Preparation method and application of photo-crosslinking zinc-doped baicalin carbon dot-based hydrogel

Abstract

The application aims to provide a preparation method and application of a photocrosslinking zinc-doped baicalin carbon dot-based hydrogel, and belongs to the field of biomedical technology.The baicalin and zinc acetate solution are used as precursors to prepare zinc-doped CDs at 180 DEG C.Meanwhile, a methacrylated gelatin precursor solution is prepared, the carbon dots are added in a certain proportion, and the zinc-doped baicalin carbon dot-based hydrogel is obtained under ultraviolet crosslinking.The carbon dot hydrogel combines the advantages of carbon dots and hydrogels, and has a certain application prospect in wound infection treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a preparation method and application of a photo-crosslinked zinc-doped scutellaria carbon point-based hydrogel. Background Art

[0002] Hydrogels are an important class of multifunctional soft materials that have attracted widespread research attention due to their wide-ranging applications in biomedicine, biomimetic smart materials, adsorption, and electrochemistry. They are typically hydrophilic and constructed as three-dimensional (3D) networks via physical or chemical crosslinking. However, conventional hydrogels still struggle to meet specialized functional requirements in many situations. To further expand the applications of hydrogels, the incorporation of additional components is necessary. By incorporating auxiliary components with diverse functionalities, hydrogels can be endowed with expanded properties. Carbon dots (CDs), an emerging class of "zero-dimensional" carbon-based materials, have also attracted significant attention in the biomedical field due to their excellent biocompatibility, superior water dispersibility, and low cytotoxicity. Furthermore, CDs possess a large number of functional groups on their surfaces, including carboxyl, amino, and aldehyde groups. This structural motif not only makes CDs highly water-soluble but also makes them ideal building blocks for hydrogel construction, where they can interact with gelators through various non-covalent interactions. Immobilizing CDs within hydrogels has been shown to be an effective strategy for avoiding the ACQ effect in aggregated states. The introduction of CDs into hydrogels has endowed them with additional properties, such as fluorescence, chirality, stimuli-responsiveness, self-healing, improved mechanical strength, and biological functionality. The combination of these two completely different types of materials not only brings structural diversity but also significantly improves many properties, resulting in new multifunctional materials. In recent years, CDs-based hydrogels have made tremendous progress in various biological applications, such as drug delivery, wound healing, and cancer therapy. Summary of the Invention

[0003] The present invention aims to provide a method for preparing and applying a photocrosslinked zinc-doped scutellaria baicalensis carbon dot-based hydrogel. Using baicalein and a zinc acetate solution as precursors, zinc-doped CDs are prepared at 180°C. A methacrylated gelatin precursor solution is also prepared, to which carbon dots are added in a specific ratio. The zinc-doped scutellaria baicalensis carbon dot-based hydrogel is then crosslinked under ultraviolet light to yield the resulting hydrogel. This carbon dot-hydrogel combines the advantages of both carbon dots and hydrogels and has promising applications in wound infection treatment.

[0004] The present invention adopts the following technical solutions:

[0005] A method for preparing a photocrosslinked zinc-doped scutellaria carbon dot-based hydrogel comprises the following steps:

[0006] The first step is to prepare zinc-doped carbon dots (Zn / CDs): wash and dry the scutellaria baicalensis, crush it completely with a grinder, and store it in a sealed bag away from light.

[0007] The crushed scutellaria powder and zinc acetate were added to deionized water and stirred on a magnetic stirrer for 30 minutes until uniformly mixed. The mixture was transferred to a 40 mL reactor and placed in an oven for reaction. After the reaction was completed, the reactor was naturally cooled and the resulting reaction solution was centrifuged at 12,000 rpm for 25 minutes to remove scutellaria powder residue and large agglomerated particles. The supernatant was filtered through a 0.22 μm filter membrane and dialyzed in ultrapure water using a dialysis bag for 24 hours, with the ultrapure water replaced every 2 hours. After concentration, the product was frozen for 10 hours and finally transferred to a freeze dryer to obtain a dark brown product, which was stored in a dark, dry place.

[0008] Step 2: Prepare methacrylated gelatin (GelMA): Add gelatin to PBS and stir continuously until completely dissolved. Then, add methacrylic anhydride dropwise for modification. The reaction is carried out at 40-60°C and 250 rpm for 2-4 hours in the dark. The reaction is terminated by diluting with 5-fold PBS. The gelatin is dialyzed in the dark for one week and freeze-dried to obtain a white foamy GelMA.

[0009] The third step is to prepare Zn / CDs-GelMA hydrogel: GelMA and Zn / CDs are dissolved in deionized water at a mass ratio of 5%-10% of gelatin to carbon dots: 0.5%-1%, stirred evenly, a photoinitiator is added, ultrasonically vibrated evenly, and placed under a 405nm ultraviolet lamp for 10-15s to obtain Zn / CDs-GelMA hydrogel.

[0010] Furthermore, the usage ratio of the scutellaria baicalensis powder, zinc acetate and deionized water in the first step is 1 g:1 g:20 mL.

[0011] Furthermore, in the first step, the temperature of the oven is 180° C. and the reaction time is 18 h.

[0012] Furthermore, the molecular weight cut-off of the dialysis bag in the first step is 500-1000Da.

[0013] Furthermore, in the second step, the usage ratio of gelatin, PBS solution, and methacrylic anhydride is 5 g:50 mL:4 mL.

[0014] Furthermore, the gelatin in the second step is type A gelatin.

[0015] Furthermore, the photoinitiator in the third step is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, and the mass concentration of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is 0.2%.

[0016] A photocrosslinked zinc-doped scutellaria carbon dot-based hydrogel is used for antibacterial and skin wound healing.

[0017] The beneficial effects of the present invention are as follows:

[0018] The zinc-doped Scutellaria baicalensis carbon dots prepared by the present invention can retain the original medicinal effects of traditional Chinese medicine to a certain extent, and have the characteristics of small dosage, high water solubility, and good biocompatibility; the zinc-doped carbon dot-based hydrogel prepared by the present invention has good cell compatibility, can provide cell adhesion sites, and is conducive to cell adhesion, diffusion and proliferation. It is particularly suitable for use as a scaffold material for tissue repair, can exert antibacterial effects, and has the characteristics of promoting angiogenesis, and is more suitable for soft tissue regeneration.

[0019] The preparation method of the present invention is simple and adopts a photocrosslinking method. The speed of forming hydrogel is fast and efficient, and can be controlled within a few seconds or even tens of seconds. At the same time, the start and end of the polymerization reaction can be controlled by controlling the switch of the light source; photocrosslinking can accurately adjust the intensity or time of light irradiation to achieve the regulation of many properties of the hydrogel, such as mechanical properties. Light-driven polymerization can be carried out in vivo in a non-invasive manner, and has good biomedical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the SEM image of the zinc-doped Scutellaria baicalensis carbon dot-based hydrogel prepared in Example 1 of the present invention.

[0021] Figure 2 The cell proliferation of the zinc-doped carbon dot-based hydrogel in Examples 1-3.

[0022] Figure 3 This is a diagram of the cell live-death experiment of GelMA and Zn / CDs-GelMA (1%) provided in Example 1 of the present invention.

[0023] Figure 4 This is a coating diagram of the zinc-doped Scutellaria baicalensis carbon dot-based hydrogel antibacterial plate prepared in Example 3 of the present invention.

[0024] Figure 5 This is a bacteria live-death diagram of the zinc-doped Scutellaria baicalensis carbon dot-based hydrogel prepared in Example 3 of the present invention (green represents live bacteria, and red represents dead bacteria). DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] Example 1

[0027] A method for preparing zinc-doped scutellaria carbon dots (Zn / CDs) is disclosed. The method comprises the following steps: first, scutellaria is cleaned, dried at low temperature, and completely crushed in a grinder. The Zn / CDs are placed in a sealed bag and stored in the dark. Then, 1 g of scutellaria powder and 1 g of zinc acetate are weighed, added to 20 ml of deionized water, and mixed evenly by magnetic stirring for 30 minutes. The mixture is then transferred to a 40 mL reactor and placed in an oven at 180° C. for 18 hours. After the reaction, the reactor is naturally cooled, and the resulting reaction solution is centrifuged at 12,000 rpm for 25 minutes to remove scutellaria powder residue and large agglomerated particles. The supernatant is filtered through a 0.22 μm filter membrane, and dialyzed in an ultrapure water environment for 24 hours using a dialysis bag (molecular weight cutoff: 500-1000 Da), during which the ultrapure water is replaced every 2 hours. After concentration, the product is frozen for 10 hours and finally transferred to a freeze dryer to obtain a dark brown product, which is then dried and stored in the dark.

[0028] 5 g of gelatin was added to 50 ml of PBS solution and stirred continuously until completely dissolved. Then, 4 ml of methacrylic anhydride was added dropwise for modification. The mixture was stirred at 50°C in the dark for 3 h. The reaction was terminated by adding 5-fold PBS dilution. The mixture was dialyzed in the dark for one week and freeze-dried to obtain white foamy methacrylated gelatin GelMA.

[0029] The freeze-dried GelMA and Zn / CDs were dissolved in deionized water at a gelatin to carbon dot mass ratio of 8%:1%, stirred evenly, and the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate LAP was added at a mass concentration of 0.2%. The mixture was homogenized by ultrasonic vibration and irradiated under a 405nm ultraviolet lamp for 10-15s to obtain Zn / CDs-GelMA hydrogel.

[0030] Example 2

[0031] A method for preparing zinc-doped scutellaria carbon dots (Zn / CDs) is disclosed. The method comprises the following steps: first, scutellaria is cleaned, dried at low temperature, and completely crushed in a grinder. The Zn / CDs are placed in a sealed bag and stored in the dark. Then, 1 g of scutellaria powder and 1 g of zinc acetate are weighed, added to 20 ml of deionized water, and mixed evenly by magnetic stirring for 30 minutes. The mixture is then transferred to a 40 mL reactor and placed in an oven at 180° C. for 18 hours. After the reaction, the reactor is naturally cooled, and the resulting reaction solution is centrifuged at 12,000 rpm for 25 minutes to remove scutellaria powder residue and large agglomerated particles. The supernatant is filtered through a 0.22 μm filter membrane, and dialyzed in an ultrapure water environment for 24 hours using a dialysis bag (molecular weight cutoff: 500-1000 Da), during which the ultrapure water is replaced every 2 hours. After concentration, the product is frozen for 10 hours and finally transferred to a freeze dryer to obtain a dark brown product, which is then dried and stored in the dark.

[0032] 5 g of gelatin was added to 50 ml of PBS solution and stirred continuously until completely dissolved. Then, 4 ml of methacrylic anhydride was added dropwise for modification. The reaction was stirred at 50°C for 3 h. 5-fold PBS was added to terminate the reaction. The gelatin was dialyzed for one week in the dark and freeze-dried to obtain white foamy methacrylated gelatin GelMA.

[0033] The freeze-dried GelMA and Zn / CDs were dissolved in deionized water at a gelatin to carbon dot mass ratio of 8%:0.8%, stirred evenly, and the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate LAP was added at a mass concentration of 0.2%. The mixture was homogenized by ultrasonic vibration and irradiated under a 405nm ultraviolet lamp for 10-15s to obtain Zn / CDs-GelMA hydrogel.

[0034] Example 3

[0035] A method for preparing zinc-doped scutellaria baicalensis carbon dots comprises preparing the zinc-doped carbon dots through a one-step hydrothermal method. First, the scutellaria baicalensis is cleaned and dried at low temperature, then completely crushed with a grinder, and placed in a sealed bag for light-proof storage. Subsequently, 1 g of scutellaria baicalensis powder and 1 g of zinc acetate are weighed, 20 ml of deionized water are added, and the mixture is magnetically stirred for 30 minutes to mix evenly. The mixture is transferred into a 40 mL reactor and placed in an oven at 180° C. for reaction for 18 hours. After the reaction, the reactor is naturally cooled, and the obtained reaction solution is centrifuged at 12,000 rpm for 25 minutes to remove scutellaria baicalensis powder residue and large agglomerated particles. The supernatant is filtered through a 0.22 μm filter membrane; dialyzed in an ultrapure water environment for 24 hours using a dialysis bag (molecular weight cutoff: 500-1,000 Da), during which the ultrapure water is replaced every 2 hours. After concentration, the mixture is frozen for 10 hours, and finally transferred to a freeze dryer to obtain a dark brown product, which is dried and stored in the dark.

[0036] 5 g of gelatin was added to 50 ml of PBS solution and stirred continuously until completely dissolved. Then, 4 ml of methacrylic anhydride was added dropwise for modification. The reaction was stirred at 50°C for 3 h. 5-fold PBS was added to terminate the reaction. The gelatin was dialyzed for one week in the dark and freeze-dried to obtain white foamy methacrylated gelatin GelMA.

[0037] The freeze-dried GelMA and Zn / CDs were dissolved in deionized water at a gelatin to carbon dot mass ratio of 8%:0.5%, stirred evenly, and the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate LAP was added at a mass concentration of 0.2%. The mixture was homogenized by ultrasonic vibration and irradiated under a 405nm ultraviolet lamp for 10-15s to obtain Zn / CDs-GelMA hydrogel.

[0038] Scanning electron microscopy images of zinc-doped carbon dot-based hydrogels in Example 1

[0039] The Zn / CDs-GelMA hydrogel prepared in Example 1 was pre-frozen in a -20°C refrigerator for 12 h, and then frozen in a -40°C vacuum freezer for 48 h to completely dehydrate and dry it. It was then cut into cylindrical sections of a certain thickness, and the sample was adhered to the surface of a scanning electron microscope sample stage using conductive glue with the cross section facing upward. It was then gold-sprayed and the cross-sectional morphology of the hydrogel was observed using a JSM-7100F field emission scanning electron microscope at a voltage of 5 kV. Figure 1 This is an SEM image of the zinc-doped Scutellaria baicalensis carbon dot-based hydrogel provided in Example 1 of the present invention.

[0040] The methacrylated gelatin and zinc-doped carbon dots prepared in Examples 1-3 were dissolved in deionized water at a therapeutic concentration ratio of 8%:0.5%-1% (0.5%, 0.8%, 1%) and stirred evenly. The photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate LAP was added at a mass concentration of 0.2%. The mixed solution was homogenized by ultrasonic vibration, and the mixed solution was filtered through a 0.2 μm sterile filter membrane, and then the mixture was filtered at 5×10 3 The cells / mL ratio was added to the bone marrow mesenchymal stem cell suspension and mixed evenly. The mixture was then injected into a 96-well plate and irradiated under a 405nm UV lamp for 10-15 seconds to obtain different Zn / CDs-GelMA (0.5%, 0.8%, 1%) hydrogels loaded with cells. The plates were cultured in an incubator at 37°C and 5% CO2. The culture medium was replaced every 48 hours during the culture period. After 1, 3, and 5 days of culture, the cell proliferation in the hydrogels was tested using CCK 8. The results are shown in the figure below. Figure 2 As shown, it can be seen that with the increase of time, cell proliferation is obvious, and each group of hydrogels has no obvious cytotoxicity.

[0041] The hydrogel precursor solution prepared in Example 1 was filtered through a 0.22 μm sterile filter membrane and then 6 Cells / mL were added to a bone marrow mesenchymal stem cell suspension and mixed thoroughly. The mixture was then injected into a confocal microplate and irradiated under 405nm UV light for 10-15 seconds to produce cell-loaded GelMA and Zn / CDs-GelMA (1%) hydrogels. Complete culture medium was then added and cultured at 37°C and 5% CO2. The culture medium was replaced every 48 hours to produce tissue-engineered 3D cell hydrogels. After 3 days of culture, the 3D cell hydrogels were removed and washed twice with PBS buffer. The washed 3D cell hydrogels were then immersed in a PBS solution containing AM and PI for staining. The gels were incubated at 37°C for 30 minutes, and the growth and distribution of cells within the 3D hydrogels were observed using a laser confocal scanning microscope. Figure 3 This is a cell live-death experiment diagram of GelMA and Zn / CDs-GelMA (1%) provided in Example 1 of the present invention. The results are shown in the attached figure. Figure 3 As shown, bone marrow mesenchymal stem cells grew well on different hydrogels. After culturing for 3 days, there were basically no dead cells on the hydrogel, indicating that the zinc-doped carbon dot hydrogel had good biocompatibility.

[0042] The methacrylated gelatin and zinc-doped carbon dots prepared in Example 1 were dissolved in deionized water at a therapeutic concentration ratio of 8%:1% and stirred evenly. A photoinitiator, lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), was added at a concentration of 0.2%. The mixture was homogenized by ultrasonic vibration and filtered through a 0.2 μm sterile filter membrane, poured into a mold, and irradiated under a 405 nm UV lamp for 10-15 seconds to produce a zinc-doped Scutellaria baicalensis carbon dot-based hydrogel. After bacterial recovery, Escherichia coli and Staphylococcus aureus bacterial suspensions were added dropwise to the solid culture medium, spread evenly with a sterile glass rod, and incubated in a 37°C incubator for 24 hours. Single colonies were then selected and placed in liquid culture medium in a 37°C shaker for bacterial amplification. The bacterial suspension was then serially diluted to select the appropriate bacterial concentration for antibacterial testing. After sterilization, the prepared hydrogels were co-cultured with different bacteria for 24 hours. The treated bacteria were then spread onto solid culture plates and incubated at 37°C for 24 hours. The antibacterial efficacy of the different hydrogels was determined by counting the number of colonies on the plates. Furthermore, live-dead dye was added to the bacterial suspensions treated with the different hydrogels and incubated at 37°C for 30 minutes. The bacterial live-dead status was observed using a fluorescence microscope to further demonstrate the antibacterial properties. Figure 4 This is a diagram of the antibacterial flat coating of zinc-doped scutellaria carbon dot-based hydrogel provided in Example 3 of the present invention. Figure 5 This is a bacteria live-death diagram of the zinc-doped Scutellaria baicalensis carbon dot-based hydrogel provided in Example 3 of the present invention (green represents live bacteria, and red represents dead bacteria). The experimental results show that the number of Staphylococcus aureus and Escherichia coli on the Zn / CDs-GelMA hydrogel solid plate culture medium is reduced, and the live-dead staining shows that the number of dead bacteria in the Zn / CDs-GelMA group is significantly increased, indicating that the Zn / CDs-GelMA hydrogel has a significant antibacterial effect.

[0043] The above embodiments of the present invention do not describe all details in detail, nor do they limit the present invention to the above embodiments. Various changes, modifications, substitutions, and variations made by those skilled in the art without departing from the principles and purpose of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. A method for preparing a photocrosslinked zinc-doped scutellaria baicalensis carbon dot-based hydrogel, characterized by: The steps include: The first step is to prepare zinc-doped carbon dots (Zn / CDs): wash and dry the scutellaria baicalensis, crush it completely with a grinder, and store it in a sealed bag away from light. The crushed scutellaria powder and zinc acetate were added to deionized water and stirred on a magnetic stirrer for 30 minutes until uniformly mixed. The mixture was transferred to a 40 mL reactor and placed in an oven for reaction. After the reaction was completed, the reactor was naturally cooled and the resulting reaction solution was centrifuged at 12,000 rpm for 25 minutes to remove scutellaria powder residue and large agglomerated particles. The supernatant was filtered through a 0.22 μm filter membrane and dialyzed in ultrapure water using a dialysis bag for 24 hours, with the ultrapure water replaced every 2 hours. After concentration, the product was frozen for 10 hours and finally transferred to a freeze dryer to obtain a dark brown product, which was stored in a dark, dry place. The dosage ratio of the scutellaria baicalensis powder, zinc acetate and deionized water is 1g:1g:20mL; The temperature of the oven is 180°C and the reaction time is 18h; Step 2: Prepare methacrylated gelatin (GelMA): Add gelatin to PBS and stir continuously until completely dissolved. Then, add methacrylic anhydride dropwise for modification. The reaction is carried out at 40-60°C and 250 rpm for 2-4 hours in the dark. The reaction is terminated by diluting with 5-fold PBS. The gelatin is dialyzed in the dark for one week and freeze-dried to obtain a white foamy GelMA. The third step is to prepare Zn / CDs-GelMA hydrogel: GelMA and Zn / CDs are dissolved in deionized water at a mass ratio of 5%-10% of gelatin to carbon dots: 0.5%-1%, stirred evenly, a photoinitiator is added, ultrasonically vibrated evenly, and placed under a 405nm ultraviolet lamp for 10-15s to obtain Zn / CDs-GelMA hydrogel.

2. The method for preparing a photocrosslinked zinc-doped scutellaria baicalensis carbon dot-based hydrogel according to claim 1, characterized in that: The molecular weight cut-off of the dialysis bag in the first step is 500-1000 Da.

3. The method for preparing a photocrosslinked zinc-doped scutellaria baicalensis carbon dot-based hydrogel according to claim 1, characterized in that: In the second step, the usage ratio of gelatin, PBS solution, and methacrylic anhydride is 5 g:50 mL:4 mL.

4. The method for preparing a photocrosslinked zinc-doped scutellaria baicalensis carbon dot-based hydrogel according to claim 1, characterized in that: The gelatin in the second step is type A gelatin.

5. The method for preparing a photocrosslinked zinc-doped scutellaria baicalensis carbon dot-based hydrogel according to claim 1, characterized in that: The photoinitiator in the third step is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, and the mass concentration of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is 0.2%.

6. Use of the photo-crosslinked zinc-doped scutellaria carbon dot-based hydrogel prepared by the preparation method according to claim 1 in the preparation of antibacterial and skin wound healing products.

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