CN115887746B - A composite hydrogel dressing with photothermal and photodynamic synergistic antibacterial capabilities - Google Patents

Abstract

The invention discloses a composite hydrogel dressing with photo-thermal and photo-dynamic synergistic antibacterial capability. Belongs to the technical field of wound dressing loaded with functional active ingredients. The wound dressing loaded by the functional active ingredient has stronger biocompatibility, tissue affinity and adhesiveness, can realize the functions of protecting an infected area, absorbing tissue seepage and the like while carrying out photo-thermal photodynamic therapy, and further enhances the application capability of photo-thermal photodynamic cooperative therapy in the field of wound therapy.

Description

A composite hydrogel dressing with photothermal and photodynamic synergistic antibacterial capabilities

Technical field

The present invention relates to the technical field of wound dressings loaded with functional active ingredients, and more specifically to a composite hydrogel dressing with photothermal and photodynamic synergistic antibacterial capabilities.

Background technique

As the largest organ of the human body, the skin is also the body’s first line of defense. Skin damage will lead to damage to the skin barrier function, making it susceptible to the influence of the internal and external environment. In severe cases, it may even cause physical disease. Wound repair of skin injuries is the best way to solve this problem. Skin wound repair refers to a series of pathophysiological processes in which local tissues are repaired through regeneration, repair and reconstruction after skin tissue is lost due to damaging factors. Infections and complications caused by poor wound repair will have serious consequences and impacts on the patient, family and society.

Skin wound repair requires first of all to avoid wound infection. Wound infection is often caused by bacterial infection. Infection may cause different degrees of cell degeneration, and may also lead to tissue necrosis and the spread of infection, resulting in chronic wounds, affecting the speed of wound healing, and even forming sepsis. ,life threatening. Topical use of antibiotics or delivery of other anti-inflammatory drugs is a common clinical treatment method. However, most antibacterial agents and antibiotics often have irritating and toxic side effects. Chronic wounds may even develop drug resistance, affecting the therapeutic effect. Unlike acute skin injuries that can heal quickly, chronic wounds cause the release of related enzymes, reactive oxygen species and inflammatory mediators due to repeated infections, reduce the activity of growth factors, and greatly reduce the healing speed. Therefore, wound repair and healing for chronic wounds has become a challenging issue.

At present, clinical wound management and treatment are mostly based on dry dressings. Dry dressings over-absorb the growth factors secreted by the human body, and excessive dryness of the wound environment will prolong the healing time and even cause secondary injuries and infections. The wet wound healing theory proposed by Winter promoted the development of wet dressings. Natural polymer (such as chitosan, sodium alginate, gelatin, etc.)-based hydrogel has become the main raw material of wet dressings. Hydrogel is a type of polymer compound with a three-dimensional network structure, which has excellent water absorption and water retention properties, biocompatibility and a similar cell growth environment. In addition, loading active ingredients such as antibacterial agents, drugs, and cytokines gives the hydrogel corresponding functional activity. Dressings for chronic wounds must not only have good antibacterial properties to prevent wound infection, but also inhibit excessive wound inflammatory reactions and collaboratively promote wound repair.

Limited by the drug resistance of bacteria and the biological toxicity of antibacterial agents, photothermal antibacterial treatment is widely favored. Compared with light of other wavelengths, near-infrared light (780~2526nm) can not only penetrate deep tissues to reach lesions, but also causes minimal damage to skin and tissues. Designing a material that uses near-infrared light and heat to inhibit bacteria can not only achieve sterilization and disinfection, but also solve the problems of narrow antibacterial spectrum, drug resistance, and toxic and side effects of traditional antibiotics. In addition, near-infrared light is beneficial to tissue regeneration and promotes wound healing. Gold nanoparticles are classic photothermal reagents in photothermal therapy. They have excellent photothermal properties because surface plasmon resonance converts excited-state photon energy into heat. Functionalizing gold nanoparticles with antibacterial properties can not only improve their photostability and biocompatibility, but also use the photothermal properties of gold nanoparticles to produce local thermal effects under near-infrared light irradiation to kill bacteria. Carbon nanomaterials are also an excellent photothermal reagent with excellent photothermal conversion properties. In addition, carbon dots also have photodynamic therapy activity, generating active oxygen free radicals through the light-induced redox process to act on wounds, inhibiting the formation of scar tissue and promoting healing.

The overuse of antibiotics has greatly diminished the effectiveness of treatments due to the emergence of drug-resistant bacteria and even superbugs. Currently, in order to achieve antibacterial effects, single photothermal therapy or photodynamic therapy is mostly used. However, the treatment temperature of single photothermal therapy for infected areas needs to exceed 70°C, which may cause damage to normal tissue. Single photodynamic therapy requires a large amount of reactive oxygen species (ROS) to kill bacteria. However, excessive ROS can cause necrosis of normal tissues by inducing inflammation. At the same time, due to the short lifespan of reactive oxygen species, the effectiveness of PDT is limited, so reactive oxygen species will not cause significant killing of enough bacteria. In addition, the production of reactive oxygen species requires the participation of oxygen. The use of oxygen during the production of reactive oxygen species will cause hypoxia in the wound tissue, and due to hypoxia, the concentration of reactive oxygen species will also decrease, further limiting the effectiveness of PDT.

If photothermal therapy and photodynamic therapy are combined to exert a synergistic effect, then under the irradiation of near-infrared light, the treatment temperature can be reduced, the demand for reactive oxygen species can be reduced, sterilization and inflammation can be achieved, and the repair and healing of wounds can be promoted. This method can not only solve the shortcomings of two single therapies, but also combine the advantages of both, achieving the effect of 1+1>2.

However, there are few reports on the application of photothermal and photodynamic synergistic therapy in wound dressings. Most of the existing products are injectable liquids, which have considerable limitations in actual wound treatment applications.

In summary, how to provide a composite hydrogel dressing with excellent photothermal and photodynamic effects, good biocompatibility and photothermal and photodynamic synergistic treatment capabilities is an urgent problem that those skilled in the art need to solve.

Contents of the invention

In view of this, the present invention provides a composite hydrogel dressing with photothermal and photodynamic synergistic antibacterial capabilities. The present invention uses carbon dots CuCCDs with photodynamic (PDT) therapeutic ability and gold nanoparticles AuNPs with photothermal therapeutic ability as photosensitizers, combined with hydrogel to prepare a composite water with photothermal photodynamic synergistic antibacterial ability. Gel, as a medical dressing for antibacterial treatment and repair promotion of chronic wounds.

In order to achieve the above objects, the present invention adopts the following technical solutions:

A kind of photosensitizer carbon dot CuCCDs, the preparation method is as follows:

Mix and stir chitosan, ethylenediamine and copper chloride dihydrate. After mixing evenly, add an appropriate amount of concentrated sulfuric acid dropwise. After cooling to room temperature, add water and stir. Then perform alcohol precipitation, centrifugation, filtration, rotary evaporation, dialysis, and freeze-drying. Get CuCCDs.

Further, the mass volume ratio of chitosan, copper chloride dihydrate, ethylenediamine and concentrated sulfuric acid is 0.4g:0.2g:6mL:4mL.

Further, the dialysis is performed for 12 hours using a cellulose dialysis bag of 500 to 1000 Da.

Application of the above-mentioned photosensitizer carbon dots CuCCDs in the preparation of hydrogel dressings.

A hydrogel dressing includes gold nanoparticles AuNPs and the above-mentioned photosensitizer carbon dots CuCCDs.

Beneficial effects achieved: Using chitosan as raw material, a new type of photosensitizer carbon dots (CuCCDs) with the ability to produce PDT under near-infrared excitation was developed, which can produce ROS through photodynamic redox reaction to achieve anti-inflammation. At the same time, chitosan was used as a reducing agent to synthesize gold nanoparticles (AuNPs) with good dispersion and stability, which can provide good photothermal capabilities under near-infrared laser irradiation and achieve thermal killing of bacteria. The present invention combines the two, taking advantage of the photothermal properties of gold nanoparticles and the photodynamic activity of carbon dots. Under the irradiation of near-infrared light, dual-light therapy exerts a synergistic effect to sterilize and reduce inflammation, repair wound damage, and improve antibacterial and wound healing. Healing properties have great potential in the field of chronic wound repair.

On this basis, CuCCDs and AuNPs were further combined with natural polymer-based hydrogels to form a multifunctional composite hydrogel that is photosensitive, antibacterial, anti-inflammatory and promotes repair. The hydrogel improves the light absorption capacity of the two photosensitizers and gives the material It has stronger photothermal-photodynamic conversion effect under near-infrared light excitation, and also has good biocompatibility, tissue affinity and adhesion.

Further, the preparation method is as follows:

(1) Preparation of photosensitizer carbon dots CuCCDs;

(2) Add chitosan to the acetic acid solution and stir until dissolved, then add HAuCl ₄ solution, mix and perform a heating reflux reaction, and cool to room temperature to obtain a gold nanoparticle AuNPs solution;

(3) Mix the AuNPs solution and the chitosan solution to obtain an AuNPs/CS mixed solution;

(4) Mix the CuCCDs solution and the gelatin solution to obtain a CuCCDs/G mixed solution;

(5) Mix the AuNPs/CS mixed solution and the CuCCDs/G mixed solution thoroughly, add the genipin solution, mix evenly and incubate at 37°C for 24 hours.

Beneficial effects achieved: The photothermal photodynamic synergistic therapeutic agent is combined with a natural polymer hydrogel with good biocompatibility, using chitosan and gelatin as the raw materials of the hydrogel. Chitosan can destroy the nucleic acid synthesis process in bacteria through polycationic interactions and has certain antibacterial capabilities. Chitosan and hydrogels have both been shown to be beneficial in wound healing. The combination of hydrogel dressing and photosensitizer further improves the light utilization rate of the photosensitizer, reduces the amount of photosensitizer required for the same effect, and helps reduce costs. Composite dressings made by combining photosensitizing therapeutic agents and hydrogels can fill the gaps in existing dressing types, and are also beneficial to promoting the practical application of photothermal and photodynamic synergistic therapy in anti-inflammation and promoting healing of wounds.

Further, in the step (2), the mass volume ratio of chitosan, acetic acid solution and HAuCl ₄ solution is 0.4g:20mL:1mL;

The volume concentration of the acetic acid solution is 1%.

Further, in the step (3), the volume ratio of the AuNPs solution and the chitosan solution is 1:3 to 1:60.

Further, in the step (4), the volume ratio of the CuCCDs solution to the gelatin solution is 1:3 to 1:30.

Further, in the step (5), the volume ratio of the AuNPs/CS mixed solution and the CuCCDs/G mixed solution is 1:2 to 2:1.

Beneficial effects achieved: The parameters defined in steps (3)-(5) can evenly disperse the two types of photosensitizers, achieve uniform structure of the entire dressing, uniform temperature rise, and balanced ROS concentration, and prevent local high temperatures and local ROS concentrations. Excessive damage to normal tissue. It can also prevent the two types of photosensitizers from interacting, causing agglomeration and structural changes, and reducing the photosensitivity activity.

Terminology explanation:

(1) Carbon dots (CDs): It is a new type of carbon nanomaterial that has attracted much attention due to its low cost, high water solubility, good photostability, good biocompatibility, efficient catalytic ability and surface modification flexibility. extensive attention. Therefore, a large number of CDs have been synthesized by designing various precursors and have been widely used. Especially in the field of nanomedicine, a small number of CDs have been demonstrated to be useful as nanophotosensitizers (nanoPSs) for photodynamic therapy (PDT).

(2) Gold nanoparticles (AuNPs): Gold nanoparticles are a type of nanomaterial that was studied earlier and are generally called colloidal gold in biological research. Its particle size is generally between 1 and 100nm, showing different colors as the particle size changes, and has a variety of applications. Photothermal therapy (PTT) under near-infrared excitation is currently widely used in the field of nanomedicine.

(3) Photothermal therapy (PTT): Photothermal agents are stimulated by light to selectively and locally heat target abnormal cells and tissues. PTT efficiency depends on the NIR absorption wavelength and coefficient of the photothermal agent and the power of the excitation light.

(4) Photodynamic therapy (PDT): Light-activated photosensitizers produce a series of photochemical reactions, producing reactive oxygen species (ROS), leading to cell death.

(5) Hydrogel: Hydrogel is a type of hydrophilic gel with a three-dimensional network structure. It can swell rapidly in water and can hold a large amount of water without dissolving in this swollen state.

(6) Chitosan (CS): Chitosan is a copolymer composed of N-glucosamine and N-acetylglucosamine comonomers, and is produced from chitin through a deacetylation reaction. Its excellent properties, such as nontoxicity, biocompatibility, biodegradability, gel-forming ability, blood, cell and tissue compatibility, thermal and chemical stability, antifungal and antibacterial activity, and low cost, make it Candidates for a variety of medical applications, including drug delivery and wound dressings.

(7) Gelatin (G): Gelatin is a product of partial hydrolysis of collagen. It has good biocompatibility and low immunogenicity. Its water absorption capacity is 5 to 10 times its own weight. It is the most attractive biological material. One of the materials. Furthermore, due to its abundant functional groups (such as amino and carboxyl groups), it can be easily modified.

(8) Genipin (GP): Genipin is the product of gardeniposide hydrolyzed by β-glucosidase. It is an excellent natural biological cross-linking agent that can be combined with proteins, collagen, gelatin and Chitosan and other cross-linked biomaterials are much less toxic than glutaraldehyde and other commonly used chemical cross-linking agents. It can also be used to treat liver disease, lower blood pressure, and relieve constipation.

It can be seen from the above technical solutions that compared with the prior art, the beneficial effects achieved by the present invention are: (1) The present invention uses the natural biological macromolecule chitosan as the raw material, and adds copper chloride to achieve copper element doping to enhance its Absorption in the near-infrared region. A new type of carbon dots with good photodynamic properties that can be excited by near-infrared laser is simply prepared through a direct oxidation method, avoiding the shortcomings of low yield and high purification cost caused by the solvothermal method (the yield of the carbon dots prepared by the method of the present invention is 20% to 25%). Compared with existing photosensitizer carbon dots, this new type of carbon dots is cheap and easy to obtain, has higher yield, strong stability, and good biocompatibility, which is conducive to its promotion and application in the biomedical field. (2) The present invention uses chitosan as a reducing agent to synthesize gold nanoparticles (AuNPs). The raw materials are green and environmentally friendly, and the operation is simple. The gold nanoparticles produced have good dispersion and stability. (3) Combining photothermal and photodynamic therapy to achieve a synergistic effect under low-power near-infrared illumination can effectively combine the advantages of both to achieve a treatment temperature of approximately 50°C in the infected area and reduce the amount of required reactive oxygen species. produce. It reduces the damage to normal tissues and overcomes the problem of insufficient bacterial killing power of a single PDT treatment, achieving good broad-spectrum bacterial killing ability. (4) The present invention uses chitosan and gelatin as raw materials and safe and non-toxic genipin as a cross-linking agent to prepare a new hydrogel dressing with good biocompatibility. The hydrogel dressing can enhance the light absorption capacity of gold nanoparticles and carbon dots, and its porous structure significantly improves the photothermal properties of AuNPs and the photodynamic properties of CuCCDs. (5) The AuNPs and CuCCDs photosensitizers in the present invention are protected by the hydrogel, which reduces the loss of the photosensitizer and improves the ability to be reused. And the combination of AuNPs and hydrogel further reduces its cytotoxicity. (6) The photothermal and photodynamic synergistic treatment composite hydrogel dressing in the present invention can quickly obtain a higher temperature through near-infrared laser irradiation, has stable photothermal conversion ability, high photothermal conversion efficiency, and can stably generate singlet oxygen, thus producing a good photothermal and photodynamic synergistic sterilization effect.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are only These are embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on the provided drawings without exerting creative efforts.

Figure 1 is a TEM and HRTEM image of CuCCDs and AuNPs prepared in Example 1 and Example 2 of the present invention. The inset is a particle size distribution histogram; where A is the TEM image of CuCCDs, B is the HRTEM image of CuCCDs, and C is the TEM image of AuNPs, and D is the HRTEM image of AuNPs;

Figure 2 is the ultraviolet absorption spectrum and fluorescence spectrum of CuCCDs at different excitation wavelengths; among them, A is the CuCCDs under the optimal conditions of the present invention (the mass ratio of chitosan and copper chloride dihydrate is 0.4g:0.2g) The UV absorption spectrum, B is the fluorescence spectrum of CuCCDs at different excitation wavelengths;

Figure 3 is the photothermal performance research results of AuNPs and AuNPs-CuCCDs@Gel in Experiment 1 of the present invention; A is the photothermal heating curve of AuNPs under 808nm (1W/cm ² ) irradiation, and B is the photothermal heating curve of different gold nanoparticles. The photothermal heating curve of the hydrogel with the addition ratio of carbon dots under 808nm (0.45W/cm ² ) irradiation;

The accompanying drawing in Figure 4 shows the research results of the photodynamic properties of CuCCDs and AuNPs-CuCCDs@Gel in Experiment 2 of the present invention; where A is the absorption spectrum of the blend of CuCCDs and DPBF under 660nm (1W/cm ² ) irradiation at different times, B is the absorption spectrum of AuNPs-CuCCDs@Gel (200:100) and DPBF blend under 660nm (1W/cm ² ) irradiation at different times;

Figure 5 is a diagram showing the photothermal photodynamic synergistic antibacterial effect of AuNPs-CuCCDs@Gel on E. coli in Experiment 3 of the present invention;

Figure 6 is a diagram showing the photothermal photodynamic synergistic antibacterial effect of AuNPs-CuCCDs@Gel on Staphylococcus aureus in Experiment 3 of the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

The medicaments required by the present invention are conventional experimental medicaments, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

Example 1 Synthesis of CuCCDs

Weigh 0.4g chitosan and 0.4g copper chloride dihydrate, place them in a 100mL beaker, add 6mL ethylenediamine, mix and stir for 15 minutes. Add 4 mL of concentrated sulfuric acid dropwise to the mixed solution. The concentration of concentrated sulfuric acid is 98wt%. After the system cooled to room temperature, 20 mL of deionized water was added and stirred for 8 h. Add 200 mL of absolute ethanol for alcohol precipitation, centrifuge for 20 minutes at a speed of 8000 rpm, filter with a filter head with a pore size of 0.45 μm, rotary evaporate at a temperature of 45°C and a vacuum of 100 mbar, concentrate the solution, and use Dialyze with a 500-1000Da cellulose dialysis bag for 12 hours, freeze-dry until completely dry, and obtain CuCCDs, which are prepared into a 1 mg/mL solution and stored at 4°C for later use.

Figure 1A is the TEM image of CuCCDs, and Figure 1B is the HRTEM image of CuCCDs.

Example 2 Synthesis of AuNPs

Weigh 0.4g of chitosan, add 20mL of 1% (V/V) acetic acid solution, and stir until fully dissolved. Then add 1 mL of 1% HAuCl ₄ and stir at room temperature for 10 min to completely disperse. The solution was refluxed at 95°C for about ten minutes to obtain a wine-red AuNPs solution, which was then cooled and stored at room temperature for later use.

Figure 1C is the TEM image of AuNPs, and Figure 1D is the HRTEM image of AuNPs.

Example 3 Preparation of CuCCDs/G mixed solution

Weigh 1g of gelatin, add 20mL of deionized water, and magnetically stir at 50°C until completely dissolved to obtain a 5% (W/V) gelatin solution. The volume ratio of the added amount of 1 mg/mL CuCCDs to the gelatin solution is (1:30, 1:6, 1:3). After mixing the two solutions, stir for 20 minutes to obtain a CuCCDs/G mixed solution.

Table 1 Ingredient list of CuCCDs/G mixed solutions with different proportions

Example 4 Preparation of AuNPs/CS mixed solution

Weigh 0.4g chitosan, add 20mL 1% (V/V) acetic acid solution, mechanically stir until completely dissolved to obtain a 2% (W/V) chitosan solution, defoam ultrasonically for 15 minutes and then refrigerate at 4°C for later use. The volume ratio of the added amount of AuNPs solution to chitosan solution is (1:60~1:3). After mixing the two solutions, stir for 20 min to obtain the AuNPs/CS mixed solution.

Table 2 Ingredient list of AuNPs/CS mixed solutions with different proportions

Example 5 Preparation of AuNPs-CuCCDs@Gel

Mix the CuCCDs/G mixed solution and the AuNPs/CS mixed solution at different volume ratios, stir magnetically for 20 min, add 150 mg/mL genipin solution, and stir magnetically for another 15 min to fully disperse the cross-linking agent. Pour the mixed solution into the mold, degas it by ultrasonic for 10 minutes, then place it in a 37°C shaker and incubate it for 24 hours to complete the cross-linking. The obtained samples were refrigerated at 4°C until use.

Table 3 Component ratio table of AuNPs-CuCCDs@Gel samples with different proportions

Example 6

The preparation method of photothermal photodynamic synergistic composite hydrogel dressing includes the following steps:

(1) Weigh 0.4g chitosan and 0.4g copper chloride dihydrate, mix with 6mL ethylenediamine at 200rpm for 15min. Add 4 mL of concentrated sulfuric acid dropwise to the mixed solution. The concentration of concentrated sulfuric acid is 98wt%. After the system cooled to room temperature, 20 mL of deionized water was added and stirred for 8 h. Add 200 mL of absolute ethanol for alcohol precipitation, centrifuge at a speed of 8000 rpm for 20 min, then filter using a filter head with a pore size of 0.45 μm, rotary evaporate at a temperature of 45°C and a vacuum of 100 mbar, concentrate the solution, and use Dialyze with a 500-1000Da cellulose dialysis bag for 12 hours, freeze-dry until completely dry, and obtain CuCCDs. Prepare a 1 mg/mL solution and store it at 4°C for later use.

(2) Weigh 0.4g of chitosan, add 20mL of 1% (V/V) acetic acid solution, and stir until fully dissolved. Then add 1 mL of 1% HAuCl ₄ and stir at room temperature for 10 min to completely disperse. The solution was refluxed at 95°C for about ten minutes to obtain an AuNPs solution, which was cooled and stored at room temperature for later use.

(3) Weigh 1g of gelatin, add 20mL of deionized water, stir magnetically at 50°C until completely dissolved to obtain a 5% (W/V) gelatin solution. The volume ratio of the added amount of 1mg/mL CuCCDs to the gelatin solution is 1:30. After mixing the two solutions, stir for 20 minutes to obtain a CuCCDs/G mixed solution.

(4) Weigh 0.4g chitosan, add 20mL 1% (V/V) acetic acid solution, stir mechanically until completely dissolved to obtain a 2% (W/V) chitosan solution, defoam ultrasonically for 15 minutes and refrigerate at 4°C for later use. . The volume ratio of the added amount of AuNPs solution to chitosan solution was 1:60. After mixing the two solutions, stir for 20 min to obtain the AuNPs/CS mixed solution.

(5) Mix the CuCCDs/G mixed solution and the AuNPs/CS mixed solution at a volume ratio of 1:1, stir magnetically for 20 min, add 150 mg/mL genipin solution, and stir magnetically for 15 min to fully disperse the cross-linking agent. Pour the mixed solution into the mold, degas it by ultrasonic for 10 minutes, then place it in a 37°C shaker and incubate it for 24 hours to complete the cross-linking. The obtained samples were refrigerated at 4°C until use.

Example 7

The preparation method of photothermal photodynamic synergistic composite hydrogel dressing includes the following steps:

(1) Weigh 0.4g chitosan and 0.2g copper chloride dihydrate, mix with 6mL ethylenediamine at 200rpm for 15min. Add 4 mL of concentrated sulfuric acid dropwise to the mixed solution. The concentration of concentrated sulfuric acid is 98wt%. After the system cooled to room temperature, 20 mL of deionized water was added and stirred for 8 h. Add 200 mL of absolute ethanol for alcohol precipitation, centrifuge for 20 minutes at a speed of 8000 rpm, filter with a filter head with a pore size of 0.45 μm, rotary evaporate at a temperature of 45°C and a vacuum of 100 mbar, concentrate the solution, and use Dialyze with a 500-1000Da cellulose dialysis bag for 12 hours, freeze-dry until completely dry, and obtain CuCCDs, which are prepared into a 1 mg/mL solution and stored at 4°C for later use.

(2) Take 0.4g chitosan, add 20mL 1% (V/V) acetic acid solution, and stir until fully dissolved. Then add 1 mL of 1% HAuCl ₄ and stir at room temperature for 10 min to completely disperse. The solution was refluxed at 95°C for about ten minutes to obtain an AuNPs solution, which was cooled and stored at room temperature for later use.

(3) Weigh 1g of gelatin, add 20mL of deionized water, stir magnetically at 50°C until completely dissolved to obtain a 5% (W/V) gelatin solution. The volume ratio of the added amount of 1mg/mL CuCCDs to the gelatin solution is 1:6. After mixing the two solutions, stir for 20 minutes to obtain a CuCCDs/G mixed solution.

(4) Weigh 0.4g chitosan, add 20mL 1% (V/V) acetic acid solution, stir mechanically until completely dissolved to obtain a 2% (W/V) chitosan solution, defoam ultrasonically for 15 minutes and refrigerate at 4°C for later use. . The volume ratio of the added amount of AuNPs solution to chitosan solution was 1:15. After mixing the two solutions, stir for 20 min to obtain the AuNPs/CS mixed solution.

(5) Mix the CuCCDs/G mixed solution and the AuNPs/CS mixed solution at a volume ratio of 1:1, stir magnetically for 20 min, add 150 mg/mL genipin solution, and stir magnetically for 15 min to fully disperse the cross-linking agent. Pour the mixed solution into the mold, degas it by ultrasonic for 10 minutes, then place it in a 37°C shaker and incubate it for 24 hours to complete the cross-linking. The obtained samples were refrigerated at 4°C until use.

Example 8

The preparation method of photothermal photodynamic synergistic composite hydrogel dressing includes the following steps:

(1) Weigh 0.4g chitosan and 0.1g copper chloride dihydrate, mix with 6mL ethylenediamine at 200rpm for 15min. Add 4 mL of concentrated sulfuric acid dropwise to the mixed solution. The concentration of concentrated sulfuric acid is 98wt%. After the system cooled to room temperature, 20 mL of deionized water was added and stirred for 8 h. Add 200 mL of absolute ethanol for alcohol precipitation, centrifuge at a speed of 8000 rpm for 20 min, then filter using a filter head with a pore size of 0.45 μm, rotary evaporate at a temperature of 45°C and a vacuum of 100 mbar, concentrate the solution, and use Dialyze with a 500-1000Da cellulose dialysis bag for 12 hours, freeze-dry until completely dry, and obtain CuCCDs. Prepare a 1 mg/mL solution and store it at 4°C for later use.

(2) Take 0.4g chitosan, add 20mL 1% (V/V) acetic acid solution, and stir until fully dissolved. Then add 1 mL of 1% HAuCl ₄ and stir at room temperature for 10 min to completely disperse. The solution was refluxed at 95°C for about ten minutes to obtain an AuNPs solution, which was cooled and stored at room temperature for later use.

(3) Weigh 1g of gelatin, add 20mL of deionized water, stir magnetically at 50°C until completely dissolved to obtain a 5% (W/V) gelatin solution. The volume ratio of the added amount of 1mg/mL CuCCDs to the gelatin solution is 1:3. After mixing the two solutions, stir for 20 minutes to obtain a CuCCDs/G mixed solution.

(4) Weigh 0.4g chitosan, add 20mL 1% (V/V) acetic acid solution, stir mechanically until completely dissolved to obtain a 2% (W/V) chitosan solution, defoam ultrasonically for 15 minutes and refrigerate at 4°C for later use. . The volume ratio of the added amount of AuNPs solution to chitosan solution was 1:3. After mixing the two solutions, stir for 20 min to obtain the AuNPs/CS mixed solution.

(5) Mix the CuCCDs/G mixed solution and the AuNPs/CS mixed solution at a volume ratio of 1:1, stir magnetically for 20 min, add 150 mg/mL genipin solution, and stir magnetically for 15 min to fully disperse the cross-linking agent. Pour the mixed solution into the mold, degas it by ultrasonic for 10 minutes, then place it in a 37°C shaker and incubate it for 24 hours to complete the cross-linking. The obtained samples were refrigerated at 4°C until use.

Experiment 1 Photothermal properties of AuNPs and AuNPs-CuCCDs@Gel

In order to study the photothermal heating ability of AuNPs under low-power 808nm near-infrared irradiation, 1 mL of AuNPs solution was placed in a centrifuge tube. The laser power is set to 1W. Use near-red light 808nm to illuminate for 10 minutes, and then turn off the near-infrared light. Use an infrared imager to record the temperature every 30 seconds, and the temperature-time curve is shown in Figure 3A.

In order to study the photothermal heating ability of AuNPs-CuCCDs@Gel under low-power 808nm near-infrared irradiation, AuNPs-CuCCDs@Gel blocks with a volume of about 2×2×2cm in different proportions (as shown in Table 3) were placed on a plastic plane. The laser power is set to 0.45W. Use near-red light 808nm to illuminate for 10 minutes, and then turn off the near-infrared light. Use an infrared imager to record the temperature every 30 seconds, and the temperature-time curve is shown in Figure 3B.

And as can be seen from Figure 3, under laser illumination with a wavelength of 808nm, both AuNPS and AuNPs-CuCCDs@Gel have good photothermal properties, and AuNPs-CuCCDs@Gel (200:100) can be used under 0.45W/cm ² power laser Under irradiation, the heating rate and maximum temperature are similar to those of AuNPs under 1W/ ^cm2 power laser irradiation, indicating that the hydrogel enhances the light utilization rate of the photosensitizer and enhances its photothermal performance.

And as can be seen from Figure 3, under laser illumination with a wavelength of 808nm, the temperature of AuNPs-CuCCDs@Gel (200:100) can effectively kill drug-resistant bacteria.

Experiment 2 Photodynamic properties of CuCCDs and AuNPs-CuCCDs@Gel

In order to study the photodynamic properties of CuCCDs under low-power 660nm near-infrared irradiation, 1.5mL of 1mg/mL CuCCDs solution and 30μL of 1mg/mL DPBF solution were mixed and placed in a quartz cuvette. The laser power was set to 1W, using near-red light 660nm for 10 min, and then turning off the near-infrared light. Use a UV spectrophotometer to record the UV absorption spectrum of the mixture in the range of 350 to 500 nm every 1 minute, and the curve is shown in Figure 4A.

In order to study the photodynamic properties of AuNPs-CuCCDs@Gel under low-power 660nm near-infrared irradiation, take V _AuNPs : V _CuCCDs = 200:100 (ACG-1 in Table 3) with a volume of about 2×2×2cm AuNPs-CuCCDs The @Gel block is placed in a quartz cuvette, and 1.5mL deionized water and 30μL 1mg/mL DPBF solution are added. The laser power was set to 1W, using near-red light 660nm for 10 min, and then turning off the near-infrared light. Using a UV spectrophotometer, record the UV absorption spectrum of the mixture in the range of 350 to 550 nm every 1 minute, and the curve is shown in Figure 4B.

As shown in Figure 4B, under laser illumination with a wavelength of 660 nm, AuNPs-CuCCDs@Gel (200:100) can significantly generate reactive oxygen species and has the ability to kill bacteria. Comparing the results in Figure 4A and Figure 4B, it can be seen that AuNPs-CuCCDs@Gel (200:100) can achieve a similar effect on DPBF under 660nm laser irradiation as that of the 1mg/mL CuCCDs solution when the amount of carbon dots added is low. degradation effect. It shows that the hydrogel enhances the light utilization efficiency of the photosensitizer and enhances its photodynamic performance.

Experiment 3 Bactericidal effect of AuNPs-CuCCDs@Gel

According to the preparation scheme discussed earlier, since it needs to reach 50°C to have better antibacterial activity, and due to the mechanical strength of the material, V _AuNPs : V _CuCCDs = 200:100 (ACG-1 in Table 3) is selected here. As a representative sample of AuNPs-CuCCDs@Gel.

Use a 24-well plate as a template to prepare Gel, AuNPs@Gel, CuCCDs@Gel, and AuNPs-CuCCDs@Gel. Prepare four of each sample, and set up an E. coli control group, an E. coli laser irradiation group, and a Staphylococcus aureus control group. , Staphylococcus aureus laser irradiation group.

After each sample was co-cultured with bacteria for 12 hours, it was placed in a six-well plate and irradiated with 10min 808nm (0.45W/cm ² ) laser and 10min 660nm (1W/cm ² ) laser respectively, during which time a thermal imaging camera was used to take pictures. Then add 5 mL of fresh liquid culture medium to each well to keep the material in a suspended state, and then culture it at 37°C for 12 hours.

Finally, take a 10 ^-5 dilution of the bacterial solution and spread it on the plate. The coated culture plate was cultured at 37°C for 12 hours, and photos were taken and colonies were counted to calculate the antibacterial rate. The results are shown in Figures 5 and 6.

As can be seen from Figures 5 and 6, compared with the Gel group, hydrogels loaded with AuNPs or CuCCDs can significantly reduce E. coli and Staphylococcus aureus, and the bactericidal effect of single photothermal is better than that of single photodynamic. Remarkably, the hydrogel loaded with both AuNPs and CuCCDs can achieve a more significant bactericidal effect, with an antibacterial rate of 91.65% against E. coli and 90.46% against Staphylococcus aureus. This result fully demonstrates that the photothermal photodynamic synergistic treatment of AuNPs-CuCCDs@Gel at low power has obvious bactericidal effect.

Each embodiment in this specification is described in a progressive manner. Each embodiment focuses on its differences from other embodiments. The same and similar parts between the various embodiments can be referred to each other.

The above description of the disclosed embodiments enables those skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be practiced in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (4)

1. A hydrogel dressing, characterized by comprising gold nanoparticles AuNPs and photosensitizer carbon dots CuCCDs; The preparation method is as follows: (1) Preparation of photosensitizer carbon dots CuCCDs; Mix chitosan, ethylenediamine and copper chloride dihydrate. After mixing evenly, add an appropriate amount of concentrated sulfuric acid dropwise. After cooling to room temperature, add water and stir. Then perform alcohol precipitation, centrifugation, filtration, rotary evaporation, dialysis, and freeze-drying. Get CuCCDs; The mass and volume ratio of chitosan, copper chloride dihydrate, ethylenediamine and concentrated sulfuric acid is 0.4g:0.2g:6mL:4mL; The dialysis is performed using a 500-1000Da cellulose dialysis bag for 12 hours; (2) Add chitosan to the acetic acid solution and stir until dissolved, then add HAuCl ₄ solution, mix and perform a heating reflux reaction, and cool to room temperature to obtain a gold nanoparticle AuNPs solution; (3) Mix the AuNPs solution and the chitosan solution to obtain an AuNPs/CS mixed solution; (4) Mix the CuCCDs solution and the gelatin solution to obtain a CuCCDs/G mixed solution; (5) Mix the AuNPs/CS mixed solution and the CuCCDs/G mixed solution thoroughly, add the genipin solution, mix evenly and incubate at 37°C for 24 hours; The volume ratio of the AuNPs/CS mixed solution to the CuCCDs/G mixed solution is 2:1. 2. The hydrogel dressing according to claim 1, wherein in the step (2), the mass volume ratio of chitosan, acetic acid solution and _HAuCl solution is 0.4g:20mL:1mL; The volume concentration of the acetic acid solution is 1%. 3. The hydrogel dressing according to claim 1, wherein in step (3), the volume ratio of the AuNPs solution to the chitosan solution is 1:3 to 1:60. 4. The hydrogel dressing according to claim 1, wherein in step (4), the volume ratio of CuCCDs solution to gelatin solution is 1:3 to 1:30.