Antibacterial hydrogel as well as preparation method and application thereof

Abstract

The invention discloses antibacterial hydrogel as well as a preparation method and application thereof. The antibacterial hydrogel comprises a carbomer hydrogel matrix and gold nanoclusters dispersed in the carbomer hydrogel matrix, wherein the gold nanocluster is crosslinked with the carbomer through a hydrogen bond. The antibacterial hydrogel is formed by crosslinking gold nanoclusters (GNCs) and carbomer (CBM) through hydrogen bonds, and the GNCs serving as an antibacterial active component can achieve an efficient antibacterial effect by destroying a bacterial membrane structure, inducing an oxidative stress reaction, interfering bacterial metabolism and the like; cBM is used as a hydrogel matrix, so that excellent adhesiveness and barrier effect are provided, the structure of GNCs can be stabilized through hydrogen-bond interaction, and the antibacterial activity of the GNCs is improved. In addition, GNCs also has an immunoregulation characteristic and can induce M2 polarization of macrophages, so that inflammatory response is reduced and wound repair is promoted.

Description

Technical Field

[0001] The invention relates to the technical field of wound dressings, and in particular to an antibacterial hydrogel and a preparation method and application thereof. Background Art

[0002] Wound infection is one of the most common complications in clinical trauma orthopedics, especially in high-energy trauma (such as severe fractures and burns). Due to the simultaneous destruction of bones, soft tissues and skin, wounds are extremely susceptible to bacterial infection. In many trauma patients, due to the destruction of the skin barrier, microorganisms are able to grow and multiply in the wound surface, thus inducing infection. Existing antibacterial dressings still have limited antibacterial effects, biocompatibility issues, and insufficient mechanical properties and functionality of the gel, which seriously affect their clinical application value.

[0003] First, limited antibacterial effect is the main problem of existing antibacterial dressings. Traditional antibacterial dressings mainly rely on antibiotics or metal ions (such as silver, copper, etc.) to inhibit bacterial growth, but with the emergence of drug-resistant strains (such as methicillin-resistant Staphylococcus aureus MRSA), the effectiveness of these antibacterial strategies has gradually weakened. Many antimicrobial agents (such as silver nanoparticles) rely only on a single bactericidal mechanism, and bacteria can develop drug resistance through biofilm formation or gene mutation.

[0004] Secondly, biocompatibility and safety issues are also important challenges for antimicrobial dressings in clinical applications. Some nano-antimicrobial agents (such as silver and zinc oxide) may be toxic to host cells and affect wound repair, while excessive reactive oxygen species (ROS) may induce damage to surrounding normal cells, aggravate the inflammatory response of the wound, and even affect the overall health of the body. Long-term use of certain metal antimicrobial materials may also lead to accumulation in the body, posing a potential risk of systemic toxicity.

[0005] Finally, the mechanical properties and functionality of gel dressings are also key factors that limit their application. Many hydrogel dressings have poor stability in a humid environment and are easily washed away or dissolved, which makes it difficult to continuously release antibacterial ingredients, affecting the antibacterial effect. At the same time, the adhesion ability of hydrogel materials is limited, making it difficult to form a stable coverage on the wound surface, affecting its long-term use. The release rate of antibacterial ingredients in some dressings is difficult to accurately control, which may result in excessive initial antibacterial ability and too rapid attenuation of the antibacterial effect in the later stage, which cannot meet the treatment needs of chronic wound infections.

[0006] Therefore, how to improve the antibacterial effect of antibacterial dressings, enhance their biocompatibility, and optimize the mechanical properties of the gel so that it can exist stably in the wound environment is a technical problem that needs to be solved urgently. Summary of the invention

[0007] The purpose of the present invention is to provide an antibacterial hydrogel and a preparation method and application thereof in view of the deficiencies in the prior art.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, an antibacterial hydrogel is provided, which includes a carbomer hydrogel matrix and gold nanoclusters dispersed in the carbomer hydrogel matrix; wherein, the gold nanoclusters are cross-linked with carbomer through hydrogen bonds.

[0010] In a second aspect, a preparation method of the above antibacterial hydrogel is provided, which includes the following steps: dissolving carbomer in deionized water to obtain a carbomer hydrogel matrix solution; then dispersing gold nanoclusters modified with 6-mercaptohexanoic acid ligands in the carbomer hydrogel matrix solution, stirring to ensure uniform dispersion, and adjusting the pH value to obtain GNCs-CBM hydrogel, that is, the antibacterial hydrogel.

[0011] Furthermore, the concentration of carbomer in the carbomer hydrogel matrix solution is 1-5 w / w%.

[0012] Still further, the concentration of carbomer in the carbomer hydrogel matrix solution is 1-3 w / w%.

[0013] Furthermore, the concentration of gold nanoclusters modified with 6-mercaptohexanoic acid ligands in the GNCs-CBM hydrogel is 0.03-0.08 mg / mL.

[0014] Still further, the concentration of gold nanoclusters modified with 6-mercaptohexanoic acid ligands in the GNCs-CBM hydrogel is 0.06-0.08 mg / mL.

[0015] Furthermore, triethanolamine is used to adjust the pH.

[0016] In a third aspect, the application of the above antibacterial hydrogel in the preparation of anti-wound infection dressings is provided.

[0017] By adopting the above technical solutions, compared with the prior art, the present invention has the following technical effects:

[0018] The antibacterial hydrogel of the present invention is formed by cross-linking gold nanoclusters (GNCs) and carbomer (CBM) through hydrogen bonds. As an antibacterial active ingredient, GNCs can achieve efficient antibacterial effects by destroying the bacterial membrane structure, inducing oxidative stress reactions, and interfering with bacterial metabolism; CBM, as the hydrogel matrix, not only provides excellent adhesion and barrier effects, but also can stabilize the structure of GNCs through hydrogen bond interactions and improve its antibacterial activity. In addition, GNCs also have immunomodulatory properties and can induce macrophage M2 polarization, thereby reducing inflammatory reactions and promoting wound repair. Description of the Drawings

[0019] Figure 1The characterization results of GNCs-CBM hydrogel are shown; among them, (a) is the actual photo of GNCs-CBM hydrogel and Blank-CBM hydrogel; (b) is the scanning electron microscope (SEM) image of GNCs-CBM hydrogel and Blank-CBM hydrogel; (cd) are the rheological experimental results of GNCs-CBM hydrogel and Blank-CBM hydrogel.

[0020] Figure 2 The results of the in vitro antibacterial property study of GNCs-CBM hydrogels are shown; among them, (a) is a macroscopic photograph of blood plate bacterial colonies in different treatment groups; (b) is a quantitative analysis of CFU counts in different treatment groups; (c) is the MRSA live-dead staining results of different treatment groups, PI (red): dead bacteria, SYTO9 (green): live bacteria, scale: 10 μm, 50 μm; (d) is the PI and SYTO9 double-staining flow cytometry results of MRSA in different treatment groups; (e) is a quantitative analysis of the bacterial PI positivity rate in the flow cytometry results; n=3, ****p<0.0001.

[0021] Figure 3 The figure shows that GNCs-CBM hydrogel promotes the repair of infected wounds; (a) is a schematic diagram of the treatment process of infected wounds by GNCs-CBM hydrogel; (b) is a photo of the wound infection conditions of different treatment groups on days 0, 1, 3, 7, and 14 after infection, with a scale of 5 mm; (c) is a schematic diagram of the wound healing area; and (d) is the statistical results of the wound healing rate of different treatment groups after treatment.

[0022] Figure 4 The results show that GNCs-CBM hydrogel repaired epidermal defects in vivo by promoting granulation tissue formation; (a) is a Masson's trichrome staining image of collagen fibers in the cross-sectional tissue of the wound surface taken on the 14th day (scale bar: 200μm, magnified image scale bar: 60μm); (b) is a quantitative analysis of the collagen fiber staining area; (c) is a quantitative analysis of the average epidermal thickness; (d) is a representative immunofluorescence image of α-SMA and CD31 double-stained sections of each group on the 14th day (scale bar: 50μm); (e) is a quantitative analysis of α-SMA-positive areas and CD31-positive areas (n=3, *p<0.05; **p<0.01).

[0023] Figure 5Shows the biocompatibility test results of GNCs-CBM hydrogel; among them, (a) is the cell viability detection of HUVEC cells, L929 cells, and RAW 264.7 cells after co-culture with GNCs-CBM hydrogel for 24 hours, n = 5, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; (b) is the hemolysis experiment of GNCs-CBM hydrogel, n = 3; (c) is the cell cytoskeleton staining of HUVEC cells and L929 cells after co-culture with GNCs-CBM hydrogel for 24 hours using TRITC-phalloidin; TRITC-phalloidin (red): actin, DAPI (blue): cell nucleus; scale bar, 75 μm.

[0024] Figure 6 Shows the in vivo biosafety evaluation results of GNCs-CBM hydrogel. Detailed implementation manners

[0025] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but it is not a limitation of the present invention. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0026] The reagents and instruments used in the following embodiments include:

[0027] Carbomer 940 (CAS No.: 76050-42-5) was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300) strain was obtained from the Microbiology Laboratory of the Sixth People's Hospital Affiliated to Shanghai Jiao Tong University. CCK-8 reagent was purchased from Dojindo Laboratories (Kumamoto, Japan). Rhodamine-labeled phalloidin was purchased from Yeasen Biotech Co., Ltd. (Shanghai, China). Trypticase soy broth was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). The LIVE / DEAD Baclight Bacterial Viability Kit and the Baclight Bacterial Membrane Potential Detection Kit were both purchased from Thermo Fisher Scientific (Massachusetts, USA).

[0028] Example 1

[0029] This example provides a preparation method of an antibacterial hydrogel, which antibacterial hydrogel includes a carbomer hydrogel matrix and gold nanoclusters dispersed in the carbomer hydrogel matrix; among them, the gold nanoclusters are crosslinked with carbomer through hydrogen bonds, and the specific preparation method is as follows:

[0030] Dissolve 1 g of carbomer 940 in 100 mL of deionized water to obtain a carbomer hydrogel matrix solution (concentration 1 w / w%); then disperse 8 mg of 6-mercaptohexanoic acid ligand-modified gold nanoclusters (MHA-GNCs) in the above carbomer hydrogel matrix solution, stir to ensure uniform dispersion, and adjust the pH value with triethanolamine solution to obtain GNCs-CBM hydrogel, that is, the antibacterial hydrogel, and store it at 4 °C for later use.

[0031] Characterize the prepared GNCs-CBM hydrogel, and the results are as Figure 1 shown. The GNCs-CBM hydrogel exhibits higher viscosity due to the presence of gold nanoclusters ( Figure 1 a). Scanning electron microscopy (SEM) analysis of the freeze-dried sample shows that particles with high electron density are evenly distributed on the porous walls of the GNCs-CBM hydrogel, while the morphology of the blank carbomer (Blank-CBM) hydrogel shows smooth pores without any particles, indicating that MHA-GNCs are evenly distributed within the hydrogel ( Figure 3 b). Evaluate the viscosity change of the hydrogel through rheological experiments. The results of the amplitude sweep test show that the storage modulus (G') is always higher than the loss modulus (G”) ( Figure 3 c), verifying the viscoelastic properties of the GNCs-CBM hydrogel. As Figure 3 shown in d, the modulus of the GNCs-CBM hydrogel is significantly higher than that of the blank control group, indicating that it forms a stronger gel network structure.

[0032] Example 2

[0033] In this example, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), spread plate counting method (SPM), Live / Dead staining method, crystal violet staining and other methods are used to systematically study the antibacterial performance and anti-biofilm ability of GNCs-CBM hydrogel in vitro.

[0034] The results are as Figure 2 shown: Record the number of colony-forming units on the plate using the dilution separation method ( Figure 2 a). The results show that the blank CBM hydrogel group has no bactericidal activity ( Figure 2 b), while the bacterial survival rate of the GNCs-CBM hydrogel group is only 8.89 ± 1.11%, which is significantly better than that of the single GNCs treatment group (17.13 ± 2.24%). Through the live-dead staining method (SYTO9 green fluorescence labels live bacteria / propidium iodide (PI) red fluorescence labels dead bacteria), it is observed that the GNCs-CBM hydrogel group shows a large area of red fluorescence distribution ( Figure 2 c), while the control group and the blank CBM group still maintain strong green fluorescence. Flow cytometry quantitative analysis ( Figure 2d) The positive rates of PI in each group were shown as follows: blank CBM group 0.53 ± 0.11%, GNCs-CBM group 88.83 ± 1.40%, GNCs group 80.98 ± 1.44%( Figure 2 e), comprehensively demonstrating that the GNCs-CBM hydrogel has a synergistic bactericidal effect.

[0035] Example 3

[0036] In this example, a mouse wound infection model was constructed to evaluate the efficacy of GNCs-CBM in vivo antibacterial and wound healing. The treatment effect of GNCs-CBM on the infected wound was recorded by taking gross pictures, and the results were as Figure 3 shown. Specifically, the GNCs-CBM hydrogel constructed in the present invention was verified for its treatment efficacy in the BALB / c mouse infected wound model( Figure 3 a). The wound healing process at specified time points (day 0, 1, 3, 7, 14) was recorded by digital photos( Figure 3 b). The comparison of relative wound areas showed that the GNCs-CBM group had the most significant wound healing effect( Figure 3 c). It is worth noting that the wound contraction rate of the GNCs-CBM hydrogel treatment group reached 15.31 ± 2.68% on the 7th day, while the control group took twice as long (16.01 ± 2.53% on the 14th day) to achieve a similar effect( Figure 3 d).

[0037] Furthermore, the late effect of the in vivo treatment of the GNCs-CBM hydrogel was verified. The tissues on the 14th day after surgery were subjected to Masson staining, and the results showed that( Figure 4 ) after treatment, the GNCs-CBM group had more deposition of newly formed dense collagen fibers, while the Blank-CBM group and the control group showed loose collagen deposition. In addition, the GNCs-CBM group had a higher vascular density in the late stage of treatment, which was verified by double immunofluorescence staining of CD31 and α-SMA and supported by the results of fluorescence relative quantitative analysis. The above results indicate that the GNCs-CBM hydrogel has the performance for treating in vivo infected wounds, can effectively eliminate bacteria and inhibit the inflammatory level, and promote the early healing of wound tissues.

[0038] Example 4

[0039] This example verified the biosafety and biocompatibility of the GNCs-CBM hydrogel.

[0040] (1) Results of in vitro cytotoxicity experiments( Figure 5)Display: The viability of GNCs-CBM gel on human dermal fibroblasts and macrophages remained above 90% within 48 h, and no obvious cytotoxicity was observed. In vivo skin irritation tests showed that the dressing did not cause obvious immune rejection or inflammatory reactions, indicating good biocompatibility.

[0041] (2) To study the in vivo safety of GNCs-CBM hydrogel, the main organs of mice were collected and sectioned for HE staining. The results showed that ( Figure 6 ), no obvious tissue damage or inflammatory changes were observed in all important organs. In conclusion, GNCs-CBM hydrogel is an infection wound dressing with excellent biocompatibility and has potential clinical application value.

[0042] The above are only the preferred embodiments of the present invention, and do not limit the implementation manners and protection scope of the present invention. For those skilled in the art, it should be realized that all equivalent replacements and obvious changes made by using the content of the specification and drawings of the present invention should be included in the protection scope of the present invention.

Claims

1. An antibacterial hydrogel, characterized in that, It includes a carbomer hydrogel matrix and gold nanoclusters dispersed in the carbomer hydrogel matrix; wherein, the gold nanoclusters are crosslinked with carbomer through hydrogen bonds.

2. A preparation method of the antibacterial hydrogel as described in claim 1, characterized in that, It includes the following steps: dissolving carbomer in deionized water to obtain a carbomer hydrogel matrix solution; then dispersing gold nanoclusters modified with 6-mercaptohexanoic acid ligands in the carbomer hydrogel matrix solution, stirring to ensure uniform dispersion, and adjusting the pH value to obtain GNCs-CBM hydrogel, that is, the antibacterial hydrogel.

3. The preparation method according to claim 2, characterized in that, The concentration of carbomer in the carbomer hydrogel matrix solution is 1-5 w / w%.

4. The preparation method according to claim 3, wherein, The concentration of carbomer in the carbomer hydrogel matrix solution is 1-3 w / w%.

5. The preparation method according to claim 2, characterized in that, The concentration of gold nanoclusters modified with 6-mercaptohexanoic acid ligands in the GNCs-CBM hydrogel is 0.03-0.08 mg / mL.

6. The preparation method according to claim 5, characterized in that, The concentration of gold nanoclusters modified with 6-mercaptohexanoic acid ligands in the GNCs-CBM hydrogel is 0.06-0.08 mg / mL.

7. The preparation method according to claim 2, wherein Triethanolamine is used to adjust the pH.

8. Use of the antibacterial hydrogel according to claim 1 in the preparation of an anti-wound infection dressing.

Images