Aromatic silver functionalized hydrogel, preparation method therefor and use thereof

Abstract

The present application discloses an aromatic silver functionalized hydrogel, a preparation method therefor, and a use thereof. Vinylpyridine carboxybetaine and vinylpyridine sulfobetaine are both zwitterions. It is proved by experiments that the two zwitterions can play a synergistic role in a hydrogel, so that the hydrogel containing the two zwitterions has better wound healing ability, and the performance of the hydrogel far exceeds that of a hydrogel containing only a single zwitterion. Silver nanowires significantly improve the conductivity of the hydrogel and facilitate wound healing; moreover, the strong antimicrobial characteristics thereof facilitate preventing wound infection and maintaining a sterile healing environment. The highly fibrillated property of carboxymethyl cellulose (CMC) greatly enhances the mechanical properties and structural stability of the hydrogel.

Description

An aromatic silver functionalized hydrogel, its preparation method and application Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to an aromatic silver functionalized hydrogel, its preparation method, and its application. Background Technology

[0002] Hydrogels have long been a focus of attention in skin wound dressings due to their unique properties, such as moisturizing and extracellular matrix-like structures. Hydrogels can also serve as delivery carriers, increasing their versatility. Many hydrogels have been developed to modulate immunomodulatory cells and promote immune responses in wound healing. Those skilled in the art strive to balance these hydrogels between inflammatory cell infiltration, regulation of macrophage polarization, enhancement of cell proliferation and migration, and to control collagen remodeling by manipulating their physicochemical properties. Studies have elucidated the effects of hydrogel physical properties such as dimensionality, hardness, porosity, morphology, electrical properties, and wettability on immune responses. However, most of these studies are limited to in vitro laboratory experiments, neglecting the immunomodulatory role of hydrogels in vivo. Summary of the Invention

[0003] This application proposes an aromatic silver functionalized hydrogel, its preparation method, and its application to solve one or more technical problems existing in the prior art.

[0004] The first aspect of this application is to provide an aromatic silver-functionalized hydrogel.

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

[0006] The third aspect of this application is to provide the use of the aromatic silver functionalized hydrogel described in the first aspect of this application in the preparation of pharmaceuticals.

[0007] The aromatic silver functionalized hydrogel described in the first aspect of this application is made from vinylpyridine carboxybetaine, vinylpyridine sulfobetaine, silver nanowires, and carboxymethyl cellulose.

[0008] The vinylpyridine carboxybetaine and vinylpyridine sulfobetaine are both zwitterions. Experiments have shown that these two zwitterions work synergistically in the hydrogel, resulting in a hydrogel containing both zwitterions exhibiting superior wound healing capabilities, far exceeding those of hydrogels containing only one zwitterion. Silver nanowires significantly improve the conductivity of the hydrogel, promoting wound healing, while their potent antibacterial properties help prevent wound infection and maintain a sterile healing environment. Furthermore, unlike typical silver nanoparticles, these silver nanowires have a one-dimensional structure, allowing for better integration into the hydrogel matrix, thus providing superior mechanical reinforcement and making the hydrogel stronger and more flexible. The highly fibrillated nature of carboxymethyl cellulose (CMC) significantly enhances the mechanical properties and structural stability of the hydrogel.

[0009] In some embodiments of the first aspect of this application, the structure of the vinylpyridine carboxybetaine is shown in formula (I):

[0010] In some application embodiments of the first aspect of this application, the method for preparing the vinylpyridine carboxybetaine is as follows: 4-vinylpyridine is dissolved in tetrahydrofuran and cooled to 0°C, β-butyrolactone is added dropwise while maintaining the temperature at 0°C to 4°C, and the mixture is stirred under a nitrogen atmosphere until the reaction is complete. The crude product is filtered, washed with diethyl ether, and dried under vacuum to obtain the product.

[0011] In some implementations of the first aspect of this application, the structure of the vinylpyridine sulfobetaine is shown in formula (II):

[0012] In some application embodiments of the first aspect of this application, the method for preparing the vinylpyridine sulfobetaine is as follows: 4-vinylpyridine is dissolved in dimethylformamide, 1,3-propane sulfonyl lactone dissolved in N,N-dimethylformamide is gradually added, the mixture is stirred at room temperature until the reaction is complete, the crude product is filtered, washed with ethyl acetate, and dried to obtain the product.

[0013] In some embodiments of the first aspect of this application, the carboxymethyl cellulose is modified carboxymethyl cellulose. The modified carboxymethyl cellulose is prepared by dissolving acrylic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 4-dimethylaminopyridine in dimethyl sulfoxide, reacting for 24 hours, then adding the mixture to a phosphate buffer solution containing dissolved carboxymethyl cellulose. The reaction product is then continuously dialyzed and lyophilized to obtain the final product.

[0014] The preparation method described in the second aspect of this application includes the following steps:

[0015] 1) Prepare vinylpyridine carboxybetaine as shown in formula (I) and vinylpyridine sulfobetaine as shown in formula (II), respectively;

[0016] 2) Dissolve the carboxymethyl cellulose in water, add the silver nanowires, and stir thoroughly;

[0017] 3) Add the vinylpyridine carboxybetaine and the vinylpyridine sulfobetaine, and after they are completely dissolved, add the initiator and the accelerator;

[0018] 4) After gelation, the hydrogel is immersed in deionized water to remove impurities, thus obtaining the aromatic silver functionalized hydrogel.

[0019] In some implementations of the second aspect of this application, the mass ratio of vinylpyridine carboxybetaine and vinylpyridine sulfobetaine in step 3) is 1:1.

[0020] In some implementations of the second aspect of this application, the initiator in step 3) is ammonium persulfate; preferably, the promoter is tetramethylethylenediamine.

[0021] The application described in the third aspect of this application refers to the use of the aromatic silver functionalized hydrogel in the preparation of a drug that promotes wound healing.

[0022] The above-described technical solution of this application has at least the following technical effects or advantages compared with the prior art:

[0023] In vivo experiments demonstrated the efficacy of the aromatic silver-functionalized hydrogel in rapidly resolving acute tissue injuries, such as full-thickness skin defects, by significantly improving the wound healing process. Specific findings included improved wound healing, promotion of angiogenesis, collagen synthesis, macrophage polarization, and granulation tissue formation. The aromatic silver-functionalized hydrogel's ability to treat acute tissue injuries and improve chronic wound healing processes makes it an ideal candidate material for various wound healing applications and other biomedical uses. Attached Figure Description

[0024] Figure 1 shows the XRD pattern of the aromatic silver functionalized hydrogel prepared in Example 1;

[0025] Figure 2 is a SEM image of the aromatic silver functionalized hydrogel prepared in Example 1;

[0026] Figure 3 shows the EDX spectrum of the aromatic silver functionalized hydrogel prepared in Example 1;

[0027] Figure 4 shows the FT-IR spectrum of the aromatic silver functionalized hydrogel prepared in Example 1;

[0028] Figure 5 shows a photograph of the wound healing and the closure rate results in Example 3;

[0029] Figure 6 shows the staining pattern of macrophage polarization in a wound section. Detailed Implementation

[0030] The following detailed description of this application is provided through embodiments to facilitate understanding of this application by those skilled in the art. It is important to note that the embodiments are merely for further illustration of this application and should not be construed as limiting the scope of protection of this application. Non-essential improvements and adjustments made to this application by those skilled in the art based on the above-described invention should still fall within the scope of protection of this application. Furthermore, all raw materials mentioned below that are not described in detail are commercially available products, and all process steps or preparation methods not mentioned in detail are process steps or preparation methods known to those skilled in the art.

[0031] Example 1: Preparation of aromatic silver functionalized hydrogel.

[0032] 1. Synthesis of acrylate-CMC:

[0033] 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, this 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 dialyzing with a molecular weight cutoff of 7 kDa for 24 hours, followed by lyophilization to obtain acrylate-CMC (A-CMC).

[0034] 2. Synthesis of vinylpyridine carboxybetaine:

[0035] 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 a nitrogen atmosphere, and the mixture was stirred at 0 °C to 4 °C for 24 hours under nitrogen. After the reaction was complete, the crude product was filtered, washed with diethyl ether, and dried under vacuum to give vinylpyridine carboxybetaine (VCB).

[0036] 3. Synthesis of vinylpyridine sulfobetaine:

[0037] 1 g of 4-vinylpyridine and 1.16 g of 1,3-propanesulfonyl lactone were dissolved separately in 5 mL of dimethylformamide (DMF), and the two solutions were then gradually mixed. The mixture was stirred at room temperature for 48 hours. After the reaction was complete, the crude product was filtered and washed three times with ethyl acetate. Subsequently, it was dried at 50 °C for 1 hour to obtain a light yellow powder, which is vinylpyridine sulfobetaine (VSB).

[0038] 4. Preparation of aromatic silver functionalized hydrogels:

[0039] 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 thorough 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 accelerator, 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 hours. After gelation, the hydrogel was immersed in deionized water to remove any residual unreacted substances and impurities, thus ensuring the purity of the final product.

[0040] Example 2: Characterization of aromatic silver functionalized hydrogel.

[0041] Phase purity of the catalyst was analyzed by wide-angle X-ray diffraction (W-XRD). W-XRD analysis was performed using a Bruker D8 diffractometer and Cu Kα radiation, with scans ranging from 10° to 80° and a Bragg angle (2π / 4). θ Field emission scanning electron microscopy (FE-SEM) was used for imaging with a Gemini SEM 300. Fourier transform infrared spectroscopy was used to analyze the functional groups of the hydrogel. NMR spectra were recorded using a Bruker AVANCE 400MHz spectrometer with deuterated solvents.

[0042] Figure 1 shows the XRD pattern of the aromatic silver-functionalized hydrogel prepared in Example 1. Notably, the diffraction peaks present at 29.6° and 21.4° indicate partial and high crystallinity of the CMC. Four distinct diffraction peaks are observed at 38.1°, 44.5°, 64.5°, and 77.5°, corresponding to the face-centered cubic (FCC) patterns of the (111), (200), (220), and (311) silver nanowires, respectively. This observation is consistent with the obtained standard data (JCPDS document number 04-0783 provided by ASTM). The silver nanowires significantly enhance the conductivity of the hydrogel. 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. The silver nanowires ensure the sustained release of silver ions, which effectively disrupts the bacterial cell wall and interferes with its metabolic processes. This sustained antibacterial effect helps prevent wound infection and maintain a sterile healing environment. The presence of silver nanowires significantly enhances the conductivity and antibacterial effects of aromatic silver-functionalized hydrogels, making them ideal candidate materials for advanced wound care applications. XRD analysis confirmed the successful incorporation of silver nanowires into the aromatic silver-functionalized hydrogels. The integration of silver not only improves the functional properties of the hydrogels but also helps create a favorable environment for rapid wound healing and tissue regeneration. Furthermore, it is noteworthy that the X-ray diffraction peaks of CMC at 29.6° and 21.4° exhibited relatively lower intensities compared to the (111) crystal plane, suggesting that zwitterionic carboxylates and sulfonates may copolymerize with CMC.

[0043] Figure 2 shows a SEM image of the aromatic silver-functionalized hydrogel prepared in Example 1. A smooth, flat texture can be observed on the hydrogel surface, with silver nanowires uniformly distributed across it. The corresponding EDX spectrum (as shown in Figure 3) and in-situ table provide 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. This combination of properties makes the aromatic silver-functionalized hydrogel a promising candidate for clinical applications, particularly as a wound dressing material.

[0044] Figure 4 shows the functional groups of the zwitterionic hydrogel with aromatic silver functionalization prepared in Example 1, characterized by FT-IR spectroscopy. In the aromatic silver functionalized hydrogel, at 2367 cm⁻¹... -1 A characteristic absorption peak was observed at [location], corresponding to pyridinium N. + Asymmetric stretching vibration of the functional group. At 1572 cm⁻¹ -1 and approximately 1298–1414 cm -1 The absorption peaks correspond to carboxylic acids (COO) and α, β, 0. -The asymmetric and symmetric stretching vibrations of the ) group confirmed the presence of the carboxylic acid group, which plays a key role in the zwitterionic properties and ion exchange capacity of the hydrogel. Furthermore, at 1141 cm⁻¹... -1 and 1034cm -1 The presence of the absorption peak is attributed to sulfate (SO3) ions. - The peaks revealed asymmetric and symmetric stretching vibrations of the sulfate group. These peaks confirmed the binding of the sulfate group, which contributes to the charge distribution of the hydrogel and enhances its interaction with biomolecules. FT-IR results confirmed the successful binding of carboxyl, sulfate, and other related functional groups into the hydrogel matrix. These functional groups are crucial to the hydrogel's properties, influencing its hydrophilicity, ionic interaction capabilities, and overall stability, making it ideal for wound healing and tissue regeneration.

[0045] Example 3: Evaluation and analysis of the effect of aromatic silver functionalized hydrogel on the wound healing process.

[0046] According to the preparation method of Example 1, two control hydrogels were prepared. The first hydrogel did not contain VSB and was named "VCB / Ag / CMC"; the second hydrogel did not contain VSB and was named "VSB / Ag / CMC".

[0047] Male KM mice aged 6–8 weeks (n = 24, weight = 30 ± 3 g) authorized by the Experimental Animal Center of Shantou University were used. All organisms were housed in a controlled environment (temperature 25 ± 1℃; 12 h / 12 ​​h light / dark cycle; humidity 60 ± 10%) with unrestricted access to water and free access to food. After anesthesia, two circular wounds with a diameter of 0.5 cm were created 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 areas were photographed with a digital camera to establish baseline measurements. The wounds were then treated differently according to the groups: the NC group was treated with PBS solution, the VCB group was covered with a 7 mm diameter disc-shaped VCB / Ag / CMC hydrogel, the VSB group was covered with a 7 mm diameter disc-shaped VSB / Ag / CMC hydrogel, and the VCS group was covered with a 7 mm diameter disc-shaped aromatic silver functionalized hydrogel. Subsequently, the hydrogel dressing was covered with a Tegaderm transparent dressing to prevent infection, and then wrapped with a thin layer of self-adhesive bandage to prevent splint damage. Mice with inconsistent treatment outcomes on two wounds were considered invalid samples. Images of mice were captured on days 0, 4, 7, 10, 14, and 17 post-treatment, and wound size was calculated using digital calipers. Wound closure rate was assessed by measuring the wound area using Image-Pro Plus software to delineate the wound edges. The wound closure rate (RW) was calculated using the following formula: RW = (S0 - S...) N ) / S0*100%

[0048] Where S0 is the wound area on day 0; S N The area of ​​the wound on day N.

[0049] Figure 5 shows photographs of wound healing in mice of each group and the results of wound closure rate assessment using Image-Pro Plus software to measure wound area and delineate wound edges. As shown in the figure, the other three groups exhibited significantly improved wound regeneration behavior compared to the NC group, with no obvious signs of inflammation or infection near the wound area. Neoepithelial proliferation extended to the core of all hydrogel-treated wounds, resulting in a reduction of the injured area. At each time point, the hydrogel containing vinylpyridine zwitterions showed a significantly higher wound closure rate than under natural conditions. The final relative wound area in the VCS group was 5.1% compared to the VCB group (9.8%), VSB group (10.5%), and NC group (17.8%). A significant difference existed between the NC and VCS groups at day 14, with p-values ​​less than 0.01 (“*” indicates p < 0.05). In summary, aromatic silver-functionalized hydrogels significantly improved wound regeneration.

[0050] Example 4: Immunohistochemical staining method to detect the effect of hydrogel on wound healing.

[0051] In Example 3, mice in each group were anesthetized with chloral acetaldehyde on day 14 and euthanized via cervical dislocation. Damaged tissue was harvested, preserved in 10% formalin solution, and embedded in paraffin to prepare wound tissue sections with a thickness of 4 micrometers. Macrophage polarization was detected in the obtained sections using the M1 macrophage marker CD68 and the M2 macrophage marker CD163, respectively. The stained sections were observed using a laser scanning confocal microscope (LSCM) from Zeiss, Germany.

[0052] Figure 6 shows the staining pattern of macrophage polarization in wound sections. Analysis in the figure reveals that, compared to natural healing and other hydrogel dressings, the VCS group exhibited a higher tendency to transform into M2 macrophages on day 7. This observation is evidenced by a significant increase in CD163 in the figure, and the finding regarding M2 / M1 macrophage expression further confirms the positive impact of aromatic silver-functionalized hydrogels on wound regeneration. These results indicate that hydrogels not only promote cell proliferation but also play an important role in regulating the immune response, which is crucial for effective healing.

[0053] The embodiments of this application have been described in detail above with reference to the accompanying drawings. However, this application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the protection scope of this application. Furthermore, unless otherwise specified, the embodiments and features described in the embodiments of this application can be combined with each other.

Claims

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

2. The aromatic silver-functionalized hydrogel of claim 1, wherein The structural formula of the ethenylpyridine carboxybetaine is shown as formula (I):

3. The aromatic silver-functionalized hydrogel of claim 2, wherein, The method for preparing the vinylpyridine carboxybetaine is as follows: 4-vinylpyridine is dissolved in tetrahydrofuran and cooled to 0°C. β-butyrolactone is added dropwise while maintaining the temperature at 0°C to 4°C. The mixture is stirred under a nitrogen atmosphere until the reaction is complete. The crude product is filtered, washed with diethyl ether, and dried under vacuum to obtain the final product.

4. The aromatic silver-functionalized hydrogel of claim 1, wherein, The structural formula of the vinylpyridine sulfobetaine is shown as formula (II):

5. The aromatic silver-functionalized hydrogel of claim 4, wherein, The method for preparing the vinylpyridine sulfobetaine is as follows: 4-vinylpyridine is dissolved in dimethylformamide, and 1,3-propane sulfonyl lactone dissolved in N,N-dimethylformamide is gradually added. The mixture is stirred at room temperature until the reaction is complete, the crude product is filtered, washed with ethyl acetate, and dried to obtain the final product.

6. The aromatic silver-functionalized hydrogel of claim 1, wherein, The carboxymethyl cellulose is a modified carboxymethyl cellulose. The modified carboxymethyl cellulose is prepared by dissolving acrylic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine in dimethyl sulfoxide. After reacting for 24 hours, the mixture is added to a phosphate buffer solution containing dissolved carboxymethyl cellulose. The reaction product is then continuously dialyzed and lyophilized to obtain the final product.

7. A process for the preparation of the aromatic silver-functionalized hydrogel according to any one of claims 1 to 6, characterized in that, Includes the following steps: 1) Prepare the vinylpyridine carboxybetaine and the vinylpyridine sulfobetaine, respectively; 2) Dissolve the carboxymethyl cellulose in water, add the silver nanowires, and stir thoroughly; 3) Add the vinylpyridine carboxybetaine and the vinylpyridine sulfobetaine, and after they are completely dissolved, add the initiator and the accelerator; 4) After gelation, the hydrogel is immersed in deionized water to remove impurities, thus obtaining the aromatic silver functionalized hydrogel.

8. The preparation method according to claim 7, characterized in that, In step 3), the mass ratio of vinylpyridine carboxybetaine to 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 promoter is tetramethylethylenediamine.

10. The use of the aromatic silver functionalized hydrogel according to any one of claims 1 to 6 in the preparation of a wound healing medicament.

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