Copper zinc gold doped nanoparticles, methods of making and antifungal applications thereof

Abstract

The present application relates to the technical field of antibacterial materials, and particularly relates to copper-zinc-gold doped nanoparticles, a preparation method thereof and antifungal application thereof.The copper-zinc-gold doped nanoparticles provided by the present application have positive charges on the surface, which can be combined with the fungal cell membrane through electrostatic adsorption, the motion performance thereof can help the nanoparticles to penetrate the biofilm, and in an acidic microenvironment, cinnamaldehyde and Cu 2+ are released, which can effectively induce the "copper death" of fungi.This death mechanism of "motion + drug / metal ion toxicity" can not only significantly enhance the removal ability of the biofilm, but more importantly, the multiple effects can help to reduce the risk of drug resistance of fungi.

Description

Technical Field

[0001] This invention relates to the field of antibacterial materials technology, and in particular to a copper-zinc-gold doped nanoparticle, its preparation method, and its antifungal applications. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Diseases caused by pathogenic fungal infections pose a serious challenge to clinical diagnosis and public health. Currently, the effectiveness of antifungal treatments is limited by multiple factors. The core issues are: the unique structure of fungal cell walls, containing components such as dextran, chitin, and mannose proteins, forms a dense network structure that acts as a physical barrier, hindering drug penetration and preventing drugs from effectively reaching their target sites; after fungi colonize host tissues or medical device surfaces, they can form biofilms that resist external attacks, weakening drug efficacy; and the long-term widespread use of traditional antifungal drugs has led to a continuous increase in drug resistance rates among common clinical pathogens such as Candida auris and Aspergillus fumigatus, posing a risk of failure to existing drug systems.

[0004] Therefore, there is an urgent need in the existing technology for a novel antifungal strategy that can effectively overcome the physical barriers of fungi, disintegrate biofilms, and deal with drug-resistant strains. Summary of the Invention

[0005] In view of this, the present invention provides copper-zinc-gold doped nanoparticles, their preparation method and antifungal applications.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing copper-zinc-gold doped nanoparticles, comprising the following steps:

[0008] (1) Dissolve a set amount of copper salt and zinc salt in an organic solvent to obtain a mixed solution of copper salt and zinc salt; dissolve a set amount of 2-aminobenzimidazole in an organic solvent to obtain a 2-aminobenzimidazole solution;

[0009] (2) Add 2-aminobenzimidazole solution to a mixed solution of copper salt and zinc salt, add L-arginine (L-Arg), and after the reaction is complete, copper-zinc-L-arginine composite nanoparticles (CZL particles) are obtained.

[0010] (3) The copper-zinc-L-arginine composite nanoparticles were dissolved in an organic solvent, mixed with solid paraffin, heated, emulsified, cooled and then dispersed in water. HAuCl4 solution was added and stirred, and then a reducing agent was added and stirred to react. After the reaction was completed, copper-zinc-gold-L-arginine composite nanoparticles (CZLA particles) were obtained.

[0011] (4) Disperse the copper-zinc-gold-L-arginine composite nanoparticles in an organic solvent, add cinnamaldehyde, stir, and let stand to obtain copper-zinc-gold doped nanoparticles (CZLAN particles).

[0012] Further, in step (1), the copper salt is a soluble copper salt; the soluble copper salt is copper nitrate or copper acetate.

[0013] Further, in step (1), the zinc salt is a soluble zinc salt; the soluble zinc salt is zinc nitrate or zinc acetate.

[0014] Furthermore, in step (1), the molar ratio of copper salt to zinc salt is 1:3-5.

[0015] Furthermore, in step (1), the organic solvent is methanol.

[0016] Furthermore, in step (2), the molar ratio of 2-aminobenzimidazole to zinc salt is 3-5:1.

[0017] Furthermore, in step (2), the molar ratio of L-arginine to 2-aminobenzimidazole is 1:15-20.

[0018] Furthermore, in step (2), after adding L-arginine, the mixture is stirred at room temperature for 0.5-2 h and then allowed to stand for 20-30 h. L-arginine, as a precursor of nitric oxide (NO), generates NO under the oxidation of hydrogen peroxide (H2O2), thereby providing gaseous power for the nanoparticles.

[0019] Furthermore, in step (2), after the reaction is complete, washing and drying operations are also included.

[0020] Furthermore, in step (3), the organic solvent is hexadecyltrimethylammonium bromide (CTAB); and ultrasonic-assisted dissolution is used.

[0021] Furthermore, in step (3), the temperature is heated to 75-85 ℃.

[0022] Furthermore, in step (3), the emulsification time is 10-30 min.

[0023] Further, in step (3), HAuCl4 solution is added and stirred for 0.5-2 h; the mass ratio of HAuCl4 to CZL particles is 1:6-8.

[0024] Further, in step (3), the reducing agent is sodium borohydride; the reduction reaction time is 10-30 min; and the molar ratio of sodium borohydride to HAuCl4 is 20-22:1. After the reducing agent is added and reacted, gold ions are reduced and deposited on the surface of CZL particles.

[0025] Furthermore, in step (4), the mass ratio of cinnamaldehyde to CZLA particles is 1:8-15. The aldehyde group of cinnamaldehyde is attached to the CZLA particles via the Schiff base reaction. Cinnamaldehyde interacts with lipids and proteins, thereby destroying the fungal cell wall and cell membrane.

[0026] Furthermore, in step (4), the mixture is stirred under an oxygen-free atmosphere at a temperature of 80-85 °C and left to stand for 20-30 h.

[0027] Furthermore, in step (4), after standing, centrifugation and ethanol washing are also included.

[0028] Secondly, the present invention provides copper-zinc-gold doped nanoparticles prepared by the method described in the first aspect.

[0029] The gold nanoparticles on the copper-zinc-gold doped nanoparticles (CZLAN particles) provided by this invention possess glucose oxidase-like properties, catalyzing the production of H₂O₂ and gluconic acid from glucose. On one hand, H₂O₂ oxidizes L-Arg to produce NO, providing the driving force for the nanoparticles to move, penetrate biomembranes, and enter fungal cells. On the other hand, the acidic environment formed by gluconic acid facilitates the breaking of imine bonds on the CZLAN particles and the release of cinnamaldehyde, while simultaneously releasing Cu. 2+ Cinnamaldehyde interacts with lipids and proteins, disrupting fungal cell walls and membranes, thus clearing a barrier for exogenous substances to enter the fungal interior; Cu 2+ It activates the copper death pathway in fungi to kill bacteria.

[0030] Thirdly, the present invention provides the application of the copper-zinc-gold doped nanoparticles described in the second aspect in antifungal activity.

[0031] Furthermore, the fungus is a pathogenic Candida, selected from one or more of Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida auris.

[0032] Furthermore, the pathogenic Candida is Candida albicans.

[0033] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0034] The gold nanoparticles in the copper-zinc-gold doped nanoparticles provided by this invention possess glucose oxidase-like properties, catalyzing the production of H₂O₂ and gluconic acid from glucose. H₂O₂ oxidizes L-Arg to produce NO, providing the driving force for the nanoparticles to move, penetrate biomembranes, and enter fungal cells. The acidic environment formed by gluconic acid facilitates the breaking of imine bonds on CZLAN and the release of cinnamaldehyde, while simultaneously releasing Cu. 2+ This invention endows nanoparticles with a powerful ability to efficiently eliminate fungi and their biofilms through multiple mechanisms, including the pharmacological activity of cinnamaldehyde, gas dynamic therapy, and copper death induction. This invention provides a novel antifungal strategy that can effectively overcome the physical barriers of fungi, disintegrate biofilms, and combat drug-resistant strains. Attached Figure Description

[0035] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0036] Figure 1 These are XRD patterns of the particles prepared according to the present invention; wherein, (A) is the XRD pattern of ZL and CZL particles; (B) is a magnified view of (A);

[0037] Figure 2 These are the infrared spectra of the CZ, CZL, and CZLAN particles of this invention;

[0038] Figure 3 This is a TEM image of the CZLAN particles of this invention;

[0039] Figure 4 This is a potential diagram of the ZL, CZL, CZLA, and CZLAN particles of the present invention;

[0040] Figure 5 These are contact angle test diagrams of ZL and CZLAN particles of the present invention, wherein (A) is the contact angle test diagram of ZL particles; and (B) is the contact angle test diagram of CZLAN particles.

[0041] Figure 6 This is a graph showing the residual amount of glucose after treatment with different concentrations of CZLAN particles.

[0042] Figure 7 This is a graph showing the change in pH value of a glucose solution after treatment with different particles.

[0043] Figure 8 This is a graph showing the NO release curves of CZLAN particles at different concentrations in the presence of glucose.

[0044] Figure 9 This is a diagram showing the trajectory of CZLAN particles in glucose solutions of different concentrations;

[0045] Figure 10 These are SEM images of Candida albicans treated with PBS and CZLAN particles; where (A) is the SEM image of Candida albicans treated with PBS; and (B) is the SEM image of Candida albicans treated with CZLAN particles.

[0046] Figure 11 These are fluorescence images of Candida albicans after incubation with PBS and CZLAN and staining with trypan blue; (A) is the fluorescence image of Candida albicans after incubation with PBS and staining with trypan blue; (B) is the fluorescence image of Candida albicans after incubation with CZLAN and staining with trypan blue.

[0047] Figure 12 This is a TEM elemental mapping of Candida albicans after treatment with PBS and CZLAN;

[0048] Figure 13 These are colony diagrams of Candida albicans from different treatment groups on Sabouraud agar plates.

[0049] Figure 14 These are confocal laser scanning images after 6 hours of co-incubation of nanoparticles with Candida albicans biofilm; (A) is PBS; (B) is CZAN; (C) is CZLAN.

[0050] Figure 15 This is a diagram showing the crystal violet staining of biofilms after treatment with different nanomaterials;

[0051] Figure 16 This is a graph showing the malondialdehyde (MDA) concentration after incubation of Candida albicans with CZLAN using a malondialdehyde (MDA) content detection kit. Detailed Implementation

[0052] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0053] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0054] Example 1

[0055] Cu(NO3)2·3H2O (357.6 mg) and Zn(NO3)2·6H2O (1761.1 mg) were dissolved in methanol (150 mL), and 2-aminobenzimidazole (3154 mg) was dissolved in 150 mL of methanol. Then, the 2-aminobenzimidazole solution was poured into the mixed solution of Cu(NO3)2·3H2O and Zn(NO3)2·6H2O with stirring, and L-arginine (254.5 mg) was added. The mixture was stirred at room temperature for 1 h and then allowed to stand for 24 h. The final product was collected and washed three times by centrifugation with methanol (10000 rpm, 5 min). Finally, the product was dried at 80 °C for 24 h to obtain CZL particles.

[0056] CZL particles (50 mg) were sonicated and dissolved in 10 mL of CTAB solution (1.25 mg / mL). The dispersion was mixed with 1.0 g of solid paraffin and heated to 80 °C. The mixture was then emulsified for 15 min using a high-speed homogenizer. To remove loose nanoparticles, the emulsion was cooled to room temperature, filtered, and washed with deionized water. The product was redispersed in 20 mL of deionized water, and 0.15 mL of HAuCl4 (126 mM) solution was added, followed by stirring for 60 min. The particles were then resuspended in 20 mL of deionized water. Subsequently, 4.0 mL of NaBH4 (100 mM) solution was added, and the reaction was stirred for 20 min. The product was filtered through filter paper and washed with pure water. After drying at 40 °C, it was dispersed in chloroform and briefly heated to dissolve the paraffin. After washing three times with chloroform, it was vacuum dried for 12 h to obtain CZLA particles.

[0057] 100 mg of CZLA nanoparticles were dispersed in 10 mL of ethanol, and 10 mg of cinnamaldehyde was added to remove oxygen. The mixture was then kept at 85 °C for 24 h under vigorous stirring. The nanoparticles were collected by centrifugation (10,000 rpm, 5 min) and washed three times with ethanol to obtain CZLA nanoparticles.

[0058] Comparative Example 1

[0059] Cu(NO3)2·3H2O (357.6 mg) and Zn(NO3)2·6H2O (1761.1 mg) were dissolved in 150 mL of methanol and stirred to form a pale blue solution. Then, an equal volume of methanol solution containing 2-aminobenzimidazole (3154 mg) was added dropwise to the above system, and the mixture was stirred for 30 min under N2 protection. After the addition of methanol, the mixture was centrifuged at 10000 rpm for 10 min to obtain CZ particles.

[0060] Comparative Example 2

[0061] Zn(NO3)2·6H2O (1761.1 mg) was dissolved in methanol (150 mL), and 2-aminobenzimidazole (3154 mg) was dissolved in methanol (150 mL). Then, the 2-aminobenzimidazole solution was poured into the Zn(NO3)2·6H2O solution with stirring, and L-arginine (254.5 mg) was added. The mixture was stirred at room temperature for 1 h and then allowed to stand for 24 h. The final product was collected and washed three times by centrifugation with methanol (10000 rpm, 5 min). Finally, the product was dried at 80 °C for 24 h to obtain ZL particles.

[0062] Example 2

[0063] 1. Fungal culture

[0064] Candida albicans was cultured in Sabouraud broth (40 g / L) at 31 °C with shaking at 180 rpm for 48 h to form colonies.

[0065] 2. Antifungal experiment with nanoparticles

[0066] When Candida albicans was in the logarithmic growth phase, it was cultured overnight in Sabouraud broth at 31 °C and 180 rpm. 200 μL of the Candida albicans suspension (10...) 7 CFU / mL), added to 200 μL PBS (phosphate buffer) or CZ or CZL or CZLA or CZLAN (20 μg / mL). -1 The suspension was incubated at 31 °C with shaking for 6 h. After serial dilution, 100 μL of the suspension was evenly spread on Sabouraud dextrose agar and incubated for 36 h. The colony count of each group was then analyzed.

[0067] 100 μL of CZLAN particles at different concentrations (0 μg / mL to 40 μg / mL) were mixed with 100 μL of Candida albicans suspension (10 μg / mL to 40 μg / mL to 10 μL ... 7 The mixture (CFU / mL) was incubated at 31 °C for 6 h, and then 100 μL of the suspension was evenly spread onto Sabouraud agar. The mixture was incubated at 31 °C for 36 h. The effects of different concentrations of CZLAN solution on colony count were observed by photographing, and the optimal inhibitory concentration of CZLAN particles was determined.

[0068] 3. Fluorescence imaging of live / dead fungi

[0069] 1 mL Candida albicans suspension (10 7Add 1 mL of PBS or CZ or CZL or CZLA or CZLAN (20 μg / mL) to the solution (CFU / mL) and incubate at 31 °C for 6 h. After washing three times by centrifugation (5000 rpm, 5 min) with PBS (1×), disperse the solution in 200 μL of sterile MOPS (3-morpholinopropanesulfonic acid) buffer, add 200 μL of staining agent SYTO 9 (SYTO 9 green fluorescent nucleic acid dye) (1:1500) and 200 μL of staining agent PI (propidium iodide) (1:1000), and incubate at 37 °C for 20 min. Wash three times by centrifugation (5000 rpm, 10 min) with sterile PBS (1×). Finally, concentrate the suspension by centrifugation to 50 μL of sterile PBS (1×) and capture fluorescence images using a fluorescence microscope.

[0070] 4. The anti-Candida albicans biofilm properties of nanoparticles

[0071] Add 1 mL / well of Candida albicans suspension to a 12-well plate (10 6 Incubate the biofilm in Sabouraud broth at 31 °C for 48 h. Then wash three times slowly with sterile PBS. Add 1 mL of PBS or CZ, CZL, CZLA, or CZLAN (20 μg / mL). Incubate at 31 °C for 6 h, remove the liquid, and wash three times slowly with sterile PBS. Add crystal violet (10 mg / mL, 200 μL / well), incubate at 31 °C for 15 min, and wash three times slowly with sterile PBS to obtain a crystal violet-stained biofilm. Observe the staining of the biofilm at the bottom of the wells.

[0072] 5. Effects of nanoparticles on the morphology of Candida albicans

[0073] 1 mL Candida albicans suspension (10 7 Add 1 mL of PBS or CZLAN (20 μg / mL) and incubate at 31 °C for 6 h. Wash three times with sterile PBS (5000 rpm, 10 min), add 10 μL of trypan blue dye (1 mg / mL), and incubate at 31 °C for 10 min. Wash three times with sterile PBS (5000 rpm, 10 min), and disperse in 100 μL of sterile PBS. Then capture fluorescence images using a fluorescence microscope.

[0074] 100 μL of Candida albicans suspension (10 7The *Candida albicans* suspension (CFU / mL) was mixed with 100 μL of CZLAN particles (20 μg / mL) and incubated at 31 °C for 4 h. 100 μL of the suspension was then washed three times by centrifugation (5000 rpm, 10 min) with sterile PBS. The lower layer was collected and incubated overnight at 4 °C in 100 μL of paraformaldehyde (4%). The mixture was then dehydrated for 5 min each time with 1 mL of ethanol at different concentrations (30%, 50%, 70%, 80%, 90%, 95%). Finally, the mixture was dehydrated twice more with 100 mL of anhydrous ethanol (5 min each time). The morphology of *Candida albicans* was observed using SEM.

[0075] Results and Discussion

[0076] 1. Characterizing nanoparticles

[0077] Figure 1 These are XRD patterns of the particles prepared according to the present invention; wherein, (A) is the XRD pattern of ZL and CZL particles; (B) is a magnified view of (A). As shown in the figure, Figure 1 (B) in the figure is a magnified view of the area within the dashed box in (A). As can be seen from the figure, the main diffraction peak of the nanoparticles shifts slightly to the left after copper ion doping. This is due to the difference in the radius between copper ions and zinc ions. Other diffraction peaks are almost unchanged, indicating that copper ions have little effect on the overall framework structure of the nanoparticles. Figure 2 The infrared spectra of the CZ, CZL, and CZLAN particles of this invention are shown in the figure, at 3360 cm⁻¹. -1 and 1640 cm -1 The peaks at 1640 cm⁻¹ are attributed to NH and CN bond vibrations, respectively, indicating that L-arginine was successfully doped into ZCL. Compared to the infrared spectra of ZCL, CZLAN shows a higher peak at 1640 cm⁻¹. -1 The peak value is significantly enhanced because the formation of imine bonds strengthens the peak value at this point. Figure 3 This is a TEM image of the CZLAN particles of the present invention. As shown in the figure, gold nanoparticles in CZLAN are dispersed on one side of CZLAN, indicating the successful synthesis of the asymmetric structure of CZLAN.

[0078] Measure the surface potential and contact angle of nanoparticles. Figure 4 The figure shows the potential diagrams of the ZL, CZL, CZLA, and CZLAN particles of this invention. The surface potential of ZL is -17.5 mV, and that of CZL is 19.3 mV. This is because the ionization energy of copper atoms is much higher than that of zinc atoms. 2+ It has more than Zn 2+Corrected redox potential. After loading gold nanoparticles and cinnamaldehyde, the positive potential of CZLAN increased slightly, which may be due to the spontaneous transfer of electrons from CZL to gold nanoparticles, which is macroscopically manifested as a positive shift in the measured potential. Figure 5 The figures show contact angle test diagrams of ZL and CZLAN particles of this invention, where (A) is the contact angle test diagram of ZL particles; and (B) is the contact angle test diagram of CZLAN particles. As shown in the figures, the contact angles of ZL and CZLAN are 48.7°±2.3° and 68.9°±2.4°, respectively, indicating that they are amphiphilic surfaces. The cell wall and cell membrane of Candida albicans are negatively charged and contain various hydrophilic and hydrophobic components. The positive potential and amphiphilic surface of CZLAN are beneficial to its interaction with fungi.

[0079] The ability of CZLAN to consume glucose was investigated. Different concentrations of CZLAN (0, 0.1, 0.5, 1.0 mg / mL) were incubated with glucose solution (12 mM) at room temperature for 40 min, followed by centrifugation at 10,000 rpm for 10 min. The glucose content in the supernatant was measured using a glucose kit. Figure 6 The figure shows the residual amount of glucose after treatment with different concentrations of CZLAN particles. As the CZLAN concentration increases, the glucose concentration in the supernatant gradually decreases. Figure 7 The graph shows the change in pH value of the solution after different particles treat glucose. As shown in the figure, PBS, CZL, CZLA, and CZLAN were incubated with glucose solution (12 mM). As the incubation time increased, the pH value of the supernatant of the CZLA group and the CZLAN group became acidic and gradually decreased. This is because the gluconic acid in the reaction product reduced the pH value of the solution, which laid the foundation for the breaking of imine bonds and the release of ions.

[0080] 2. Study the relationship between CZLAN nanoparticles and NO generation.

[0081] Figure 8 The figure shows the NO release curves of different concentrations of CZLAN particles in the presence of glucose. As shown in the figure, after different concentrations of CZLAN were incubated with glucose (12.0 mM), the amount of NO produced gradually increased with the increase of CZLAN concentration, indicating that CZLAN catalyzes the production of H2O2 from glucose, which is conducive to the generation of NO, and the reaction shows a positive correlation with concentration.

[0082] 3. Study the motion behavior of nanoparticles

[0083] The motion of CZLAN nanoparticles in glucose solutions of different concentrations (0, 1.0, 5.0 and 10.0 mg / mL) was investigated. Figure 9The figure shows the trajectory of CZLAN nanoparticles in glucose solutions of different concentrations. As the trajectory is observed, CZLAN nanoparticles exhibit significant Brownian motion in a 0 mg / mL glucose solution. With increasing glucose concentration, the distance covered by the CZLAN nanoparticles gradually increases. These phenomena are attributed to the CZLAN nanoparticles catalyzing the formation of H₂O₂ from glucose, which in turn oxidizes L-Arg to produce NO gas, propelling the nanoparticles' movement.

[0084] 4. Antibacterial properties

[0085] Figure 10 The images show SEM images of Candida albicans treated with PBS and CZLAN particles; (A) is the SEM image of Candida albicans treated with PBS; and (B) is the SEM image of Candida albicans treated with CZLAN particles. The changes on the surface of Candida albicans after treatment with PBS or CZLAN nanoparticles are compared, as shown in the figure. It can be seen from the figure that the surface wrinkles and collapses of Candida albicans treated with CZLAN nanoparticles increased significantly. To verify the destructive effect of cinnamaldehyde on the fungal cell wall, Candida albicans was co-incubated with CZLAN nanoparticles for 6 h. Figure 11 These are fluorescence images of *Candida albicans* after incubation with PBS and CZLAN followed by trypan blue staining; (A) shows the fluorescence image of *Candida albicans* after incubation with PBS followed by trypan blue staining; (B) shows the fluorescence image of *Candida albicans* after incubation with CZLAN followed by trypan blue staining. The images show that the green fluorescence signal in the CZLAN group was significantly weakened after trypan blue staining. This phenomenon indicates that the release of cinnamaldehyde successfully disrupted the integrity of the fungal cell wall. Figure 12 This is a TEM elemental mapping image of Candida albicans treated with PBS and CZLAN. Elemental analysis of the nanoparticle-treated Candida albicans was performed using an energy dispersive spectroscopy (EDS) instrument. The results showed that, compared with the PBS control group, the CZLAN-treated group exhibited a significant Cu element signal within the cells. This indicates that CZLAN nanoparticles can effectively enter the cell interior, successfully achieving the delivery and enrichment of Cu element in Candida albicans. Figure 13 The figure shows the colony diagrams of Candida albicans from different treatment groups on Sabouraud agar. The plate method was used to visually compare the bactericidal potential of 20 μg / mL ZL, CZL, CZLA, or CZLAN nanoparticles against Candida albicans, and it was found that CZLAN had the strongest bactericidal ability.

[0086] 5. Anti-biofilm properties

[0087] Effectively removing Candida albicans biofilm is key to disrupting the first line of defense against Candida albicans. Figure 14These are confocal laser scanning images after co-incubation of nanoparticles with *Candida albicans* biofilm for 6 h; (A) is PBS; (B) is CZAN; and (C) is CZLAN. As shown in the figure, the *Candida albicans* biofilm, stained with SYTO 9, exhibits strong green fluorescence in the PBS-treated biofilm, indicating that its biofilm structure is intact and dense, with high total amount and activity. In contrast, the green fluorescence intensity of the CZAN nanoparticle group is slightly decreased, indicating that it has a slight biofilm removal ability. The CZLAN nanoparticle group shows sporadic and weak green fluorescent spots, indicating that the biofilm thickness is also significantly reduced and the structure is severely damaged. These results strongly demonstrate that CZLAN nanoparticles have excellent anti-biofilm efficacy and can effectively remove mature fungal biofilms.

[0088] The effects of nanoparticles on the growth of Candida albicans biofilms were further evaluated using crystal violet staining. PBS or different nanoparticles were added at the initial stage of Candida albicans biofilm culture. After 48 h of culture, the Candida albicans cells themselves and the extracellular polymer matrix were stained with crystal violet to reflect the total biomass of the biofilm. Figure 15 The figures show the staining of crystal violet after treatment with different nanomaterials. As shown, compared to the PBS group, the ZL nanoparticle group exhibits a denser and more complete purple structure, indicating that its inhibitory effect on biofilms is limited. In contrast, the CZLAN nanoparticle group shows almost no visible purple adhesion, clearly revealing its strong biofilm scavenging ability.

[0089] 6. Mechanism of action of CZLAN nanoparticles with Candida albicans

[0090] Copper is an essential element for the growth and reproduction of fungi, and Candida albicans possesses a complex regulatory system to maintain intracellular copper levels. 2+ The steady state of Cu 2+ Excessive amounts can kill fungi. The accumulation of lipid peroxides (LPO) is a significant indicator of copper poisoning. Figure 16 This figure shows the malondialdehyde (MDA) concentration in *Candida albicans* after incubation with CZLAN, as determined using a malondialdehyde (MDA) content detection kit. As shown, the MDA level, a LPO decomposition product, significantly increased after CZLAN nanoparticle treatment, indicating that CZLAN nanoparticles induced a greater degree of lipid peroxidation. These results provide direct evidence of oxidative damage induced by CZLAN in *Candida albicans*.

[0091] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing copper-zinc-gold doped nanoparticles, characterized in that, Includes the following steps: (1) Dissolve a set amount of copper salt and zinc salt in an organic solvent to obtain a mixed solution of copper salt and zinc salt; dissolve a set amount of 2-aminobenzimidazole in an organic solvent to obtain a 2-aminobenzimidazole solution; (2) Add 2-aminobenzimidazole solution to a mixed solution of copper salt and zinc salt, add L-arginine, and after the reaction is complete, copper-zinc-L-arginine composite particles, namely CZL particles, are obtained. (3) Dissolve CZL particles in an organic solvent, mix with solid paraffin, heat, emulsify, cool and then disperse in water, add HAuCl4 solution and stir, then add reducing agent and stir to react. After the reaction is complete, copper zinc gold-L-arginine composite nanoparticles, namely CZLA particles, are obtained. (4) Disperse CZLA particles in an organic solvent, add cinnamaldehyde, stir, and let stand to obtain copper-zinc-gold doped nanoparticles. In step (1), the molar ratio of copper salt to zinc salt is 1:3-5; In step (2), the molar ratio of 2-aminobenzimidazole to zinc salt is 3-5:1; the molar ratio of L-arginine to 2-aminobenzimidazole is 1:15-20. In step (4), the mass ratio of cinnamaldehyde to CZLA particles is 1:8-15.

2. The preparation method according to claim 1, characterized in that, In step (1), the copper salt is a soluble copper salt; the soluble copper salt is copper nitrate or copper acetate.

3. The preparation method according to claim 1, characterized in that, In step (1), the zinc salt is a soluble zinc salt; the soluble zinc salt is zinc nitrate or zinc acetate; And / or, in step (1), the organic solvent is methanol.

4. The preparation method according to claim 1, characterized in that, In step (2), after adding L-arginine, stir at room temperature for 0.5-2 h and let stand for 20-30 h; And / or, in step (2), after the reaction is complete, washing and drying operations are also included.

5. The preparation method according to claim 1, characterized in that, In step (3), the temperature is heated to 75-85 ℃; And / or, in step (3), the emulsification time is 10-30 min.

6. The preparation method according to claim 1, characterized in that, In step (3), HAuCl4 solution is added and stirred for 0.5-2 hours; the mass ratio of HAuCl4 to CZL particles is 1:6-8. And / or, in step (3), the reducing agent is sodium borohydride; the reduction reaction time is 10-30 min; and the molar ratio of sodium borohydride to HAuCl4 is 20-22:

1.

7. The preparation method according to claim 1, characterized in that, In step (4), the mixture is stirred in an oxygen-free atmosphere at a temperature of 80-85 °C and left to stand for 20-30 h.

8. Copper-zinc-gold doped nanoparticles prepared by the method for preparing copper-zinc-gold doped nanoparticles according to any one of claims 1-7.

9. The application of the copper-zinc-gold doped nanoparticles as described in claim 8 in the preparation of antifungal drugs, characterized in that, The fungus is a pathogenic Candida, which is selected from one or more of Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida auris.

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