Preparation method and application of injectable antibacterial hydrogel

Abstract

The invention discloses a preparation method and application of injectable antibacterial hydrogel, and belongs to the field of material preparation.The injectable antibacterial hydrogel with good self-healing performance is prepared by doping MPDA NPs loaded with PUE into OSA / CMCs-based hydrogel, the injectable antibacterial hydrogel shows good mechanical performance, meanwhile, the injectable antibacterial hydrogel has a good photothermal effect due to doping of MPDA-PUE NPs, and the injectable antibacterial hydrogel has good self-healing performance. According to the present invention, the low-temperature PTT and the PUE controlled-release and slow-release effects are perfectly combined, such that the antibacterial effect of the low-temperature PTT and the anti-oxidation, immunoreaction regulation and angiogenesis promotion capabilities of the PUE are perfectly combined; the hydrogel shows good mechanical performance in related characterization, and meanwhile it is proved that the hydrogel has good performance in the aspects of antibiosis, immunoreaction regulation and angiogenesis promotion in follow-up experiments.

Description

Technical Field

[0001] The invention discloses a preparation method and application of an injectable antibacterial hydrogel, belonging to the field of material preparation. Background Art

[0002] Wound infection is a serious clinical challenge, often accompanied by the threat of persistent inflammatory response and increased bacterial resistance. A variety of materials with good antibacterial properties have been developed for the treatment of infected wounds. As one of the emerging wound dressings, hydrogels have a high degree of biomimetic properties and good biocompatibility because their physical properties are similar to the structure of the extracellular matrix and can well simulate the biological microenvironment of damaged tissues. At the same time, due to the high water content and porosity of hydrogels, this advantage enables them to provide a protective barrier for the wound surface while ensuring the smooth exchange of metabolic waste at the wound site, which can well promote the rapid repair of the defect site. Therefore, they have received more and more attention in wound dressings, bone tissue engineering, biopharmaceuticals and clinical treatment.

[0003] Although most wound dressing materials currently have relatively ideal biocompatibility, most of them have shortcomings such as being unable to perfectly fill irregular wounds, being unable to maintain for a long time, having poor anti-infection ability and being prone to drug resistance, and having relatively simple functions. How to improve their plasticity, adhesion, and tolerance while ensuring biocompatibility while enhancing their ability to kill bacteria and regulate the immune microenvironment has become one of the main problems that need to be solved urgently.

[0004] Currently, most antibacterial dressings enhance their antibacterial ability through more effective antibacterial drugs, such as various more efficient antibiotics, metal ions such as silver and copper, and various nanoparticles, etc., but ignore the side effects of these antibacterial agents such as drug toxicity and the production of large amounts of reactive oxygen. Summary of the invention

[0005] In view of the defects of the prior art, the present invention proposes a preparation method and application of an injectable antibacterial hydrogel, which solves the problems of existing hydrogel dressings with relatively single functions, poor physical properties and inability to match irregular wounds well. The multifunctional hydrogel has good morphological controllability, adhesion, resistance, and can clear infection, regulate immune response and promote angiogenesis.

[0006] The technical solution of the present invention is as follows:

[0007] According to a first aspect of an embodiment of the present invention, there is provided a method for preparing an injectable antibacterial hydrogel, comprising:

[0008] Step 1: Deionized water and anhydrous ethanol are respectively taken into a beaker and stirred evenly by a magnetic stirrer, and then Pluronic F127 and mesitylene are added in sequence, and then ultrasonic vibration is performed to form a white emulsion, and then tris(hydroxymethyl)aminomethane hydrochloride is added, and dopamine hydrochloride is added after stirring, and the pH is adjusted to 8.5, and stirred at room temperature in the dark for 24 hours;

[0009] Step 2: Collect the liquid, centrifuge and discard the supernatant, collect the mesoporous polydopamine nanoparticles at the bottom of the tube, wash them three times with water, a mixture of anhydrous ethanol and acetone, and weigh them after freeze-drying to calculate the yield;

[0010] Step 3: dissolving puerarin powder and mesoporous polydopamine nanoparticles in anhydrous ethanol, stirring the mixed solution for 4 hours in the dark, then collecting the liquid, centrifuging to collect the mesoporous polydopamine@puerarin, and then washing with deionized water for 3 times;

[0011] Step 4: Sodium alginate was dissolved in deionized water, and then sodium periodate was added, and the mixture was stirred at room temperature in the dark for 6 hours, and then ethylene glycol was added to terminate the reaction. The final crude polymer was dialyzed in deionized water for 3 days to obtain oxidized sodium alginate, which was freeze-dried for later use;

[0012] Step 5: Dissolve carboxymethyl chitosan and oxidized sodium alginate in phosphate buffered saline,

[0013] Mesoporous polydopamine nanoparticles or mesoporous polydopamine@puerarin nanoparticles are dispersed in oxidized sodium alginate and mixed evenly. The oxidized sodium alginate solution and the carboxymethyl chitosan solution are mixed evenly and allowed to stand for 2-5 minutes to obtain a composite hydrogel.

[0014] Preferably, the composite hydrogel comprises: carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel.

[0015] Preferably, in step 1: deionized water is 65 ml, anhydrous ethanol is 60 ml, Pluronic F127 is 360 mg, mesitylene is 415 μL, ultrasonic oscillation is 30 min, tris(hydroxymethyl)aminomethane hydrochloride is 90 mg, and after stirring for 10 min, 60 mg of dopamine hydrochloride is added.

[0016] Preferably, in step 2: centrifugation is performed at 12000 rpm for 15 min.

[0017] Preferably, in step 3: the mass ratio of puerarin powder, mesoporous polydopamine nanoparticles and anhydrous ethanol is 2:1, and mesoporous polydopamine@puerarin is collected by centrifugation at 12000 rpm.

[0018] Preferably, in step 4: 1 g of sodium alginate is dissolved in 100 ml of deionized water, and then 1.08 g of sodium periodate is added, stirred at room temperature in the dark for 6 hours, and then 1.5 ml of ethylene glycol is added to terminate the reaction.

[0019] Preferably, in step 5: 2-5% carboxymethyl chitosan and 10-20% oxidized sodium alginate are respectively dissolved in phosphate buffered saline, and the oxidized sodium alginate solution and the carboxymethyl chitosan solution are mixed evenly at a concentration ratio of 2:3.

[0020] Preferably, in step 5: the concentration of the mesoporous polydopamine nanoparticles in the composite hydrogel is 0.5 mg / ml.

[0021] According to a second aspect of an embodiment of the present invention, there is provided an injectable antibacterial hydrogel prepared by the preparation method described in the first aspect.

[0022] According to a third aspect of the embodiments of the present invention, there is provided use of the injectable antibacterial hydrogel described in the second aspect in a wound healing material or a medicine.

[0023] The present invention has the following beneficial effects compared with the prior art:

[0024] The present invention discloses a preparation method and application of an injectable antibacterial hydrogel. By incorporating MPDANPs loaded with PUE into OSA / CMCs-based hydrogel, an injectable hydrogel with good self-healing properties is prepared, showing good mechanical properties. At the same time, the incorporation of MPDA@PUE NPs gives it a good photothermal effect and a controlled release and sustained release effect on PUE, perfectly combining the antibacterial effect of low-temperature PTT and the antioxidant, immune response regulation, and angiogenesis promotion capabilities of PUE. The hydrogel has shown good mechanical properties in relevant characterizations, and has been proven in subsequent experiments to have good performance in antibacterial, immune response regulation, and angiogenesis promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The present invention is a transmission electron microscope image of mesoporous polydopamine nanoparticles according to an exemplary embodiment.

[0026] Figure 2 The present invention is a transmission electron microscopy image of mesoporous polydopamine@puerarin nanoparticles according to an exemplary embodiment.

[0027] Figure 3 The present invention is a scanning electron microscope comparison diagram of carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel according to an exemplary embodiment.

[0028] Figure 4 The present invention shows the swelling curves of carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel according to an exemplary embodiment.

[0029] Figure 5 The present invention is a schematic diagram showing the injectability of mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel according to an exemplary embodiment.

[0030] Figure 6 The present invention is a schematic diagram of the adhesion of mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel according to an exemplary embodiment.

[0031] Figure 7 The present invention is a schematic diagram of the self-healing performance of mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel according to an exemplary embodiment.

[0032] Figure 8 The present invention is a temperature change curve of mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel doped with mesoporous polydopamine nanoparticles of different concentrations according to an exemplary embodiment.

[0033] Fig. 9 The present invention is a temperature change curve of a mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel with a mesoporous polydopamine nanoparticle concentration of 0.5 mg / mL under near-infrared light irradiation of different powers according to an exemplary embodiment.

[0034] Fig.10 The present invention is based on an exemplary embodiment showing the release curve of puerarin from mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel with and without near-infrared light irradiation.

[0035] Fig.11 The present invention is a graph of live / dead cell fluorescence staining results of a control group, carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light, and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light after 1 and 3 days of cell culture according to an exemplary embodiment.

[0036] Fig.12The present invention is a graph of cell activity detection after 1 and 3 days of cell culture in a control group, carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near infrared light, and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near infrared light according to an exemplary embodiment.

[0037] Fig.13 The present invention is a blank control, a negative control, a carboxymethyl chitosan / oxidized sodium alginate hydrogel, a mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, a mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel, and a mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate+near-infrared light group intracellular ROS fluorescence staining diagram according to an exemplary embodiment;

[0038] Fig.14 The present invention is a graph of quantification results of intracellular ROS in blank control, negative control, carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light group shown according to an exemplary embodiment.

[0039] Fig.15 The present invention is an immunofluorescence staining diagram of the expression of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α and anti-inflammatory related factors IL-10, Arg-1 and TGF-β1 in cells in the blank control, negative control, carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light group according to an exemplary embodiment.

[0040] Fig.16 The present invention is a graph of the quantitative results of immunofluorescence intensity of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α and anti-inflammatory related factors IL-10, Arg-1 and TGF-β1 in cells of blank control, negative control cells, carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light group according to an exemplary embodiment.

[0041] Fig.17 The present invention is a graph showing the colony formation of Staphylococcus aureus and Escherichia coli biofilms on agar plates in the control group, carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light, and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light groups according to an exemplary embodiment.

[0042] Fig.18 The present invention is a graph of quantitative results of colony formation of S. aureus and E. coli biofilms on agar plates in the control group, carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light, and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light groups according to an exemplary embodiment.

[0043] Fig.19 The present invention is a graph showing the results of a human umbilical vein endothelial cell scratch experiment in a control group, carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel, and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light group according to an exemplary embodiment.

[0044] Fig. 20 The present invention is based on an exemplary embodiment showing the quantitative analysis results of the human umbilical vein endothelial cell scratch test in the control group, carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light group.

[0045] Fig.21 The present invention is a graph showing protein fluorescence staining of endothelial cell-specific markers HIF-1α, CD31 and VEGF in a control group, carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light group according to an exemplary embodiment. DETAILED DESCRIPTION

[0046] The following is based on the attached Figure 1-21 The present invention is further described:

[0047] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] In the description of the present invention, it should be noted that the terms "center", "up", "down", "left", "right", "vertical", "horizontal", "inside", "outside", etc. indicate directions or positional relationships based on the directions or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operated in a specific direction. Therefore, they cannot be understood as limitations on the present invention.

[0049] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0050] Embodiment 1

[0051] According to an exemplary embodiment, a method for preparing an injectable antibacterial hydrogel is shown, and the specific steps are as follows:

[0052] Step 1: Take 65 ml of deionized water and 60 ml of anhydrous ethanol respectively and put them into a beaker and stir them evenly with a magnetic stirrer. Then add 360 mg of Pluronic F127 and 415 μL of mesitylene in turn, and then ultrasonically vibrate for 30 minutes to form a white emulsion. Then add 90 mg of tris(hydroxymethyl)aminomethane hydrochloride, stir for 10 minutes, then add 60 mg of dopamine hydrochloride, adjust the pH to 8.5, and stir at room temperature in the dark for 24 hours.

[0053] Step 2: Collect the liquid, centrifuge at 12000 rpm for 15 min and discard the supernatant, collect the mesoporous polydopamine nanoparticles at the bottom of the tube, wash them three times with water, a mixture of anhydrous ethanol and acetone, respectively, and weigh them after freeze-drying to calculate the yield. Figure 1 This is a transmission electron microscopy image of mesoporous polydopamine nanoparticles. Figure 2This is a transmission electron microscopy image of mesoporous polydopamine@puerarin nanoparticles. The transmission electron microscopy results show that the mesoporous polydopamine nanoparticles are in a uniform spherical shape with a mesoporous structure on the surface. Compared with mesoporous polydopamine, the mesoporous structure of mesoporous polydopamine@puerarin nanoparticles is fuzzy and the surface is covered with a rough thin layer, which indicates that puerarin is successfully loaded in the mesopores and surface of the mesoporous polydopamine nanoparticles.

[0054] Step 3: Puerarin powder and mesoporous polydopamine nanoparticles were dissolved in anhydrous ethanol at a mass ratio of 2:1, the mixed solution was stirred for 4 hours in the dark, and then the liquid was collected, and the mesoporous polydopamine@puerarin was collected by centrifugation at 12000 rpm for 15 min, and then washed with deionized water for 3 times.

[0055] Step 4: Dissolve 1 g of sodium alginate in 100 ml of deionized water, then add 1.08 g of sodium periodate, stir at room temperature in the dark for 6 hours, then add 1.5 ml of ethylene glycol to terminate the reaction, and dialyze the final crude polymer in deionized water for 3 days to obtain oxidized sodium alginate, which is freeze-dried for later use;

[0056] Step 5: Dissolve carboxymethyl chitosan and oxidized sodium alginate in phosphate buffered saline to prepare solutions with concentrations of 2-5% and 10-20%, respectively.

[0057] Mesoporous polydopamine nanoparticles or mesoporous polydopamine@puerarin nanoparticles are dispersed in oxidized sodium alginate solution and mixed evenly. The oxidized sodium alginate solution and the carboxymethyl chitosan solution are mixed evenly at a concentration ratio of 2:3, and the mixture is allowed to stand for 2-5 minutes to obtain a composite hydrogel. The composite hydrogel includes: carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel. The concentration of mesoporous polydopamine nanoparticles in the composite hydrogel is 0.5 mg / ml.

[0058] Figure 3 This is a scanning electron microscope comparison of carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel. The SEM image shows that the prepared hydrogel presents a uniform porous structure that is interconnected, which is conducive to the penetration of oxygen and the absorption of wound exudate, and some mesoporous polydopamine@puerarin nanoparticles can be observed in the mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel.

[0059] Figure 4Figure 1 is the swelling curve of carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel. It can be seen that the hydrogel reaches swelling equilibrium after about 36 hours, and the swelling rate of mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel is lower than that of G, indicating that the interaction between nanoparticles and polymer chains can limit their excessive expansion.

[0060] Figure 5 This is a schematic diagram of the injectability of mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel. The hydrogel can be injected into the culture dish evenly and continuously with a syringe, and the words "A" and "B" can be easily written, proving that the prepared hydrogel has good injectability and can better match various irregular wounds.

[0061] Figure 6 This is a schematic diagram of the adhesion of mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel. Figure 7 This is a schematic diagram of the self-healing performance of mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel. Mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel can maintain a good shape on repeatedly twisted pig skin and does not fall off, proving that the hydrogel has good adhesion. The colorless and black hydrogels were cut separately. After the two halves came into contact without the influence of external forces, they could be merged into a complete gel and would not break after being pulled, proving that the hydrogel has good self-healing properties. Good adhesion and self-healing properties are conducive to its maintenance on the wound surface and resistance to external damage.

[0062] Figure 8 The present invention is a temperature change curve of mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel doped with different concentrations of mesoporous polydopamine nanoparticles. Fig. 9 The temperature change curve of mesoporous polydopamine@puerarin / carboxymethyl chitosan / sodium alginate hydrogel with a mesoporous polydopamine nanoparticle concentration of 0.5 mg / mL under near-infrared light irradiation of different powers. The results show that the temperature change of the hydrogel is dependent on the concentration of mesoporous polydopamine and the near-infrared light power. The heat generated by the photothermal effect can enhance tissue regeneration and cause bacterial cell membrane rupture and protein inactivation, but higher temperatures (about 50°C) may also cause the death of normal cells. Therefore, the laser power intensity we chose for subsequent experiments was 1W / cm 2 .

[0063] Fig.10The release curves of puerarin from mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel with and without near-infrared light irradiation. The near-infrared light group has a higher drug release rate, indicating that the delivery system can promote drug release under near-infrared light irradiation.

[0064] This application prepares an injectable hydrogel with good self-healing properties by incorporating PUE-loaded MPDA NPs into OSA / CMCs-based hydrogels. It exhibits good mechanical properties. At the same time, the incorporation of MPDA@PUE NPs gives it a good photothermal effect and a controlled release of PUE, perfectly combining the antibacterial effect of low-temperature PTT and the antioxidant, immune response regulation, and angiogenesis-promoting abilities of PUE. The hydrogel has shown good mechanical properties in relevant characterizations, and has been shown in subsequent experiments to have good performance in antibacterial, immune response regulation, and angiogenesis promotion.

[0065] Embodiment 2

[0066] Fig.11 The present invention is a graph of live / dead cell fluorescence staining results of a control group, carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light, and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light after 1 and 3 days of cell culture according to an exemplary embodiment. Fig.12 The present invention is a graph of cell activity detection after 1 and 3 days of cell culture in a control group, carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near infrared light, and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near infrared light according to an exemplary embodiment.

[0067] The results showed that each group of cells had good cell morphology or structure, few dead cells were observed, and the cell survival rate of each group was over 90%. After 3 days, the mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light group's hydrogels significantly promoted cell proliferation, indicating that mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel can promote cell proliferation.

[0068] Embodiment 3

[0069] Fig.13 The present invention is a blank control, a negative control, a carboxymethyl chitosan / oxidized sodium alginate hydrogel, a mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, a mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel, and a mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate+near-infrared light group intracellular ROS fluorescence staining diagram according to an exemplary embodiment; Fig.14 The present invention is a graph of quantification results of intracellular ROS in blank control, negative control, carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light group shown according to an exemplary embodiment.

[0070] The results showed that the content of reactive oxygen species in each hydrogel group was reduced, and the lowest fluorescence intensity was detected in the cells of the mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light group, which was almost consistent with the uninduced blank control group, proving that the prepared hydrogel has good antioxidant capacity and can effectively remove reactive oxygen species in cells under near-infrared light irradiation.

[0071] Embodiment 4

[0072] Fig.15 The present invention is an immunofluorescence staining diagram of the expression of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α and anti-inflammatory related factors IL-10, Arg-1 and TGF-β1 in cells of a blank control, a negative control, a carboxymethyl chitosan / oxidized sodium alginate hydrogel, a mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, a mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel and a mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel+near-infrared light group according to an exemplary embodiment, Fig.16 The present invention is a graph of quantitative results of immunofluorescence intensity of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α and anti-inflammatory related factors IL-10, Arg-1 and TGF-β1 in cells in the blank control, negative control, carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light group according to an exemplary embodiment.

[0073] The results showed that the expression levels of various inflammatory-related factors IL-1β, IL-6 and TNF-α (pro-inflammatory) and IL-10, Arg-1 and TGF-β1 (anti-inflammatory) of macrophages can reflect the effect of the sample on the inflammatory response. The negative control group stimulated by bacterial cell wall component lipopolysaccharide expressed high pro-inflammatory factors, while the mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light groups significantly inhibited the expression of these inflammatory factors. The mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light group had the most significant effect, while the expression of anti-inflammatory factors showed an opposite trend.

[0074] Embodiment 5

[0075] Fig.17 The present invention is a graph showing the colony formation of Staphylococcus aureus and Escherichia coli biofilms on agar plates in the control group, carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel+near-infrared light, and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel+near-infrared light groups according to an exemplary embodiment. Fig.18 The present invention is a graph of quantitative results of colony formation of S. aureus and E. coli biofilms on agar plates in the control group, carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light, and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light groups according to an exemplary embodiment.

[0076] The results showed that mesoporous polydopamine nanoparticles had good photothermal therapy effects. The mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light groups showed the strongest antibacterial ability. The antibacterial effect of the mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light group on E. coli was more significant than that on S. aureus.

[0077] Embodiment 6

[0078] Fig.19The present invention is a graph showing the results of a human umbilical vein endothelial cell scratch test on a control group, carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel, and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel+near infrared light group according to an exemplary embodiment. Fig. 20 The present invention is based on an exemplary embodiment showing the quantitative analysis results of the human umbilical vein endothelial cell scratch test in the control group, carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light group.

[0079] The results showed that the mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel and the mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light groups caused the highest level of cell migration, indicating that puerarin can promote the migration of human umbilical vein endothelial cells to the greatest extent.

[0080] Embodiment 7

[0081] Fig.21 The present invention is a graph showing protein fluorescence staining of endothelial cell-specific markers HIF-1α, CD31 and VEGF in a control group, carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light group according to an exemplary embodiment.

[0082] The results showed that the fluorescence intensity of three vascular-related proteins CD31, HIF-1a and VEGF in the mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light groups was significantly stronger than that in the other groups without puerarin, among which the mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel + near-infrared light group had the most significant effect, indicating that it can promote the formation of blood vessels to the greatest extent. .

[0083] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily realized. Therefore, without departing from the general concept defined by the claims and equivalent scope, the present invention is not limited to the specific details and the illustrations shown and described here.

Claims

1. A method for preparing an injectable antibacterial hydrogel, characterized in that: include: Step 1: Deionized water and anhydrous ethanol are respectively taken into a beaker and stirred evenly by a magnetic stirrer, and then Pluronic F127 and mesitylene are added in sequence, and then ultrasonic vibration is performed to form a white emulsion, and then tris(hydroxymethyl)aminomethane hydrochloride is added, and dopamine hydrochloride is added after stirring, and the pH is adjusted to 8.5, and stirred at room temperature in the dark for 24 hours; Step 2: Collect the liquid, centrifuge and discard the supernatant, collect the mesoporous polydopamine nanoparticles at the bottom of the tube, wash them three times with water, a mixture of anhydrous ethanol and acetone, and weigh them after freeze-drying to calculate the yield; Step 3: dissolving puerarin powder and mesoporous polydopamine nanoparticles in anhydrous ethanol, stirring the mixed solution for 4 hours in the dark, then collecting the liquid, centrifuging to collect the mesoporous polydopamine@puerarin, and then washing with deionized water for 3 times; Step 4: Sodium alginate was dissolved in deionized water, and then sodium periodate was added, and the mixture was stirred at room temperature in the dark for 6 hours, and then ethylene glycol was added to terminate the reaction. The final crude polymer was dialyzed in deionized water for 3 days to obtain oxidized sodium alginate, which was freeze-dried for later use; Step 5: Dissolve carboxymethyl chitosan and oxidized sodium alginate in phosphate buffered saline respectively, disperse mesoporous polydopamine nanoparticles or mesoporous polydopamine@puerarin in oxidized sodium alginate and mix evenly, mix the oxidized sodium alginate solution and the carboxymethyl chitosan solution evenly, and let stand for 2-5 minutes to obtain a composite hydrogel.

2. The method for preparing the injectable antibacterial hydrogel according to claim 1, characterized in that: The composite hydrogel includes: carboxymethyl chitosan / oxidized sodium alginate hydrogel, mesoporous polydopamine / carboxymethyl chitosan / oxidized sodium alginate hydrogel and mesoporous polydopamine@puerarin / carboxymethyl chitosan / oxidized sodium alginate hydrogel.

3. The method for preparing the injectable antibacterial hydrogel according to claim 1 or 2, characterized in that: In the step 1, deionized water is 65 ml, anhydrous ethanol is 60 ml, Pluronic F127 is 360 mg, mesitylene is 415 μL, ultrasonic oscillation is 30 min, tris(hydroxymethyl)aminomethane hydrochloride is 90 mg, and after stirring for 10 min, 60 mg of dopamine hydrochloride is added.

4. The method for preparing the injectable antibacterial hydrogel according to claim 3, characterized in that: In the step 2: centrifugation is performed at 12000 rpm for 15 min.

5. The method for preparing the injectable antibacterial hydrogel according to claim 4, characterized in that: In the step 3, the mass ratio of puerarin powder, mesoporous polydopamine nanoparticles and anhydrous ethanol is 2:1, and mesoporous polydopamine@puerarin is collected by centrifugation at 12000 rpm.

6. The method for preparing the injectable antibacterial hydrogel according to claim 5, characterized in that: In the step 4: 1 g of sodium alginate was dissolved in 100 ml of deionized water, and then 1.08 g of sodium periodate was added, stirred at room temperature in the dark for 6 hours, and then 1.5 ml of ethylene glycol was added to terminate the reaction.

7. The method for preparing the injectable antibacterial hydrogel according to claim 5 or 6, characterized in that: In the step 5: dissolving 2-5% carboxymethyl chitosan and 10-20% oxidized sodium alginate in phosphate buffered saline respectively, and mixing the oxidized sodium alginate solution and the carboxymethyl chitosan solution in a concentration ratio of 2:

3.

8. The method for preparing the injectable antibacterial hydrogel according to claim 7, characterized in that: In step 5: the concentration of the mesoporous polydopamine nanoparticles in the composite hydrogel is 0.5 mg / ml.

9. An injectable antibacterial hydrogel prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the injectable antibacterial hydrogel according to claim 9 in wound healing materials or medicines.

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