Sprayable hydrogel for healing bacterial infection wounds and preparation method thereof

Abstract

A sprayable hydrogel for wound healing caused by bacterial infection and a preparation method thereof, comprising the steps of preparing g-C3N4 nanosheets with melamine as a precursor, and then in-situ reducing HAuCl4·3H2O on the g-C3N4 nanosheets to prepare AuNPs-C3N4 nanozymes; preparing oxidized hyaluronic acid by a sodium periodate oxidation method; incubating the AuNPs-C3N4 nanozyme solution with a glucose solution and then adding a Mn-containing 2+ The manganese salt solution and bicarbonate solution were then mixed with the oxidized hyaluronic acid solution to obtain a dispersion, and the dispersion was sprayed with the carboxymethyl chitosan solution to produce the AuNPs-C3N4 / Glu / Mn 2+ @Carboxymethyl chitosan-OHA hydrogel. The preparation process of this method is simple, and the prepared hydrogel has excellent in vitro antibacterial efficiency, good biocompatibility and bacterial adsorption, which can effectively remove bacteria from wound infection sites and promote wound healing.

Description

Technical Field

[0001] The invention belongs to the technical field of wound healing, and particularly relates to a sprayable hydrogel for healing bacterially infected wounds and a preparation method thereof. Background Art

[0002] The skin is the largest organ in the human body and an important barrier to protect the body. As the most vulnerable organ, skin injury is a complex process. During the wound repair process, damaged skin tissue may be infected with bacteria, and in severe cases, the wound cannot heal. In recent years, antibacterial strategies based on nanozymes, metal ions, antibacterial drugs, and photothermal and photodynamic therapy have been widely reported. In nature, enzymes are widely involved in combating invading pathogens, especially in the plant and animal kingdoms. Natural enzymes have attracted widespread attention due to their high catalytic efficiency and good biocompatibility, but they are expensive and easily inactivated. Nanozymes are a class of nanomaterials with enzyme-like properties, which show great potential in various applications as a substitute for natural enzymes. Therefore, the development and design of functional nanomaterials with enzyme-like catalytic activity has become an effective alternative. However, during the treatment process, the rapid release of nanozymes in exposed wounds reduces the retention time of drugs at the wound site, reduces the therapeutic effect, and even induces off-target side effects. In addition, traditional wound dressings, such as gauze and bandages, have the functions of stopping bleeding and protecting wounds, but are not ideal for promoting wound healing. During frequent dressing changes, dehydration and secondary wound infection are likely to occur. Synthesizing innovative wound dressings with strong antibacterial ability, no drug resistance, and good biocompatibility is an urgent problem to be solved in this field.

[0003] In recent years, the application of hydrogels with good biocompatibility in wound healing has received increasing attention. Hydrogels have a three-dimensional network structure, good moisture retention and the ability to absorb wound exudate, which can promote the diffusion of loaded substances and have broad application prospects in the biomedical field. In addition, loading some functional nanodrugs into hydrogels for wound healing can not only overcome the problems of fast drug release and low efficiency of fixed values ​​at the wound site, but also reshape the wound microenvironment. By adhering hydrogels to the wound surface of bacterial infection, the sustained release of drugs and long-term retention of the wound surface can be achieved, reducing potential off-target side effects, improving the treatment effect, and accelerating wound repair. However, existing antibacterial hydrogels often have potential toxic side effects on normal tissues due to non-selective killing. In addition, the movement of the wound site can cause the dressing to tear or even be damaged, which requires the dressing to have good self-healing properties. Summary of the invention

[0004] The purpose of the present invention is to provide a sprayable hydrogel for healing bacterially infected wounds and a preparation method thereof. The method has a simple preparation process, and the prepared hydrogel has excellent in vitro antibacterial efficiency, good biocompatibility and bacterial adsorption, can effectively remove bacteria from wound infection sites, and promote wound healing.

[0005] To achieve the above object, the present invention provides a method for preparing a sprayable hydrogel for healing bacterially infected wounds, comprising the following steps:

[0006] S1, preparation of g-C3N4 nanosheets using melamine as precursor;

[0007] S2, in situ reduction of HAuCl4·3H2O on the g-C3N4 nanosheets prepared in step S1 to prepare AuNPs-C3N4 nanozyme;

[0008] S3, preparing oxidized hyaluronic acid by sodium periodate oxidation method;

[0009] S4, prepare AuNPs-C3N4 nanozyme solution, glucose solution, Mn-containing 2+ Manganese salt solution, bicarbonate solution, oxidized hyaluronic acid solution and carboxymethyl chitosan solution; AuNPs-C3N4 nanozyme solution was incubated with glucose solution and then added with Mn 2+ The manganese salt solution and bicarbonate solution were then mixed with the oxidized hyaluronic acid solution to obtain a dispersion, and the dispersion was sprayed with the carboxymethyl chitosan solution to produce a Schiff base reaction to obtain AuNPs-C3N4 / Glu / Mn 2+ @Carboxymethyl chitosan-OHA hydrogel.

[0010] Preferably, in step S1, melamine is used as a precursor and calcined repeatedly at 550° C. to obtain g-C3N4.

[0011] Furthermore, in step S1, melamine is used as a precursor, the temperature is increased to 550°C at a heating rate of 2°C / min and calcined for 4 hours, then ground after cooling, and then heated to 550°C at a heating rate of 5°C / min and calcined for 1 hour. After cooling to room temperature, the temperature is then increased to 550°C at a heating rate of 2°C / min and calcined for 1 hour. After cooling, the temperature is finally increased to 550°C at a heating rate of 5°C / min and calcined for 1 hour to obtain g-C3N4 nanosheets.

[0012] Preferably, in step S2, the mass ratio of the gold element in HAuCl4·3H2O to the g-C3N4 nanosheets is 1:(1-4).

[0013] Furthermore, in step S2, the AuNPs-C3N4 nanozyme uses g-C3N4 nanosheets as a carrier and sodium borohydride as a reducing agent to reduce chloroauric acid.

[0014] Furthermore, the specific steps of step S2 are: ultrasonically dispersing the g-C3N4 nanosheets in deionized water to form a uniform dispersion; taking the uniform dispersion, preparing gold nanoparticles with a size of about 2.0-3.0 nm on the surface of the g-C3N4 nanosheets by sodium borohydride reduction method, and obtaining AuNPs-C3N4 nanozyme after centrifugal washing and drying.

[0015] Furthermore, in step S3, a sodium periodate solution as an oxidant is added to the hyaluronic acid solution, and after continuous stirring, ethylene glycol is added to terminate the reaction, and the reaction is continued to quench excess sodium periodate, and the sponge-like oxidized hyaluronic acid is purified and freeze-dried to obtain.

[0016] Preferably, in step S3, the stirring time of sodium periodate and hyaluronic acid is 18-24 hours, and the reaction is carried out at room temperature in the dark; the mass volume ratio of hyaluronic acid to ethylene glycol is 1 g: (1-1.2) mL, and the continuous reaction time is 50-70 min.

[0017] Preferably, in step S4, the Schiff base reaction time, i.e., the gelation time of the hydrogel, is 80-100 s, and the mass ratio of the oxidized hyaluronic acid to the carboxymethyl chitosan is (1-2):1.

[0018] In order to achieve the above-mentioned purpose of the invention, the present invention also provides a sprayable hydrogel for healing bacterially infected wounds prepared by the above-mentioned preparation method.

[0019] The present invention uses Schiff base reaction to prepare hydrogel, and the obtained hydrogel is loaded with AuNPs-C3N4 nanozyme, glucose and Mn 2+ , which can release AuNPs-C3N4 nanozymes with glucose oxidase-like activity at the bacterial infection wound site, which can in situ generate hydrogen peroxide and drive Mn 2+ The mediated Fenton-like reaction produces hydroxyl radicals (·OH), which produce antimicrobial active substances and eliminate Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. Moreover, the in situ formed hydrogel has the characteristics of pH-responsive degradation, long-term nanozyme retention, sustained release of loaded nanozymes and rapid in situ wound coverage, which can relieve skin wound inflammation and accelerate the healing of bacterially infected wounds.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a hydrogel with excellent in vitro antibacterial efficiency, good biocompatibility and bacterial adsorption. The hydrogel has the above functions while reducing toxic side effects, promoting collagen deposition and tissue regeneration, meeting the needs of promoting skin wound healing, and has broad application prospects in bacterial wound healing. This patent constructs a hydrogel with self-healing properties through Schiff base bonds, which has the advantages of mild reaction conditions, fast reaction speed, and adaptability to biomaterials. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a transmission electron microscopic image of the AuNPs-C3N4 nanozyme prepared in the embodiment of the present invention;

[0023] Figure 2 This is a statistical diagram of the particle size of gold nanoparticles in the AuNPs-C3N4 nanozyme prepared in the embodiment of the present invention;

[0024] Figure 3 The AuNPs-C3N4 / Glu / Mn prepared in the embodiment of the present invention 2+ @Photo of the formation process of carboxymethyl chitosan-OHA hydrogel;

[0025] Figure 4 The AuNPs-C3N4 / Glu / Mn prepared in the embodiment of the present invention 2+ @Degradation of carboxymethyl chitosan-OHA hydrogel under different pH conditions;

[0026] Figure 5 The AuNPs-C3N4 / Glu / Mn prepared in the embodiment of the present invention 2+ @Degradation rate of carboxymethyl chitosan-OHA hydrogel at different reaction times;

[0027] Figure 6 The AuNPs-C3N4 / Glu / Mn prepared in the embodiment of the present invention 2+ The effect diagram of the removal of Staphylococcus aureus;

[0028] Figure 7 The AuNPs-C3N4 / Glu / Mn prepared in the embodiment of the present invention 2+ The effect of eliminating methicillin-resistant Staphylococcus aureus is shown in the figure;

[0029] Figure 8 The AuNPs-C3N4 / Glu / Mn prepared in the embodiment of the present invention 2+ @Schematic diagram of the cytotoxicity of carboxymethyl chitosan-OHA hydrogel to fibroblasts;

[0030] Fig. 9 The AuNPs-C3N4 / Glu / Mn prepared in the embodiment of the present invention2+ @Chart of carboxymethyl chitosan-OHA hydrogel on the healing of bacterially infected skin wounds in mice. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] The raw materials and reagents used in the following examples, unless otherwise specified, are all commercially available.

[0033] Example

[0034] A method for preparing a sprayable hydrogel for healing bacterially infected wounds, comprising the following steps:

[0035] S1. Preparation of g-C3N4 nanosheets using melamine as a precursor, the specific steps are as follows:

[0036] S1-1, take 2g of melamine and heat it to 550℃ at room temperature at a heating rate of 2℃ / min and calcine for 4h to obtain bright yellow g-C3N4;

[0037] S1-2, grind the cooled bright yellow g-C3N4, then heat it to 550°C at a heating rate of 5°C / min and calcine it for 1 h, cool it to room temperature, and then heat it to 550°C at a heating rate of 2°C / min and calcine it for 1 h to obtain multilayer g-C3N4;

[0038] S1-3, heating the cooled multilayer g-C3N4 to 550°C at a heating rate of 5°C / min and calcining for 1 h to obtain g-C3N4 nanosheets;

[0039] S2, in situ reduction of HAuCl4·3H2O on the g-C3N4 nanosheets prepared in step S1 to prepare AuNPs-C3N4 nanozyme, the specific steps are as follows:

[0040] S2-1, ultrasonically disperse g-C3N4 nanosheets in deionized water to form a uniform dispersion with a concentration of 0.1 mg / mL; ultrasonically treat 10 mL of 0.1 mg / mL g-C3N4 suspension for 10 min (120w), add HAuCl4·3H2O under stirring, and the mass ratio of gold element to g-C3N4 nanosheets is 1:2 to obtain AuNPs-C3N4 nanosheets;

[0041] S2-2, after stirring for 1 min, 25 μL of 0.1 M NaBH4 solution prepared with ice water was added to prepare gold nanoparticles on the surface of g-C3N4 nanosheets by sodium borohydride reduction method;

[0042] S2-3, the reaction suspension was centrifuged, washed and freeze-dried to obtain AuNPs-C3N4 nanozyme;

[0043] S3. Preparing oxidized hyaluronic acid (OHA) by sodium periodate oxidation method, the specific steps are as follows:

[0044] S3-1, 1g HA (M W =100-200 kDa) was dissolved in 100 mL of deionized water and stirred for about 10 min to obtain a uniform solution;

[0045] S3-2, add 5 mL of 2.5 mM NaIO4 solution to the solution prepared in step S3-1, and stir in the dark at room temperature for 24 h;

[0046] S3-3, add 1.2 mL of ethylene glycol to the solution in step S3-2 to terminate the reaction, continue stirring for 1 h to quench the remaining NaIO4; dialysis and purification of the solution (MWCO = 3500 Da) for 72 h, and freeze-dry to obtain white sponge-like OHA;

[0047] S4. Preparation of AuNPs-C3N4 / Glu / Mn by Schiff base reaction 2+ @Carboxymethyl chitosan-OHA hydrogel;

[0048] S4-1, 1 mg of AuNPs-C3N4 nanozyme was dissolved in 1 mL of deionized water; 0.396 mg of MnCl2·4H2O was dissolved in 1 mL of deionized water; 0.09 g of glucose was dissolved in 1 mL of deionized water; 0.168 mg of NaHCO3 was dissolved in 1 mL of deionized water; 40 mg of carboxymethyl chitosan was dissolved in 1 mL of PBS; 40 mg of OHA was dissolved in 1 mL of PBS;

[0049] S4-2, after incubating 100 μL of AuNPs-C3N4 nanozyme solution with 100 μL of Glu solution, 90 μL of MnCl2·4H2O solution and 10 μL of NaHCO3 solution were added, and then 250 μL of OHA solution was added and mixed to obtain a dispersion;

[0050] S4-3, spraying the dispersion prepared in step S4-2 with 250 μL of carboxymethyl chitosan solution to produce AuNPs-C3N4 / Glu / Mn 2+ @Carboxymethyl chitosan-OHA hydrogel.

[0051] The AuNPs-C3N4 nanozyme prepared in step S2 of this embodiment was characterized by using a transmission electron microscope to observe the morphology and particle size of the AuNPs-C3N4 nanozyme, and using Nano measure to measure the particle size of the gold nanoparticles. Figure 1 and Figure 2 .from Figure 1It can be seen that the ultrasmall gold nanoparticles are evenly distributed on the g-C3N4 nanosheets, indicating the successful preparation of AuNPs-C3N4 nanozymes; Figure 2 As can be seen in the figure, the size of the gold nanoparticles is about 2.0-3.0 nm. Figure 3 The AuNPs-C3N4 / Glu / Mn prepared in step S4 of this embodiment 2+ @The formation process of carboxymethyl chitosan-OHA hydrogel is recorded. It can be seen from the figure that the two hydrogel precursors can be quickly cross-linked to form a hydrogel about 95s after being sprayed on each other.

[0052] The AuNPs-C3N4 / Glu / Mn prepared in this example 2+ @Carboxymethyl chitosan-OHA hydrogel degradation test: AuNPs-C3N4 / Glu / Mn 2+ @Carboxymethyl chitosan-OHA hydrogel was immersed in phosphate buffer at pH = 6, 7, and 8, and the AuNPs-C3N4 / Glu / Mn 2+ @ pH-responsive degradation of carboxymethyl chitosan-OHA hydrogel, see Figure 4 , and calculated the degradation rate of hydrogel at different reaction times. Figure 5 .

[0053] Figure 4 and Figure 5 The experimental results showed that the degradation occurred faster at a lower pH of 6, which was due to the decomposition of the supramolecular structure and imine bonds under acidic conditions. 2+ @Carboxymethyl chitosan-OHA hydrogel continuously degrades and releases active substances to generate OH, which effectively fights bacterial infection.

[0054] The product prepared by the present invention has an antibacterial effect. 2+ The hydrogel prepared by carboxymethyl chitosan solution and oxidized hyaluronic acid solution only plays the role of encapsulating the effective antibacterial components. 2+ Antibacterial (Staphylococcus aureus and MRSA) test verification:

[0055] Staphylococcus aureus antibacterial experiment:

[0056] Step (1): Take 100 μL 1 mg / mL AuNPs-C3N4 and 100 μL 0.5 M Glu and incubate for about 3 hours, then add 90 μL 2 mM MnCl2·4H2O and 10 μL 2 mM NaHCO3

[0057] Step (2): add 100 μL of Staphylococcus aureus with OD 600 = 0.05 to step (1) and incubate for about 6 hours as the experimental group;

[0058] Step (3): 100 μL of Staphylococcus aureus with an OD 600 of 0.05 was added to an equal volume of deionized water as in step (1) and incubated for about 6 hours as a control group;

[0059] Step (4): Dilute the suspensions in steps (2) and (3) and apply them to plates. After incubation at 37°C for 24 hours, count the single colonies on the LB solid medium. Figure 6 ;

[0060] Figure 6 The experimental results show that the AuNPs-C3N4 / Glu / Mn 2+ Compared with the control group, it can effectively eliminate bacteria.

[0061] Antibacterial test for methicillin-resistant Staphylococcus aureus:

[0062] Step (1): Take 100 μL 1 mg / mL AuNPs-C3N4 and 100 μL 0.5 M Glu and incubate for about 3 hours, then add 90 μL 2 mM MnCl2·4H2O and 10 μL 2 mM NaHCO3;

[0063] Step (2): add 100 μL of OD 600 = 0.05 MRSA to step (1) and incubate for about 6 hours;

[0064] Step (3): 100 μL of OD 600 = 0.05 methicillin-resistant Staphylococcus aureus was added to an equal volume of deionized water as in step (1) and incubated for about 6 hours as a control group;

[0065] Step (4): Dilute the suspensions from steps (2) and (3) and plate them. After incubating at 37°C for 24 hours, count the single colonies on the LB solid medium. Figure 7 ;

[0066] Figure 7 The experimental results show that the AuNPs-C3N4 / Glu / Mn 2+ Compared with the control group, it can effectively eliminate bacteria.

[0067] The hydrogel prepared in this example was subjected to a fibroblast toxicity test: after culturing fibroblasts (seeding concentration of 50,000 per milliliter, 100 μL per well) in a 96-well plate for 24 hours, 100 μL of a high-glucose medium containing AuNPs-C3N4 nanozyme, MnCl2·4H2O, carboxymethyl chitosan, OHA, and hydrogel extract was added (wherein the concentration of AuNPs-C3N4 nanozyme was 5, 10, 20, 50, 100, 200 μg / mL, MnCl2·4H2O, carboxymethyl chitosan, OHA, and hydrogel extract was added). The concentrations of nCl2·4H2O were 0.05, 0.1, 0.2, 0.5, 1, and 2 mM, the concentrations of carboxymethyl chitosan were 0.2, 0.5, 1, 2, and 4 mg / mL, the concentrations of OHA were 0.05, 0.1, 0.2, 0.5, and 1 mg / mL, and the dilution factors of the hydrogel extract were 10, 100, 500, 1000, and 5000, respectively). The cells were incubated at 37°C and 5% CO2 for 24 h. The cytotoxicity of the materials was evaluated by MTT assay. The results are shown in Table 2. Figure 8 ;

[0068] Figure 8 The experimental results show that, compared with the control group, the materials involved in the embodiments of the present invention have almost no damage to cells and have good biocompatibility.

[0069] The hydrogel prepared in this example was tested for wound healing effect in mice: a number of 6-week-old C57BL / 6 female mice in good health were selected to construct a 1 cm 2 The wounds were inoculated with Staphylococcus aureus. After 24 hours, phosphate buffer (control group), AuNPs-C3N4 / Glu / Mn 2+ @CO gel was sprayed evenly on the wound, and the wound area was observed and counted. The results are shown in Fig. 9 .

[0070] Fig. 9 The experimental results show that the hydrogel prepared in this example has an excellent effect of promoting wound healing, that is, the hydrogel of the present invention can significantly accelerate the healing of bacterially infected skin wounds compared with the control group.

Claims

1. A method for preparing a sprayable hydrogel for healing bacterially infected wounds, characterized in that: The following steps are involved: S1, preparation of g-C3N4 nanosheets using melamine as precursor; S2, in situ reduction of HAuCl4∙3H2O on the g-C3N4 nanosheets prepared in step S1 to prepare AuNPs-C3N4 nanozymes; the mass ratio of gold element in HAuCl4∙3H2O to g-C3N4 nanosheets is 1:(1-4); S3, preparing oxidized hyaluronic acid by sodium periodate oxidation method; S4, prepare AuNPs-C3N4 nanozyme solution, glucose solution, Mn-containing 2+ Manganese salt solution, bicarbonate solution, oxidized hyaluronic acid solution and carboxymethyl chitosan solution; AuNPs-C3N4 nanozyme solution was incubated with glucose solution and then added with Mn 2+ The manganese salt solution and bicarbonate solution were then mixed with the oxidized hyaluronic acid solution to obtain a dispersion, and the dispersion was sprayed with the carboxymethyl chitosan solution to produce a Schiff base reaction to obtain AuNPs-C3N4 / glucose / Mn 2+ @Carboxymethyl chitosan-oxidized hyaluronic acid hydrogel; the mass ratio between oxidized hyaluronic acid and carboxymethyl chitosan is (1-2):

1.

2. The method for preparing a sprayable hydrogel for wound healing caused by bacterial infection according to claim 1, characterized in that: In step S1, melamine is used as a precursor and calcined repeatedly at 550°C to obtain g-C3N4 nanosheets.

3. The method for preparing a sprayable hydrogel for wound healing caused by bacterial infection according to claim 2, characterized in that: In step S1, melamine is used as a precursor, the temperature is increased to 550°C at a heating rate of 2°C / min and calcined for 4 h, then ground after cooling, and then heated to 550°C at a heating rate of 5°C / min and calcined for 1 h. After cooling to room temperature, the temperature is then increased to 550°C at a heating rate of 2°C / min and calcined for 1 h. After cooling, the temperature is finally increased to 550°C at a heating rate of 5°C / min and calcined for 1 h to obtain g-C3N4 nanosheets.

4. The method for preparing a sprayable hydrogel for wound healing caused by bacterial infection according to claim 1 or 2, characterized in that: In step S2, the AuNPs-C3N4 nanozyme uses g-C3N4 nanosheets as a carrier and sodium borohydride as a reducing agent to reduce chloroauric acid.

5. The method for preparing a sprayable hydrogel for wound healing caused by bacterial infection according to claim 4, characterized in that: The specific steps of step S2 are: ultrasonically dispersing the g-C3N4 nanosheets in deionized water to form a uniform dispersion; taking the uniform dispersion, preparing gold nanoparticles with a size of 2.0-3.0 nm on the surface of the g-C3N4 nanosheets by sodium borohydride reduction method, and obtaining AuNPs-C3N4 nanozyme after centrifugal washing and drying.

6. The method for preparing a sprayable hydrogel for wound healing caused by bacterial infection according to claim 1 or 2, characterized in that: In step S3, a sodium periodate solution as an oxidant is added to the hyaluronic acid solution, and after continuous stirring, ethylene glycol is added to terminate the reaction, and the reaction is continued to quench excess sodium periodate, and then purified and freeze-dried to obtain sponge-like oxidized hyaluronic acid.

7. The method for preparing a sprayable hydrogel for wound healing caused by bacterial infection according to claim 6, characterized in that: In step S3, the stirring time of sodium periodate and hyaluronic acid is 18-24 h, and the reaction is carried out at room temperature in the dark; the mass volume ratio of hyaluronic acid to ethylene glycol is 1 g: (1-1.2) mL, and the continuous reaction time is 50-70 min.

8. The method for preparing a sprayable hydrogel for wound healing caused by bacterial infection according to claim 1 or 2, characterized in that: In step S4, the Schiff base reaction time, i.e., the gelation time of the hydrogel, is 80-100 s.

9. A sprayable hydrogel for healing bacterially infected wounds prepared by the preparation method according to any one of claims 1 to 8.

Images