Gold nano-composite antibacterial material based on hyaluronic acid modification as well as preparation method and application of gold nano-composite antibacterial material

Abstract

The invention relates to the field of gold nano antibacterial materials, and mainly relates to a gold nano composite antibacterial material based on hyaluronic acid modification and a preparation method and application thereof. A preparation method of a gold nano composite antibacterial material based on hyaluronic acid modification comprises the following steps: adding acetic acid and sophorolipid into a hyaluronic acid solution to obtain a mixed solution; adding a chloroauric acid solution into the mixed solution, and stirring to obtain a room-temperature solution; adding a sodium cyanoborohydride solution into the room-temperature solution under a violent stirring condition; after the sodium cyanoborohydride solution is added, reducing the stirring speed and stirring to obtain a reaction solution; and carrying out high-speed centrifugation, resuspension, filtration and sterilization on the reaction liquid to obtain the hyaluronic acid modification-based gold nano composite antibacterial material. The material not only has excellent broad-spectrum antibacterial performance and is especially effective to drug-resistant bacteria, but also has significantly improved biocompatibility, and is expected to reduce adverse effects on wound healing. Meanwhile, the method disclosed by the invention is relatively simple and controllable in process and suitable for large-scale preparation.

Description

Technical Field

[0001] This application relates to the field of gold nanoparticle antibacterial materials, and mainly to a gold nanoparticle composite antibacterial material based on hyaluronic acid modification, its preparation method and application. Background Technology

[0002] With the increasing severity of bacterial resistance, the antibacterial effects of traditional antibiotics are gradually weakening. Developing novel, highly efficient, low-toxicity antibacterial materials that are less prone to inducing resistance has become a current research hotspot. Gold nanoparticles, due to their unique physicochemical properties, such as surface plasmon resonance, good biocompatibility, and modifiability, demonstrate enormous application potential in the field of antibacterial research.

[0003] However, current gold nanoparticles suffer from poor stability, easy aggregation, insufficient biocompatibility, and cumbersome preparation processes, limiting their practical application in the biomedical field. Therefore, surface modification of gold nanoparticles is necessary to improve their properties.

[0004] Hyaluronic acid is a natural macromolecular polysaccharide with good biocompatibility, biodegradability, and non-immunogenicity. It can also specifically bind to the CD44 receptor on the cell surface, exhibiting a certain degree of targeting. Gold nanoparticles synthesized based on hyaluronic acid can not only improve the stability and biocompatibility of gold nanoparticles but also endow them with targeted antibacterial properties, potentially addressing the shortcomings of existing gold nanoparticle antibacterial materials.

[0005] Currently, there are some studies on hyaluronic acid-modified gold nanomaterials, but most of these studies have limited antibacterial effects and applicability, and the preparation methods suffer from problems such as harsh reaction conditions, cumbersome steps, and high costs. Therefore, developing a hyaluronic acid-modified gold nanocomposite antibacterial material with a simple preparation process, low cost, excellent antibacterial properties, and multifunctionality is of significant practical importance. Summary of the Invention

[0006] To address the shortcomings of existing gold nanoparticle antibacterial materials in terms of biocompatibility and wound healing promotion, and to meet the need for combating drug-resistant bacteria, this application aims to provide a hyaluronic acid-modified gold nanoparticle composite antibacterial material, its preparation method, and its applications. This material not only possesses excellent broad-spectrum antibacterial properties, especially effective against drug-resistant bacteria, but also exhibits significantly improved biocompatibility, potentially reducing adverse effects on wound healing. Furthermore, this invention provides a method for preparing this composite material, which is relatively simple, controllable, and suitable for large-scale production.

[0007] The technical solution of this application is as follows: A method for preparing a hyaluronic acid-modified gold nanocomposite antibacterial material includes the following steps: Hyaluronic acid is dissolved in water to obtain a hyaluronic acid solution. Acetic acid and sophorolipid are added to the hyaluronic acid solution to obtain a mixed solution. Add chloroauric acid solution to the mixed solution and stir to obtain a room temperature solution; Under vigorous stirring, sodium cyanoborohydride solution was added to the room temperature solution; After the sodium cyanoborohydride solution has been added, reduce the stirring speed and continue stirring at a low speed to obtain the reaction solution; The reaction solution is centrifuged at high speed, resuspended, filtered, and sterilized to obtain the gold nanocomposite antibacterial material based on hyaluronic acid modification.

[0008] Sophorolipids exist primarily as lactones and unionized acidic sophorolipids. In the system, after specific coordination with acetic acid, the hydrophobic portion of the sophorolipid molecule becomes dominant, enhancing hydrophobic interactions. This change allows sophorolipids to form smaller, denser, and more stable micelle structures. In subsequent steps such as adding chloroauric acid solution, these stable micelle structures effectively disperse nanoparticles. Without a good dispersion mechanism during nanoparticle formation, nanoparticles are prone to aggregation, leading to increased and uneven particle size, thus affecting material properties. The stable micelle structures formed by sophorolipids effectively prevent the aggregation of forming gold nanoparticles, resulting in smaller and more uniform gold nanoparticles, significantly improving material stability.

[0009] Adding sodium cyanoborohydride solution to the room temperature solution under vigorous stirring helps the sodium cyanoborohydride to disperse rapidly and uniformly in the solution, allowing it to fully contact and react with components such as chloroauric acid. Sodium cyanoborohydride acts as a reducing agent here, reducing gold ions in chloroauric acid to gold atoms, thereby forming gold nanoparticles. Hyaluronic acid also plays an important role in this process; it can interact with the surface of the gold nanoparticles through its chemical groups, adsorbing onto the surface and further stabilizing the gold nanoparticles, preventing their aggregation. Subsequent low-speed stirring further stabilizes the reaction system, avoiding damage to the formed gold nanoparticles caused by vigorous stirring, and also promotes a complete reaction, allowing hyaluronic acid to better modify the surface of the gold nanoparticles.

[0010] Furthermore, the concentration of the hyaluronic acid solution in the entire reaction system is 0.02~0.40 mol / L; The amount of acetic acid added is in a volume ratio of 1:100 to 1:500 with the volume of the hyaluronic acid solution. The amount of sophorolipid added is in a molar ratio of 1:10 to 1:30 with that of hyaluronic acid.

[0011] When the concentration of hyaluronic acid solution is too high, the viscosity of the solution increases significantly, which affects the diffusion of components and the reaction rate in the reaction system. Excessive hyaluronic acid molecules may form an excessive network structure in the solution, hindering the effective contact between chloroauric acid and the reducing agent sodium cyanoborohydride, leading to uneven formation of gold nanoparticles and even incomplete local reactions, thus affecting the performance stability of the final material. Therefore, controlling the concentration of hyaluronic acid solution at 0.02-0.40 mol / L ensures sufficient and appropriate amounts of hyaluronic acid molecules participate in the reaction, achieving effective modification of gold nanoparticles and enabling the material to possess both good biological properties and stable performance.

[0012] Furthermore, the concentration of chloroauric acid in the entire reaction system is 0.05~0.20 mol / L; After adding the chloroauric acid solution to the mixed solution, stir at 500-900 rpm for 5-30 minutes to obtain the room temperature solution.

[0013] Furthermore, the concentration of the sodium cyanoborohydride solution in the entire reaction system is 0.01~0.30 mol / L.

[0014] Furthermore, the speed of the vigorous stirring is 1000~3000 rpm; The low-speed stirring time is 5 to 10 hours, and the low-speed stirring speed is 500 to 900 rpm.

[0015] Furthermore, the high-speed centrifugation speed is 12000~18000 rpm; the high-speed centrifugation time is 10-30 minutes.

[0016] This application also provides a gold nanocomposite antibacterial material based on hyaluronic acid modification, comprising gold nanoparticles as the core and hyaluronic acid molecules modified on the surface.

[0017] The gold nanocomposite antibacterial material based on hyaluronic acid modification provided in this application has a special structure. It mainly works by using hyaluronic acid as both a dispersant to protect the dispersion of gold nanoparticles and to exert antibacterial effects through synergistic interaction with gold nanoparticles.

[0018] Gold nanoparticles and surface-modified hyaluronic acid molecules work synergistically. The gold nanoparticles provide a powerful antibacterial physical mechanism, such as the photothermal effect and the high activity sites resulting from their small size; while the hyaluronic acid molecules ensure the material's application through biocompatibility, biodegradability, and targeting. The combination of these two components results in a hyaluronic acid-modified gold nanocomposite antibacterial material that not only possesses excellent antibacterial properties, capable of combating multidrug-resistant bacteria and even methicillin-resistant Staphylococcus aureus (MRSA), but also exhibits good biocompatibility and targeting, making it an excellent medical antibacterial material and providing a new and effective approach to solving the current problem of bacterial resistance.

[0019] Furthermore, the average particle size of the gold nanoparticles ranges from 1 to 60 nm.

[0020] This application also provides the application of hyaluronic acid-modified gold nanocomposite antibacterial materials in the preparation of drugs or related medical products for the treatment or prevention of bacterial infections.

[0021] Furthermore, the bacterial infection includes infections caused by one or more pathogens such as Staphylococcus aureus, Escherichia coli, methicillin-resistant Staphylococcus aureus, and multidrug-resistant Escherichia coli.

[0022] Compared with the prior art, this application has the following beneficial effects: 1. The gold nanocomposite antibacterial material based on hyaluronic acid modification prepared in this application has excellent antibacterial properties and can resist multidrug-resistant bacteria, even methicillin-resistant Staphylococcus aureus (MRSA).

[0023] 2. The gold nanocomposite antibacterial material based on hyaluronic acid modification prepared in this application has good biocompatibility and low toxicity to human cells, making it an excellent medical antibacterial material. Attached Figure Description

[0024] Figure 1 This is a TEM image of the gold nanocomposite antibacterial material modified with hyaluronic acid, which is Example 1 of this application.

[0025] Figure 2 This is a graph showing the effect of hyaluronic acid-modified gold nanocomposite antibacterial material on the permeability of Escherichia coli cell membranes in Example 1 of this application (fluorescence staining method).

[0026] Figure 3 The images show TEM and SEM images of the destructive effect of the hyaluronic acid-modified gold nanocomposite antibacterial material on the bacterial cell wall in Example 1 of this application. Detailed Implementation

[0027] This application provides a hyaluronic acid-modified gold nanocomposite antibacterial material, its preparation method, and its application. To make the objectives, technical solutions, and effects of this application clearer and more explicit, the following provides a more detailed description. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0028] This application provides a method for preparing a hyaluronic acid-modified gold nanocomposite antibacterial material, comprising the following steps: (1) Dissolve hyaluronic acid (HA) in deionized water to prepare a hyaluronic acid solution. Add acetic acid and sophorolipid to the hyaluronic acid solution in sequence, mix thoroughly to obtain a mixed solution.

[0029] Acetic acid is used to adjust the pH of the system and play a stabilizing role, while sophorolipids help with the subsequent dispersion of nanoparticles.

[0030] Sophorolipids are produced by biodegradation, have excellent biocompatibility, extremely low toxicity, minimal skin irritation, and are very safe for oral administration.

[0031] The concentration of hyaluronic acid solution in the entire reaction system is 0.02~0.40 mol / L, preferably 0.10 mol / L.

[0032] Preferably, in the hyaluronic acid solution, the amount of hyaluronic acid (n) : volume of water (V) = 1 : 7 (mol / L).

[0033] Acetic acid addition: The volume ratio of acetic acid to hyaluronic acid solution is 1:100 to 1:500, preferably 1:200.

[0034] The amount of sophorolipid added: the molar ratio with hyaluronic acid is 1:10 to 1:30, preferably 1:20.

[0035] Sophorolipids mainly exist as lactones and unionized acidic sophorolipids. The special combination of sophorolipids and acetic acid in the system enables the hydrophobic part of the sophorolipid molecule to dominate. The strong hydrophobic effect can form smaller, denser and more stable micelle structures, which is beneficial for dispersing nanoparticles and making the obtained nanoparticles more stable.

[0036] (2) Add chloroauric acid (HAuCl4) solution to the mixed solution, place the mixed system at room temperature (25℃), and stir at room temperature to obtain a room temperature solution.

[0037] Maintaining the reaction system at room temperature during this step ensures the reducing power of the subsequent reducing agent, which is beneficial for controlling the nucleation of nanoparticles.

[0038] The chloroauric acid solution is obtained by dissolving chloroauric acid in deionized water and mixing thoroughly.

[0039] The concentration of HAuCl4 in the entire reaction system is 0.05~0.20 mol / L, preferably 0.10 mol / L.

[0040] Preferably, in the chloroauric acid (HAuCl4) solution, the amount of HAuCl4 (n) : volume of water (V) = 1:1 (mol / L).

[0041] Stirring speed: 500~900 rpm; optimal speed: 800 rpm.

[0042] Stirring time at room temperature: 5~30 minutes, preferably 20 minutes.

[0043] (3) Under vigorous stirring, a freshly prepared sodium cyanoborohydride (NaBH3CN) solution was slowly added dropwise to the room temperature solution. After the addition was complete, the stirring speed was reduced, and stirring was continued at room temperature for a period of time to allow the reaction to be complete and the Au-HA nanoparticles to be initially stabilized, thus obtaining the reaction solution.

[0044] The concentration of NaBH3CN solution in the entire reaction system is 0.01~0.30 mol / L, preferably 0.20 mol / L (it must be prepared and used immediately).

[0045] Preferably, in the NaBH3CN solution, the amount of NaBH3CN (n) : volume of water (V) = 1:1 (mol / L).

[0046] Vigorous stirring speed: 1000~3000 rpm, with 1500 rpm being the optimal speed.

[0047] Stirring at low speed at room temperature after dropwise addition: 5-10 hours, preferably 8 hours.

[0048] (4) The reaction solution is centrifuged at high speed and resuspended in deionized water to remove unreacted small molecule raw materials, by-products and excess reagents. After resuspension, the solution is filtered and sterilized to obtain a pure and sterile Au-HA nanocomposite aqueous dispersion, which is a gold nanocomposite antibacterial material modified with hyaluronic acid. The product should be stored at 4°C to maintain stability.

[0049] High-speed centrifugation speed: 12000~18000 rpm, preferably 15000 rpm. Centrifugation time: 10-30 minutes, preferably 20 minutes.

[0050] Sterilization method: Filtration sterilization is employed. A microporous membrane with a pore size of 0.22 μm is preferred for filtration sterilization.

[0051] This application also provides a gold nanocomposite antibacterial material based on hyaluronic acid modification. This hyaluronic acid-modified gold nanocomposite antibacterial material consists of gold nanoparticles as the core and hyaluronic acid molecules modified on the surface.

[0052] The average particle size of the gold nanoparticles ranges from 1 to 60 nm, preferably from 3 to 15 nm. Modification with hyaluronic acid imparts good water dispersibility, stability, and biocompatibility to the material, and may exert antibacterial effects through mechanisms such as disrupting bacterial cell walls / membranes.

[0053] This application also provides the application of hyaluronic acid-modified gold nanocomposite antibacterial materials in the preparation of drugs or related medical products for the treatment or prevention of bacterial infections.

[0054] Preferably, the bacterial infection includes, but is not limited to, infections caused by one or more of the following pathogens: Staphylococcus aureus; Escherichia coli; Methicillin-resistant Staphylococcus aureus (MRSA); Multidrug-resistant Escherichia coli (MDR E. coli).

[0055] The present application will be further described below through specific embodiments.

[0056] Example 1 A method for preparing a hyaluronic acid-modified gold nanocomposite antibacterial material includes the following steps: Dissolve hyaluronic acid (HA, 200 mg, 1.0 mmol) in 7 mL of deionized water, add 50 μL of acetic acid and 34 mg of sophorolipid and mix thoroughly. Then add HAuCl4 solution (410 mg, 1.0 mmol, pre-dissolved in 1 mL of deionized water) and stir at room temperature for about 20 minutes.

[0057] Next, while stirring vigorously at 1500 rpm, a sodium cyanoborohydride (NaBH3CN) solution (124 mg, 2.0 mmol, dissolved in 2 mL of deionized water, freshly prepared to avoid oxidation) was slowly added dropwise, and the solution slowly turned brown.

[0058] Then reduce the stirring speed (800 rpm) and continue stirring at room temperature for 8 hours to ensure the reduction reaction is complete, so that the nanoparticles tend to be stable and fully modified by HA.

[0059] After the reaction was complete, the solution was centrifuged for 20 minutes (15,000 rpm) using a high-speed centrifuge to remove the supernatant; it was then resuspended in 10 ml of deionized water, and the process was repeated twice. Finally, the solution was filtered through a 0.22 μm microporous membrane (Millipore) to achieve sterilization.

[0060] Collect the filtrate to obtain a pure and sterile Au-HA nanocomposite aqueous dispersion (a gold nanocomposite antibacterial material based on hyaluronic acid modification). Seal it and store it in a refrigerator at 4°C for later use.

[0061] Take a small sample (Au-HA nanocomposite aqueous dispersion) for TEM observation. Figure 1 The results showed that gold nanoparticles with uniform particle size and good dispersibility were successfully synthesized, with the main particle size distribution range being 3-15 nm.

[0062] Experimental Example 1 This experimental example illustrates the inhibitory effect of hyaluronic acid-modified gold nanocomposite antibacterial materials on bacteria.

[0063] This experiment used the micro-broth dilution method to evaluate the antibacterial efficacy of the hyaluronic acid-modified gold nanocomposite antibacterial material of Example 1 and to determine its minimum inhibitory concentration (MIC).

[0064] Disposable 96-well plates were used as the reaction vessel in the experiment. The specific procedures are as follows: The well plates were divided into groups, with the first well of each group set as the highest drug concentration group, with an initial volume of 200 μL. Each of the remaining wells was pre-filled with 100 μL of culture medium, and then a concentration gradient was established using a serial dilution method—100 μL of liquid was aspirated from the first well and added to the next well, mixing thoroughly until the drug concentration in the next well was reduced to half that of the first well; this process was repeated for each well, until the penultimate well was mixed and 100 μL was aspirated and discarded. The last well served as a negative control with a drug concentration of 0 μg / mL.

[0065] The test strains were cultured to the logarithmic growth phase, and the bacterial suspension concentration was adjusted to 10⁵ CFU / mL with fresh culture medium. 10 μL of bacterial suspension was added to each well. The plates were incubated at 37°C for 24 hours, with three replicates for each concentration to ensure experimental reliability. After incubation, bacterial growth was assessed by observing the turbidity of the liquid in the wells, and the MIC value was determined.

[0066] The experimental results are shown in Table 1.

[0067] Table 1. Inhibitory effect of hyaluronic acid-modified gold nanocomposite antibacterial materials on bacteria (unit: μg / mL)

[0068] Experimental Example 2 This experimental example illustrates the effect of hyaluronic acid-modified gold nanocomposite antibacterial materials on bacterial cell membrane permeability.

[0069] Bacterial cell membrane permeability experiment: First, logarithmically growing Escherichia coli was incubated with the hyaluronic acid-modified gold nanocomposite antibacterial material (4 μg / mL) from Example 1 in a culture medium for 3 hours.

[0070] Then, the bacterial cells are collected by centrifugation and washed with a buffer solution (such as PBS).

[0071] Next, SYTOX Green dye (1µM) was added to the bacterial suspension and incubated at room temperature in the dark for 15 minutes.

[0072] Finally, the stained bacterial solution was directly taken for observation under a fluorescence microscope.

[0073] At this point, bacteria with damaged cell membranes will emit bright green fluorescence as the dye enters and binds to their DNA. This can be visually assessed by comparing them with a negative control group that has not been treated with antibacterial materials.

[0074] The degree of change in membrane permeability, such as Figure 2 As shown.

[0075] Experimental Example 3 This experimental example illustrates the destructive effects of hyaluronic acid-modified gold nanocomposite antibacterial materials on cell walls.

[0076] Cell wall destruction experiment: Staphylococcus aureus and the hyaluronic acid-modified gold nanocomposite antibacterial material (8 μg / mL) of Example 1 were co-incubated in a culture medium for 3 hours.

[0077] Staphylococcus aureus PBS solution was obtained using the method described in the bacterial cell membrane permeability experiment of Example 2 above. Its microstructure was stabilized by double fixation with glutaraldehyde and osmium tetroxide.

[0078] Subsequently, for SEM, the sample needs to be dehydrated in a gradient, dried to maintain its natural morphology, and finally sprayed with a thin gold film under vacuum to enhance conductivity; while for TEM, which requires higher resolution, the sample needs to be embedded in resin for polymerization after dehydration, then cut into 50-100 nanometer thin slices using an ultrathin slicer and placed on a copper grid.

[0079] The results are as follows Figure 3 As shown, bacteria treated with nanomaterials all exhibited significant lysis.

[0080] In summary, hyaluronic acid-modified gold nanocomposite antibacterial materials can cause cell membrane structure damage, ultimately leading to bacterial cell lysis.

[0081] It should be understood that the application of this application is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of this application.

Claims

1. A method for preparing a gold nanocomposite antibacterial material based on hyaluronic acid modification, characterized in that, Includes the following steps: Hyaluronic acid is dissolved in water to obtain a hyaluronic acid solution. Acetic acid and sophorolipid are added to the hyaluronic acid solution to obtain a mixed solution. Add chloroauric acid solution to the mixed solution and stir to obtain a room temperature solution; Under vigorous stirring, sodium cyanoborohydride solution was added to the room temperature solution; After the sodium cyanoborohydride solution has been added, reduce the stirring speed and continue stirring at a low speed to obtain the reaction solution; The reaction solution is centrifuged at high speed, resuspended, filtered, and sterilized to obtain the gold nanocomposite antibacterial material based on hyaluronic acid modification.

2. The preparation method of the gold nanocomposite antibacterial material based on hyaluronic acid modification according to claim 1, characterized in that, The concentration of the hyaluronic acid solution in the entire reaction system is 0.02~0.40 mol / L; The amount of acetic acid added is in a volume ratio of 1:100 to 1:500 with the volume of the hyaluronic acid solution. The amount of sophorolipid added is in a molar ratio of 1:10 to 1:30 with that of hyaluronic acid.

3. The method for preparing the hyaluronic acid-modified gold nanocomposite antibacterial material according to claim 1, characterized in that, The concentration of chloroauric acid in the entire reaction system is 0.05~0.20 mol / L; After adding the chloroauric acid solution to the mixed solution, stir at 500-900 rpm for 5-30 minutes to obtain the room temperature solution.

4. The method for preparing the hyaluronic acid-modified gold nanocomposite antibacterial material according to claim 1, characterized in that, The concentration of the sodium cyanoborohydride solution in the entire reaction system is 0.01~0.30 mol / L.

5. The method for preparing the hyaluronic acid-modified gold nanocomposite antibacterial material according to claim 1, characterized in that, The speed of the vigorous stirring is 1000~3000 rpm; The low-speed stirring time is 5 to 10 hours, and the low-speed stirring speed is 500 to 900 rpm.

6. The method for preparing the hyaluronic acid-modified gold nanocomposite antibacterial material according to claim 1, characterized in that, The speed of the high-speed centrifuge is 12,000 to 18,000 rpm; the time of the high-speed centrifuge is 10 to 30 minutes.

7. A hyaluronic acid-modified gold nanocomposite antibacterial material prepared according to the preparation method of the hyaluronic acid-modified gold nanocomposite antibacterial material according to any one of claims 1-6, characterized in that, It includes gold nanoparticles as the core and hyaluronic acid molecules modified on the surface.

8. The gold nanocomposite antibacterial material based on hyaluronic acid modification according to claim 7, characterized in that, The average particle size of the gold nanoparticles ranges from 1 to 60 nm.

9. The use of a hyaluronic acid-modified gold nanocomposite antibacterial material according to any one of claims 7-8 in the preparation of a drug or related medical product for treating or preventing bacterial infections.

10. The application of the hyaluronic acid-modified gold nanocomposite antibacterial material according to claim 9 in the preparation of drugs or related medical products for treating or preventing bacterial infections, characterized in that, The bacterial infections include those caused by one or more pathogens such as Staphylococcus aureus, Escherichia coli, methicillin-resistant Staphylococcus aureus, and multidrug-resistant Escherichia coli.

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