CN113289053A

CN113289053A - Antibacterial hydrogel wound dressing loaded with two-dimensional material and nanoparticles and preparation method thereof

Info

Publication number: CN113289053A
Application number: CN202110519576.9A
Authority: CN
Inventors: 郭瑞; 冯龙宝; 刘玉; 蓝咏
Original assignee: Guangzhou Bioscience Co ltd
Current assignee: Guangzhou Bioscience Co ltd
Priority date: 2021-05-12
Filing date: 2021-05-12
Publication date: 2021-08-24
Anticipated expiration: 2041-05-12
Also published as: CN113289053B

Abstract

The invention relates to the technical field of biomedical engineering materials, and discloses an antibacterial hydrogel wound dressing loaded with a two-dimensional material and nanoparticles and a preparation method thereof²⁺The nanoparticles being capable of reacting with boric acid to release Zn²⁺The nanoparticles of (3) are preferably ZnO nanoparticles. The antibacterial hydrogel wound dressing disclosed by the invention utilizes the loaded two-dimensional boron alkene nanosheet to generate ROS (reactive oxygen species) through the photodynamic propertySexual oxygen) to realize antibacterial property, and simultaneously the loaded ZnO nanoparticles are decomposed to generate Zn with good antibacterial property²⁺Under the synergistic effect of the two components, the antibacterial effect of the hydrogel wound dressing is greatly improved, and in addition, Zn²⁺Can also promote the migration and proliferation of fibroblasts, and has excellent promoting effect on the healing of wounds.

Description

Antibacterial hydrogel wound dressing loaded with two-dimensional material and nanoparticles and preparation method thereof

Technical Field

The invention relates to the technical field of medical biomaterials, in particular to an antibacterial hydrogel wound dressing loaded with a two-dimensional material and nanoparticles and a preparation method thereof.

Background

Bacterial infections can interfere greatly with healing of both acute and chronic wounds, and are one of the most difficult challenges in clinical wound care. According to the world health organization, approximately 1500 million people die each year from bacterial infections. The skin, the largest organ of the human body, is highly susceptible to bacterial infection after injury due to prolonged exposure. After the wound surface is infected, excessive inflammatory reaction is caused firstly, the wound is simply treated in a common disinfection mode, although the activity of bacteria can be weakened temporarily, the disinfection effect is not thorough, and a large amount of bacterial biofilms remained at colony accumulation positions can still make the bacteria roll up to earth and restore the activity again. This forms a great interference in the initial inflammatory phase of wound repair, greatly prolongs the inflammatory cycle, and also seriously affects the subsequent repair phase. The traditional antibacterial mode is to use antibiotics, the antibiotics can effectively inhibit and kill most bacteria, but the bacteria can generate drug resistance after long-term use of the antibiotics, the dosage can be increased only if the antibacterial effect is achieved by continuously using the antibiotics, and the health problem is aggravated if the administration mode is improper.

The ideal wound dressing not only can promote wound healing, but also has good biological safety, can not cause secondary damage to the wound after being directly contacted with the wound, can effectively play a role in stopping bleeding, maintains the balance of body fluid and electrolyte at the wound part, and absorbs excessive seepage at the wound. In addition, good dressings should have analgesic and antibacterial effects on wounds, thereby promoting wound healing and reducing scar formation. Conventional wound dressings for the treatment of infected wounds typically involve the use of high concentrations of antibiotics at high frequencies, which often have varying degrees of side effects on the human body. In recent years, some wound dressings, such as chitosan-based dressings, collagen dressings, and some silver-or zinc-loaded dressings, have been used to some extent clinically and commercially, but their therapeutic effects have been expected to be at some distance from expectations. The hydrogel has high water content, flexible and plastic shape, mechanical property fitting wound and good biocompatibility, and is considered as a wound repair clinical application material with great potential. Firstly, the hydrogel is a gel substance filled with water in a cross-linked network, and the porous cross-linked network structure of the hydrogel enables the hydrogel to have good swelling performance, so that a certain oxygen content is provided for a gel dressing system, and the swelling performance of the hydrogel can absorb redundant exudate in a wound, maintain the moist environment of a focus part and promote the repair of the wound. In recent years, antibiotic-loaded nanoparticles also show a good preventive effect in anti-infection, but the nanoparticles have certain cytotoxicity, and antibiotics are easy to generate drug resistance and have obvious damage to living organs. Based on these imperfect treatments, the development of new, more effective antibacterial drugs remains an urgent need in clinical applications.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nanoparticles and the preparation method thereof.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

an antibacterial hydrogel wound dressing loaded with a two-dimensional material and nanoparticles comprises a hydrogel body, two-dimensional borolene nanosheets loaded in the hydrogel body and a material capable of reacting with boric acid to release Zn²⁺The nanoparticles of (1).

As a preferred embodiment of the antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nanoparticles, the dressing can be reacted with boric acidShould release Zn²⁺The nanoparticles of (a) are ZnO nanoparticles.

As a preferable embodiment of the antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nanoparticles, the two-dimensional boroalkene nanosheet is layered, the average particle size is 300-500 nm, the Polymer Dispersibility Index (PDI) is 0.32, and the width is 100-400 nm.

As a preferred embodiment of the antibacterial hydrogel wound dressing loaded with two-dimensional material and nanoparticles of the present invention, the hydrogel body is composed of methacrylated gelatin, oxidized dextran and a cross-linking agent, and the cross-linking agent is phenyl-2, 4, 6-trimethylbenzoylphosphonate lithium.

The antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nanoparticles firstly takes methacrylic acid gelatin (GelMA) and oxidized dextran (oDex) as main networks, and takes phenyl-2, 4, 6-trimethylbenzoyl phosphonate Lithium (LAP) as a cross-linking agent, and the cross-linking is initiated under the irradiation of visible light to form stable hydrogel which is used as a carrier part of the whole dressing system. The GelMA/oDex hydrogel system can be firstly dripped on a wound in a solution form, and then is quickly gelatinized in situ by illumination, so that the GelMA/oDex hydrogel system can be perfectly attached to the shape and the structure of the wound. The synthesized hydrogel has the strength and hardness matched with the skin through the modes of proportion adjustment and the like, can stably exist after being attached to a wound, can play the function of extracellular matrix, can be attached to the wound to maintain the moist environment of the wound, absorbs excessive skin seepage, is gradually degraded into a substance without toxic and side effects along with the repair of the damaged skin, and can be absorbed or smoothly discharged out of the body. Two-dimensional borolene nano-sheets (Borophene) added into the gel system have photodynamic effect under the irradiation of near-infrared laser with the wavelength of 660nm, and simultaneously, the loaded ZnO nano-particles have photocatalytic effect under the irradiation of near-infrared light, so that the loaded ZnO nano-particles can be decomposed to release Zn²⁺In Zn²⁺The antibacterial effect of the hydrogel wound dressing of the invention is enhanced under the synergistic effect of the above, and in addition, Zn²⁺Also can promote proliferation and migration of fibroblast, and promote wound healing. In vitro antibacterial experimentsThe hydrogel loaded with the Borophene nanosheets and the ZnO nanoparticles has a remarkable antibacterial effect on both gram-positive and gram-negative bacteria. In addition, the addition of the nano particles not only plays an antibacterial function, but also enhances the mechanical properties of the hydrogel.

The invention also provides a preparation method of the antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nanoparticles, which comprises the following steps:

(1) dispersing two-dimensional boron alkene nano-sheets in PBS buffer solution, and then adding the solution which can react with boric acid to release Zn²⁺To obtain a solution a;

(2) taking the solution A, adding methacrylated gelatin into the solution A to obtain a solution B, taking the solution A, and adding oxidized dextran into the solution A to obtain a solution C;

(3) and (3) putting the solution B and the solution C into the same centrifugal tube, adding phenyl-2, 4, 6-trimethylbenzoyl phosphonate lithium serving as a cross-linking agent, fully and uniformly mixing, and initiating cross-linking under the irradiation of visible light to obtain the antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nano particles.

As a preferred embodiment of the preparation method of the antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nanoparticles, the step (1) specifically comprises the following steps: dispersing the two-dimensional boron alkene nano-sheets in PBS buffer solution to prepare 0.25-1 mg/mL two-dimensional boron alkene nano-sheet-PBS solution, and reacting with boric acid to release Zn²⁺The nano particles are added into the two-dimensional boron alkene nano sheet-PBS solution, so that the two-dimensional boron alkene nano sheet-PBS solution can react with boric acid to release Zn²⁺The concentration of the nano-particles reaches 0.5-2 mg/mL, and a solution A is obtained.

As a preferred embodiment of the preparation method of the antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nanoparticles, in the step (1), the concentration of the two-dimensional borolene nano sheet-PBS solution is 0.5mg/mL, and the two-dimensional borolene nano sheet-PBS solution can react with boric acid to release Zn²⁺The concentration of the nanoparticles of (2) is 1 mg/mL.

As a preferred embodiment of the preparation method of the antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nanoparticles, the step (2) specifically comprises the following steps: dissolving methacrylated gelatin in the solution A to prepare a solution B with the concentration of 1-15% w/v, taking another solution A, and dissolving oxidized dextran in the solution A to prepare a solution C with the concentration of 1-10% w/v.

As a preferred embodiment of the preparation method of the antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nanoparticles, the step (3) specifically comprises the following steps: and (3) respectively putting 0.5mL of the solution B and 0.4mL of the solution C into the same centrifugal tube, adding 0.1mL of 8-12% w/v phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate as a cross-linking agent, performing vortex oscillation, fully mixing uniformly, and irradiating 1-5 min by using blue light with the wavelength of 365-760 nm to initiate cross-linking, thereby obtaining the antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nanoparticles.

As a preferred embodiment of the preparation method of the antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nanoparticles, in the step (3), the wavelength of the blue light is 420nm, and the irradiation time of the blue light is 3 min.

In the antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nanoparticles, the two-dimensional borolene nanosheet (Borophene) has a photodynamic effect under the irradiation of near-infrared laser with the wavelength of 660nm, can generate a local ROS (reactive oxygen species) environment, destroys a biological film and a structure of bacteria in the environment around a wound surface, accelerates the degradation of borolene under the irradiation of near-infrared light, degrades the borolene into boric acid, and reacts with the ZnO nanoparticles loaded at the same time to decompose the boroboric acid to generate Zn²⁺，Zn²⁺The antibacterial effect of the hydrogel wound dressing is further enhanced by cooperating with the photodynamic effect of the borolene, so that the inflammatory reaction is reduced, and the healing of the wound is promoted.

The invention also utilizes methacrylated gelatin (GelMA)/oxidized dextran (oDex) hydrogel as a carrier, and uses the borolene nano-sheets (Borophene) and ZnO nano-particles in combination with the GelMA/oDex hydrogel. Different from the traditional nanoparticle solution, the nanoparticles encapsulated in the GelMA/oDex hydrogel can stay in situ for a long time and are gradually released along with the degradation of the hydrogel, so that the administration times and possible in-vivo circulating toxicity problems of the nanoparticles can be obviously reduced.

Compared with the in vitro and in vivo antibacterial hydrogel reported in the past, the antibacterial hydrogel wound dressing prepared by the invention has the mechanical property and the antibacterial property which are closer to the modulus of skin. The in vitro result shows that the antibacterial hydrogel wound dressing has good biocompatibility and antibacterial property.

In conclusion, the antibacterial hydrogel wound dressing prepared by the invention has a great application prospect in wound infection, particularly in the aspects of wound healing of severe wound and open wound infection.

Compared with the prior art, the invention has the beneficial effects that:

(1) according to the invention, the antibacterial property is realized by loading the two-dimensional boron alkene nanosheet (Borophene) and generating ROS (reactive oxygen species) by utilizing the photodynamic property of the loaded two-dimensional boron alkene nanosheet, so that the problem of bacterial infection in the wound healing process can be effectively solved, and the method is the first application practice of the two-dimensional boron material in the field of biomedical engineering materials, especially in the aspect of hydrogel wound dressings.

(2) Compared with the existing wound antibacterial dressing at home and abroad, the antibacterial hydrogel wound dressing prepared by the invention not only utilizes the two-dimensional borolene nano-sheet (Borophene) to exert antibacterial performance through photodynamic effect, but also decomposes the loaded ZnO nano-particles to generate Zn with good antibacterial performance²⁺Under the synergistic effect of the two components, the antibacterial effect of the hydrogel wound dressing is greatly improved; furthermore, Zn²⁺Can also promote the migration and proliferation of fibroblasts, and has excellent promoting effect on the healing of wounds.

Drawings

FIG. 1 is FTIR spectra of Gtn, GelMA, Dex and oDex samples in an effective example of the present invention;

FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of four samples of Gtn, GelMA, Dex and oDex in an effective example of the invention;

FIG. 3 is a graph showing the measurement results of particle size and PDI of two-dimensional borolene nanoplatelets in an effect example of the present invention;

FIG. 4 is a TEM representation of two-dimensional borolene nanoplatelets in an effect example of the present invention;

FIG. 5 is a graph showing the results of rheological measurements on hydrogels in an effect example of the present invention;

FIG. 6 is a graph showing the results of the test for the anti-deformability of the hydrogel in the working examples of the present invention;

FIG. 7 is a graph showing the results of a biocompatibility test of a hydrogel in an effect example of the present invention;

FIG. 8 is a graph showing the antibacterial effect of hydrogels with different compositions in the effect example of the present invention.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.

Example 1: preparation of methacrylated gelatin (GelMA)

Dissolving 0.5-2.0 g of gelatin in 20mL of deionized water, heating in a water bath at 50 ℃, and stirring until the gelatin is completely dissolved. After the gelatin is completely dissolved, 0.5-1 mL of methacrylic anhydride is dropwise added into the gelatin solution by a rubber dropper. And magnetically stirring the mixed solution at the rotating speed of 500-800 rpm/min under the condition of water bath at 50 ℃ for reaction for 1-4 h, transferring the mixed solution into a dialysis bag (the molecular weight cutoff is 3500Da), and dialyzing the mixed solution in deionized water for 5 days to remove unreacted anhydride completely. And finally, centrifuging the reaction solution at the rotating speed of 3000-8000 rpm/min for 2-5 min, collecting supernatant, freezing at-80 ℃, putting the supernatant into a freeze dryer, and freeze-drying to obtain floccule, namely the target product of methacrylic acid gelatin (GelMA). The degree of substitution of the resulting methacrylated gelatin (GelMA) was 62%.

In this embodiment, the gelatin is preferably used in an amount of 1.0 g; the dosage of the methacrylic anhydride is preferably 0.6 mL;

the magnetic stirring rotating speed is preferably 500rpm/min, and the magnetic stirring reaction time is preferably 2 hours;

the centrifugal rotating speed is preferably 6000rpm/min, and the centrifugal time is preferably 3 min.

Example 2: preparation of oxidized dextran (oDex)

Dissolving 1.0-3.0 g of dextran in 200mL of deionized water, and stirring until the dextran is completely dissolved. Adding 1.0-2.0 g of sodium periodate into the dextran solution under the condition of keeping out of the sun, and magnetically stirring for reaction for 3-4 hours at the rotating speed of 500-800 rpm/min at the room temperature in the absence of the sun. And finally, adding 2mL of ethylene glycol solution into the reaction solution, and continuously stirring for 0.5-2 h to remove excessive sodium periodate so as to stop the reaction. After completion of the reaction, the reaction solution was collected and transferred to a cellulose dialysis bag (molecular weight cut-off 3500Da) and dialyzed in deionized water for 5 days. And (3) freezing the dialyzed solution at-80 ℃, and putting the solution into a freeze dryer, wherein the obtained spongy substance after freeze drying is the target product, namely the oxidized dextran (oDex). The resulting oxidized dextran (oDex) had an oxidation degree of 94.5%.

In this embodiment, the amount of dextran is preferably 1.0 g; the dosage of the sodium periodate is preferably 1.0 g;

the rotation speed of the light-proof magnetic stirring is preferably 500rpm/min, and the light-proof magnetic stirring time is preferably 4 hours;

the time for continuing stirring after the magnetic stirring in the dark is preferably 1.5 h.

Example 3: synthesis of two-dimensional borolene nano-sheet (Borophene)

Weighing 10-30 mg of boron powder, dispersing in 30-60 mL of a saturated sodium hydroxide solution of methyl pyrrolidone (NMP), and then carrying out ultrasonic treatment on the dispersion for 6-12 hours under the ice bath condition by using a cell crusher. Centrifuging the turbid liquid obtained by ultrasonic at the speed of 3000-8000 rpm/min for 5-10 minutes to remove large particles of the non-peeled boron powder, and collecting the upper brown clear liquid, namely the dispersion liquid of the two-dimensional boron alkene nanosheets. The method is used for preparing the two-dimensional boron alkene nano-sheet by using a liquid phase stripping method, and the yield is about 42%.

In this embodiment, the amount of the boron powder is preferably 20 mg; the dosage of the saturated sodium hydroxide solution of the methyl pyrrolidone (NMP) is preferably 50 mL;

the ultrasonic time is preferably 12 hours;

the centrifugal rotating speed is preferably 6000rpm/min, and the centrifugal time is preferably 10 minutes.

Example 4: synthesis of ZnO nanoparticles

Weighing 0.5-2 g Zn (Ac)₂·2H₂Dissolving O in 10-50 mL of ethanol, adding the solution into a hydrothermal synthesis reactor with the volume of 50mL, continuously reacting for 6-12 h at the temperature of 80-120 ℃, and freeze-drying the completely reacted solution to obtain powder, namely ZnO nano powder.

In this embodiment, the Zn (Ac)₂·2H₂The amount of O used is preferably 1 g; the dosage of the ethanol is preferably 30 mL;

the hydrothermal synthesis reaction time in the hydrothermal synthesis reactor is preferably 8h, and the hydrothermal synthesis reaction temperature is preferably 100 ℃.

Example 5: preparation of methacrylated gelatin (GelMA)/oxidized dextran (oDex) hydrogels

100mg of the methacrylated gelatin (GelMA) prepared in example 1 was dissolved in 1mL of PBS buffer to obtain a 10% w/v solution of methacrylated gelatin (GelMA), 40mg of the oxidized dextran (oDex) prepared in example 2 was dissolved in 1mL of PBS buffer to obtain a 4% w/v solution of oxidized dextran (oDex), 0.5mL of 10% w/v solution of GelMA and 0.4mL of 4% w/voDex solution of PBS were added to the same EP tube, 0.1mL of 10% w/v solution of phenyl-2, 4, 6-trimethylbenzoyllithium phosphonate (LAP) was added as a cross-linking agent, and the mixture was vortexed to mix the two solutions thoroughly. And finally, using a blue flashlight with the wavelength of 365-760 nm to irradiate for 1-5 min to initiate crosslinking, thereby obtaining the methacrylic acid gelatin (GelMA)/oxidized dextran (oDex) hydrogel.

In the present embodiment, the wavelength of the blue-light flashlight is preferably 420mm, and the time for light irradiation to initiate crosslinking is preferably 3 min.

Example 6: preparation of methacrylated gelatin (GelMA)/dextran oxide (oDex) @ two-dimensional borolene nano-sheet (Borophene)/zinc oxide (ZnO) hydrogel

Weighing 1mg of the two-dimensional borolene nanosheet (Borophene) prepared in example 3, dispersing the nanosheet in 2mL of PBS buffer solution to prepare a Borophene-PBS solution with a concentration of 0.5mg/mL, and weighing 2mg of the ZnO nanopowder prepared in example 4, and adding the ZnO nanopowder into the Borophene-PBS solution. 100mg of the methacrylated gelatin (GelMA) prepared in example 1 was dissolved in 1mL of the above-mentioned Borophene-PBS solution to obtain a 10% w/v GelMA solution in Borophene-PBS, and 40mg of oxidized dextran (oDex) was dissolved in 1mL of the above-mentioned Borophene-PBS solution to obtain a 4% w/v oDex solution in Borophene-PBS. 0.5mL of a 10% w/v GelMA Borophene-PBS solution and 0.4mL of a 4% w/v oDex Borophene-PBS solution were placed in the same EP tube, 0.1mL of 10% w/v lithium phenyl-2, 4, 6-trimethylbenzoylphosphonate (LAP) was added as a cross-linking agent, and the mixture was vortexed to mix the two solutions thoroughly. And finally, using a blue flashlight with the wavelength of 365-760 nm to perform illumination for 1-5 min to initiate crosslinking, so as to obtain methacrylic acid gelatin (GelMA)/dextran oxide (oDex) @ two-dimensional borane nanosheet (Borophene)/zinc oxide (ZnO) hydrogel.

Effect embodiment:

1. FT-IR characterization

Samples to be tested Gtn (nickel tricarbamoyl perchlorate), GelMA (methacrylated gelatin), Dex (dextran) and oDex (oxidized dextran) were prepared by potassium bromide tableting, and the infrared spectrum was measured using a Fourier infrared spectrometer.

As shown in FIG. 1, FTIR spectra showed that the peak intensity was at 1029cm^-1Is provided with a CH₂The bending vibration absorption peak of the group indicates that methacrylic groups exist in the chemical structure of GelMA; the IR spectrum of oDex compared with Dex is at 1728cm^-1A C ═ O stretching vibration peak appeared, indicating that sodium periodate successfully oxidized the hydroxyl groups in dextran to aldehyde groups.

2. Nuclear magnetism

GelMA and oDex are precisely weighed and respectively dissolved in deuterated heavy water by 5mg, the chemical structure is more precisely determined by a nuclear magnetic resonance hydrogen spectrogram, and the obtained spectrogram is shown in figure 2. Two chemical shifts of 5.35 and 5.58ppm correspond to methylene groups in the methacrylic groups of GelMA; and the aldehyde group in the oDex appears in the range of 4.35-5.82 ppm. Indicating the successful preparation of methacrylated gelatin and oxidized dextran.

3. DLS characterization of two-dimensional borolene nanoplatelets (Borophene)

The particle size and potential of the two-dimensional boroalkene nanoplate prepared in example 3 were measured using a dynamic light scattering nanolaser particle sizer. The measurement result is shown in fig. 3, and fig. 3 shows that the average particle size of the prepared two-dimensional boron alkene nano-sheet is 392.6nm, and the PDI is 0.32, which indicates that the prepared two-dimensional boron alkene nano-sheet has uniform particle size distribution and the size meets the expected requirement.

4. TEM characterization of two-dimensional borolene nanoplatelets (Borophene)

In order to more accurately determine the structure and shape of the two-dimensional boroalkene nanosheet prepared in example 3, the microstructure thereof was observed using a transmission electron microscope. As shown in FIG. 4, TEM results show that the boron alkene nanosheets are layered, low in thickness and strong in light transmittance, and the width of the single nanosheet is about 200nm, which is consistent with DLS results.

5. Rheological characterization of hydrogels

The rheological properties of the resulting methacrylated gelatin (GelMA)/oxidized dextran (oDex) hydrogel were characterized using a rotational rheometer.

The test was divided into four groups, group 1 being test group 1, the test sample being a 6% w/v GelMA hydrogel; group 2 is test group 2, the test sample is 6% (w/v) GelMA/2% (w/v) oDex hydrogel; group 3 is test group 3, the test sample is 6% (w/v) GelMA/2% (w/v) oDex/Borophene hydrogel; panel 4 is test panel 4 and the test samples are 6% (w/v) GelMA/2% (w/v) oDex/Borophene/Zn.

The test was performed on parallel vertebral plates 20mm in diameter. The rigidity of the hydrogel was measured by time sweep test, as shown in fig. 5(b), at a constant strain of 1%, a frequency of 1Hz, and a measurement temperature of 25 ℃; the stability of the hydrogel was determined by frequency scanning, as shown in FIG. 5(a), with a constant strain of 0.5% and a frequency varying from 0.1Hz to 10Hz, at a test temperature of 25 ℃.

The result shows that the prepared hydrogel can be stably formed, the storage modulus is larger than the loss modulus, and after the hydrogel is stably formed, the modulus does not change along with time any more, which shows that the crosslinking network of the gel is completely formed.

6. Compressive modulus characterization of hydrogels

An ideal wound dressing should have good resistance to deformation to better conform to the wound site. The prepared hydrogel was subjected to static and cyclic compression tests using a universal tester to characterize its mechanical properties.

As shown in fig. 6(a) - (d), the results show that the prepared hydrogel has good deformation resistance, besides the GelMA/oDex pure hydrogel carrier, the gel system loaded with borolene (Borophene) and ZnO also has good deformation resistance, and when the strain is 60%, the three can recover to the original shape after 50 cycles of compression. In addition, the modulus and toughness of the gel can be obviously improved after the nano particles are added, and when the deformation reaches 60%, the modulus of a gel system can reach 45Kpa at most, which is close to the modulus of the skin.

7. Biocompatibility testing

Cultured L929 cells (mouse fibroblasts) were digested with 0.25% pancreatin and suspended at a density of 2X 10 per well⁴one/mL cell suspension was seeded in 48-well plates. Taking out original culture solution after culturing for 12h, and respectively adding 500 mu L of experimental material leaching liquor into each dish, wherein the experimental material leaching liquor is GelMA hydrogel leaching liquor, GelMA/oDex/borophene/Zn hydrogel leaching liquor, and only 500 mu L of complete culture medium is added as a blank control group. Each group is provided with at least 5 holes. The liquid is changed every 24h, and the experiment is carried out for 24 h. The specific operation method comprises the following steps:

cell survival rate: the viability of the cells was quantified using CCK-8. Taken at specified time intervalsDischarging corresponding pore plate, adding 100 μ L CCK-8 working solution into each pore, and incubating with carbon dioxide (containing 5% CO) at 37 deg.C₂) After 1-2 h of medium incubation, measuring the absorbance (OD) at the wavelength of 450nm by using an enzyme-labeling instrument, and calculating the cell survival rate according to the following formula:

cell survival (%) ═ OD_{Experimental group}/OD_{Control group}×100％

As shown in fig. 7, all the cells of the hydrogel group (GelMA group, GelMA/oDex/borophene/Zn group) exhibited higher viability at 24h of culture time as compared to the control group (control group). From fig. 7, it can be seen that the cell survival rates of the hydrogel groups are all greater than 90%, the cytotoxicity is all 0-1 grade, and the hydrogel groups are not toxic to cells, so that the prepared hydrogel is considered to be non-cytotoxic.

8. Antibacterial experiments

(1) Recovery of strain and preparation of bacterial suspension

The frozen gram positive bacteria (s. aureus) were thawed and cultured in solid LB medium for recovery. Picking out single colony growing well after recovery every other day, inoculating on liquid LB culture medium, culturing at 37 deg.C for 24 hr, diluting with normal saline, counting by plate colony counting method, and making into bacteria with concentration of 1 × 10⁶CFU/mL of laboratory bacterial suspension.

(2) Co-cultivation of materials

And respectively adding 900 mu L of the bacterial suspension into four selected holes in a sterile pore plate, then sequentially dropwise adding a PBS buffer solution, Gel, Gel + Borophene Gel and Gel + Borophene + ZnO Gel, then irradiating for 5 minutes by using a near infrared laser with the wavelength of 660nm, and then putting the pore plate in a biochemical incubator at 37 ℃ for co-culture for 4 hours. Then each group of culture solution is taken out from a sterile operating platform for dilution 10⁴After doubling, 100. mu.L of each was inoculated on LB agar plates and then placed back into a 37 ℃ biochemical incubator for inverted culture for 24 hours.

As shown in FIG. 8, the results show that a large amount of Staphylococcus aureus grows in a group without an antibacterial substance (i.e., the Control group in FIG. 8), even in a pure hydrogel group without an antibacterial material (i.e., the Gel group), the normal growth of bacteria is hardly influenced under the irradiation of single near infrared light, the material has obvious inhibition and killing effects on the growth of the Staphylococcus aureus under the irradiation of the hydrogel loaded with boron alkene and the near infrared light due to the photodynamic action, and the antibacterial rate can reach more than 99 percent through calculation. Meanwhile, under the irradiation of near infrared light, the hydrogel loaded with the boron alkene and ZnO nanoparticles has the antibacterial rate of 100 percent due to the synergistic antibacterial action of the boron alkene and ZnO nanoparticles.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and substitutions can be made without departing from the technical principle of the present invention, and these modifications and substitutions should also be regarded as the protection scope of the present invention.

Claims

1. An antibacterial hydrogel wound dressing loaded with a two-dimensional material and nanoparticles is characterized by comprising a hydrogel body, two-dimensional borolene nanosheets loaded in the hydrogel body and a material capable of reacting with boric acid to release Zn²⁺The nanoparticles of (1).

2. The two-dimensional material and nanoparticle-loaded antimicrobial hydrogel wound dressing of claim 1, wherein the hydrogel is capable of reacting with boric acid to release Zn²⁺The nanoparticles of (a) are ZnO nanoparticles.

3. The antibacterial hydrogel wound dressing loaded with two-dimensional materials and nanoparticles according to claim 1, wherein the two-dimensional borolene nanosheets are layered, have an average particle size of 300-500 nm, a polymer dispersibility index of 0.32, and have a width of 100-400 nm.

4. The two-dimensional material and nanoparticle-loaded antimicrobial hydrogel wound dressing of claim 1, wherein the hydrogel body is composed of methacrylated gelatin, oxidized dextran, and a cross-linking agent that is lithium phenyl-2, 4, 6-trimethylbenzoylphosphonate.

5. A preparation method of the antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nanoparticles, which is disclosed by any one of claims 1 to 4, is characterized by comprising the following steps:

6. The method for preparing the antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nanoparticles according to claim 5, wherein the step (1) specifically comprises the following steps: dispersing the two-dimensional boron alkene nano-sheets in PBS buffer solution to prepare 0.25-1 mg/mL two-dimensional boron alkene nano-sheet-PBS solution, and reacting with boric acid to release Zn²⁺The nano particles are added into the two-dimensional boron alkene nano sheet-PBS solution, so that the two-dimensional boron alkene nano sheet-PBS solution can react with boric acid to release Zn²⁺The concentration of the nano-particles reaches 0.5-2 mg/mL, and a solution A is obtained.

7. The preparation method of the antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nanoparticles, according to claim 6, wherein in the step (1), the concentration of the two-dimensional borolene nano sheet-PBS solution is 0.5mg/mL, and the two-dimensional borolene nano sheet-PBS solution can react with boric acid to release Zn²⁺The concentration of the nanoparticles of (2) is 1 mg/mL.

8. The method for preparing the antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nanoparticles according to claim 5, wherein the step (2) specifically comprises the following steps: dissolving methacrylated gelatin in the solution A to prepare a solution B with the concentration of 1-15% w/v, taking another solution A, and dissolving oxidized dextran in the solution A to prepare a solution C with the concentration of 1-10% w/v.

9. The method for preparing the antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nanoparticles according to claim 5, wherein the step (3) specifically comprises the following steps: and (3) respectively putting 0.5mL of the solution B and 0.4mL of the solution C into the same centrifugal tube, adding 0.1mL of 8-12% w/v phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate as a cross-linking agent, performing vortex oscillation, fully mixing uniformly, and irradiating 1-5 min by using blue light with the wavelength of 365-760 nm to initiate cross-linking, thereby obtaining the antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nanoparticles.

10. The method for preparing the antibacterial hydrogel wound dressing loaded with the two-dimensional material and the nanoparticles according to claim 9, wherein in the step (3), the wavelength of the blue light is 420nm, and the irradiation time of the blue light is 3 min.