Aromatic silver functionalized hydrogel as well as preparation method and application thereof

Abstract

The invention discloses aromatic silver functionalized hydrogel as well as a preparation method and application thereof. Vinylpyridine carboxyl betaine and vinylpyridine sulphobetaine are both zwitterions, and experiments prove that the two zwitterions can play a synergistic role in the hydrogel, so that the hydrogel has better wound healing capacity, and the performance of the hydrogel is far better than that of hydrogel only containing a single zwitterion. The conductivity of the hydrogel is remarkably improved by the silver nanowires, wound healing is facilitated, and meanwhile, wound infection is prevented and a sterile healing environment is kept due to the powerful antibacterial characteristic. Due to the high fibrillation property of carboxymethyl cellulose (CMC), the mechanical property and the structural stability of the hydrogel are greatly enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to an aromatic silver functionalized hydrogel and a preparation method and application thereof. Background Art

[0002] Hydrogels have long been of interest for skin wound dressings due to their unique properties, such as moisture retention and extracellular matrix-like structure. They can also serve as delivery vehicles, increasing their versatility. Many hydrogels have been developed to modulate immunomodulatory cells and promote immune responses in wound healing. These hydrogels strive to strike a balance between inflammatory cell infiltration, modulating macrophage polarization, enhancing cell proliferation and migration, and controlling collagen remodeling by manipulating their physicochemical properties. The effects of physical properties such as dimensionality, hardness, porosity, morphology, electrical properties, and wettability on immune responses have been elucidated. However, most of these studies have been limited to laboratory experiments conducted in vitro, ignoring the immunomodulatory role played by hydrogels in vivo. Summary of the invention

[0003] The present invention provides an aromatic silver functionalized hydrogel and a preparation method and application thereof, so as to solve one or more technical problems existing in the prior art.

[0004] The first aspect of the present invention is to provide an aromatic silver functionalized hydrogel.

[0005] The second aspect of the present invention is to provide a method for preparing the aromatic silver functionalized hydrogel described in the first aspect of the present invention.

[0006] The third aspect of the present invention is to provide the use of the aromatic silver functionalized hydrogel described in the first aspect of the present invention in the preparation of medicines.

[0007] The raw materials of the aromatic silver functionalized hydrogel described in the first aspect of the present invention include vinylpyridine carboxybetaine, vinylpyridine sulfobetaine, silver nanowires and carboxymethyl cellulose.

[0008] The vinylpyridine carboxybetaine and the vinylpyridine sulfobetaine are both zwitterions. Experimental verification shows that the two zwitterions can play a synergistic role in the hydrogel, have better wound healing ability, and the performance is far superior to the hydrogel containing only a single zwitterion. Silver nanowires significantly improve the conductivity of the hydrogel, which is beneficial to wound healing. At the same time, the strong antibacterial properties help prevent wound infection and maintain a sterile healing environment. In addition, unlike general silver nanoparticles, the silver nanowires have a one-dimensional structure and can be better integrated into the hydrogel matrix, thereby providing superior mechanical reinforcement, making the hydrogel stronger and more flexible. The highly fibrillated nature of carboxymethyl cellulose (CMC) greatly enhances the mechanical properties and structural stability of the hydrogel.

[0009] In some application embodiments of the first aspect of the present invention, the structural formula of the vinylpyridine carboxy betaine is as shown in formula (I):

[0010] Formula (I).

[0011] In some application embodiments of the first aspect of the present invention, the preparation method of the vinylpyridine carboxybetaine is: dissolving 4-vinylpyridine in tetrahydrofuran and cooling to 0°C, adding β-butyrolactone dropwise, maintaining a nitrogen atmosphere of 0~4°C and stirring until the reaction is completed, filtering the crude product, washing with ether, and drying under vacuum.

[0012] In some application embodiments of the first aspect of the present invention, the structural formula of the vinylpyridine sulfobetaine is as shown in formula (II):

[0013] Formula (II).

[0014] In some application embodiments of the first aspect of the present invention, the preparation method of the vinylpyridine sulfobetaine is: dissolving 4-vinylpyridine in dimethylformamide, gradually adding 1,3-propane sultone dissolved in N,N-dimethylformamide, stirring at room temperature until the reaction is completed, filtering the crude product, washing with ethyl acetate, and drying to obtain the product.

[0015] In some application embodiments of the first aspect of the present invention, the carboxymethyl cellulose is modified carboxymethyl cellulose. The preparation method of the modified carboxymethyl cellulose is: dissolving acrylic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine in dimethyl sulfoxide, reacting for 24 hours, adding to a phosphate buffer solution in which carboxymethyl cellulose is dissolved, and the reaction product is subjected to continuous dialysis and freeze-dried to obtain the modified carboxymethyl cellulose.

[0016] The preparation method described in the second aspect of the present invention comprises the following steps: 1) preparing the vinylpyridine carboxy betaine represented by formula (I) and the vinylpyridine sulfonyl betaine represented by formula (II) respectively; 2) dissolving the carboxymethyl cellulose in water, adding the silver nanowires, and stirring thoroughly; 3) adding the vinylpyridine carboxy betaine and the vinylpyridine sulfobetaine, and adding an initiator and an accelerator after they are completely dissolved; 4) After gelation is achieved, immersing the hydrogel in deionized water to remove impurities, thereby obtaining the aromatic silver functionalized hydrogel.

[0017] In some application embodiments of the second aspect of the present invention, in step 3), the mass ratio of the vinylpyridine carboxybetaine to the vinylpyridine sulfobetaine is 1:1.

[0018] In some application embodiments of the second aspect of the present invention, in step 3), the initiator is ammonium persulfate; preferably, the accelerator is tetramethylethylenediamine.

[0019] The application described in the third aspect of the present invention refers to the use of the aromatic silver functionalized hydrogel for preparing a drug for promoting wound healing.

[0020] Compared with the prior art, the above technical solution of the present invention has at least the following technical effects or advantages: Through in vivo experiments, the efficacy of the aromatic silver functionalized hydrogel in rapidly resolving acute tissue injury, such as a full-thickness skin defect model, was not only verified but also demonstrated by significantly improving the wound healing process. Specific findings include specific improvements in wound healing by promoting angiogenesis, collagen synthesis, macrophage polarization, and the formation of granulation tissue. The ability of the aromatic silver functionalized hydrogel to address acute tissue injury and improve the healing process of chronic wounds makes it an ideal candidate for a variety of wound healing applications and other biomedical uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the XRD pattern of the aromatic silver functionalized hydrogel; Figure 2 is a SEM image of the aromatic silver functionalized hydrogel; Figure 3 is an EDX spectrum of the aromatic silver functionalized hydrogel; Figure 4 is the FT-IR spectrum of the aromatic silver functionalized hydrogel; Figure 5 The wound healing photos and closure rate results in Example 3; Figure 6 Staining images of macrophage polarization in wound sections. DETAILED DESCRIPTION

[0022] The present invention is described in detail below through examples to facilitate understanding of the present invention by persons skilled in the art. It is necessary to point out that the examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Non-essential improvements and adjustments made to the present invention by persons skilled in the art based on the above-mentioned invention content should still fall within the scope of protection of the present invention. At the same time, the raw materials mentioned below that are not described in detail are all commercially available products, and the process steps or preparation methods that are not mentioned in detail are all process steps or preparation methods known to those skilled in the art.

[0023] Example 1: Preparation of the aromatic silver functionalized hydrogel.

[0024] 1. Synthesis of Acrylate-CMC: 0.1 mL of acrylic acid, 0.3 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and 0.04 g of 4-dimethylaminopyridine (DMAP) were dissolved in 2 mL of dimethyl sulfoxide (DMSO) and allowed to react for 24 hours. Subsequently, the solution was added to 30 mL of carboxymethyl cellulose (CMC) in phosphate buffer. The reaction was carried out at room temperature for another 24 hours. The resulting product was subjected to continuous dialysis with a molecular weight cutoff of 7 kDa for 24 hours and then lyophilized to obtain acrylate-CMC (A-CMC).

[0025] 2. Synthesis of Vinylpyridine Carboxybetaine: 1 g of 4-vinylpyridine was dissolved in 5 mL of tetrahydrofuran and cooled to 0°C. Then 0.65 g of β-butyrolactone was added dropwise under nitrogen atmosphere, and the mixture was stirred at 0-4°C for 24 hours under nitrogen. After the reaction was completed, the crude product was filtered, washed with ether, and dried under vacuum to obtain vinylpyridine carboxybetaine (VCB).

[0026] 3. Synthesis of Vinylpyridine Sulfobetaine: 1 g of 4-vinylpyridine and 1.16 g of 1,3-propane sultone were dissolved in 5 mL of dimethylformamide (DMF) respectively, and then the two solutions were gradually mixed. The mixture was stirred at room temperature for 48 hours. After the reaction was completed, the crude product was filtered and washed three times with ethyl acetate. Subsequently, it was placed at 50 ° C and dried for 1 hour to obtain a light yellow powder, which is vinylpyridine sulfobetaine (VSB).

[0027] 4. Preparation of the aromatic silver functionalized hydrogel: 1 g of A-CMC was dissolved in 24 mL of water, and then 1 mL of silver nanowire solution (purchased from Merck, Chile) was added dropwise while ensuring adequate stirring. Subsequently, 0.25 g of VCB and 0.25 g of VSB were added to the mixture. After the components were completely dissolved, the free radical polymerization process was initiated by adding 10 mg of ammonium persulfate (APS) and 5 L of tetramethylethylenediamine (TEMED) as initiator and promoter, respectively. The resulting hydrogel sample was carefully transferred to a Petri dish, where the polymerization process was allowed to proceed at 70 °C for 24 h. After gelation, the hydrogel was immersed in deionized water to remove any remaining unreacted substances and impurities, thereby ensuring the purity of the final product.

[0028] Example 2, characterization of the aromatic silver functionalized hydrogel.

[0029] The phase purity of the catalysts was determined using wide angle X-ray diffraction (W-XRD) patterns, which were performed using a Bruker D8 diffractometer with Cu Kα radiation, scanning from 10° to 80° with a Bragg angle (2 θ ). Field emission scanning electron microscopy (FE-SEM) mapping was performed using a Gemini SEM 300. The functional groups of the hydrogels were analyzed using Fourier transform infrared spectroscopy. NMR spectra were recorded using a Bruker AVANCE 400 MHz spectrometer using deuterated solvents.

[0030] Figure 1 The XRD pattern of the aromatic silver functionalized hydrogel prepared in Example 1. It is worth noting that the diffraction peaks at 29.6° and 21.4° indicate partial and high crystallinity of CMC. There are four obvious diffraction peaks at 38.1°, 44.5°, 64.5° and 77.5°, corresponding to the face-centered cubic (FCC) patterns of (111), (200), (220) and (311) silver nanowires, respectively. This observation is consistent with the standard data obtained (JCPDS file number 04-0783 provided by ASTM). Silver nanowires significantly improve the conductivity of the hydrogel. The enhanced conductivity is particularly beneficial for wound healing, where electrical stimulation can promote cell migration and proliferation, thereby accelerating the healing process. Secondly, the potent antibacterial properties of silver are crucial in this case. Silver nanowires ensure the continuous release of silver ions, which effectively destroys the cell wall of bacteria and interferes with their metabolic processes. This sustained antibacterial effect helps prevent wound infection and maintain a sterile healing environment. The presence of silver nanowires significantly enhanced the electrical conductivity and antibacterial effect of the aromatic silver functionalized hydrogel, making it an ideal candidate for advanced wound care applications. XRD analysis confirmed the successful incorporation of silver nanowires into the aromatic silver functionalized hydrogel. The integration of silver not only improved the functional properties of the hydrogel but also helped to create a favorable environment for rapid wound healing and tissue regeneration. In addition, it is worth noting that the X-ray diffraction peaks of CMC at 29.6° and 21.4° showed relatively lower intensities compared to the crystal plane of (111), indicating that zwitterionic carboxylates and sulfonates may copolymerize with CMC.

[0031] Figure 2 is a SEM image of the aromatic silver functionalized hydrogel. It can be observed that the hydrogel surface presents a smooth and flat texture, and the silver nanowires are evenly distributed on the surface of the aromatic silver functionalized hydrogel. The corresponding EDX spectrum (such as Figure 3The in situ table provides the atomic and weight percentages of Ag and other elements present in the hydrogel. The incorporation of these silver nanowires not only enhances the smoothness and stability of the hydrogel surface, but also contributes to its bactericidal properties. The combination of these properties makes the aromatic silver functionalized hydrogel a promising candidate for clinical applications, especially as a wound dressing material.

[0032] Figure 4 The functional groups of the aromatic silver functionalized hydrogel zwitterionic hydrogel were characterized by FT-IR spectroscopy. In the aromatic silver functionalized hydrogel, at 2367 cm -1 A characteristic absorption peak was observed at + Asymmetric stretching vibration of the group. At 1572 cm -1 and about 1298~1414 cm -1 The absorption peaks correspond to carboxylic acid (COO - ) groups, confirming the presence of carboxylic acid groups that play a key role in the zwitterionic nature and ion exchange capacity of the hydrogel. In addition, the -1 and 1034 cm -1 The presence of the absorption peak at - ) groups. These peaks confirm the incorporation of sulfate groups, which contribute to the charge distribution of the hydrogel and enhance the interaction with biomolecules. FT-IR results confirm the successful incorporation of carboxylates, sulfates, and other related functional moieties into the hydrogel matrix. These functional groups are critical to the performance of the hydrogel, affecting its hydrophilicity, ionic interaction capabilities, and overall stability, making it very suitable for use in wound healing and tissue regeneration.

[0033] Example 3, evaluation and analysis of the effect of the aromatic silver functionalized hydrogel on the wound healing process.

[0034] Two control hydrogels were prepared according to the preparation method of Example 1. The first one did not contain VSB, and the prepared hydrogel was called "VCB / Ag / CMC"; the second one did not contain VCB, and the prepared hydrogel was called "VSB / Ag / CMC".

[0035] Male KM mice (n=24, weight=30±3 g) aged 6-8 weeks were used, authorized by the Experimental Animal Center of Shantou University. All organisms were housed in a controlled environment (temperature 25±1℃; 12h / 12h light-dark cycle; humidity 60±10%) with unrestricted access to water and complete free diet. After anesthesia, two circular wounds with a diameter of 0.5 cm were made on the shaved back of each mouse. The 24 injured mice were divided into NC, VCB, VSB, and VCS groups, with 6 mice in each group. The wound area was photographed with a digital camera to establish baseline measurements. The wounds were then treated in different ways according to the group: the NC group used PBS solution to treat the wounds, the VCB group used a 7 mm diameter disc-shaped VCB / Ag / CMC hydrogel to cover the wounds, the VSB group used a 7 mm diameter disc-shaped VSB / Ag / CMC hydrogel to cover the wounds, and the VCS group used a 7 mm diameter disc-shaped aromatic silver functionalized hydrogel to cover the wounds. Subsequently, the hydrogel dressing was covered with a Tegaderm transparent dressing to prevent infection and wrapped with a thin layer of self-adhesive bandage to prevent splint injury. If the treatment effect of two wounds in the same mouse was inconsistent, it was considered an invalid sample. The images of the mice were captured on days 0, 4, 7, 10, 14, and 17 after treatment, and the wound size was calculated with a digital vernier caliper. The closure rate of the wound was evaluated by measuring the wound area using Image-Pro Plus software to depict the wound edge. The wound closure rate (RW) was calculated using the following formula: RW=(S0-S N ) / S0*100% Among them, S0 is the wound area on day 0; S N is the wound area on day N.

[0036] Figure 5 The figure shows the wound healing of the hydrogel and the result of measuring the wound area using Image-Pro Plus software to depict the wound edge to evaluate the closure rate of the wound. As can be seen from the figure, compared with the NC group, the other three groups showed significantly improved wound regeneration behavior, and there were no obvious signs of inflammation or infection near the wound area. The proliferation of the new epidermis extended to the core of all hydrogel-treated wounds, resulting in a reduction in the injured area. At each time point, the hydrogel containing vinylpyridine zwitterions showed a significantly high wound closure rate under natural conditions. The final relative wound area of ​​the VCS group was 5.1% compared with the VCB group (9.8%), the VSB group (10.5%) and the NC group (17.8%). On day 14, there was a significant difference between the NC group and the VCS group, with a p value of less than 0.01 ("*" indicates p < 0.05). In general, the aromatic silver functionalized hydrogel significantly improved wound regeneration.

[0037] Example 4: Immunohistochemical staining was used to detect the effect of the hydrogel on wound healing.

[0038] In Example 3, each group of mice was anesthetized with chloral on the 14th day and euthanized by cervical dislocation. The damaged tissue was harvested, preserved with 10% formalin solution and inserted into paraffin, and wound tissue sections with a thickness of 4 microns were prepared. The obtained sections were respectively measured for macrophage polarization using M1 macrophage marker CD68 and M2 marker CD163. The stained sections were observed using a laser scanning confocal microscope (LSCM) from Zeiss, Germany.

[0039] Figure 6 Figure 2 is a staining diagram of macrophage polarization in wound sections. Analysis in the figure shows that the VCS group showed a higher tendency to transform into M2 macrophages on day 7 compared with natural healing and other hydrogel dressings. This observation is derived from the significant increase in green in the figure. The finding on M2 / M1 macrophage expression once again confirms the positive effect of the aromatic silver functionalized hydrogel in the wound regeneration process. These results show that the hydrogel not only promotes cell proliferation, but also plays an important role in regulating the immune response, which is crucial for effective healing.

[0040] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An aromatic silver functionalized hydrogel, characterized in that: The raw materials include vinylpyridine carboxybetaine, vinylpyridine sulfobetaine, silver nanowires and carboxymethyl cellulose.

2. The aromatic silver functionalized hydrogel according to claim 1, characterized in that: The structural formula of the vinylpyridine carboxy betaine is shown in formula (I): Formula (I).

3. The aromatic silver functionalized hydrogel according to claim 2, characterized in that: The preparation method of the vinylpyridine carboxybetaine is as follows: 4-vinylpyridine is dissolved in tetrahydrofuran and cooled to 0°C, β-butyrolactone is added dropwise, and a nitrogen atmosphere of 0-4°C is maintained and stirred until the reaction is completed, and the crude product is filtered, washed with ether, and dried under vacuum to obtain the obtained product.

4. The aromatic silver functionalized hydrogel according to claim 1, characterized in that: The structural formula of the vinylpyridine sulfobetaine is shown in formula (II): Formula (II).

5. The aromatic silver functionalized hydrogel according to claim 4, characterized in that: The preparation method of the vinylpyridine sulfobetaine is as follows: 4-vinylpyridine is dissolved in dimethylformamide, 1,3-propane sultone dissolved in N,N-dimethylformamide is gradually added, the reaction is stirred at room temperature until the reaction is completed, the crude product is filtered, washed with ethyl acetate, and dried to obtain the product.

6. The aromatic silver functionalized hydrogel according to claim 1, characterized in that: The carboxymethyl cellulose is modified carboxymethyl cellulose. The preparation method of the modified carboxymethyl cellulose is as follows: acrylic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 4-dimethylaminopyridine are dissolved in dimethyl sulfoxide, react for 24 hours, and then added into a phosphate buffer solution containing carboxymethyl cellulose. The reaction product is subjected to continuous dialysis and freeze-dried to obtain the modified carboxymethyl cellulose.

7. The method for preparing the aromatic silver functionalized hydrogel according to any one of claims 1 to 6, characterized in that: Includes steps: 1) preparing the vinylpyridine carboxy betaine and the vinylpyridine sulfobetaine respectively; 2) dissolving the carboxymethyl cellulose in water, adding the silver nanowires, and stirring thoroughly; 3) adding the vinylpyridine carboxy betaine and the vinylpyridine sulfobetaine, and adding an initiator and an accelerator after they are completely dissolved; 4) After gelation is achieved, immersing the hydrogel in deionized water to remove impurities, thereby obtaining the aromatic silver functionalized hydrogel.

8. The preparation method according to claim 7, characterized in that: Step 3) The mass ratio of the vinylpyridine carboxybetaine to the vinylpyridine sulfobetaine is 1:

1.

9. The preparation method according to claim 7, characterized in that: Step 3) The initiator is ammonium persulfate; preferably, the accelerator is tetramethylethylenediamine.

10. Use of the aromatic silver functionalized hydrogel according to any one of claims 1 to 6 in the preparation of a drug for promoting wound healing.

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