Methoxyphenol derivative modified gold nanoparticles, preparation method and application thereof

Abstract

The present application relates to the technical field of antibacterial materials, in particular to methoxy phenolic derivative modified gold nanoparticles and a preparation method and application thereof.The methoxy phenolic derivative modified gold nanoparticles provided by the present application have spectral antibacterial properties, and have good antibacterial effects on gram-positive bacteria and gram-negative bacteria.In addition, the methoxy phenolic derivative modified gold nanoparticles provided by the present application can inhibit the efflux pump and transmembrane transport dependent on proton motive force, forming an 'inhibitory' effect on the multi-drug resistance mechanism.The π-π and hydrogen bonds provided by the methoxy phenolic aromatic skeleton are conducive to the multi-point weak binding with polysaccharides / proteins in extracellular polymers, weaken the viscoelasticity of the matrix network, inhibit the formation of bacterial membranes and promote the depolymerization of existing bacterial membranes.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of antibacterial materials, and in particular to methoxy phenol derivative modified gold nanoparticles and a preparation method and application thereof. BACKGROUND

[0002] Bacterial drug resistance is increasingly serious, which weakens the efficacy of traditional antibiotics, leads to rising treatment costs and prolonged recovery periods. The global surge in multi-drug resistant (MDR) bacterial infections poses a serious threat to public health, prompting researchers to seek innovative antibacterial strategies.

[0003] The development of antibacterial drugs is currently mainly based on two aspects, one is the discovery of natural antibacterial ingredients, and the other is the artificial synthesis of antibacterial drugs. The former requires a large amount of separation, purification and analysis work, which is complex and difficult to popularize. The latter mainly focuses on structural modification of existing drugs, but it is easy to make bacteria resistant in a short period of time. Nanomaterials provide an excellent platform for the research and development of antibacterial drugs, mainly based on the following characteristics: nanoparticles are comparable in size to biological macromolecules, which can effectively carry drugs into bacteria and interact with related targets; nanoparticles have a high specific surface area, which can significantly increase the effective concentration of the loaded drugs through multivalent effects; the surface properties of nanoparticles are active, which can modify various functional molecules to improve the physical properties of drugs and achieve targeted drug therapy.

[0004] Among them, gold nanomaterials (AuNPs) are of concern due to their unique physicochemical properties, with advantages such as efficient preparation, good antibacterial performance, high biological safety, and difficulty in inducing drug resistance. Preliminary research work has confirmed that AuNPs can destroy cell wall structure, interfere with bacterial metabolism, and inhibit bacterial proliferation.

[0005] However, the current gold nanomaterials have a narrow antibacterial spectrum, only having inhibitory effect on single gram-positive or gram-negative bacteria, lack of broad-spectrum; cannot "reverse" bacterial resistance to antibacterial drugs; and are difficult to inhibit bacterial biofilm formation or promote the disaggregation of existing biofilms. SUMMARY

[0006] To overcome the above problems, the present application provides methoxy phenol derivative modified gold nanoparticles and a preparation method and application thereof.

[0007] To achieve the above technical purposes, the present application adopts the following technical solutions:

[0008] In a first aspect of the present application, methoxy phenol derivative modified gold nanoparticles are provided, which are coated with methoxy phenol derivative ligands on the surface of gold nanoparticles, and the gold nanoparticles are connected to the methoxy phenol derivative ligands through gold-sulfur bonds;

[0009] The structural formula of the methoxy phenolic derivative ligand is shown in the following formula (I):

[0010] ;

[0011] Formula (I);

[0012] R1 is selected from one of the following formula (II) or formula (III):

[0013] ;

[0014] Formula (II);

[0015] ;

[0016] Formula (III);

[0017] R2 is selected from one of the following formula (IV), formula (V) or formula (VI):

[0018] ;

[0019] Formula (IV);

[0020] ;

[0021] Formula (V);

[0022] ;

[0023] Formula (VI).

[0024] In one or more embodiments, the methoxy phenolic derivative modified gold nanoparticles are circular, and the particle size is 5-6 nm.

[0025] In a second aspect of the present application, a preparation method of the methoxy phenolic derivative modified gold nanoparticles of the first aspect is provided, comprising the following steps:

[0026] The methoxy phenolic derivative ligand is dissolved in an organic solvent, sodium citrate is added, and after being mixed uniformly, gold salt is added, and mixed again uniformly; then sodium borohydride is added, and the reaction is stirred, and the solid is collected to obtain the methoxy phenolic derivative modified gold nanoparticles.

[0027] In one or more embodiments, the organic solvent is selected from N,N-dimethylformamide (DMF).

[0028] In one or more embodiments, the concentration of the methoxy phenolic derivative ligand in the organic solvent is 2.5-4 mmol / L, preferably 3.2 mmol / L.

[0029] In one or more embodiments, the gold salt is selected from chloroauric acid.

[0030] In one or more embodiments, the molar ratio of the methoxy phenolic derivative ligand and the gold salt is (0.8-1.2):(0.8-1.2), preferably 1:1.

[0031] In one or more embodiments, the molar ratio of the gold salt and sodium borohydride is 1:(2.5-4).

[0032] In one or more embodiments, the concentration of sodium citrate in the organic solvent is 2.5-4 mmol / L, preferably 3.2 mmol / L.

[0033] In one or more embodiments, the preparation method of the methoxy phenolic derivative ligand comprises the following steps:

[0034] Compound 1 is reacted with N-hydroxysuccinimide (NHS) to synthesize compound 2;

[0035] Compound 2 is reacted with 1,6-hexanediamine to synthesize compound 3;

[0036] Compound 3 is reacted with compound 4, compound 5 and compound 6 to synthesize the methoxy phenolic derivative ligand;

[0037] wherein, the structural formula of compound 1 is as follows:

[0038] ;

[0039] The structural formula of compound 2 is as follows:

[0040] ;

[0041] The structural formula of compound 3 is as follows:

[0042] ;

[0043] The structural formula of compound 4 is as follows:

[0044] ;

[0045] The structural formula of compound 5 is as follows:

[0046] ;

[0047] The structural formula of compound 6 is as follows:

[0048] ;

[0049] R1 in compound 1 and compound 2 is selected from one of the following formula (II) or formula (III):

[0050] ;

[0051] Formula (II);

[0052] ;

[0053] Formula (III);

[0054] R2 in compound 5 is selected from one of the following Formula (IV), Formula (V) or Formula (VI):

[0055] ;

[0056] Formula (IV);

[0057] ;

[0058] Formula (V);

[0059] ;

[0060] Formula (VI).

[0061] Preferably, the method for synthesizing compound 2 by reacting compound 1 with N-hydroxysuccinimide (NHS) specifically comprises:

[0062] Compound 1 and N-hydroxysuccinimide are dispersed in dichloromethane, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) is added as a condensing agent to synthesize compound 2 by reaction.

[0063] Further preferably, the molar ratio of compound 1, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1: (1-1.5): (1-1.5).

[0064] Further preferably, the temperature of the reaction is 0-4 ℃.

[0065] Preferably, the method for synthesizing compound 3 by reacting compound 2 with 1,6-hexanediamine specifically comprises:

[0066] A condensing agent triethylenediamine (DABCO) is dispersed in dichloromethane, and then 1,6-hexanediamine is added, and after being mixed uniformly, compound 2 is added to synthesize compound 3 by reaction.

[0067] Further preferably, the molar ratio of condensing agent triethylenediamine (DABCO), 1,6-hexanediamine and compound 2 is 3: (6-8): 2.

[0068] Further preferably, the temperature of the reaction is 0-4 ℃.

[0069] Preferably, the method for synthesizing the methoxy phenolic derivative ligand by reacting compound 3 with compound 4, compound 5 and compound 6 specifically comprises:

[0070] Compound 3 is dispersed with compound 4, compound 5 and compound 6 in methanol, and the reaction obtains the methoxy phenolic derivative ligand.

[0071] Further preferably, the molar ratio of compound 3 to compound 4, compound 5 and compound 6 is (0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2).

[0072] Further preferably, the reaction temperature is 40-50 ℃, and the reaction time is 40-60 h.

[0073] In a third aspect, the application provides a use of the methoxy phenolic derivative modified gold nanoparticle of the first aspect or the methoxy phenolic derivative modified gold nanoparticle prepared by the preparation method of the second aspect in the preparation of an antibacterial product.

[0074] In one or more embodiments, the antibacterial product comprises an antibacterial drug.

[0075] In one or more embodiments, the target bacteria of the antibacterial product comprise gram-positive bacteria or gram-negative bacteria.

[0076] In a fourth aspect, the application provides an antibacterial product comprising the methoxy phenolic derivative modified gold nanoparticle of the first aspect or the methoxy phenolic derivative modified gold nanoparticle prepared by the preparation method of the second aspect.

[0077] In one or more embodiments, the antibacterial product comprises an antibacterial drug.

[0078] In one or more embodiments, the target bacteria of the antibacterial product comprise gram-positive bacteria or gram-negative bacteria.

[0079] The application has the following beneficial effects:

[0080] 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

[0081] 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.

[0082] Figure 1 A method for preparing ligands of methoxyphenol derivatives;

[0083] Figure 2 The hydrogen NMR spectrum of GNP-1;

[0084] Figure 3 The hydrogen NMR spectrum of GNP-2;

[0085] Figure 4 The hydrogen NMR spectrum of GNP-3;

[0086] Figure 5 The hydrogen NMR spectrum of GNP-4;

[0087] Figure 6 The hydrogen NMR spectrum of GNP-5;

[0088] Figure 7 The hydrogen NMR spectrum of GNP-6;

[0089] Figure 8Transmission electron microscope images of gold nanoparticles modified with methoxy phenolic derivatives (GNP-1-Au~GNP-6-Au); wherein 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, f is GNP-6-Au;

[0090] Figure 9 Dynamic light scattering particle size analysis of gold nanoparticles modified with methoxy phenolic derivatives (GNP-1-Au~GNP-6-Au);

[0091] Figure 10 Surface zeta potential of gold nanoparticles modified with methoxy phenolic derivatives (GNP-1-Au~GNP-6-Au);

[0092] Figure 11 Hydrophobicity analysis of the surface of gold nanoparticles modified with methoxy phenolic derivatives (GNP-1-Au~GNP-6-Au);

[0093] Figure 12 Minimum inhibitory concentration (MIC) of gold nanoparticles modified with methoxy phenolic derivatives (GNP-1-Au~GNP-6-Au);

[0094] Figure 13 Minimum bactericidal concentration (MBC) of gold nanoparticles modified with methoxy phenolic derivatives (GNP-1-Au~GNP-6-Au);

[0095] Figure 14 Results of the effect of gold nanoparticles modified with methoxy phenolic derivatives (GNP-1-Au~GNP-6-Au) on the membrane integrity of bacteria, determined by propidium iodide (PI) probe uptake;

[0096] Figure 15 Results of the release of extracellular adenosine triphosphate (ATP) after treatment with gold nanoparticles modified with methoxy phenolic derivatives (GNP-1-Au~GNP-6-Au);

[0097] Figure 16 Results of the membrane potential depolarization effect, determined by fluorescent probe;

[0098] Figure 17 Results of the effect of gold nanoparticles modified with methoxy phenolic derivatives (GNP-1-Au~GNP-6-Au) on the proton motive force (PMF)-dependent efflux pumps and transmembrane transport;

[0099] Figure 18 Results of the effect of gold nanoparticles modified with methoxy phenolic derivatives (GNP-1-Au~GNP-6-Au) on the membrane integrity of E. coli (ATCC 8739) and S. aureus (ATCC 6538) in the presence of the antibiotic ampicillin; E. coli) Staphylococcus aureus (S. aureus) S. aureus ) Inhibition effect on biofilm formation

[0100] Figure 19 The results of the change of total sugar content in the extracellular polymeric substance (EPS) of the biofilm after treatment of methoxy phenol derivative modified gold nanoparticles (GNP-1-Au~GNP-6-Au). DETAILED DESCRIPTION

[0101] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, 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 application belongs.

[0102] It is to be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments consistent with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0103] In order to enable persons skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples.

[0104] Example 1

[0105] Figure 1 The preparation method of the methoxy phenol derivative ligand is described in reference Figure 1 , and the methoxy phenol derivative ligand shown in formula (I), i.e. GNP-1~GNP-6, is synthesized.

[0106] The structural formula of GNP-1~GNP-6 is shown in Table 1 below.

[0107] Table 1 Structural formula of GNP-1~GNP-6

[0108]

[0109] Specifically:

[0110] The structural formula of GNP-1 is:

[0111] ;

[0112] The structural formula of GNP-2 is:

[0113] ;

[0114] The structural formula of GNP-3 is:

[0115] ;

[0116] The structural formula for GNP-4 is:

[0117] ;

[0118] The structural formula for GNP-5 is:

[0119] ;

[0120] The structural formula for GNP-6 is:

[0121] .

[0122] 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.

[0123] 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.

[0124] 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%).

[0125] The nuclear magnetic hydrogen spectrum of GNP-1 is shown in Figure 2 .

[0126] The preparation method of GNP-2 to GNP-6 is the same as that of GNP-1, with the difference that different substituents R1 or R2 are replaced.

[0127] GNP-2 is a light yellow powder, and the yield is 45.7%; the nuclear magnetic hydrogen spectrum of GNP-2 is shown in Figure 3 .

[0128] GNP-3 is a light yellow powder, and the yield is 33.8%; the nuclear magnetic hydrogen spectrum of GNP-3 is shown in Figure 4 .

[0129] GNP-4 is a light yellow powder, and the yield is 55.2%; the nuclear magnetic hydrogen spectrum of GNP-4 is shown in Figure 5 .

[0130] GNP-5 is a light yellow powder, and the yield is 45.1%; the nuclear magnetic hydrogen spectrum of GNP-5 is shown in Figure 6 .

[0131] GNP-6 is a light yellow powder, and the yield is 50.2%; the nuclear magnetic hydrogen spectrum of GNP-6 is shown in Figure 7 .

[0132] Example 2

[0133] Preparation of methoxy phenolic derivative modified gold nanoparticles (GNP-1-Au to GNP-6-Au):

[0134] 0.19 mmol of sodium borohydride was dispersed in 10 mL of deionized water to obtain an aqueous sodium borohydride solution;

[0135] 0.064 mmol of methoxy phenolic derivative ligand (GNP-1 to GNP-6) was dissolved in 20 mL of DMF, 0.064 mmol of trisodium citrate was added and stirred at room temperature for 30 min, then 0.064 mmol of chloroauric acid was added and stirred at room temperature for 30 min, the aqueous sodium borohydride solution was added dropwise, and the stirring was continued at room temperature overnight. The methoxy phenolic derivative modified gold nanoparticles (GNP-1-Au to GNP-6-Au) were separated by centrifugation (15000 rad / min).

[0136] Figure 8 The transmission electron microscope images of methoxy phenolic derivative modified gold nanoparticles (GNP-1-Au to GNP-6-Au) can be seen from Figure 8 , which are approximately circular, and the average particle size is 5-6 nm.

[0137] Figure 9 Dynamic light scattering particle size analysis of methoxy phenolic derivative modified gold nanoparticles (GNP-1-Au~GNP-6-Au), the dynamic hydrated particle size of methoxy phenolic derivative modified gold nanoparticles (GNP-1-Au~GNP-6-Au) is about 75~91 nm.

[0138] Figure 10 Surface Zeta potential of methoxy phenolic derivative modified gold nanoparticles (GNP-1-Au~GNP-6-Au), from Figure 10 As can be seen from the above table, the surface of methoxy phenolic derivative modified gold nanoparticles (GNP-1-Au~GNP-6-Au) is negatively charged, and the Zeta potential is about -7~+2 mV.

[0139] Example 3

[0140] Hydrophilic-hydrophobic analysis of the surface of methoxy phenolic derivative modified gold nanoparticles (GNP-1-Au~GNP-6-Au):

[0141] Mix equal volumes of deionized water and n-octanol solution and stir for 24 h; let the mixed solution stand until it is layered, the upper layer is water-saturated n-octanol solution, and the lower layer is n-octanol-saturated water solution, separate the two phases and store separately for use.

[0142] Add a certain volume of n-octanol-saturated water solution, and then add 0.3 mg of methoxy phenolic derivative modified gold nanoparticles (GNP-1-Au~GNP-6-Au) to a total volume of 1 mL. After mixing, add an equal volume of 1 mL of water-saturated n-octanol solution and place it in a shaking bed for 24 h.

[0143] After shaking, remove the centrifuge tube and let it stand for a period of time until the two-phase separation boundary is clear. Use a pipette to remove a certain volume of the two-phase solution and transfer it to different 10 mL cuvettes. Dry at 120 ℃ under vacuum for 4 h until the solution is completely volatilized. Add 500 μL of freshly prepared aqua regia to the cuvettes and digest for 12 h, then add high-purity water to a final volume of 10 mL.

[0144] Prepare a gold standard curve: use a gold standard solution (1000 ppm) to prepare a series of gold standard solutions with different concentrations, as shown in Table 2. Use an inductively coupled plasma mass spectrometer (ICP-MS) to detect the content of gold in the n-octanol phase and the water phase. The obtained concentration is substituted into the following formula to calculate the octanol-water partition coefficient of methoxy phenolic derivative modified gold nanoparticles (GNP-1-Au~GNP-6-Au).

[0145] Log P = Log(C O / C W );

[0146] 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.

[0147] Table 2. Method for preparing standard working curves

[0148]

[0149] Figure 11 The 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.

[0150] Example 4

[0151] Determination of the minimum inhibitory concentration (MIC) of methoxyphenol derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au):

[0152] 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 The end-point OD was obtained by a microplate reader. Negative / blank controls were used to confirm "growth / no contamination" and did not generate MIC values. Independent replicates were >3; the geometric mean was taken for the MIC of each sample across multiple strains; results are shown in Table 2, which shows that the methoxy phenolic derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) exhibited measurable minimum inhibitory concentrations (MICs) against both Gram-negative bacteria (E. coli, P. aeruginosa, and A. baumannii) and Gram-positive bacteria (S. aureus and E. faecalis), indicating that the methoxy phenolic derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) had broad-spectrum antibacterial effects. The MICs of different samples were different, suggesting that the structure of the ligand could significantly affect its antibacterial potency, and the sample with a lower MIC had stronger antibacterial activity. Figure 12 Figure 12 As shown in Table 2, the methoxy phenolic derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) exhibited measurable minimum inhibitory concentrations (MICs) against both Gram-negative bacteria (E. coli, P. aeruginosa, and A. baumannii) and Gram-positive bacteria (S. aureus and E. faecalis), indicating that the methoxy phenolic derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) had broad-spectrum antibacterial effects. E. coli S. aureus As shown in Table 2, the methoxy phenolic derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) exhibited measurable minimum inhibitory concentrations (MICs) against both Gram-negative bacteria (E. coli, P. aeruginosa, and A. baumannii) and Gram-positive bacteria (S. aureus and E. faecalis), indicating that the methoxy phenolic derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) had broad-spectrum antibacterial effects.

[0153] Example 5

[0154] Determination of the minimum bactericidal concentration (MBC) of the methoxy phenolic derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au):

[0155] The reagents and materials were the same as in Example 4 (samples / medium / strains / controls), and in addition, tryptone soya agar (TSA) plates were required. The MIC test was completed according to Example 5 to the end-point reading, and at the MIC end-point, 10 μL was taken from each well, 10-fold gradient dilution was performed with drug-free fresh medium, and then the TSA plates were coated and incubated at 35±2 °C overnight. The plate count of 100 CFU was selected and the original well surviving bacterial amount was converted; the end-point CFU of the negative control was used as the basis. MBC determination: defined as the minimum concentration that killed ≥99.9% relative to the negative control. Independent replicates were >3; results are shown in Table 3, which shows that the methoxy phenolic derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) exhibited corresponding minimum bactericidal concentrations (MBCs) against both Gram-negative bacteria (E. coli, P. aeruginosa, and A. baumannii) and Gram-positive bacteria (S. aureus and E. faecalis), indicating that the methoxy phenolic derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) not only inhibited bacterial growth, but also achieved a bactericidal effect within a certain concentration range. Figure 13 Figure 13 As shown in Table 3, the methoxy phenolic derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) exhibited corresponding minimum bactericidal concentrations (MBCs) against both Gram-negative bacteria (E. coli, P. aeruginosa, and A. baumannii) and Gram-positive bacteria (S. aureus and E. faecalis), indicating that the methoxy phenolic derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) not only inhibited bacterial growth, but also achieved a bactericidal effect within a certain concentration range. E. coli S. aureus As shown in Table 3, the methoxy phenolic derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) exhibited corresponding minimum bactericidal concentrations (MBCs) against both Gram-negative bacteria (E. coli, P. aeruginosa, and A. baumannii) and Gram-positive bacteria (S. aureus and E. faecalis), indicating that the methoxy phenolic derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) not only inhibited bacterial growth, but also achieved a bactericidal effect within a certain concentration range. Figure 12 As shown in Table 3, the methoxy phenolic derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) exhibited corresponding minimum bactericidal concentrations (MBCs) against both Gram-negative bacteria (E. coli, P. aeruginosa, and A. baumannii) and Gram-positive bacteria (S. aureus and E. faecalis), indicating that the methoxy phenolic derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) not only inhibited bacterial growth, but also achieved a bactericidal effect within a certain concentration range. ​​​​

[0156] Example 6

[0157] Iodide propidium (PI) probe uptake assay to determine the effect of methoxy phenolic derivative modified gold nanoparticles (GNP-1-Au~GNP-6-Au) on the membrane integrity of bacteria:

[0158] E. coli (ATCC 25922) E. coli ) and Staphylococcus aureus (ATCC 29213) S. aureus ) were inoculated in CA-MHB from single colony, and cultured at 35±2 ℃ with shaking until the logarithmic growth phase (OD 600 about 0.3~0.6). The bacteria were diluted to about 1×10 7 CFU / mL with sterile PBS buffer. Methoxy phenolic derivative modified gold nanoparticles (GNP-1-Au~GNP-6-Au) were added to the bacterial solution at a predetermined concentration (1×MIC) and incubated at 37 ℃ for 60 min. Then iodide propidium (PI) probe was added to a final concentration of 8 μg / mL, and incubated in the dark for 15 min. The PI signal (Ex / Em about 535 / 617 nm) was detected by a fluorescence microplate reader, and the relative uptake rate was calculated by normalizing the negative control (without methoxy phenolic derivative modified gold nanoparticles (GNP-1-Au~GNP-6-Au)) to 1. The detection was repeated at least 3 times, and the mean value±standard error (SEM) was used to represent the results. As shown in Figure 14 compared with the control group, the relative uptake rate of PI in the methoxy phenolic derivative modified gold nanoparticles (GNP-1-Au~GNP-6-Au) treatment group increased, among which the GNP-4-Au treatment group showed more significant results, indicating that it had a stronger effect on the membrane integrity and permeability of the two types of bacteria.

[0159] Example 7

[0160] Extracellular adenosine triphosphate (ATP) release assay to determine the change in membrane flux:

[0161] E. coli (ATCC 25922) E. coli ) and Staphylococcus aureus (ATCC 29213) S. aureus ) were inoculated in CA-MHB from single colony, and cultured at 35±2 ℃ with shaking until the logarithmic growth phase (OD 600 about 0.3~0.6). The bacteria were diluted to about 1×10 7CFU / 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.

[0162] Example 8

[0163] Fluorescent probes are used to measure the depolarization effect of bacterial membrane potential.

[0164] 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.

[0165] Example 9

[0166] Ethidium bromide / Nile red accumulation experiment assessment of proton-dependent efflux pump suppression:

[0167] 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 17 As 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.

[0168] Example 10

[0169] 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:

[0170] 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 each methoxy phenolic derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) treatment group was normalized to 1 with the negative control and a columnar chart was drawn. The results, as shown in Figure 18 Figure 6, showed that the inhibition degree of biofilm formation by two types of bacteria was different for different methoxy phenolic derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au), and the biofilm biomass of the GNP-4-Au treatment group decreased more obviously, indicating that it had stronger anti-biofilm potential.

[0171] Example 11

[0172] The effect of methoxy phenolic derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) on the generation of extracellular matrix was evaluated by determining the total sugar content in extracellular polymeric substance (EPS):

[0173] After the biofilm was constructed according to the method shown in Example 10, the biofilm in each well was collected and resuspended with PBS buffer. The extracellular polymeric substance (EPS) was released into the solution by vortex oscillation (1000 rpm, 1 min), and then the cell debris was removed by low-speed centrifugation (1000 g, 5 min) and the supernatant was retained as the EPS crude extract. The total sugar content in EPS was quantified by the phenol-sulfuric acid method: the total sugar concentration of the sample was converted by the glucose standard curve, the absorbance (490 nm) was recorded and the background was deducted. The total sugar content in each treatment group was normalized to 1 with the negative control, and the relative total sugar rate in EPS (mean ± SEM, n≥3) was obtained. The results, as shown in Figure 19 Figure 7, showed that the total sugar content in EPS of two types of bacteria was reduced to different degrees by methoxy phenolic derivative-modified gold nanoparticles (GNP-1-Au~GNP-6-Au) treatment, and the decrease was more significant in the GNP-4-Au treatment group, suggesting that it might enhance the anti-biofilm effect by weakening the matrix generation or promoting the matrix depolymerization.

[0174] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Methoxyphenol derivative-modified gold nanoparticles characterized in that, The methoxy phenolic derivative ligand is coated on the surface of the gold nanoparticles, and the gold nanoparticles are connected with the methoxy phenolic derivative ligand through a gold-sulfur bond. The methoxy phenolic derivative ligand has a structural formula as shown in the following formula (I), Formula (I); R1 is selected from one of the following formula (II) or formula (III): ； Formula (II); ； Formula (III); R2 is selected from one of the following formula (IV), formula (V) or formula (VI): ； Formula (IV); ； Formula (V); ； Formula (VI).

2. The method of producing methoxyphenol derivative-modified gold nanoparticles according to claim 1, characterized by, The method comprises the following steps: The methoxy phenolic derivative ligand is dissolved in an organic solvent, sodium citrate is added, and the mixture is uniformly mixed, then gold salt is added, and the mixture is uniformly mixed again, then sodium borohydride is added, and the mixture is stirred to react, and the solid is collected to obtain the methoxy phenolic derivative modified gold nanoparticles; The molar ratio of the methoxy phenolic derivative ligand to the gold salt is (0.8-1.2):(0.8-1.2); The molar ratio of the gold salt to the sodium borohydride is 1:(2.5-4); The concentration of the sodium citrate in the organic solvent is 2.5-4 mmol / L.

3. The production method according to claim 2, wherein The organic solvent is selected from N,N-dimethylformamide; The concentration of the methoxy phenolic derivative ligand in the organic solvent is 2.5-4 mmol / L.

4. The production method according to claim 2, wherein The gold salt is selected from chloroauric acid.

5. The production method according to claim 2, wherein The preparation method of the methoxy phenolic derivative ligand comprises the following steps: Compound 1 is reacted with N-hydroxysuccinimide (NHS) to synthesize compound 2; Compound 2 is reacted with 1,6-hexanediamine to synthesize compound 3; Compound 3 is reacted with compound 4, compound 5 and compound 6 to synthesize the methoxy phenolic derivative ligand; The structural formula of compound 1 is as follows: ； The structural formula of compound 2 is as follows: ； The structural formula of compound 3 is as follows: ； The structural formula of compound 4 is as follows: ； The structural formula of compound 5 is as follows: ； The structural formula of compound 6 is as follows: ； R1 in compound 1 and compound 2 is selected from one of the following formula (II) or formula (III): ； Formula (II); ； Formula (III); R2 in compound 5 is selected from one of the following formula (IV), formula (V) or formula (VI): ； Formula (IV); ； Formula (V); ； Formula (VI).

6. The production method according to claim 5, wherein The method for synthesizing compound 2 by reacting compound 1 with N-hydroxysuccinimide specifically comprises the following steps: Compound 1 and N-hydroxysuccinimide are dispersed in dichloromethane, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 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 method for synthesizing compound 3 by reacting compound 2 with 1,6-hexanediamine specifically comprises the following steps: The condensing agent triethylenediamine is dispersed in dichloromethane, and then 1,6-hexanediamine is added, the mixture is uniformly mixed, and then compound 2 is added to synthesize compound 3; The molar ratio of the condensing agent triethylenediamine, 1,6-hexanediamine and compound 1 is 3:(6-8):

2.

7. The production method according to claim 5, wherein The method for synthesizing the methoxy phenolic derivative ligand by reacting compound 3 with compound 4, compound 5 and compound 6 specifically comprises the following steps: The compound 3 is dispersed in methanol with compound 4, compound 5 and compound 6 to obtain methoxy phenolic derivative ligand by reaction; The molar ratio of compound 3 to compound 4, compound 5 and compound 6 is (0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2).

8. Use of the methoxy phenolic derivative modified gold nanoparticles of claim 1 or the methoxy phenolic derivative modified gold nanoparticles prepared by the preparation method of any one of claims 2-7 in the preparation of an antibacterial product.

9. Use according to claim 8, wherein the compound is ###0002### The antibacterial product includes an antibacterial drug; The target bacteria of the antibacterial product include gram-positive bacteria or gram-negative bacteria.

10. An antibacterial product, characterized by The methoxy phenolic derivative modified gold nanoparticles of claim 1 or the methoxy phenolic derivative modified gold nanoparticles prepared by the preparation method of any one of claims 2-7.

Images