the sponge before and after water absorption respectively; H0 and H1 are the heights of the hydrogel before and after water absorption respectively, with the unit of mm.

[0095] Result description: The swelling ratio and volume swelling ratio of the hydrogel are affected by EPLMA. The greater the EPLMA content, the higher the swelling ratio and the greater the volume swelling ratio. The addition of GOx and C@Z@T has little effect on the swelling ratio and volume swelling ratio.

[0096] 3. Hemolysis rate analysis of hydrogel

[0097] Table 4 Hemolysis rate of hydrogel

[0098]

[0099] Note: For Comparative Example 3, the liquid is not tested.

[0100] Hemolysis rate test method: First, centrifuge fresh whole blood at 2000 rpm for 15 min to remove serum. Wash it 5 times repeatedly with PBS to ensure pure red blood cells are obtained. Dilute the pure red blood cells with PBS solution to obtain a red blood cell suspension with a concentration of 2% (v / v). Then add 1 ml of the red blood cell suspension to the sample to be tested (100 μL of the gelled sample) and incubate it at 37 °C for 1 h. Centrifuge this mixture at 1600 rpm for 10 min and then measure the absorbance value of the supernatant at a wavelength of 524 nm. In addition, use distilled water as the positive control and PBS as the negative control. The hemolysis rate (H) of the sponge is calculated according to the following formula: H(%) = (OD sample -OD negative ) / (OD positive -OD negative )×100, where H - hemolysis rate, unit %; OD sample - absorbance value of the sample group; OD negative - absorbance value of the negative control group; OD positive- Absorbance value of the positive control group;

[0101] Results show that the hemolysis rate of the hydrogels is all < 5%, meeting the requirements of biomedical materials. Due to the excellent antibacterial properties of TA in the C@Z@T nanoparticles, they will also have a weak impact on red blood cells, but all within a controllable range.

[0102] 4. Antibacterial property analysis of the hydrogel

[0103] Table 5 In vitro antibacterial rate (%)

[0104]

[0105] Test method: After resuscitating and culturing Escherichia coli and Staphylococcus aureus until the logarithmic growth phase, centrifuge, collect the bacteria and resuspend them in physiological saline. The final bacterial suspension concentration is 1×10 8 CFU / mL. After adding 500 μL of the corresponding hydrogel solution to a 24-well plate, cure it into a gel under ultraviolet light. Add 500 μL of physiological saline to the control group. Subsequently, add 100 μL of the diluted bacterial suspension and 1 mL of sterile physiological saline to each well, incubate at 37 °C for 24 hours, perform gradient dilution and then spread it on an LB agar plate for counting.

[0106] Results show that EPLMA and C@Z@T act synergistically to exert a dual antibacterial effect with excellent results. In addition, rCol1MA-PBA is a protein and has no effect on killing bacteria. As an inherent antibacterial material, EPL has broad-spectrum antibacterial activity against bacteria and fungi. After grafting MA, the antibacterial activity of EPL decreases significantly. Therefore, in the composite antibacterial hydrogel of the embodiments of the present invention, C@Z@T plays a major antibacterial role, and the higher the addition amount, the stronger the antibacterial effect. This is mainly because both ZIF-8 and TA in the core-shell nanozyme C@Z@T have good antibacterial activities, and they will damage the cell membrane structure of microorganisms, causing the interruption of cell material, energy, and information transmission, and ultimately leading to cell death.

[0107] 5. In vitro antioxidant property analysis of the hydrogel

[0108] Table 6 DPPH free radical scavenging rate (%)

[0109]

[0110] Test method: Prepare the test samples according to the instructions of the commercially available DPPH free radical scavenging ability detection kit. After adding the extraction solutions of the corresponding examples and comparative examples to the DPPH solution, incubate in the dark for 30 minutes, detect the absorbance at 515 nm with a UV-visible spectrophotometer, and calculate the free radical scavenging rate.

[0111] Results description: The addition of C@Z@T further enhances the antioxidant effect of the hydrogel and endows the hydrogel with the ability to scavenge free radicals. This is mainly because the TA molecular structure has multiple phenolic hydroxyl groups (-OH), which gives it a strong hydrogen-donating ability. Therefore, it has outstanding performance in the antioxidant activity of scavenging free radicals.

[0112] From the above results, it can be seen that the performance of each hydrogel sample in the embodiments of the present invention is better, especially the sample in Embodiment 3. Figure 6 This is a schematic diagram of the curing of the composite hydrogel in the embodiment of the present invention into a gel. Figure 7 This is the SEM image of the hydrogel after curing. It can be seen that the cured hydrogel presents a three-dimensional network structure with uniform pores.

[0113] In the antibacterial composite hydrogel system of the present invention, the concentrations of rCol1MA-PBA and EPLMA mainly affect the storage modulus of the substrate. The swelling performance is mainly affected by EPLMA. EPLMA and C@Z@T act synergistically to exert a dual antibacterial effect. The antioxidant performance is mainly affected by the addition of the core-shell nanozyme. Each component synergizes with each other and supports each other, having good antibacterial and antioxidant properties, and can be used for wound dressings, especially suitable for diabetic chronic wounds.

[0114] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0115] The above-described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent should be subject to the appended claims.

Claims

1. An antibacterial composite hydrogel loaded with nanoparticles, characterized in that: The hydrogel network body is composed of phenylboronic acid grafted methacryloyl type I collagen rCol1MA-PBA and methacryloyl polylysine EPLMA, and cross-linked with glucose oxidase GOx, and is loaded with core-shell nanozyme C@Z@T. The core-shell nanozyme C@Z@T is composed of a spherical CeO2 nanoparticle core and a zeolite imidazolate framework material ZIF-8 shell, and tannic acid TA coated on the shell.

2. The antibacterial composite hydrogel according to claim 1, characterized in that: The hydrogel contains 10wt%-20wt% rCol1MA-PBA, 7.5wt%-12.5wt% EPLMA, 10 mg / mL GOx, and 50-200 μg / mL C@Z@T.

3. The antibacterial composite hydrogel according to claim 1, characterized in that: The rCol1MA-PBA is prepared by the following method: Dissolve 2 g of type I recombinant collagen in 100 mL of water and stir until completely dissolved; 1.0 g 3-aminophenylboronic acid, 1.0 g EDC and 0.5 g NHS were dissolved in 50 mL water and 10 mL DMSO solution, and then the mixture was added to the type I recombinant collagen solution, reacted at room temperature for 3 days, dialyzed with a 3500 Da dialysis bag, and freeze-dried to obtain rCol1-PBA; Weigh 1.0 g of rCol1-PBA and dissolve it in 100 mL of deionized water. After it is fully dissolved, add 1 mL of methacrylic anhydride and react at room temperature for 4 h. Dialyze with an 8000 Da dialysis bag and lyophilize to obtain rCol1MA-PBA.

4. The antibacterial composite hydrogel according to claim 1, characterized in that: The EPLMA is prepared by nucleophilic amination ring-opening reaction of polylysine EPL and polymethyl methacrylate MA, and the molar ratio of MA to EPL is 11:

1.

5. The antibacterial composite hydrogel according to claim 1, characterized in that: The particle size of the core-shell nanozyme C@Z@T is 20-700 nm, and the zeta potential is -26.9±0.721 mV.

6. The antibacterial composite hydrogel according to claim 5, characterized in that: The preparation method of the core-shell nanozyme C@Z@T comprises the following steps: S1 Preparation of CeO2@ZIF-8 nanoparticles; The spherical CeO2 nanoparticles and PVP are respectively dissolved in an organic solvent and mixed evenly, and then the product is immediately dispersed in a 2-methylimidazole / organic solvent solution after centrifugation until it is completely mixed; then, zinc salt is added, mixed and reacted to obtain the CeO2@ZIF-8 nanoparticles; Preparation of S2 CeO2@ZIF-8@TA nanozyme; The prepared CeO2@ZIF-8 nanoparticles are dispersed evenly in water, and then tannic acid is added to disperse evenly, and then centrifugally washed and dried to obtain the core-shell nanozyme.

7. The antibacterial composite hydrogel according to claim 6, characterized in that: In the step S1, the reaction temperature is 24-37°C, spherical CeO2 nanoparticles: PVP = 30-40mg: 0.5-1.5g; in the step S2, the reaction temperature is 25-35°C, CeO2@ZIF-8 nanoparticles: tannic acid = 0.1-0.5g: 0.05-0.15g.

8. The method for preparing the antibacterial composite hydrogel according to any one of claims 1 to 7, characterized in that: The steps include: A solution containing a photoinitiator is prepared, and then rCol1MA-PBA and EPLMA are dissolved therein. After they are completely dissolved, GOx is added and vortexed to completely dissolve and evenly disperse them. Subsequently, C@Z@T core-shell nanozymes are added and stirred to disperse. Then, cross-linking is performed under ultraviolet light under vacuum conditions to obtain the antibacterial composite hydrogel.

9. Use of the antibacterial composite hydrogel according to any one of claims 1 to 8 in wound dressings.

10. The use according to claim 9, wherein the wound dressing is a diabetic wound dressing.

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