Methoxyphenol derivative modified gold nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to the technical field of antibacterial materials, in particular to gold nanoparticles modified by methoxyphenol derivatives as well as a preparation method and application of the gold nanoparticles. The gold nanoparticles modified by the methoxyphenol derivatives, provided by the invention, have a spectral antibacterial property, and have a good antibacterial effect on gram-positive bacteria and gram-negative bacteria. In addition, the gold nanoparticles modified by the methoxyphenol derivatives provided by the invention can inhibit efflux pump and transmembrane transport depending on proton dynamic potential, and a reverse inhibition effect on a multidrug resistance mechanism is formed. Pi-Pi and hydrogen bonds provided by the methoxyphenol aromatic skeleton are beneficial to multi-point weak combination with polysaccharide / protein in the extracellular polymeric substance, so that the viscoelasticity of a matrix network is weakened, the formation of a bacterial membrane is inhibited, and the depolymerization of the existing bacterial membrane is promoted.

Description

Technical Field

[0001] This invention relates to the field of antibacterial materials technology, specifically to gold nanoparticles modified with methoxyphenol derivatives, their preparation methods, and applications. Background Technology

[0002] The growing problem of bacterial resistance is diminishing the effectiveness of traditional antibiotics, leading to increased treatment costs and prolonged recovery periods. The surge in multidrug-resistant (MDR) bacterial infections globally poses a serious threat to public health, prompting researchers to seek innovative antimicrobial strategies.

[0003] The development of antibacterial drugs is currently based on two main aspects: the discovery of natural antibacterial components and the artificial synthesis of antibacterial drugs. The former requires extensive separation, purification, and analysis, involving numerous and complex steps, making widespread application difficult. The latter currently focuses on structural modification of existing drugs, but this can easily lead to bacterial resistance in a short period. Nanomaterials provide an excellent platform for antibacterial drug development, mainly due to the following characteristics: nanoparticles are similar in size to biomolecules, enabling them to effectively carry drugs into bacteria and interact with relevant targets; nanoparticles have a high specific surface area, which can significantly increase the effective concentration of the loaded drug through multivalent effects; and nanoparticles have active surface properties, allowing modification of various functional molecules to improve the physical properties of drugs and achieve targeted therapy.

[0004] Among them, gold nanomaterials (AuNPs) have attracted attention due to their unique physicochemical properties, possessing advantages such as high efficiency in preparation, good antibacterial properties, high biosafety, and low likelihood of inducing drug resistance. Preliminary research has preliminarily confirmed that AuNPs can disrupt cell wall structure, interfere with bacterial metabolism, and inhibit bacterial proliferation.

[0005] However, current gold nanomaterials have a narrow antibacterial spectrum, inhibiting only single Gram-positive or Gram-negative bacteria, lacking broad-spectrum activity; they cannot "reverse" bacterial resistance to antimicrobial drugs; and they are difficult to inhibit bacterial biofilm formation or promote the depolymerization of existing biofilms. Summary of the Invention

[0006] To overcome the above problems, this invention provides gold nanoparticles modified with methoxyphenol derivatives, their preparation methods, and applications.

[0007] To achieve the above technical objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides gold nanoparticles modified with methoxyphenol derivatives, wherein the gold nanoparticles are coated with methoxyphenol derivative ligands, and the gold nanoparticles and the methoxyphenol derivative ligands are connected by gold-sulfur bonds. The structural formula of the methoxyphenol derivative ligand is shown in formula (Ⅰ) below. ; Equation (I); Wherein, R1 is selected from one of the following formulas (II) or (III): ; Formula (II); ; Formula (Ⅲ); R2 is selected from one of the following formulas: (Ⅳ), (Ⅴ), or (Ⅵ): ; Formula (Ⅳ); ; Formula (V); ; Formula (VI).

[0008] In one or more embodiments, the methoxyphenol derivative-modified gold nanoparticles are spherical with a particle size of 5-6 nm.

[0009] A second aspect of the present invention provides a method for preparing gold nanoparticles modified with methoxyphenol derivatives as described in the first aspect, comprising the following steps: Methoxyphenol derivative ligands were dissolved in an organic solvent, sodium citrate was added, and the mixture was stirred until homogeneous. Then, gold salt was added and the mixture was stirred until homogeneous again. Subsequently, sodium borohydride was added, the reaction was stirred, and the solid was collected to obtain gold nanoparticles modified with methoxyphenol derivatives.

[0010] In one or more embodiments, the organic solvent is selected from N,N-dimethylformamide (DMF).

[0011] In one or more embodiments, the concentration of the methoxyphenol derivative ligand in the organic solvent is 2.5~4 mmol / L, preferably 3.2 mmol / L.

[0012] In one or more embodiments, the gold salt is selected from chloroauric acid.

[0013] In one or more embodiments, the molar ratio of the methoxyphenol derivative ligand to the gold salt is (0.8~1.2):(0.8~1.2), preferably 1:1.

[0014] In one or more embodiments, the molar ratio of gold salt to sodium borohydride is 1:(2.5~4).

[0015] In one or more embodiments, the concentration of sodium citrate in the organic solvent is 2.5 to 4 mmol / L, preferably 3.2 mmol / L.

[0016] In one or more embodiments, the preparation method of the methoxyphenol derivative ligand includes the following steps: Compound 1 was reacted with N-hydroxysuccinimide (NHS) to synthesize compound 2; Compound 2 reacts with 1,6-hexanediamine to synthesize compound 3; Compound 3 reacts with compounds 4, 5 and 6 to synthesize methoxyphenol derivative ligands; The structural formula of compound 1 is shown below: ; The structural formula of compound 2 is shown below: ; The structural formula of compound 3 is shown below: ; The structural formula of compound 4 is shown below: ; The structural formula of compound 5 is shown below: ; The structural formula of compound 6 is shown below: ; In compounds 1 and 2, R1 is selected from one of formulas (II) or (III): ; Formula (II); ; Formula (Ⅲ); In compound 5, R2 is selected from one of the following formulas: (Ⅳ), (Ⅴ), or (Ⅵ): ; Formula (Ⅳ); ; Formula (V); ; Formula (VI).

[0017] Preferably, the method for synthesizing compound 2 by reacting compound 1 with N-hydroxysuccinimide (NHS) specifically includes: Compound 1 and N-hydroxysuccinimide were dispersed in dichloromethane, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) was added as a condensing agent to synthesize compound 2.

[0018] More preferably, the molar ratio of compound 1, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:(1~1.5):(1~1.5).

[0019] More preferably, the reaction temperature is 0~4 ℃.

[0020] Preferably, the method for synthesizing compound 3 by reacting compound 2 with 1,6-hexanediamine specifically includes: The condensing agent triethylenediamine (DABCO) was dispersed in dichloromethane, and then 1,6-hexanediamine was added. After mixing evenly, compound 2 was added to react and synthesize compound 3.

[0021] More preferably, the molar ratio of the condensing agent triethylenediamine (DABCO), 1,6-hexanediamine and compound 2 is 3:(6~8):2.

[0022] More preferably, the reaction temperature is 0~4 ℃.

[0023] Preferably, the method for synthesizing methoxyphenol derivative ligands by reacting compound 3 with compounds 4, 5, and 6 specifically includes: Compound 3 was dispersed in methanol along with compounds 4, 5 and 6, and the reaction yielded methoxyphenol derivative ligands.

[0024] More preferably, the molar ratio of compound 3 to compounds 4, 5 and 6 is (0.8~1.2):(0.8~1.2):(0.8~1.2):(0.8~1.2).

[0025] More preferably, the reaction temperature is 40~50 °C and the reaction time is 40~60 h.

[0026] A third aspect of the present invention provides the application of gold nanoparticles modified with methoxyphenol derivatives as described in the first aspect or gold nanoparticles modified with methoxyphenol derivatives prepared by the preparation method described in the second aspect in the preparation of antibacterial products.

[0027] In one or more embodiments, the antimicrobial product includes an antimicrobial drug.

[0028] In one or more embodiments, the target bacteria of the antimicrobial product include Gram-positive bacteria or Gram-negative bacteria.

[0029] A fourth aspect of the present invention provides an antibacterial product comprising gold nanoparticles modified with methoxyphenol derivatives as described in the first aspect or gold nanoparticles modified with methoxyphenol derivatives prepared by the preparation method described in the second aspect.

[0030] In one or more embodiments, the antimicrobial product includes an antimicrobial drug.

[0031] In one or more embodiments, the target bacteria of the antimicrobial product include Gram-positive bacteria or Gram-negative bacteria.

[0032] The beneficial effects of this invention are as follows: The methoxyphenol derivative-modified gold nanoparticles provided by this invention exhibit broad-spectrum antibacterial activity, demonstrating excellent antibacterial effects against both Gram-positive bacteria (such as Staphylococcus aureus) and Gram-negative bacteria (such as Escherichia coli). The increased surface hydrophobicity and hydrogen bond donor (HBD) density of the methoxyphenol derivative-modified gold nanoparticles enhance weak interactions with acyl chains / head groups, promoting hydrophobic insertion and local dissemination of the outer membrane / cell membrane. Against Gram-negative bacteria, the weakened outer membrane barrier further improves accessibility to intracellular targets. Furthermore, the microscopic proton-coupled electron transfer (PCET) involving the phenolic hydroxyl groups in methoxyphenol derivative ligands can alter local proton transport and electron flow on the membrane surface. This is manifested as an increase in depolarization in the determination of bacterial membrane potential depolarization effect by fluorescent probes 3,3'-dipropylthiocyanine iodide (DiSC3(5)) or bis(1,3-dibarbituric acid)-trimethyloxenolol (DiBAC4(3)), thereby inhibiting proton-dependent efflux pumps and transmembrane transport, forming a "reverse inhibition" effect against multidrug resistance mechanisms. The π-π and hydrogen bonds provided by the aromatic skeleton of methoxyphenol derivative ligands facilitate multi-point weak binding with polysaccharides / proteins in extracellular polymers (EPS), weakening the viscoelasticity of the matrix network; inhibiting bacterial membrane formation and promoting the depolymerization of existing bacterial membranes. Attached Figure Description

[0033] 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.

[0034] Figure 1 A method for preparing ligands of methoxyphenol derivatives; Figure 2 The hydrogen NMR spectrum of GNP-1; Figure 3 The hydrogen NMR spectrum of GNP-2; Figure 4 The hydrogen NMR spectrum of GNP-3; Figure 5The hydrogen NMR spectrum of GNP-4; Figure 6 The hydrogen NMR spectrum of GNP-5; Figure 7 The hydrogen NMR spectrum of GNP-6; Figure 8 Transmission electron microscope images of gold nanoparticles (GNP-1-Au~GNP-6-Au) modified with methoxyphenol derivatives; where a is GNP-1-Au, b is GNP-2-Au, c is GNP-3-Au, d is GNP-4-Au, e is GNP-5-Au, and f is GNP-6-Au. Figure 9 Dynamic light scattering particle size analysis of gold nanoparticles (GNP-1-Au~GNP-6-Au) modified with methoxyphenol derivatives; Figure 10 The surface Zeta potential of gold nanoparticles (GNP-1-Au~GNP-6-Au) modified with methoxyphenol derivatives; Figure 11 Hydrophilicity and hydrophobicity analysis of the surface of gold nanoparticles (GNP-1-Au~GNP-6-Au) modified with methoxyphenol derivatives; Figure 12 Minimum inhibitory concentration (MIC) of gold nanoparticles modified with methoxyphenol derivatives (GNP-1-Au~GNP-6-Au). Figure 13 Minimum bactericidal concentration (MBC) of gold nanoparticles modified with methoxyphenol derivatives (GNP-1-Au~GNP-6-Au). Figure 14 The results of measuring the effect of propidium iodide (PI) probe uptake on bacterial membrane integrity by methoxyphenol derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au); Figure 15 The results of extracellular adenosine triphosphate (ATP) release after treatment with gold nanoparticles modified with methoxyphenol derivatives (GNP-1-Au~GNP-6-Au); Figure 16 Results of bacterial membrane potential depolarization effect measured using fluorescent probes; Figure 17 The results show the effects of methoxyphenol derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) on proton-dependent dynamic potential (PMF) efflux pumps and transmembrane transport. Figure 18 Gold nanoparticles modified with methoxyphenol derivatives (GNP-1-Au~GNP-6-Au) are effective against Escherichia coli (E. coli). E. coli ) and Staphylococcus aureus ( S. aureus Inhibitory effect on biofilm formation; Figure 19 Results of changes in total sugar content in extracellular polymeric substances (EPS) of biomembranes after treatment with gold nanoparticles modified with methoxyphenol derivatives (GNP-1-Au~GNP-6-Au). Detailed Implementation

[0035] 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 in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0037] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0038] Example 1 Figure 1 For the preparation method of methoxyphenol derivative ligands, refer to Figure 1 The methoxyphenol derivative ligands shown in formula (Ⅰ) are GNP-1 to GNP-6.

[0039] The structural formulas of GNP-1 to GNP-6 are shown in Table 1 below.

[0040] Table 1. Structural Formulas of GNP-1 to GNP-6

[0041] Specifically: The structural formula of GNP-1 is: ; The structural formula for GNP-2 is: ; The structural formula of GNP-3 is: ; The structural formula for GNP-4 is: ; The structural formula for GNP-5 is: ; The structural formula for GNP-6 is: .

[0042] Under ice bath conditions, 0.05 mol of 4-carboxypyridine was dispersed in 100 mL of dichloromethane (DCM). After mixing thoroughly, 0.06 mol of N-hydroxysuccinimide (NHS) and 0.06 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) were added while stirring. The reaction was maintained under ice bath conditions. The reaction was monitored by thin-layer chromatography (TLC). After 6 h of reaction, the solution gradually became clear. The reaction was then stopped, filtered, and a dichloromethane solution of compound 2 was obtained, which was directly used in the next step of the reaction.

[0043] Under ice bath conditions, 0.075 mol of the condensing agent triethylenediamine (DABCO) was dispersed in 50 mL of dichloromethane (DCM). After thorough mixing, 24.6 mL of 1,6-hexanediamine was added while stirring. Once the reaction system was homogeneous and stable, a dichloromethane solution of compound 2 was slowly added dropwise to the reaction system over a period of 2 h. The reaction was continued for 24 h, during which the reaction system gradually thickened. The reaction was monitored by TLC. After the reaction was completed, the solvent was removed by rotary evaporation, and the crude product was repeatedly washed with water. The crude product was then purified by column chromatography using 300-mesh silica gel with a dichloromethane (DCM):ethyl acetate (EA) mixture of 1:1 (volume ratio) as the developing solvent, yielding an orange-red solid product, compound 3.

[0044] 5 mmol of compound 3, 5 mmol of vanillin, 5 mmol of compound 4, and 5 mmol of compound 6 were dispersed in 10 mL of methanol, mixed thoroughly, and reacted at 320 K for 48 h. The reaction was monitored by TLC. After the reaction was completed, most of the solvent was removed by rotary evaporation, and the mixture was purified by column chromatography using 300-mesh silica gel. The developing solvent was a 1:1 (volume ratio) mixture of petroleum ether and ethyl acetate (EA); GNP-1 was obtained (yield 63.79%).

[0045] The hydrogen NMR spectrum of GNP-1 is as follows: Figure 2 As shown.

[0046] The preparation methods for GNP-2 to GNP-6 are the same as those for GNP-1, the difference being the substitution of different substituents R1 or R2.

[0047] GNP-2 is a pale yellow powder with a yield of 45.7%; the 1H NMR spectrum of GNP-2 is shown below. Figure 3 As shown.

[0048] GNP-3 is a pale yellow powder with a yield of 33.8%; the 1H NMR spectrum of GNP-3 is shown below. Figure 4 As shown.

[0049] GNP-4 is a pale yellow powder with a yield of 55.2%; the 1H NMR spectrum of GNP-4 is shown below. Figure 5 As shown.

[0050] GNP-5 is a pale yellow powder with a yield of 45.1%; the 1H NMR spectrum of GNP-5 is shown below. Figure 6 As shown.

[0051] GNP-6 is a pale yellow powder with a yield of 50.2%; the 1H NMR spectrum of GNP-6 is shown below. Figure 7 As shown.

[0052] Example 2 Preparation of gold nanoparticles modified with methoxyphenol derivatives (GNP-1-Au~GNP-6-Au): Disperse 0.19 mmol sodium borohydride in 10 mL of deionized water to obtain an aqueous solution of sodium borohydride; 0.064 mmol of methoxyphenol derivative ligands (GNP-1~GNP-6) were dissolved in 20 mL of DMF, 0.064 mmol of trisodium citrate was added and the mixture was stirred at room temperature for 30 min, followed by the addition of 0.064 mmol of chloroauric acid and stirring at room temperature for another 30 min. Sodium borohydride aqueous solution was then added dropwise, and the mixture was stirred overnight at room temperature. The methoxyphenol derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) were obtained by centrifugation (15000 rad / min).

[0053] Figure 8 Transmission electron microscopy images of gold nanoparticles (GNP-1-Au~GNP-6-Au) modified with methoxyphenol derivatives, from... Figure 8 As can be seen, the gold nanoparticles modified with methoxyphenol derivatives (GNP-1-Au~GNP-6-Au) are approximately spherical with an average particle size of 5~6 nm.

[0054] Figure 9 Dynamic light scattering particle size analysis of gold nanoparticles (GNP-1-Au~GNP-6-Au) modified with methoxyphenol derivatives showed that the dynamic hydrated particle size of the gold nanoparticles (GNP-1-Au~GNP-6-Au) was approximately 75~91 nm.

[0055] Figure 10 The surface Zeta potential of gold nanoparticles (GNP-1-Au~GNP-6-Au) modified with methoxyphenol derivatives is derived from... Figure 10As can be seen, the surfaces of the gold nanoparticles (GNP-1-Au~GNP-6-Au) modified with methoxyphenol derivatives are all negatively charged, with a Zeta potential of approximately -7 to +2 mV.

[0056] Example 3 Hydrophilicity and hydrophobicity analysis of the surface of gold nanoparticles (GNP-1-Au~GNP-6-Au) modified with methoxyphenol derivatives: Mix equal volumes of deionized water and n-octanol solution and stir for 24 h; allow the mixture to stand until it separates into layers. The upper layer is a water-saturated n-octanol solution and the lower layer is a water-saturated aqueous solution of n-octanol. Separate the two phases and store them separately for later use.

[0057] Add a certain volume of saturated aqueous solution of n-octanol, then add 0.3 mg of gold nanoparticles modified with methoxyphenol derivatives (GNP-1-Au~GNP-6-Au) to a total volume of 1 mL. After mixing by inverting the container, add an equal volume of 1 mL of water-saturated n-octanol solution and shake on a shaker for 24 h.

[0058] After shaking, remove the centrifuge tubes and allow them to stand for a period of time until the separation boundary between the two phases is clear. Use a pipette to take a certain volume of the two-phase solution and transfer it to different 10 mL colorimetric tubes. Dry at 120 °C under vacuum for 4 h until the solution has completely evaporated. Add 500 μL of freshly prepared aqua regia to the colorimetric tubes, digest for 12 h, and then add high-purity water to make up to 10 mL.

[0059] Preparation of gold standard curves: A series of gold standard solutions with concentration gradients were prepared using a gold standard solution (1000 ppm), as shown in Table 2 below. The gold content in the n-octanol and aqueous phases was detected using inductively coupled plasma mass spectrometry (ICP-MS). The obtained concentrations were substituted into the following formula to calculate the octanol-water partition coefficient of the methoxyphenol derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au).

[0060] Log P = Log(C O / C W ); Where Log P is the n-octanol-water partition coefficient, C O C represents the concentration of gold in the n-octanol phase. W This represents the concentration of gold in the aqueous phase.

[0061] Table 2. Standard Working Curve Preparation Method

[0062] Figure 11The hydrophilicity / hydrophobicity analysis results of the surface of gold nanoparticles (GNP-1-Au~GNP-6-Au) modified with methoxyphenol derivatives are as follows: Figure 11 As can be seen, the n-octanol-water partition coefficient (Log P) of gold nanoparticles modified with methoxyphenol derivatives (GNP-1-Au~GNP-6-Au) varies significantly among different ligand structures, indicating that the hydrophilicity and hydrophobicity of the particle surface can be controlled by changing the substituent combination of the methoxyphenol derivative ligands. Among them, the samples with higher Log P indicate that their surface is more hydrophobic, which is more conducive to multi-point weak interactions with bacterial membrane acyl chains or head groups and promotes membrane insertion, providing an interfacial property basis for the subsequent differences in antibacterial / bactericidal abilities.

[0063] Example 4 Determination of the minimum inhibitory concentration (MIC) of methoxyphenol derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au): Methoxyphenol derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) (dispersed in pure water or phosphate-buffered saline (PBS) buffer, 1 mg / mL). Culture medium: cationic-regulated Mueller-Hinton broth (CA-MHB). Strains: *Escherichia coli* (… E. coli ) and Staphylococcus aureus ( S. aureus Positive control: Ciprofloxacin (CIP, used for...) E. coli ) and vancomycin (VAN, used for S. aureus Prepare fresh working solution according to the instructions. Negative control: culture medium + bacteria (without drugs); blank control: culture medium (without inoculation). Pick a single colony and inoculate it into CA-MHB, incubate overnight at 35 °C; dilute to an initial inoculation volume of 5 × 10⁻⁶. 5 CFU / mL; add an equal volume of bacterial suspension (100 µL) to each well, ensuring a final volume of 200 µL after drug addition. Incubate at 35±2 °C for 20 h. S. aureus (Up to 24 h); endpoint OD is obtained using a microplate reader. Negative / blank controls are used to confirm "growth / no contamination" and do not generate MIC values. ≥3 independent replicates; geometric mean of MICs for each sample across multiple strains is taken; results are as follows... Figure 12 As shown, from Figure 12 As can be seen, methoxyphenol derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) are effective against Gram-negative bacteria (GNP-1-Au~GNP-6-Au). E. coli ) and Gram-positive bacteria ( S. aureusAll samples exhibited measurable minimum inhibitory concentrations (MICs), indicating that the methoxyphenol derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) have broad-spectrum antibacterial activity. The MICs of different samples varied, suggesting that the ligand structure can significantly affect their antibacterial efficacy, with samples having lower MICs exhibiting stronger antibacterial activity.

[0064] Example 5 Determination of the minimum bactericidal concentration (MBC) of methoxyphenol derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au): The reagents and materials were the same as in Example 4 (sample / culture medium / strain / control), except for tryptone soy agar (TSA) plates. Following Example 5, the MIC test was performed to the endpoint reading. At the MIC endpoint, 10 µL was taken from each well and serially diluted 10-fold with drug-free fresh culture medium. The diluted samples were then plated onto tryptone soy agar (TSA) plates and incubated overnight at 35 ± 2 °C. Plates with 100 CFU were counted, and the original well viable cell count was calculated; the negative control endpoint CFU was used as the baseline. MBC determination: defined as the lowest concentration that resulted in ≥99.9% relative kill rate compared to the negative control. ≥3 independent replicates were performed; results are as follows. Figure 13 As shown, from Figure 13 As can be seen, methoxyphenol derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) have the effect of... E. coli and S. aureus All samples yielded a corresponding minimum bactericidal concentration (MBC), indicating that the methoxyphenol derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) not only inhibit bacterial growth but also achieve bactericidal effects within a certain concentration range; the MBC sizes varied among different samples, and were consistent with... Figure 12 The corresponding trend of MIC shows that the difference in interfacial properties caused by the ligand structure further determines the effective concentration threshold for its transformation from "bacteriostasis" to "bactericidal", providing functional endpoint support for subsequent mechanisms such as membrane integrity disruption, ATP leakage, membrane potential depolarization, and efflux pump inhibition.

[0065] Example 6 The effect of propidium iodide (PI) probe uptake on bacterial membrane integrity of methoxyphenol derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au): E. coli ( E. coli ) and Staphylococcus aureus ( S. aureus Single colonies were inoculated into CA-MHB and cultured at 35±2 ℃ with shaking until the logarithmic growth phase (OD200). 600 (Approximately 0.3~0.6), diluted to approximately 1×10⁻⁶ using sterile PBS buffer.7 CFU / mL. Methoxyphenol-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) were added to the bacterial culture at a preset concentration (1×MIC) and incubated at 37 °C for 60 min. Propidium iodide (PI) probe was then added to a final concentration of 8 μg / mL and incubated in the dark for 15 min. The PI signal (Ex / Em approximately 535 / 617 nm) was detected using a fluorescent microplate reader, with unmodified gold nanoparticles (GNP-1-Au~GNP-6-Au) serving as a negative control. Relative uptake was calculated by normalizing to 1. The assay was repeated at least three times, and results are expressed as mean ± standard error (SEM). Results are as follows: Figure 14 As shown, compared with the control, the relative PI uptake rate of the gold nanoparticles modified with methoxyphenol derivatives (GNP-1-Au~GNP-6-Au) increased, with the GNP-4-Au treatment group showing a more significant effect, suggesting that it has a strong effect on disrupting membrane integrity / enhancing permeability of the two types of bacteria.

[0066] Example 7 Measurement of membrane flux changes by extracellular adenosine triphosphate (ATP) release: E. coli ( E. coli ) and Staphylococcus aureus ( S. aureus Single colonies were inoculated into CA-MHB and cultured at 35±2 ℃ with shaking until the logarithmic growth phase (OD200). 600 (Approximately 0.3~0.6), diluted to approximately 1×10⁻⁶ using sterile PBS buffer. 7 CFU / mL. Methoxyphenol derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) were added to the bacterial culture at a preset concentration (1×MIC) and incubated at 37 °C for 60 min. After the reaction, the supernatant was collected by centrifugation (10000 g, 4 min) at 4 °C for quantitative detection of extracellular ATP. A commercially available ATP bioluminescence detection kit (luciferase-luciferin system) was used, and the reaction system was prepared according to the instructions. The luminescence intensity (RLU) was recorded. The negative control (without sample) was normalized to 1, and the relative release rate of extracellular ATP in each treatment group was calculated and plotted as mean ± SEM (n≥3). The results are shown below. Figure 15 As shown, the treatment groups of gold nanoparticles modified with methoxyphenol derivatives (GNP-1-Au~GNP-6-Au) can significantly increase the extracellular ATP release level. Among them, the GNP-4-Au treatment group has a higher overall release level, which is consistent with its trend of enhancing membrane permeability / disrupting membrane structure.

[0067] Example 8 Fluorescent probes are used to measure the depolarization effect of bacterial membrane potential. E. coli ( E. coli ) and Staphylococcus aureus ( S. aureus Single colonies were inoculated into CA-MHB and cultured at 35±2 ℃ with shaking until the logarithmic growth phase (OD200). 600 (Approximately 0.3~0.6), diluted to approximately 1×10⁻⁶ using sterile PBS buffer. 7 CFU / mL. Add fluorescent probes 3,3'-dipropylthiocyanate dicarbonyl iodide (DiSC3(5)) or bis(1,3-dibarbituric acid)-trimethyloxenolol (DiBAC4(3)), and incubate in the dark for 20 min to allow the fluorescent probes to reach a steady-state distribution; if necessary, 0.1 M KCl can be added to stabilize the baseline. Then, add methoxyphenol derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) (concentration 1×MIC), and monitor fluorescence changes in real time (DiBAC4(3) Ex / Em: 622 / 670 nm, DiSC3(5) Ex / Em: 492 / 515 nm). A negative control was set up for quality control; fluorescence changes were normalized to the control group to obtain the relative depolarization rate, and a bar chart was plotted. Results are as follows: Figure 16 As shown, the treatment groups of gold nanoparticles modified with methoxyphenol derivatives (GNP-1-Au~GNP-6-Au) can induce different degrees of membrane depolarization. Among them, the depolarization signal of the GNP-4-Au treatment group is more prominent, indicating that its interference with membrane potential and proton dynamic potential is more obvious.

[0068] Example 9 Ethidium bromide / Nile red accumulation experiment assessment of proton-dependent efflux pump suppression: E. coli ( E. coli ) and Staphylococcus aureus ( S. aureus Single colonies were inoculated into CA-MHB and cultured at 35±2 ℃ with shaking until the logarithmic growth phase (OD200). 600 (Approximately 0.3~0.6), diluted to approximately 1×10⁻⁶ using sterile PBS buffer. 7 CFU / mL. Ethidium bromide (final concentration 2 μg / mL) or Nile red (final concentration 2 μM) was added as efflux pump substrate probes; after incubation at 37 °C for 15 min, methoxyphenol derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) (concentration 1×MIC) were added, and incubation continued for 20 min. Intracellular fluorescence accumulation was recorded using a fluorescence microplate reader (ethidium bromide Ex / Em 530 / 600 nm, Nile red Ex / Em 552 / 636 nm). The fluorescence signals of each methoxyphenol derivative-modified gold nanoparticle (GNP-1-Au~GNP-6-Au) treatment group were normalized to 1 with respect to the control group to obtain the relative fluorescence accumulation rate. The results are as follows. Figure 17As shown, treatment with gold nanoparticles modified with methoxyphenol derivatives (GNP-1-Au~GNP-6-Au) can increase the accumulation of probes in bacteria, suggesting that they have a certain inhibitory effect on proton-dependent efflux pumps and transmembrane transport. The accumulation enhancement is more obvious in the GNP-4-Au treatment group.

[0069] Example 10 The inhibitory effect of methoxyphenol derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) on biofilm formation was determined by crystal violet (CV) method: Escherichia coli (E. coli) was inoculated into 96-well plates. E. coli ) and Staphylococcus aureus ( S. aureus (Initial vaccination dose 1×10) 7 CFU / mL was added to Luria-Bertani medium. Simultaneously, methoxyphenol-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) were added at a sub-inhibitory concentration (approximately 1 / 2 × MIC) to avoid excessive interference with the growth of planktonic bacteria. After static incubation for 24 h to form a biofilm, the supernatant was discarded, and the cells were gently washed three times with PBS buffer to remove unattached cells. The cells were stained with 0.1 wt% crystal violet for 15 min, and after washing away the free dye, the bound dye was dissolved in 30% ethanol (v / v) for OD measurement. 570 The relative biofilm biomass of the gold nanoparticles modified with methoxyphenol derivatives (GNP-1-Au~GNP-6-Au) was obtained and plotted as a bar chart after normalization to 1 with the negative control as the base. Results are as follows: Figure 18 As shown, gold nanoparticles modified with different methoxyphenol derivatives (GNP-1-Au~GNP-6-Au) showed different degrees of inhibition on biofilm formation in the two types of bacteria. Among them, the biofilm biomass of the GNP-4-Au treatment group decreased more significantly, indicating that it has stronger anti-biofilm potential.

[0070] Example 11 The effect of methoxyphenol derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) on extracellular matrix formation was evaluated by determining the total sugar content in extracellular polymeric substances (EPS). After constructing the biofilm as shown in Example 10, the biofilm from each well was collected and resuspended in PBS buffer. Extracellular polymeric substances (EPS) were released into the solution by vortexing (1000 rpm, 1 min). Cell debris was then removed by low-speed centrifugation (1000 g, 5 min), and the supernatant was retained as the crude EPS extract. The total sugar content in EPS was quantified using the phenol-sulfuric acid method: the total sugar concentration of the sample was converted using a glucose standard curve, the absorbance (490 nm) was recorded, and background subtraction was performed. The total sugar content in each treatment group was normalized to 1 with the negative control, yielding the relative total sugar percentage in EPS (mean ± SEM, n ≥ 3). The results are as follows: Figure 19 As shown, treatment with methoxyphenol derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) can reduce the total sugar content in EPS of two types of bacteria to varying degrees. The GNP-4-Au treatment group showed a more significant decrease, suggesting that it may enhance the anti-biofilm effect by weakening matrix generation or promoting matrix depolymerization.

[0071] 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. Gold nanoparticles modified with methoxyphenol derivatives, characterized in that, It involves coating gold nanoparticles with methoxyphenol derivative ligands, wherein the gold nanoparticles and methoxyphenol derivative ligands are connected by gold-sulfur bonds. The structural formula of the methoxyphenol derivative ligand is shown in formula (Ⅰ) below. ； Equation (I); Wherein, R1 is selected from one of the following formulas (II) or (III): ； Formula (II); ； Formula (Ⅲ); R2 is selected from one of the following formulas: (Ⅳ), (Ⅴ) or (Ⅵ): ； Formula (Ⅳ); ； Formula (V); ； Formula (VI).

2. The method for preparing methoxyphenol derivative-modified gold nanoparticles according to claim 1, characterized in that, Includes the following steps: Methoxyphenol derivative ligands were dissolved in an organic solvent, sodium citrate was added, and the mixture was stirred until homogeneous. Then, gold salt was added and the mixture was stirred until homogeneous again. Subsequently, sodium borohydride was added, the reaction was stirred, and the solid was collected to obtain gold nanoparticles modified with methoxyphenol derivatives. The molar ratio of methoxyphenol derivative ligands to gold salts was (0.8~1.2):(0.8~1.2). The molar ratio of gold salt to sodium borohydride is 1:(2.5~4). The concentration of sodium citrate in organic solvents is 2.5~4 mmol / L.

3. The preparation method according to claim 2, characterized in that, The organic solvent is selected from N,N-dimethylformamide; The concentration of the methoxyphenol derivative ligand in the organic solvent is 2.5~4 mmol / L.

4. The preparation method according to claim 2, characterized in that, The gold salt is selected from chloroauric acid.

5. The preparation method according to claim 2, characterized in that, The preparation method of methoxyphenol derivative ligands includes the following steps: Compound 1 was reacted with N-hydroxysuccinimide (NHS) to synthesize compound 2; Compound 2 reacts with 1,6-hexanediamine to synthesize compound 3; Compound 3 reacts with compounds 4, 5 and 6 to synthesize methoxyphenol derivative ligands; The structural formula of compound 1 is shown below: ； The structural formula of compound 2 is shown below: ； The structural formula of compound 3 is shown below: ； The structural formula of compound 4 is shown below: ； The structural formula of compound 5 is shown below: ； The structural formula of compound 6 is shown below: ； In compounds 1 and 2, R1 is selected from one of formulas (II) or (III): ； Formula (II); ； Formula (Ⅲ); In compound 5, R2 is selected from one of the following formulas: (Ⅳ), (Ⅴ), or (Ⅵ): ； Formula (Ⅳ); ； Formula (V); ； Formula (VI).

6. The preparation method according to claim 5, characterized in that, The specific methods for synthesizing compound 2 by reacting compound 1 with N-hydroxysuccinimide include: Compound 1 and N-hydroxysuccinimide were dispersed in dichloromethane, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added as a condensing agent to synthesize compound 2. The molar ratio of compound 1, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:(1~1.5):(1~1.5). The specific methods for synthesizing compound 3 by reacting compound 2 with 1,6-hexanediamine include: The condensing agent triethylenediamine was dispersed in dichloromethane, then 1,6-hexanediamine was added, and after mixing evenly, compound 2 was added to react and synthesize compound 3. The molar ratio of the condensing agent triethylenediamine, 1,6-hexanediamine and compound 1 is 3:(6~8):

2.

7. The preparation method according to claim 5, characterized in that, The specific methods for synthesizing methoxyphenol derivative ligands by reacting compound 3 with compounds 4, 5, and 6 include: Compound 3 was dispersed in methanol along with compounds 4, 5 and 6, and the reaction yielded methoxyphenol derivative ligands. The molar ratio of compound 3 to compounds 4, 5 and 6 is (0.8~1.2):(0.8~1.2):(0.8~1.2):(0.8~1.2).

8. The application of the gold nanoparticles modified with methoxyphenol derivatives as described in claim 1 or the gold nanoparticles modified with methoxyphenol derivatives prepared by the preparation method according to any one of claims 2 to 7 in the preparation of antibacterial products.

9. The application as described in claim 8, characterized in that, The antibacterial products include antibacterial drugs; The target bacteria of the antimicrobial product include Gram-positive bacteria or Gram-negative bacteria.

10. An antibacterial product, characterized in that, This includes gold nanoparticles modified with methoxyphenol derivatives as described in claim 1, or gold nanoparticles modified with methoxyphenol derivatives prepared by the preparation method described in any one of claims 2 to 7.

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