Antibacterial hydrogel for promoting wound healing based on mesoporous dopamine loaded alpha-N heterocyclic thiosemicarbazone as well as preparation method and application of antibacterial hydrogel

Abstract

The invention relates to a self-healing injectable dual-network hydrogel (GHUS-mPDA at HL) which is used for wound healing and has photo-thermal antibacterial activity and is synthesized on the basis of mesoporous polydopamine nanoparticles (mPDA), thiosemicarbazone (HL), ureido pyrimidone modified gelatin (GHU) and sodium alginate (SA). Sodium alginate and ureido pyrimidone modified gelatin containing quadruple hydrogen bonds have adhesion and antibacterial properties, and the hydrogel is endowed with self-repairing capability, injectability, adhesion and antibacterial properties by crosslinking. Mesoporous polydopamine nanoparticles (mPDA) with a photothermal effect and thiosemicarbazone (HL) with efficient antibacterial ability endow the hydrogel with excellent photothermal antibacterial performance. In addition, the hydrogel has good biocompatibility. Mouse skin incision model experiments show that the GHUS-mPDA-coated HL hydrogel can effectively promote full-thickness wound healing, and the GHUS-mPDA-coated HL hydrogel has great potential in the aspect of promoting wound healing.

Description

Technical Field The present invention belongs to the technical field of biomedical hydrogels, and in particular relates to a photothermal antibacterial hydrogel based on mesoporous dopamine loaded with α-N heterocyclic thiosemicarbazone for promoting wound healing, a preparation method thereof, and applications thereof in antibacterial and wound healing promotion. Background Art

[0002] Wound healing is a complex physiological process that requires a high degree of coordination between different types of cells, chemokines, cytokines, and various growth factors in sequential healing steps. Currently, bacterial infection is the primary challenge in wound treatment because the formation of exudate caused by bacterial infection hinders wound repair. The problem of bacterial infection caused by pathogenic bacteria (Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium, Streptococcus, etc.) is one of the biggest challenges that urgently needs to be solved globally. Antibiotics continue to play an important role in the clinical treatment of bacterial infections. However, the overuse and misuse of antibiotics have led to multidrug resistance (MDR) and extremely resistant bacteria. Due to the difficulty in developing new antibiotics and discovering new targets, there is an urgent need to construct antibacterial biomaterials to alleviate the antimicrobial resistance of pathogenic bacteria.

[0003] α-N-heterocyclic thiosemicarbazones have a wide range of pharmacological activities, such as antitumor, antibacterial, antiviral and antimalarial. The strength of their antibacterial and anticancer activities is affected by the methylation of the pyridine amino group and the terminal amino group. Secondly, α-N-heterocyclic thiosemicarbazones have excellent ability to coordinate various metal ions, which can increase the selective permeability of bacterial cell membranes by chelating divalent cations required on the cell surface, thereby improving their antibacterial activity. Despite the rapid development of α-N-heterocyclic thiosemicarbazone drugs, there are still many pharmacological defects such as low water solubility and high cytotoxicity. The use of gel technology for drug delivery is an attractive strategy to change the chemical or biological activity of drugs. TSCs are a key part of the drug design process because they are able to form coordination bonds and hydrogen bonds.

[0004] Hydrogels are promising antimicrobial materials with a hydrophilic three-dimensional network structure formed by physical interactions or chemical covalent bonds, which can accommodate a variety of materials, including nanoparticles and polymers. Hydrogels can be made of natural (such as chitosan, folic acid and gelatin) or synthetic polymers (such as polydopamine, polyethylene glycol and polyvinyl alcohol). Due to the typical biodegradability, biocompatibility and tunable mechanical properties of hydrogels, they have been widely used in biomedical fields such as wound dressings, drug delivery, tissue engineering, contact lenses and implantable devices. However, traditional hydrogels cannot effectively cover irregular wounds. Injectable hydrogels have recently attracted increasing attention due to their ability to fill and cover spaces of any shape. Hydrogels can be prepared by physical crosslinking (i.e., shear thinning) and chemical crosslinking methods. Physically and chemically crosslinked injectable self-healing hydrogels have received extensive attention in wound dressings due to their rapid gelation process and good self-healing ability. Therefore, constructing antimicrobial hydrogels loaded with α-N-heterocyclic thiosemicarbazones to alleviate the antimicrobial resistance of pathogenic bacteria is an effective strategy to improve the therapeutic effect of wound dressings. In addition, photothermal therapy (PTT) kills bacteria by directly destroying their structure rather than engaging in metabolism. In addition to direct disinfection, high temperatures can also disrupt the structure of biofilms, enhancing the effectiveness of various other antimicrobial strategies by increasing bacterial sensitivity. On the other hand, the heat generated during PTT can act as a trigger to release thiosemicarbazones from the carrier to fight bacteria.

[0005] The present invention focuses on the problem of bacterial infection affecting wound healing, and develops a hydrogel with good biocompatibility and high-efficiency photothermal antibacterial performance, and photothermally promotes wound healing in mice. The present invention uses a photothermal antibacterial hydrogel based on mesoporous dopamine loaded with α-N heterocyclic thiosemicarbazone. Dopamine hydrochloride monomers undergo spontaneous oxidative polymerization, and form mPDA nanoparticles with intramolecular / intermolecular crosslinking through non-covalent interactions such as hydrogen bonds, π-π stacking, hydrophobic interactions, and charge transfer. Thiosemicarbazone is loaded onto mesoporous mPDA nanoparticles with photothermal effect. Urea-pyrimidone-modified gelatin (GHU) can form a hydrogel network of quadruple hydrogen bonds, which forms an interpenetrating double network hydrogel with sodium alginate. Experimental verification shows that the antibacterial effect of GHUS-mPDA@HL hydrogel is significantly improved, and it has good injectability, self-healing and adhesion. The present invention then conducted a dead-live fluorescence staining test, and the test results showed that the hydrogel cells have excellent biocompatibility. In vivo animal studies showed that the GHUS-mPDA@HL light-exposed group had the ability to promote epithelialization compared with the control group, and had broad application prospects in the field of wound healing. Summary of the invention

[0006] The purpose of the present invention is to overcome the defects of the prior art and provide a photothermal antibacterial hydrogel based on mesoporous dopamine loaded with α-N heterocyclic thiosemicarbazone to promote wound healing. The preparation method of the hydrogel is simple and has good adhesion, injectability, self-healing property, biocompatibility, photothermal antibacterial performance, and wound healing promotion performance.

[0007] The present invention also provides a method for preparing the photothermal antibacterial hydrogel and its application in antibacterial and wound healing promotion.

[0008] To achieve the above object, the present invention adopts the following technical solution: A method for preparing an antibacterial hydrogel dressing for promoting wound healing based on mesoporous dopamine loaded with α-N heterocyclic thiosemicarbazone, comprising the following steps: 1) Dissolve tert-butyl (2-formylpyridin-3-yl)carbamate and 4,4-dimethyl-3-thiosemicarbazide in an ethanol-water mixture to obtain system A, add concentrated hydrochloric acid dropwise, stir and heat under reflux for 2-4 h, then cool to room temperature, adjust the pH of the system to alkaline, stir, filter, and stand to precipitate crystals HL; 2) Dissolve poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) F127 in an ethanol-water mixture, then add dopamine hydrochloride (DA) and mesitylene (TMB), the solution turns milky white, then adjust the pH of the system to alkaline, stir in the dark for 12-16 hours, centrifuge, and wash to obtain powdered mPDA; 3) The HL solution and the mPDA solution were stirred at room temperature for 6-24 hours, centrifuged, and washed to obtain the mesoporous dopamine complex mPDA@HL loaded with α-N heterocyclic thiosemicarbazone; 4) Synthesis of ureidopyrimidone-modified gelatin (GHU), the specific scheme is as follows: under an inert atmosphere, 1,6-hexane diisocyanate (HDI) is dispersed in dimethyl sulfoxide (DMSO), then the temperature is raised to 90-110°C and 2-aminopyrimidin-4(1H)-one (UPy) is added to react for 12-20 h, after the reaction is completed, the temperature is lowered to room temperature, n-hexane is added to the reactant, filtered, washed, and dried to obtain the product UPy-HDI (abbreviated as HU); Under an inert atmosphere, gelatin (G) and UPy-HDI are dispersed in DMSO, and then heated to 90-110°C for reaction for 12-20 hours. After the reaction is completed, the temperature is lowered to room temperature, and the reaction product is added to pre-frozen (-15 ~ -20°C) acetone, filtered to obtain a white solid; the solid is resuspended in deionized water and dialyzed (molecular weight cutoff not exceeding 10,000), and finally freeze-dried to obtain the purified polymer GHU; 5) Vortex-mix sodium alginate, GHU, mPDA@HL and water to obtain a composite hydrogel material GHUS-mPDA@HL.

[0009] Specifically, in step 1), the molar ratio of (2-formylpyridin-3-yl)carbamic acid tert-butyl ester to 4,4-dimethyl-3-thiosemicarbazide is less than 1:1, preferably 0.5-1:1. The pH of the system can be adjusted to alkaline by adding NaHCO3 solution, such as pH between 8-11.

[0010] Furthermore, in step 1), 75-50 μL of concentrated hydrochloric acid is added dropwise to every 1 mL of system A. In the ethanol-water mixture, the volume ratio of ethanol to water is preferably 5-8:1.

[0011] Specifically, in step 2), the mass ratio of F127 to dopamine hydrochloride is 4:1-3; the mass ratio of F127 to mesitylene is 1:1-20. The preferred order of adding dopamine hydrochloride and TMB is: first add dopamine hydrochloride and react for 20-30 minutes, then add TMB. In step 2), the ethanol-water mixture can be obtained by mixing ethanol and water in any proportion, preferably in a volume ratio of 1-5:1.

[0012] Furthermore, in step 2), the pH of the system is adjusted to 8-11 by adding L-arginine.

[0013] Specifically, in step 3), the mass ratio of HL to mPDA is 1:0.5-2.

[0014] Specifically, in step 4), the molar ratio of hexamethylene diisocyanate to 2-aminopyrimidine-4(1H)-one is 6-8:1; and the mass ratio of gelatin to UPy-HDI is 19:0.5-2.

[0015] Specifically, in step 5), the mass ratio of sodium alginate, GHU, and mPDA@HL is 60-70:60-70:1.

[0016] The present invention provides an antibacterial hydrogel dressing for promoting wound healing based on mesoporous dopamine loaded α-N heterocyclic thiosemicarbazone obtained by the above preparation method. The antibacterial hydrogel has excellent adhesion, injectability and self-healing properties.

[0017] The present invention also provides the use of the hydrogel dressing in antibacterial or wound healing promotion. The hydrogel dressing has excellent photothermal antibacterial properties and the effect of accelerating wound healing in mice.

[0018] The present invention overcomes the pharmacological defects of α-N heterocyclic thiosemicarbazone, such as low water solubility and high cytotoxicity. Photothermal therapy (PTT), which uses photothermal agents to convert laser light energy into thermal energy, has become a new strategy with great antibacterial potential. It physically eliminates microorganisms through a variety of thermal effects, including protein / enzyme denaturation, cell membrane rupture, cell cavitation, and cell fluid evaporation. In addition, as an antibacterial tool, it has many advantages: targeted selectivity, deep tissue / biomembrane penetration, remote controllability, non-invasiveness, rapid effectiveness, and few side effects. Secondly, hydrogels are used to maintain a moist microenvironment, allow water and oxygen penetration, absorb excess exudate, and shield microorganisms. Function is the basis and important function of wound healing by promoting fibroblast proliferation, keratinocyte migration, and final re-epithelialization.

[0019] The present invention synthesizes a double network hydrogel composed of hydrogen bonds; one network involves sodium alginate and the other network involves UPy parts to form a quadruple hydrogen bond hydrogel. This type of double hydrogen bond network makes the matrix injectable, self-healing and adhesive. Dopamine hydrochloride monomers are polymerized by spontaneous oxidation, and mPDA nanoparticles with intramolecular / intermolecular crosslinking are formed through non-covalent interactions such as hydrogen bonds, π-π stacking, hydrophobic interactions and charge transfer. α-N heterocyclic thiosemicarbazone has a terminal dimethyl structure that gives it excellent antibacterial properties. It can bind to bacterial cell membranes to rupture them and kill bacteria. In addition, α-N heterocyclic thiosemicarbazone has antioxidant properties and can promote cell proliferation at trace levels. Therefore, GHUS-mPDA@HL hydrogel has the ability to scavenge reactive oxygen and accelerate wound healing.

[0020] Wound infection is an escalating medical problem that poses a threat to global public health and personal health. As a new sterilization strategy, photothermal antibacterial technology has the advantages of excellent sterilization efficiency, high safety, and low risk of emergence of drug-resistant bacteria. Combining inherent antibacterial activity with light-assisted antibacterial therapy to develop a new multifunctional dressing with synergistic and efficient antibacterial effect and promote wound healing has attractive advantages in the field of clinical treatment of wound infection. The present invention synthesizes a self-healing injectable double network hydrogel (GHUS-mPDA@HL) with photothermal antibacterial activity for wound healing based on mesoporous polydopamine nanoparticles (mPDA), thiosemicarbazone (HL), ureidopyrimidone-modified gelatin (GHU) and sodium alginate (SA). Sodium alginate and ureidopyrimidone-modified gelatin containing quadruple hydrogen bonds have adhesive antibacterial properties, and crosslinking gives the hydrogel self-healing ability, injectability, adhesion. And antibacterial properties. Mesoporous polydopamine nanoparticles (mPDA) with photothermal effect and thiosemicarbazone (HL) with high antibacterial ability give the hydrogel excellent photothermal antibacterial properties. In addition, the hydrogel has good biocompatibility. The mouse skin incision model experiment showed that GHUS-mPDA@HL hydrogel can effectively promote full-thickness wound healing, indicating its great potential in promoting wound healing.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1) The present invention synthesizes a double network hydrogel composed of hydrogen bonds. One network involves sodium alginate and the other network involves UPy parts, forming a quadruple hydrogen bond hydrogel. This type of double hydrogen bond network makes the matrix injectable, self-healing and adhesive.

[0022] 2) The polydopamine (PDA) used in the present invention has the advantages of simple preparation, good photothermal conversion effect, high biocompatibility, easy functionalization and adhesion, etc. Taking advantage of these advantages, dopamine is widely used to synthesize PDA nanosystems with excellent photothermal effect, good biocompatibility and high drug loading capacity.

[0023] 3) The present invention overcomes the pharmacological defects of the present solution, such as low water solubility and high cytotoxicity of α-N heterocyclic thiosemicarbazone, and broadens its application field.

[0024] 4) The composite hydrogel prepared by the present invention has high photothermal sterilization efficiency and good biocompatibility, and can maintain a high therapeutic effect for a certain period of time.

[0025] 5) The hydrogel prepared by the present invention is injectable, self-healing, and adhesive, can perfectly fit the wound, and has the ability to accelerate wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A diagram briefly describing the synthesis route of the photothermal antibacterial hydrogel based on mesoporous dopamine loaded α-N heterocyclic thiosemicarbazone of the present invention; Figure 2 This is an electron microscope image of mesoporous polydopamine nanoparticles mPDA; Figure 3 Nitrogen adsorption isotherm of mPDA (a) and pore size distribution curve of mPDA (b); Figure 4 IR spectra of DA, mPDA, HL, and mPDA@HL; Figure 5 is the infrared spectrum of G, HU, and GHU; Figure 6 Electron microscopy images of GHUS and GHUS-mPDA@HL hydrogels; Figure 7 The gel infusion in air for GHUS-mPDA@HL hydrogel allows printing of different shapes and their adhesion; Figure 8 The photothermal effect of GHUS-mPDA@HL hydrogel at different powers; Fig. 9 The effect of GHUS-mPDA@HL hydrogel on Escherichia coli ( E. coli ) and Staphylococcus aureus ( S. aureus )'s photothermal antibacterial activity; Fig.10 Live and dead fluorescence staining images of HL, GHUS hydrogel, and GHUS-mPDA@HL hydrogel; Fig.11 The wound healing effect diagram of the blank group, 3M wound dressing, GHUS hydrogel, GHUS-mPDA@HL hydrogel and GHUS-mPDA@HLNIR hydrogel (a) and the wound healing rate of each experimental group (b). DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0028] In the following examples, the raw materials used are common commercial products or can be prepared by conventional techniques in the art; for example, 2-aminopyrimidine-4(1H)-one (UPy, also called isocytosine, CAS: 108-53-2), 1,6-hexamethylenediisocyanate (HDI), gelatin (Gelatin, abbreviated as G), sodium alginate (SA), L-arginine, F127, and TMB were purchased from Inokai Chemical Co., Ltd.; tert-butyl (2-formylpyridin-3-yl)carbamate and 4,4-dimethyl-3-thiosemicarbazide were purchased from Beijing Bailingwei Technology Co., Ltd.

[0029] In the following examples, the antibacterial properties and wound healing experiments involved may be measured using conventional techniques in the art unless otherwise specified.

[0030] Room temperature refers to 25±5° C. Concentrated hydrochloric acid refers to common commercially available concentrated hydrochloric acid with a concentration of 36% to 38%.

[0031] Embodiment 1: Photothermal antibacterial hydrogel (GHUS-mPDA@HL) based on mesoporous dopamine loaded α-N heterocyclic thiosemicarbazone to promote wound healing, wherein the chemical name of the α-N heterocyclic thiosemicarbazone ligand is: HL = 3-aminopyridine-2-carboxaldehyde-N(4)-dimethylthiosemicarbazone. The preparation process of the hydrogel is as follows: (1) Preparation of α-N-heterocyclic thiosemicarbazone ligand (HL) Preparation of HL = 3-aminopyridine-2-carboxaldehyde-N(4)-dimethylthiosemicarbazone: Dissolve (2-formylpyridin-3-yl)carbamic acid tert-butyl ester (0.2934 g, 1.32 mmol) and 4,4-dimethyl-3-thiosemicarbazone (0.1528 g, 1.454 mmol) in 6 mL of ethanol-water mixed solution (V 乙醇 :V 水 =5:1), add 300 μL of concentrated hydrochloric acid, stir and reflux for 3 h. Then cool to room temperature, add NaHCO3 solution (0.1778 g of NaHCO3 dissolved in 8 ml of water) to adjust the pH to about 8.5, stir at room temperature for 30 min. Filter, let the filtrate stand, and yellow crystals HL will precipitate after about 3 days.

[0032] (2) Synthesis of mesoporous polydopamine nanoparticles mPDA Dissolve 36 mg of F127 in 12 mL of ethanol-water mixture (V 乙醇 :V 水 =1:1), then add 9 mg dopamine hydrochloride (DA), stir for 20 min until completely dissolved, then slowly add 0.3 mL (0.2061 g) TMB to the mixed solution under ultrasonic conditions, the solution turns milky white, then add 1 mL (9 mg / mL) of L-arginine to adjust the pH to about 8.5, stir at room temperature in the dark for 12 h, finally centrifuge and wash to obtain black powder mPDA.

[0033] (3) Synthesis of mesoporous dopamine composite mPDA@HL loaded with α-N-heterocyclic thiosemicarbazone An equal volume of 1 mg / mL HL solution and 1 mg / mL mPDA solution were mixed at room temperature and stirred for reaction for 12 h. The mixture was then centrifuged and washed with distilled water to obtain the mesoporous dopamine complex mPDA@HL loaded with α-N heterocyclic thiosemicarbazone.

[0034] (4) Synthesis of ureidopyrimidone-modified gelatin GHU The specific scheme is as follows: open the nitrogen pipeline and introduce nitrogen for ≥30 minutes to establish a nitrogen atmosphere in the flask. Pour HDI (2.243mL, 0.014 mol) and DMSO (30 mL) into the flask, then immerse all the flasks below the neck in a heated and stirred oil bath, wait until it is heated to 100°C, and add UPy (0.22 g, 0.002 mol) into the flask. After reacting for 16 h, turn off the heating stirrer, turn off the nitrogen after the temperature drops to room temperature, then add 50 mL of n-hexane to the reactant, stir for another 1 min, then filter, wash, and dry to obtain the product UPy-HDI (abbreviated as HU).

[0035] Open the nitrogen line and pass nitrogen for ≥30 minutes to establish a nitrogen atmosphere in the flask. Add gelatin (4.75 g), HU (0.25 g) and DMSO (30 mL) to the flask, turn on the stirrer and heat to 100°C for 16 h. After the reaction is completed and the temperature drops to room temperature, turn off the nitrogen, then add the above solution to 300 mL of pre-frozen (-18 ~ -20°C) acetone through a dropper, stir for another 1 min, filter to obtain a white solid, resuspend the solid in deionized water and dialyze for 3 days (molecular weight cutoff, MWCO: 10000), and finally freeze-dry at -80°C to obtain the purified polymer GHU.

[0036] (5) GHU (0.4 g), sodium alginate (SA, 0.4 g) and mPDA@HL 6 mg were added to 6 mL of H2O and vortexed to obtain GHUS-mPDA@HL hydrogel. The specific synthesis process is as follows: Figure 1 shown.

[0037] In addition, GHU (0.4 g) and sodium alginate (SA, 0.4 g) were added to 6 mL of H2O and vortexed to obtain GHUS hydrogel, which was used as a control.

[0038] Embodiment 2: Mesoporous polydopamine nanoparticles (mPDA) were prepared by soft template method. Their structural characterization results are shown in Figure 2 shown. Figure 2 The results showed that mPDA had uniform pore size, clear spherical morphology and mesoporous structure.

[0039] The porous information of mPDA was studied by N2 adsorption experiment. The N2 adsorption-desorption isotherms of mPDA samples showed a typical Langmuir IV isotherm, indicating the existence of a mesoporous structure. Figure 3 As shown in Figure 1, in the range of P / P0 of 0.6-1.0, a wide H4 type hysteresis loop caused by capillary condensation was found, which is a typical feature of mesoporous materials (Figure a). The specific surface area of ​​mPDA is 38.741 m 2 g −1 , the pore size is 7.2315 nm (Figure b).

[0040] The differences in the chemical composition of dopamine hydrochloride DA, mPDA and mPDA@HL were further analyzed by FTIR experiments. Figure 4 For DA, 3000−3400 cm −1 The broad peaks can be attributed to the structure of intermolecular hydrogen bonds, with peaks at 2433.75, 2539.81, 2640.09, and 2750.52 cm −1 It is due to the stretching vibration of aromatic amino groups. After polymerization, the −1 These four peaks in the range disappeared, which may be due to the conversion of amino groups into secondary amines during this process. -1 The absorption peaks at 1199 cm -1 The absorption peaks belong to NN stretching vibration, 794 cm -1 The absorption peak belongs to C=S stretching vibration. In the spectrum of mPDA@HL, the typical characteristic absorption peak of HL appears, indicating that HL is successfully loaded into mPDA.

[0041] Embodiment 3: The FTIR spectra of gelatin G, HU, and GHU are shown in Figure 2. Figure 5 As shown, the spectrum of HU is at 2361 cm -1 The absorption peak of 3310 cm-1 in the GHU polymer spectrum indicates the complete reaction of the isocyanate groups. −1 The decrease in the intensity of the broad peak also confirmed the grafting of UPy fragments onto the polymer chains.

[0042] After the gel was freeze-dried, the SEM images of GHUS hydrogel and GHUS-mPDA@HL hydrogel were as follows: Figure 6As shown. In both figures, a continuous porous network structure can be observed, indicating that the hydrogel was successfully prepared. Secondly, after amplification, small mPDA@HL particles exist in the GHUS-mPDA@HL hydrogel, indicating that mPDA@HL is successfully loaded into the hydrogel.

[0043] Figure 7 The gel injected in air for GHUS-mPDA@HL hydrogel allows printing of different shapes and their adhesion. To demonstrate injectability, the hydrogel can be freely injected through the needle without clogging and formed into different shapes, which indicates great potential in implant therapy. The hydrogel strip sticks to the finger and dynamically adapts to the movement of the finger. The hydrogel strip can be peeled off the finger with only a small amount of residue left at the edge of the adhered hydrogel.

[0044] In order to evaluate the photothermal effect, the present invention was conducted at different powers (1, 1.2, 1.1, 1.6, 1.8, 2.0 W / cm 2 ) was used to measure the photothermal effect within 5 min. The results are shown in Figure 8 The results show that the temperature of GHUS-mPDA@HL hydrogel increases with the increase of power density, and the temperature rise (ΔT) of the hydrogel increases with the increase of NIR light power. Figure 8 As shown, the ΔT values ​​of the hydrogels were 25.9, 28.1, 33.8, 37.7, 39.8, and 41.6 °C (1.0, 1.2, 1.4, 1.6, 1.8, and 2.0 W / cm 2 ).

[0045] Embodiment 4: The colony counting method was used to evaluate the antibacterial properties of the composite hydrogel. The present invention investigated the antibacterial properties of the blank control group (Con), GHUS, GHUS-mPDA@HL hydrogel, and GHUS-mPDA@HL hydrogel + NIR against Escherichia coli ( E. coli ) and Staphylococcus aureus ( S. aureus )'s antibacterial effect, the results are shown in Fig. 9 .like Fig. 9 As shown in the figure, by exploring the antibacterial properties of GHUS, GHUS-mPDA@HL hydrogel, and GHUS-mPDA@HL+NIR hydrogel, the results show that the three hydrogel materials have certain antibacterial effects on Escherichia coli and Staphylococcus aureus, and GHUS-mPDA@HL+NIR hydrogel has the best antibacterial effect. The antibacterial effect of the GHUS-mPDA@HL+NIR hydrogel group is significantly improved. The reason is that the increase in photothermal temperature can also kill bacteria. Secondly, the temperature rises after light irradiation, which is conducive to the release of HL, which can destroy the integrity of the bacterial outer membrane by chelating the necessary divalent cations on the cell surface, thereby increasing the antibacterial activity.

[0046] Embodiment 5: 1×10 cells were seeded into each well of a 96-well cell culture plate. 4 L929 cells were added with 100 μL of DMEM medium containing 10% FBS. After 6 h of culture, the cells were observed under a microscope to be attached to the wall, so the original culture medium was discarded and replaced with culture medium extracts containing HL, GHUS, and GHUS-mPDA@HL hydrogels (obtained by incubating 0.5 mg HL, GHUS or GHUS-mPDA@HL hydrogel with 1 mL of DMEM medium containing 10% FBS at room temperature for 12 h). The control group (Control) was replaced with fresh DMEM medium containing 10% FBS. Three replicate wells were set up for each group, and 100 μL of PBS buffer was added to the wells around the above operation to reduce the impact of the volatilization of the liquid in the wells on the experimental results. The biocompatibility of the hydrogel was evaluated by culturing at 37°C for 24, 48, and 72 hours. The results are shown in the table. Fig.10 .like Fig.10 As shown in the figure, Calcein and propidium iodide (PI) were used to fluorescently stain live cells and dead cells, respectively. After being cultured in the DMEM extract of HL, there was a large amount of red in the fluorescent staining, and the number of green live cells was small, and the cell survival rate decreased. The results showed that HL had greater toxicity. GHUS had less toxicity. After being cultured in the DMEM extract of GHUS-mPDA@HL, most of the cells were green spindle-shaped, indicating that the hydrogel was non-toxic to normal cells. This shows that GHUS-mPDA@HL hydrogel can be used as an antibacterial wound dressing.

[0047] Embodiment 6: Generally, wound area is the most intuitive evaluation index for evaluating the skin repair effect of different groups. The wounds were directly treated with the control group (untreated, Con), 3M wound dressing, GHUS hydrogel, GHUS-mPDA@HL hydrogel, and GHUS-mPDA@HL hydrogel + NIR group. The results are shown in Fig.11 .like Fig.11 As shown in a. There were significant differences in wound size on the 2nd, 5th, and 10th days after surgery. There was no infection or rejection during the treatment, indicating that GHUS-mPDA@HL hydrogel + NIR has good therapeutic effect and biocompatibility. After 10 days of treatment, the wound healing rates of the untreated group, 3M wound dressing group, GHUS hydrogel, and GHUS-mPDA@HL hydrogel were 69.95%, 82.75%, 88.73%, and 86.5%, respectively, while the wound healing rate of the GHUS-mPDA@HL hydrogel + NIR group reached 99.07%. Fig.11 As shown in b.

[0048] In summary, the present invention focuses on the problem of bacterial infection and wound infection, and develops a photothermal antibacterial hydrogel based on mesoporous dopamine loaded with α-N heterocyclic thiosemicarbazone, which is a double network hydrogel composed of hydrogen bonds, one network involving sodium alginate and the other network involving UPy part, forming a quadruple hydrogen bond hydrogel. This type of double hydrogen bond network makes the matrix injectable, self-healing, and adhesive. Dopamine hydrochloride monomers are spontaneously oxidatively polymerized to form mPDA nanoparticles with intramolecular / intermolecular crosslinks through non-covalent interactions such as hydrogen bonds, π-π stacking, hydrophobic interactions, and charge transfer. α-N heterocyclic thiosemicarbazone has a terminal dimethyl structure that gives it excellent antibacterial properties and can bind to bacterial cell membranes to rupture them and kill bacteria. In addition, α-N heterocyclic thiosemicarbazone has antioxidant properties and can promote cell proliferation at trace levels. Therefore, GHUS-mPDA@HL hydrogel has the ability to remove reactive oxygen and accelerate wound healing. Based on the combination of mPDA nanoparticle photothermal therapy and the synergistic antibacterial effect of HL, GHUS-mPDA@HL hydrogel + NIR has a good synergistic bactericidal effect on Escherichia coli and Staphylococcus aureus. In addition, the full-thickness skin defect model also showed that GHUS-mPDA@HL hydrogel + NIR can effectively accelerate the wound healing process. Overall, this study provides a simple and promising method for the preparation of multifunctional hydrogel dressings for skin wound healing.

Claims

1. A method for preparing an antibacterial hydrogel dressing based on mesoporous dopamine loaded with α-N heterocyclic thiosemicarbazone to promote wound healing, characterized in that: The steps include: 1) Dissolve tert-butyl (2-formylpyridin-3-yl)carbamate and 4,4-dimethyl-3-thiosemicarbazide in an ethanol-water mixture to obtain system A, add concentrated hydrochloric acid dropwise, stir and heat under reflux for 2-4 h, cool to room temperature, adjust the pH of the system to alkaline, stir, filter, and stand to precipitate crystals HL; 2) Dissolve F127 in an ethanol-water mixture, then add dopamine hydrochloride and mesitylene, then adjust the pH of the system to alkaline, stir in the dark for 12-16 h, centrifuge, and wash to obtain powdered mPDA; 3) The HL solution and the mPDA solution were stirred at room temperature for 6-24 h, centrifuged, and washed to obtain the mesoporous dopamine complex mPDA@HL loaded with α-N heterocyclic thiosemicarbazone; 4) Under an inert atmosphere, 1,6-hexanediisocyanate is dispersed in DMSO, then the temperature is raised to 90-110°C and 2-aminopyrimidine-4(1H)-one is added to react for 12-20 h. After the reaction is completed, the temperature is lowered to room temperature, n-hexane is added to the reactants, filtered, washed, and dried to obtain the product UPy-HDI; Under an inert atmosphere, gelatin and UPy-HDI were dispersed in DMSO, and then heated to 90-110°C for reaction for 12-20 h. After the reaction, the temperature was lowered to room temperature, and the reaction product was added to pre-frozen acetone, filtered, and the solid was resuspended in deionized water and dialyzed, and finally freeze-dried to obtain the polymer GHU. 5) Mix sodium alginate, GHU, mPDA@HL and water to obtain.

2. The preparation method according to claim 1, characterized in that In step 1), the molar ratio of the (2-formylpyridin-3-yl)carbamic acid tert-butyl ester to 4,4-dimethyl-3-thiosemicarbazide is 0.5-1:

1.

3. The preparation method according to claim 1, characterized in that: In step 1), add 75-50 μL of concentrated hydrochloric acid to every 1 mL of system A.

4. The preparation method according to claim 1, characterized in that: In step 2), the mass ratio of F127 to dopamine hydrochloride is 4:1-3; the mass ratio of F127 to mesitylene is 1:1-20.

5. The preparation method according to claim 1, characterized in that: In step 2), the pH of the system is adjusted to 8-11 by adding L-arginine.

6. The preparation method according to claim 1, characterized in that: In step 3), the mass ratio of HL to mPDA is 1:0.5-2.

7. The preparation method according to claim 1, characterized in that: In step 4), the molar ratio of hexamethylene diisocyanate to 2-aminopyrimidine-4(1H)-one is 6-8:1; and the mass ratio of gelatin to UPy-HDI is 19:0.5-2.

8. The preparation method according to claim 1, characterized in that: In step 5), the mass ratio of sodium alginate, GHU and mPDA@HL is 60-70:60-70:

1.

9. An antibacterial hydrogel dressing for promoting wound healing based on mesoporous dopamine loaded with α-N heterocyclic thiosemicarbazone obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the hydrogel dressing according to claim 9 in antibacterial or wound healing promotion.

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