A catechol derivative ligand and gold nanoparticles modified with the same and applications thereof

Abstract

This invention relates to the field of nanomaterials technology, specifically to a catechol derivative ligand and its modified gold nanoparticles and their applications. The catechol derivative-modified gold nanoparticles provided by this invention can act as bioreducing agents, specifically by increasing the GSH / GSSG ratio and NADPH / NADP ratio in tumor cells. + By adjusting the ratio, reducing extracellular H2O2 content, increasing GPx content, and decreasing MDA content, a reducing stress state is formed, thus enabling it to act as a biological reducing agent. Under reducing stress, it can not only inhibit the proliferation rate of tumor cells but also activate mitomycin C, enhancing the therapeutic effect of mitomycin C on tumors.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, specifically to a catechol derivative ligand and its modified gold nanoparticles and their applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance 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] Cancer is a major global health problem and has become the leading cause of death for people under 70 years old in the vast majority of countries and regions. Surgery, radiotherapy, and chemotherapy are the three most commonly used methods for cancer treatment; however, surgery can only remove tumors that are visible to the naked eye, and it is difficult to eliminate invisible subclinical lesions; traditional chemotherapy often suffers from poor targeting and dose-limited toxicity; traditional radiotherapy not only destroys cancer cells, but also causes some damage to surrounding healthy tissues.

[0004] Mitomycin C (MMC) is a drug derived from actinomycetes (…). Streptomyces caespitosus Mitomycin C, an antitumor antibiotic isolated from the culture medium of tumor cells, is a cell cycle-nonspecific antitumor drug with a broad antitumor spectrum. Its mechanism of action involves depolymerizing the DNA of tumor cells and simultaneously inhibiting DNA replication, thereby suppressing tumor cell division. Although mitomycin C has a broad antitumor spectrum and rapid action, its therapeutic index is not high. This is because mitomycin C is a typical bioreduction-activating drug, requiring activation by reductase systems such as quinone oxidoreductase 1 (NQO1) and reduced coenzyme II (NADPH)-cytochrome P450 reductase to produce active intermediates capable of alkylation or cross-linking with DNA. However, the intracellular bioreduction-activation process is complex and unstable.

[0005] Therefore, there is an urgent need for a biological reducing agent to regulate cellular reduction stress, activate mitomycin C, and thus enhance the therapeutic effect of mitomycin C on tumors. Summary of the Invention

[0006] To overcome the above problems, the present invention provides a catechol derivative ligand and its modified gold nanoparticles and their applications.

[0007] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a catechol derivative ligand having the following structural formula (I):

[0009]

[0010] Equation (I);

[0011] Wherein, R1 is selected from one of the following formulas (II) or (III):

[0012]

[0013] Formula (II);

[0014]

[0015] Formula (Ⅲ);

[0016] Wherein, R2 is selected from one of the following formulas (Ⅳ), (Ⅴ), or (Ⅵ):

[0017]

[0018] Formula (Ⅳ);

[0019]

[0020] Formula (V);

[0021]

[0022] Formula (VI).

[0023] A second aspect of the present invention provides a method for preparing the catechol derivative ligand described in the first aspect, comprising the following steps:

[0024] (1) Compound 1 was reacted with N-hydroxysuccinimide (NHS) to synthesize compound 2;

[0025] (2) Compound 2 reacts with Compound 7 to synthesize Compound 3;

[0026] (3) Compound 3 reacts with compounds 4, 5 and 6 to synthesize catechol derivative ligands;

[0027] The structural formula of compound 1 is shown below:

[0028] ;

[0029] The structural formula of compound 2 is shown below:

[0030] ;

[0031] The structural formula of compound 3 is shown below:

[0032] ;

[0033] The structural formula of compound 4 is shown below:

[0034] ;

[0035] The structural formula of compound 5 is shown below:

[0036] ;

[0037] The structural formula of compound 6 is shown below:

[0038] ;

[0039] The structural formula of compound 7 is shown below:

[0040]

[0041] In compounds 1, 2, and 3, R1 is selected from one of formulas (II) or (III):

[0042]

[0043] Formula (II);

[0044]

[0045] Formula (Ⅲ);

[0046] In compound 5, R2 is selected from one of the following formulas: (Ⅳ), (Ⅴ), or (Ⅵ):

[0047]

[0048] Formula (Ⅳ);

[0049]

[0050] Formula (V);

[0051]

[0052] Formula (VI).

[0053] Preferably, in step (1), the method for synthesizing compound 2 by reacting compound 1 with N-hydroxysuccinimide (NHS) includes:

[0054] 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.

[0055] 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).

[0056] More preferably, the reaction temperature is 0~4 ℃.

[0057] Preferably, in step (2), the method for synthesizing compound 3 by reacting compound 2 with compound 7 specifically includes:

[0058] The condensing agent triethylenediamine (DABCO) was dispersed in dichloromethane, then compound 7 was added, and after mixing evenly, compound 2 was added to react and synthesize compound 3.

[0059] More preferably, the molar ratio of the condensing agent triethylenediamine (DABCO), compound 7 and compound 2 is 3:(6~8):2.

[0060] More preferably, the reaction temperature is 0~4 ℃.

[0061] Preferably, in step (2), the method for synthesizing the catechol derivative ligand by reacting compound 3 with compounds 4, 5, and 6 includes:

[0062] Compound 3 was dispersed in methanol along with compounds 4, 5 and 6 to obtain catechol derivative ligands.

[0063] 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).

[0064] More preferably, the reaction temperature is 40~50 °C and the reaction time is 40~60 h.

[0065] A third aspect of the present invention provides a catechol derivative-modified gold nanoparticle, wherein the surface of the gold nanoparticle is coated with a catechol derivative ligand, and the gold nanoparticle and the catechol derivative ligand are connected by a gold-sulfur bond.

[0066] The catechol derivative ligand is the catechol derivative ligand described in the first aspect or the catechol derivative ligand prepared by the preparation method described in the second aspect.

[0067] A fourth aspect of the present invention provides a method for preparing catechol derivative-modified gold nanoparticles as described in the third aspect, comprising the following steps:

[0068] The catechol derivative ligand was 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 aqueous solution was added, the reaction was stirred, and the solid was collected to obtain catechol derivative-modified gold nanoparticles.

[0069] In one or more embodiments, the organic solvent is selected from N,N-dimethylformamide (DMF).

[0070] In one or more embodiments, the concentration of the catechol derivative ligand in the organic solvent is 2.5 to 4 mmol / L, preferably 3.2 mmol / L.

[0071] In one or more embodiments, the gold salt is selected from chloroauric acid.

[0072] In one or more embodiments, the molar ratio of the catechol derivative ligand to the gold salt is (0.8~1.2):(0.8~1.2), preferably 1:1.

[0073] In one or more embodiments, the molar ratio of the gold salt to sodium borohydride is 1:(2.5~4).

[0074] In one or more embodiments, the concentration of the sodium borohydride aqueous solution is 16-26 mmol / L.

[0075] 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.

[0076] A fifth aspect of the present invention provides the application of catechol derivative-modified gold nanoparticles as described in the third aspect or catechol derivative-modified gold nanoparticles prepared by the preparation method described in the fourth aspect as a bioreducing agent.

[0077] In one or more embodiments, the application includes:

[0078] 1) Increase the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in tumor cells;

[0079] 2) Increase the ratio of reduced coenzyme II (NADPH) to reduced coenzyme II oxidized state (NADP) in tumor cells. + )ratio;

[0080] 3) Reduce the extracellular H2O2 content of tumor cells;

[0081] 4) Increase the content of glutathione peroxidase (GPx) in tumor cells;

[0082] 5) Reduces the content of lipid peroxide malondialdehyde (MDA) in tumor cells.

[0083] Preferably, the tumor cells are A549 cells.

[0084] A sixth aspect of the present invention provides the use of catechol derivative-modified gold nanoparticles as described in the third aspect or catechol derivative-modified gold nanoparticles prepared by the preparation method described in the fourth aspect, in combination with mitomycin C, in the preparation of antitumor drugs.

[0085] In one or more embodiments, the tumor includes one or more of breast cancer, colorectal cancer, liver cancer, rhabdomyosarcoma, cervical cancer, lung cancer, and bladder cancer, preferably lung cancer.

[0086] In one or more embodiments, the mass ratio of the catechol derivative-modified gold nanoparticles to mitomycin C is (45~55):1.

[0087] A seventh aspect of the present invention provides an antitumor drug comprising catechol derivative-modified gold nanoparticles as described in the third aspect or catechol derivative-modified gold nanoparticles prepared by the preparation method described in the fourth aspect, and mitomycin C.

[0088] In one or more embodiments, the mass ratio of the catechol derivative-modified gold nanoparticles to mitomycin C is (45~55):1.

[0089] The beneficial effects of this invention are as follows:

[0090] The catechol derivative-modified gold nanoparticles provided by this invention can act as bioreducing agents, specifically by increasing the GSH / GSSG ratio and NADPH / NADP ratio in tumor cells. + By adjusting the ratio, reducing extracellular H2O2 content, increasing GPx content, and decreasing MDA content, a reducing stress state is formed, thus enabling it to act as a biological reducing agent. Under reducing stress, it can not only inhibit the proliferation rate of tumor cells but also activate mitomycin C, enhancing the therapeutic effect of mitomycin C on tumors.

[0091] The catechol group in the catechol derivative ligand itself has a typical reversible redox pair (catechol / o-quinone). Under physiological or near-physiological conditions, it can undergo proton-coupled electron transfer (PCET) to be oxidized to o-quinone and release electrons / protons, thus exhibiting mild reducing properties. At the same time, o-quinone can be reduced back to catechol by intracellular reducing systems (such as the NADPH-associated reductase system), making this group a redox cycle unit. When catechol derivative ligands are anchored to the surface of gold nanoparticles via gold-sulfur bonds, on the one hand, the high density of catechol sites on the surface of gold nanoparticles can form a locally enriched electron donor microenvironment at the nano-bio interface, reducing the energy barrier for interfacial electron transfer and increasing the effective collision / exchange frequency of reducing species on the surface; on the other hand, the conductivity and surface states of gold nanoparticles can promote electron delocalization and charge transfer of ligands, making the oxidation process of catechol to o-quinone more likely to occur at the interface, and further driving the reduction and scavenging of oxidizing species such as peroxides / free radicals, thus serving as a biological reducing agent. Attached Figure Description

[0092] 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.

[0093] Figure 1 Method for preparing catechol derivative ligands;

[0094] Figure 2 The hydrogen NMR spectrum of LFGNP-1;

[0095] Figure 3 The hydrogen NMR spectrum of LFGNP-2;

[0096] Figure 4 The hydrogen NMR spectrum of LFGNP-3;

[0097] Figure 5 The hydrogen NMR spectrum of LFGNP-4;

[0098] Figure 6 The hydrogen NMR spectrum of LFGNP-5;

[0099] Figure 7 The hydrogen NMR spectrum of LFGNP-6;

[0100] Figure 8Transmission electron microscope images of catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au), where a is LFGNP-1-Au, b is LFGNP-2-Au, c is LFGNP-3-Au, d is LFGNP-4-Au, e is LFGNP-5-Au, and f is LFGNP-6-Au;

[0101] Figure 9 A graph showing the relative ratio of GSH to GSSG;

[0102] Figure 10 NADPH / NADP + Relative ratio chart;

[0103] Figure 11 A graph showing the relative content of H2O2;

[0104] Figure 12 A graph showing the relative content of GPx;

[0105] Figure 13 A graph showing the relative content of MDA;

[0106] Figure 14 A graph showing the relative DNA content of each group;

[0107] Figure 15 The graph shows the cell viability of each group;

[0108] Figure 16 A graph showing the relative ratio of GSH to GSSG;

[0109] Figure 17 NADPH / NADP + Relative ratio chart. Detailed Implementation

[0110] 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.

[0111] 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.

[0112] 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.

[0113] Example 1

[0114] Figure 1 The preparation method of catechol derivative ligands is as follows: Figure 1 The method shown synthesizes the catechol derivative ligands shown in formula (Ⅰ), namely LFGNP-1 to LFGNP-6.

[0115] The structural formulas of LFGNP-1 to LFGNP-6 are shown in Table 1 below.

[0116] Table 1. Structural formulas of LFGNP-1 to LFGNP-6

[0117]

[0118] Specifically, the structural formula of LFGNP-1 is:

[0119] ;

[0120] The structural formula of LFGNP-2 is:

[0121] ;

[0122] The structural formula of LFGNP-3 is:

[0123] ;

[0124] The structural formula of LFGNP-4 is:

[0125] ;

[0126] The structural formula of LFGNP-5 is:

[0127] ;

[0128] The structural formula of LFGNP-6 is:

[0129] .

[0130] Preparation of LFGNP-1:

[0131] (1) Under ice bath conditions, 0.05 mol of 4-carboxyphenylboronic acid was dispersed in 100 mL of dichloromethane (DCM). After mixing evenly, 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 conditions were 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 stopped, filtered, and a dichloromethane solution of compound 2 was obtained. The solution was then directly introduced into the next step of the reaction.

[0132] (2) Under ice bath conditions, 0.075 mol of the condensing agent triethylenediamine (DABCO) was dispersed in 50 mL of dichloromethane (DCM). After mixing thoroughly, 24.6 mL of compound 7 was added while stirring. After 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 became viscous. The reaction was monitored by TLC. After the reaction was completed, the solvent was removed by rotary evaporation, and the crude product was washed repeatedly with water. The crude product was purified by column chromatography using 300-mesh silica gel. The developing solvent was a 1:1 (volume ratio) mixture of dichloromethane (DCM) and ethyl acetate (EA). Compound 3 was obtained.

[0133] (3) 5 mmol of compound 3, 5 mmol of compound 4, 5 mmol of 3,4-dihydroxybenzaldehyde 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 product was purified by column chromatography using 300-mesh silica gel. The developing solvent was a mixed solvent system of petroleum ether and ethyl acetate (EA) = 1:1 (volume ratio); LFGNP-1 was obtained.

[0134] The preparation methods for LFGNP-2 to LFGNP-6 are the same as those for LFGNP-1, the difference being the substitution of different substituents R1 or R2.

[0135] Figure 2 The hydrogen NMR spectrum of LFGNP-1;

[0136] Figure 3 The hydrogen NMR spectrum of LFGNP-2;

[0137] Figure 4 The hydrogen NMR spectrum of LFGNP-3;

[0138] Figure 5 This is the 1H NMR spectrum of LFGNP-4.

[0139] Figure 6The hydrogen NMR spectrum of LFGNP-5;

[0140] Figure 7 This is the 1H NMR spectrum of LFGNP-6.

[0141] Example 2

[0142] Preparation of catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au):

[0143] 0.064 mmol of catechol derivative ligands (LFGNP-1~LFGNP-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. Then, 0.064 mmol of chloroauric acid was added and the mixture was stirred at room temperature for 30 min. 10 mL of 19 mmol / L sodium borohydride aqueous solution was added dropwise, and the mixture was stirred overnight at room temperature. The solid was collected by centrifugation (15000 rad / min) and washed three times each with DMF and ultrapure water to obtain catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au), which were dispersed in ultrapure water for later use.

[0144] Figure 8 Transmission electron microscope images of catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au), from... Figure 8 As can be seen, the catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) are all approximately spherical, with clear particle outlines, concentrated size distribution, and no large-area aggregation.

[0145] Table 2 shows the hydration size, surface zeta potential, polydispersity index (PDI), hydrophilicity / hydrophobicity analysis (n-octanol-water partition coefficient Log P) and elemental analysis results of catechol derivative-modified gold nanoparticles (LFGNP-1-Au ~ LFGNP-6-Au).

[0146] Table 2 Characterization of catechol derivative-modified gold nanoparticles (LFGNP-1-Au ~ LFGNP-6-Au)

[0147]

[0148] As shown in Table 2, the catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) exhibit a consistent core platform and tunable surface properties, demonstrating good overall aqueous dispersibility and colloidal stability. Their hydrated particle size is concentrated in the range of 73.4~88.5 nm, and their polydispersity index (PDI) is 0.075~0.244, indicating that the catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) have a relatively concentrated particle size distribution in the aqueous phase with no obvious large particle aggregation peaks, indicating that the system is in a stable dispersible state. The surface Zeta potential of the catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) is -21.6~-36.7 mV, exhibiting a moderately negative charge. This potential range provides effective electrostatic repulsion and interface stabilization, thus facilitating long-term dispersion in an aqueous environment and reducing the risk of spontaneous aggregation. Meanwhile, elemental analysis results showed that the surface ligand loading of gold nanoparticles modified with different catechol derivatives (LFGNP-1-Au~LFGNP-6-Au) was on the same order of magnitude (approximately 247~344 ligands per particle, corresponding to a surface density of approximately 3.40~4.75 ligands / nm). 2 The results indicate that the catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) show relatively small differences in the "ligand loading number." This suggests that the differences in the interfacial hydration layer and weak interaction network, caused by the structure of the catechol derivative ligands themselves (such as differences in π-electron density, polarizability, and hydrogen bond donor / acceptor microenvironment due to substituents), can lead to subsequent differences in cell phenotypes and biological effects. Furthermore, the octanol-water partition system (LogP) ranges from -1.35 to -0.78, generally indicating a hydrophilic bias. However, measurable differences exist between the different catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au). This further demonstrates that the hydrophilicity / hydrophobicity and interfacial physicochemical properties of materials can be controlled by regulating the structural characteristics of the catechol derivative ligands, providing a physicochemical basis for subsequent cellular-level reductive stress regulation and related anti-tumor applications.

[0149] Among them, the hydrophilicity and hydrophobicity analysis of the surface of catechol derivative-modified gold nanoparticles (LFGNP-1-Au ~ LFGNP-6-Au) was performed:

[0150] 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.

[0151] Add a certain volume of saturated aqueous solution of n-octanol, then add 0.3 mg of catechol derivative-modified gold nanoparticles (LFGNP-1-Au ~LFGNP-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.

[0152] 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.

[0153] 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 3 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 catechol derivative-modified gold nanoparticles (LFGNP-1-Au ~ LFGNP-6-Au).

[0154] Log P = Log (C O / C W );

[0155] 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.

[0156] Table 3. Standard Working Curve Preparation Method

[0157]

[0158] Example 3

[0159] Catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) increase the GSH / GSSG ratio in A549 cells:

[0160] After digesting and counting A549 cells, the following steps were performed: 1×10⁻⁶ cells were counted. 5Two mL of cell suspension was seeded into 6-well plates at a density of cells / mL and incubated for 24 h. Then, catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) dispersions were added to a final concentration of 50 μg / mL, and incubation was continued for another 24 h. The culture medium was discarded, and the cells were washed twice with pre-cooled phosphate-buffered saline (PBS). Lysis buffer containing N-ethylmaleimide (NEM) was added, and the contents of reduced GSH and oxidized GSSG were determined according to the instructions of a commercial GSH / GSSG colorimetric / fluorescence kit. The results were normalized to serum total protein (BCA protein) (nmol / mg protein). Three biological replicates were set for each sample, and each experiment was independently repeated at least three times. The results were normalized to 1 with the control group to obtain the GSH / GSSG relative ratio.

[0161] The results are as follows Figure 9 As shown, catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) can significantly increase the GSH / GSSG ratio in A549 cells (p<0.05).

[0162] Example 4

[0163] Catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) increased NADPH / NADP in A549 cells. + ratio:

[0164] After digesting and counting A549 cells, the following steps were performed: 1×10⁻⁶ cells were counted. 5 Two mL of cell suspension was seeded into 6-well plates at a density of cells / mL and incubated statically for 24 h. Then, a dispersion of catechol-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) was added to a final concentration of 50 μg / mL, and incubation continued for another 24 h. The culture medium was then discarded on ice, and NADPH and NADP were extracted using an acid / base biphasic extraction reagent. + Read the NADP(H) ratio assay kit instructions on a microplate reader and convert the concentrations accordingly. Calculate the NADPH / NADP ratio after normalizing the data to protein content. + The ratio was calculated by setting up three biological replicates for each sample and conducting each experiment independently at least three times. The results were normalized to 1 with the control group as the result, and the NADPH / NADP ratio was obtained. + Relative ratio.

[0165] The results are as follows Figure 10 As shown, catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) can significantly increase the NADPH / NADP ratio in A549 cells. +The ratio suggests that the cellular reducing equivalent reserve is regulated through a surface hydrogen bond donor network.

[0166] Example 5

[0167] Catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) increase extracellular H2O2 content in A549 cells:

[0168] After digesting and counting A549 cells, the following steps were performed: 1×10⁻⁶ cells were counted. 5 Two mL of cell suspension was seeded into 6-well plates at a density of cells / mL and incubated for 24 h. Then, a dispersion of catechol-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) with a final concentration of 50 μg / mL was added, and the plates were incubated for another 24 h. The supernatant from each well was collected and transferred to a black 96-well plate. Amplex Red working solution (50 μM Amplex Red + 0.1 U / mL horseradish peroxidase (HRP)) was added to each well, and the plates were incubated at 37 °C in the dark for 30 min. The fluorescence intensity was read at 560 / 590 nm (Ex / Em), and a catalase supplementation control was set up to verify the signal specificity. Three biological replicates were set up for each sample, and each experiment was independently repeated at least three times. The results were normalized to 1 with the control group to obtain the relative H2O2 content.

[0169] The results are as follows Figure 11 As shown, catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) can significantly increase the extracellular H2O2 content of A549 cells, demonstrating that they can regulate cellular redox homeostasis.

[0170] Example 6

[0171] Catechol derivative-modified gold nanoparticles (LFGNP-1-Au ~ LFGNP-6-Au) increased GPx content in A549 cells:

[0172] After digesting and counting A549 cells, the following steps were performed: 1×10⁻⁶ cells were counted. 5Two mL of cell suspension was seeded into 6-well plates at a density of cells / mL and incubated statically for 24 h. Then, a dispersion of catechol-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) with a final concentration of 50 μg / mL was added, and the plates were incubated for another 24 h. After discarding the supernatant, the supernatant was lysed with ice-cold lysis buffer. The glutathione peroxidase (GPx) activity assay kit (coupled glutathione reductase (GR) / NADPH method) was used to continuously monitor the rate of decrease of NADPH absorbance at 340 nm and convert it to U / mg protein. Three biological replicates were set up for each sample, and each experiment was independently repeated at least three times. The results were normalized to 1 with the control group to obtain the relative GPx content.

[0173] The results are as follows Figure 12 As shown, catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) can significantly increase the GPx content in A549 cells, demonstrating that catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) can enhance the activity of cellular antioxidant enzymes.

[0174] Example 7

[0175] Catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) reduced MDA content in A549 cells:

[0176] After digesting and counting A549 cells, the following steps were performed: 1×10⁻⁶ cells were counted. 5 Two mL of cell suspension was seeded into 6-well plates at a density of cells / mL and incubated statically for 24 h. Then, a dispersion of catechol-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) with a final concentration of 50 μg / mL was added, and the plates were incubated for another 24 h. After discarding the culture medium, the cells were lysed with lysis buffer containing butylated hydroxytoluene (BHT). The cells were then analyzed according to the thiobarbituric acid (TBARS) / MDA assay kit, and a reading at 532 nm was obtained. The results were normalized to BCA protein quantification (nmol / mg protein). Three biological replicates were set up for each sample, and each experiment was independently repeated at least three times. The results were normalized to 1 with the control group to obtain the relative MDA content.

[0177] The results are as follows Figure 13 As shown, catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) can significantly reduce MDA content in A549 cells.

[0178] Example 8

[0179] Catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) inhibited A549 cell proliferation:

[0180] After digesting and counting A549 cells, the cells were counted at a rate of 5 × 10⁻⁶. 3 Cells were seeded at a density of 100 μL of complete culture medium per well in black permeable 96-well plates and incubated statically for 24 h. The supernatant was then discarded, and complete culture medium containing catechol-modified gold nanoparticles (LFGNP-1-Au ~ LFGNP-6-Au) at a final concentration of 50 μg / mL was added to each well. The negative control group received an equal volume of complete culture medium, and incubation continued for 48 h. After incubation, the culture medium was discarded, and the cells were gently washed once with pre-cooled PBS buffer. Cell lysis buffer and CyQUANT DNA working solution were added, and the cells were incubated at room temperature for 30 min in the dark to allow the dye to fully bind to the cellular DNA. Fluorescence intensity was read in the corresponding channels using a microplate reader (CyQUANT system: Ex / Em = 485 / 530 nm). Six replicates were set up for each group, and three independent replicates were performed. Results were normalized to 1 (control group) to obtain the relative DNA content of each group.

[0181] The results are as follows Figure 14 As shown, compared with the negative control group, the DNA content in the catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) treatment group decreased to varying degrees, suggesting that the catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) can produce a measurable inhibitory effect on A549 cell proliferation within the sublethal dose window, and the degree of inhibition differs among different particles, indicating that the differences in their surface hydrogen bond donor microenvironment and interfacial physicochemical properties can be used for regulated intervention of cell proliferation phenotype.

[0182] The aforementioned "sublethal dose" refers to the dose window within which catechol-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au), when applied alone for a given treatment time, do not cause large-scale cell death or irreversible disintegration, and the overall cell survival remains within an acceptable range, but are still able to induce measurable cellular stress responses and affect functional phenotypes. The purpose of using a sublethal dose is twofold: firstly, to avoid "false positive inhibition" caused by the inherent cytotoxicity of the material; and secondly, to ensure that the decrease in DNA content, inhibition of proliferation, and changes in reducing stress indicators can be attributed to the material's regulatory effect on cellular reducing stress / metabolic homeostasis.

[0183] Example 9

[0184] Catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) combined with mitomycin C for antitumor effects:

[0185] After digesting and counting A549 cells, the following steps were performed: 1×10⁻⁶ cells were counted. 5 Two mL of cell suspension was seeded into 6-well plates at a density of cells / mL and incubated statically for 24 h. Cells were then divided into: a control group (without catechol-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) and mitomycin C), mitomycin C monotherapy groups (final concentrations of 0.1 μg / mL, 0.5 μg / mL, and 1.0 μg / mL), catechol-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) monotherapy group (final concentration of 50 μg / mL), and a combination therapy group. The combination therapy group included catechol-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) (final concentration of 50 μg / mL) and mitomycin C (final concentration of 1 μg / mL). After incubation for 24–48 h, the absorbance of each group was read on an ELISA reader using a commercially available CCK-8 cell viability assay reagent, and the relative cell viability was calculated by normalizing the control group to 1. Each group was set up with 6 replicates and 3 independent replicate experiments were performed.

[0186] To verify that the decrease in cell viability caused by catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) combined with mitomycin C is related to the regulation of reducing stress, A549 cells (1×10⁻⁶) were seeded in 6-well plates under the same conditions as in Examples 3 and 4. 5 Gold nanoparticles (2 mL per well) adhered to the wells for 24 h. After 24 h of adhesion, the following treatments were administered for 24 h: control, mitomycin C monotherapy (1.0 μg / mL), and catechol derivative-modified gold nanoparticles (LFGNP-1-Au ~ LFGNP-6-Au) (final concentration 50 μg / mL). After treatment, the following indicators were measured and normalized according to the methods described in Examples 3 and 4: GSH / GSSG ratio, NADPH / NADP ratio. + Relative ratio. Results are as follows: Figure 15 As shown, when mitomycin C was used in combination with catechol-modified gold nanoparticles (LFGNP-1~LFGNP-6), the survival rate of A549 cells was significantly lower than that of mitomycin C alone or catechol-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) alone; at the same time, the combination therapy group showed a more significant decrease in the GSH / GSSG ratio and NADPH / NADP ratio. + The ratio showed a reduction stress tendency change consistent with that of Examples 3 and 4. Figure 16 and Figure 17 The above results suggest that catechol derivative-modified gold nanoparticles (LFGNP-1-Au~LFGNP-6-Au) can activate mitomycin C and enhance its therapeutic effect on tumors by inducing / enhancing tumor cell reductive stress through the surface hydrogen bond donor microenvironment.

[0187] 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 catechol derivative-modified gold nanoparticle, characterized in that, It involves coating the surface of gold nanoparticles with catechol derivative ligands, wherein the gold nanoparticles and the catechol derivative ligands are connected by gold-sulfur bonds. The catechol derivative ligands are selected from LFGNP-1, LFGNP-2, LFGNP-4, and LFGNP-5; The structural formula of LFGNP-1 is: ； The structural formula of LFGNP-2 is: ； The structural formula of LFGNP-4 is: ； The structural formula of LFGNP-5 is: 。 2. The method for preparing catechol derivative-modified gold nanoparticles according to claim 1, characterized in that, Includes the following steps: The catechol derivative ligand was 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 aqueous solution was added, the reaction was stirred, and the solid was collected to obtain catechol derivative-modified gold nanoparticles.

3. The application of the catechol derivative-modified gold nanoparticles of claim 1 or the catechol derivative-modified gold nanoparticles prepared by the preparation method of claim 2 in combination with mitomycin C in the preparation of antitumor drugs.

4. The application as described in claim 3, characterized in that, The tumor is selected from one or more of the following: breast cancer, colorectal cancer, liver cancer, rhabdomyosarcoma, cervical cancer, lung cancer, and bladder cancer. The mass ratio of the catechol derivative-modified gold nanoparticles to mitomycin C is (45~55):

1.

5. An antitumor drug, characterized in that, The catechol derivative-modified gold nanoparticles as described in claim 1, or the catechol derivative-modified gold nanoparticles prepared by the preparation method described in claim 2, and mitomycin C.

6. The antitumor drug as described in claim 5, characterized in that, The mass ratio of the catechol derivative-modified gold nanoparticles to mitomycin C is (45~55):1.

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