Aromatic amine derivative-modified gold nanoparticles, their preparation method and application

Abstract

This invention relates to the field of biomedical technology, specifically to a gold nanoparticle modified with an aromatic amine derivative, its preparation method, and its application. The gold nanoparticle modified with an aromatic amine derivative provided by this invention can increase the endoplasmic reticulum stress level of non-small cell lung cancer (NSCLC) cells and enhance their sensitivity to chemotherapy drugs, synergistically improving the mortality rate of NSCLC cells in conjunction with chemotherapy drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a gold nanoparticle modified with an aromatic amine derivative, its preparation method, and its application. Background Technology

[0002] Lung cancer is a common primary malignant tumor of the lung, with most cases originating from the bronchial mucosal epithelium. The basic pathological types of lung cancer are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with more than 80% of lung cancers being non-small cell lung cancer.

[0003] Currently, the main treatment for non-small cell lung cancer is surgical resection combined with radiotherapy and chemotherapy. Chemotherapy mainly includes targeted therapy with known targets and conventional chemotherapy drugs with unknown targets. However, during chemotherapy, many lung cancer patients develop chemotherapy drug resistance, which greatly affects the treatment outcome.

[0004] Endoplasmic reticulum (ER) stress refers to a state of intracellular protein imbalance caused by dysfunction of the endoplasmic reticulum (ER), which disrupts protein folding and modification processes within the ER. ER stress is triggered when cells face environmental stresses such as nutrient deficiency, insufficient oxygen supply, or calcium imbalance. ER stress is typically addressed through a mechanism called the ER stress response. An important ER stress response mechanism involves the activation of three non-coding RNA-mediated signaling pathways known as the unfolded protein response (UPR). UPR maintains intracellular homeostasis, alleviates stress, and promotes cell survival by reducing protein burden, increasing ER volume, and clearing accumulated unfolded proteins. However, when stress is excessively severe or prolonged, such as with chemotherapy, the unrepairable overload of ER stress can activate pathways such as apoptosis, autophagy, or necrosis, inducing programmed cell death. Because rapid cell division requires a large supply of protein synthesis, cancer cells, compared to ordinary cells, often acquire a more active UPR signaling pathway and a higher overall response to endoplasmic reticulum stress-induced death through gene mutations. As a result, they can still survive and proliferate under various stimulating stresses such as hypoxia, nutrient deficiency, chemotherapy drug stimulation, ovulation, and DNA damage, ultimately leading to the formation of chemotherapy drug tolerance and greatly reducing the patient's survival rate.

[0005] Therefore, there is an urgent need for an endoplasmic reticulum stress inducer to enhance the endoplasmic reticulum stress sensitivity in lung cancer. Summary of the Invention

[0006] To overcome the above problems, the present invention provides an aromatic amine derivative-modified gold nanoparticle, its preparation method and application.

[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 gold nanoparticle modified with an aromatic amine derivative, wherein the gold nanoparticle is coated with an aromatic amine derivative ligand, and the gold nanoparticle and the aromatic amine derivative ligand are connected by a gold-sulfur bond.

[0009] The structural formula of the aromatic amine derivative ligand is shown in formula (Ⅰ) below:

[0010]

[0011] Equation (I);

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

[0013]

[0014] Formula (II);

[0015]

[0016] Formula (Ⅲ);

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

[0018]

[0019] Formula (Ⅳ);

[0020]

[0021] Formula (V).

[0022] A second aspect of the present invention provides a method for preparing gold nanoparticles modified with aromatic amine derivatives as described in the first aspect, comprising the following steps:

[0023] Aromatic amine 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 aqueous solution was added, the reaction was stirred, and the solid was collected to obtain gold nanoparticles modified with aromatic amine derivatives.

[0024] In one or more embodiments, the organic solvent is selected from N,N-dimethylformamide (DMF).

[0025] In one or more embodiments, the concentration of the aromatic amine derivative ligand in the organic solvent is 2.5~4 mmol / L, preferably 3.2 mmol / L.

[0026] In one or more embodiments, the gold salt is selected from chloroauric acid.

[0027] In one or more embodiments, the molar ratio of the aromatic amine derivative ligand to the gold salt is (0.8~1.2):(0.8~1.2), preferably 1:1.

[0028] In one or more embodiments, the molar ratio of the gold salt to sodium borohydride is 1:(2.5~4).

[0029] In one or more embodiments, the concentration of the sodium borohydride aqueous solution is 16-26 mmol / L.

[0030] 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.

[0031] In one or more embodiments, the method for preparing the aromatic amine derivative ligand includes the following steps:

[0032] (1) Compound 1 was reacted with N-hydroxysuccinimide (NHS) to synthesize compound 2;

[0033] (2) Compound 2 reacts with 1,4-p-phenylenediamine to synthesize compound 3;

[0034] (3) Compound 3 reacts with compounds 4, 5 and 6 to synthesize aromatic amine derivative ligands;

[0035] The structural formula of compound 1 is shown below:

[0036] ;

[0037] The structural formula of compound 2 is shown below:

[0038] ;

[0039] The structural formula of compound 3 is shown below:

[0040] ;

[0041] The structural formula of compound 4 is shown below:

[0042] ;

[0043] The structural formula of compound 5 is shown below:

[0044] ;

[0045] The structural formula of compound 6 is shown below:

[0046] ;

[0047] In compounds 1, 2, and 3, R1 is selected from one of formulas (II) or (III):

[0048]

[0049] Formula (II);

[0050]

[0051] Formula (Ⅲ);

[0052] In compound 5, R2 is selected from one of formulas (Ⅳ) or (Ⅴ):

[0053]

[0054] Formula (Ⅳ);

[0055]

[0056] Formula (V).

[0057] Preferably, in step (1), the method for synthesizing compound 2 by reacting compound 1 with N-hydroxysuccinimide (NHS) includes:

[0058] 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.

[0059] 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).

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

[0061] Preferably, in step (2), the method for synthesizing compound 3 by reacting compound 2 with 1,4-p-phenylenediamine specifically includes:

[0062] 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.

[0063] More preferably, the molar ratio of the condensing agent triethylenediamine (DABCO), 1,6-hexanediamine and compound 2 is 3:(6~8):2.

[0064] More preferably, the reaction temperature is 0~4 ℃.

[0065] Preferably, in step (2), the method for synthesizing aromatic amine derivative ligands by reacting compound 3 with compounds 4, 5, and 6 includes:

[0066] Compound 3 was dispersed in methanol along with compounds 4, 5 and 6, and the reaction yielded aromatic amine derivative ligands.

[0067] 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).

[0068] More preferably, the reaction temperature is 40~50 °C and the reaction time is 40~60 h.

[0069] A third aspect of the present invention provides the application of gold nanoparticles modified with aromatic amine derivatives as described in the first aspect or gold nanoparticles modified with aromatic amine derivatives prepared by the preparation method described in the second aspect as endoplasmic reticulum stress inducers.

[0070] In one or more embodiments, the application is at least one of the following 1) to 4);

[0071] 1) Increase the endoplasmic reticulum stress level in non-small cell lung cancer cells;

[0072] 2) Prepare products that enhance endoplasmic reticulum stress levels in non-small cell lung cancer cells;

[0073] 3) Improve the sensitivity of non-small cell lung cancer cells to chemotherapy drugs;

[0074] 4) Prepare products that improve the sensitivity of non-small cell lung cancer cells to chemotherapy drugs.

[0075] Preferably, in 3) and 4), the chemotherapeutic agent is cisplatin or a pharmaceutically acceptable salt thereof.

[0076] A fourth aspect of the present invention provides the use of gold nanoparticles modified with aromatic amine derivatives as described in the first aspect or gold nanoparticles modified with aromatic amine derivatives prepared by the preparation method described in the second aspect, in combination with chemotherapeutic drugs, in the preparation of drugs for non-small cell lung cancer.

[0077] In one or more embodiments, the chemotherapeutic agent is cisplatin or a pharmaceutically acceptable salt thereof.

[0078] In one or more embodiments, the mass ratio of the aromatic amine derivative-modified gold nanoparticles to the chemotherapeutic drug is (0.5~20):1, preferably (1~10):1.

[0079] A fifth aspect of the present invention provides a pharmaceutical composition for treating non-small cell lung cancer, comprising gold nanoparticles modified with aromatic amine derivatives as described in the first aspect or gold nanoparticles modified with aromatic amine derivatives prepared by the preparation method described in the second aspect, and a chemotherapeutic drug.

[0080] In one or more embodiments, the chemotherapeutic agent is cisplatin or a pharmaceutically acceptable salt thereof.

[0081] In one or more embodiments, the mass ratio of the aromatic amine derivative-modified gold nanoparticles to the chemotherapeutic drug is (0.5~20):1, preferably (1~10):1.

[0082] The beneficial effects of this invention are as follows:

[0083] The aromatic amine derivative-modified gold nanoparticles provided in this invention can increase the endoplasmic reticulum stress level of non-small cell lung cancer (NSCLC) cells and enhance their sensitivity to chemotherapeutic drugs, synergistically increasing the mortality rate of NSCLC cells. In A549 cells treated with aromatic amine derivative-modified gold nanoparticles, the expression of glucose-regulated protein 78 (GRP78) and C / EBP homolog (CHOP) was upregulated, demonstrating that the aromatic amine derivative-modified gold nanoparticles can increase the endoplasmic reticulum stress level of NSCLC cells and activate the UPR endoplasmic reticulum-like kinase (PERK)-eukaryotic translation initiation factor 2α (eIF2α)-activated transcription factor 4 (ATF4) branch, the inositol demand enzyme 1-X box binding protein 1 (1RE1-XBP1) splicing branch, and the activated transcription factor 6 (ATF6) branch, causing protein homeostasis breakdown, reducing and increasing the sensitivity of NSCLC cells to chemotherapeutic drugs, and promoting apoptosis of A549 cells. Attached Figure Description

[0084] 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.

[0085] Figure 1 Methods for preparing ligands of aromatic amine derivatives;

[0086] Figure 2 The hydrogen NMR spectrum of FAGNP-1;

[0087] Figure 3 The hydrogen NMR spectrum of FAGNP-2;

[0088] Figure 4 The hydrogen NMR spectrum of FAGNP-3;

[0089] Figure 5 The hydrogen NMR spectrum of FAGNP-4;

[0090] Figure 6 Transmission electron microscope images of gold nanoparticles (FAGNP-1-Au~FAGNP-4-Au) modified with aromatic amine derivatives; where A is FAGNP-1-Au, B is FAGNP-2-Au, C is FAGNP-3-Au, and D is FAGNP-4-Au.

[0091] Figure 7 The relative expression results of GRP78 or CHOP proteins after treatment with gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au);

[0092] Figure 8 The relative ratio of p-eIF2α / eIF2α and the relative expression of ATF4 after treatment with gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au);

[0093] Figure 9 The relative ratio of XBP1s / (XBP1s+XBP1u) after treatment with gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au) is shown in the figure.

[0094] Figure 10 The relative amount of N-ATF6 after treatment with gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au);

[0095] Figure 11 The relative expression results of Ub and p62 after treatment with gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au);

[0096] Figure 12 The figure shows the synergistic effect of aromatic amine derivative-modified gold nanoparticles (FAGNP-1-Au~FAGNP-4-Au) and the chemotherapy drug cisplatin on improving the mortality rate of A549 cells. Detailed Implementation

[0097] 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.

[0098] 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.

[0099] 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.

[0100] Example 1

[0101] Figure 1 The preparation method of ligands for aromatic amine derivatives is as follows: Figure 1 The method shown synthesizes the aromatic amine derivative ligands shown in formula (Ⅰ), namely FAGNP-1 to FAGNP-4.

[0102] The structural formulas of FAGNP-1 to FAGNP-4 are shown in Table 1 below.

[0103] Table 1 Structural Formulas of FAGNP-1 to FAGNP-4

[0104]

[0105] Specifically:

[0106] The structural formula of FAGNP-1 is:

[0107] ;

[0108] The structural formula of FAGNP-2 is:

[0109] ;

[0110] The structural formula of FAGNP-3 is:

[0111] ;

[0112] The structural formula of FAGNP-4 is:

[0113] .

[0114] Preparation of FAGNP-1:

[0115] (1) Under ice bath conditions, 0.05 mol indole-3-carboxylic acid was dispersed in 100 mL dichloromethane (DCM). After mixing evenly, 0.06 mol N-hydroxysuccinimide (NHS) and 0.06 mol 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.

[0116] (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, 32.6 mL of 1,4-p-phenylenediamine was added while stirring. After the reaction system was homogeneous and stable, the 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.

[0117] (3) 5 mmol of compound 3, 5 mmol of thiophenol-2-carboxaldehyde, 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 product was purified by column chromatography using 300-mesh silica gel. The developing solvent was a mixed solvent system of petroleum ether: ethyl acetate (EA) = 1:1 (volume ratio); FAGNP-1 was obtained.

[0118] The preparation methods for FAGNP-2 to FAGNP-4 are the same as those for FAGNP-1, the difference being the substitution of different substituents R1 or R2.

[0119] Figure 2 The hydrogen NMR spectrum of FAGNP-1;

[0120] Figure 3 The hydrogen NMR spectrum of FAGNP-2;

[0121] Figure 4 The hydrogen NMR spectrum of FAGNP-3;

[0122] Figure 5 This is the hydrogen NMR spectrum of FAGNP-4.

[0123] Example 2

[0124] Preparation of gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au):

[0125] 0.064 mmol of aromatic amine derivative ligands (FAGNP-2~FAGNP-4) were dissolved in 20 mL of DMF. 0.064 mmol of sodium 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 aromatic amine derivative-modified gold nanoparticles (FAGNP-1-Au~FAGNP-4-Au), which were dispersed in ultrapure water for later use.

[0126] Figure 6 Transmission electron microscopy images of gold nanoparticles (FAGNP-1-Au~FAGNP-4-Au) modified with aromatic amine derivatives, from... Figure 6 It can be seen that the gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au) are all approximately spherical, with uniform particle morphology and an average particle size of about 6 nm. No obvious large-scale aggregation was observed.

[0127] Table 2 shows the hydration size, surface Zeta potential, distribution coefficient (PDI) in high-purity water, hydrophilicity / hydrophobicity analysis (n-octanol-water partition coefficient Log P) and elemental analysis results of the gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au).

[0128] Table 2 Characterization of gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au)

[0129]

[0130] As shown in Table 2, the gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au to FAGNP-4-Au) exhibit the characteristics of "consistent core platform and tunable surface properties" in terms of physicochemical properties: their hydrated particle size is concentrated in the range of 72.2–98.3 nm, and their PDI is 0.143–0.245, indicating good dispersion and system stability in aqueous phase; the surface Zeta potential is 4.9–13.6 mV, exhibiting a slightly positive charge, which is beneficial for interaction with the cell membrane interface. Meanwhile, the surface ligand loading of the gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au to FAGNP-4-Au) is on the same order of magnitude (approximately 264–293 ligands per particle, corresponding to a surface density of approximately 3.64–3.76 ligands / nm). 2 This indicates that the differences in biological effects among different samples mainly stem from differences in the structure of aromatic amine derivative ligands rather than differences in ligand loading. Furthermore, the LogP values ​​showed significant differences within the range of 1.49–2.45 (e.g., lower LogP for FAGNP-1-Au and higher LogP for FAGNP-3-Au), demonstrating that the hydrophilicity / hydrophobicity of particle surfaces can be regulated by altering the π-electron density and polarizability of aromatic amine ligands. This provides a physicochemical basis for subsequent differences in cellular uptake and endoplasmic reticulum stress regulation.

[0131] The hydrophilicity / hydrophobicity analysis process of the surface of gold nanoparticles (FAGNP-1-Au~FAGNP-4-Au) modified with aromatic amine derivatives was as follows:

[0132] 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.

[0133] Add a certain volume of saturated aqueous solution of n-octanol, then add 0.3 mg of gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-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.

[0134] 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.

[0135] 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 aromatic amine derivative-modified gold nanoparticles (FAGNP-1-Au~FAGNP-4-Au).

[0136] Log P = Log (C O / C W );

[0137] 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.

[0138] Table 3. Standard Working Curve Preparation Method

[0139]

[0140] Example 3

[0141] Aromatic amine derivative-modified gold nanoparticles (FAGNP-1-Au~FAGNP-4-Au) induce endoplasmic reticulum stress in A549 cells:

[0142] 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 cultured at 37 °C and 5% CO2 for 24 h. Then, a dispersion of gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au) was added to a final concentration of 50 μg / mL, and the plates were cultured for another 24 h. After treatment, the culture medium was discarded and the plates were washed twice with pre-cooled phosphate-buffered saline (PBS). Total protein was extracted by adding lysis buffer, and the levels of GRP78 and CHOP proteins were detected by Western blot. All results were normalized to 1 for the untreated negative control group. Each sample was tested three times, and the experiment was biologically replicated and independently repeated at least three times.

[0143] The results are as follows Figure 7 As shown, compared with the negative control, the gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au) significantly upregulated the expression of GRP78 and CHOP proteins within the sublethal dose range, demonstrating that the gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au) can increase the endoplasmic reticulum stress level in non-small cell lung cancer cells.

[0144] To demonstrate that the treatment conditions for the aromatic amine derivative-modified gold nanoparticles used in this embodiment fall within the sub-lethal dose range, and to define their lethal threshold in A549 cells, cell viability was used as the evaluation index for verification. (See cisplatin-synergistic viability bar chart). Figure 12 As shown in the figure, under the condition of cisplatin = 0 μmol / L (i.e. no chemotherapy drugs added), after treatment with aromatic amine derivative modified gold nanoparticles (FAGNP-1~FAGNP-4), the cell survival rate remained at a high level of 88%~92%, which was close to that of the blank control group. This indicates that the amount of aromatic amine derivative modified gold nanoparticles used in this example does not have a significant acute lethal effect on the cells. Therefore, this amount can be defined as a sublethal dose and used for subsequent endoplasmic reticulum stress and UPR pathway detection experiments.

[0145] Example 4

[0146] Aromatic amine derivative-modified gold nanoparticles (FAGNP-1-Au~FAGNP-4-Au) activate the PERK-eIF2α-ATF4- branch, IRE1-XBP1- splicing branch, and ATF6 branch of UPR:

[0147] (1) Aromatic amine derivative-modified gold nanoparticles (FAGNP-1-Au~FAGNP-4-Au) activate the PERK-eIF2α-ATF4- branch of UPR:

[0148] 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 cultured at 37 °C and 5% CO2 for 24 h. Then, a dispersion of gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au) was added to a final concentration of 50 μg / mL, and the plates were cultured for another 24 h. After treatment, the culture medium was discarded and the plates were washed twice with pre-cooled phosphate-buffered saline (PBS). Total protein was extracted by adding lysis buffer, and the levels of eukaryotic translation initiation factor 2α (eIF2α), phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α), and activated transcription factor 4 (ATF4) were detected by Western blot. The p-eIF2α / eIF2α ratio and ATF4 gray value were quantitatively normalized. Each sample was tested three times, and the experiment was biologically replicated and independently repeated at least three times.

[0149] The results are as follows Figure 8As shown, compared with the negative control, the gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au) can significantly increase the p-eIF2α / eIF2α ratio and upregulate ATF4 expression, demonstrating that the gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au) activate the PERK-eIF2α-ATF4- branch of UPR.

[0150] (2) Aromatic amine derivative-modified gold nanoparticles (FAGNP-1-Au~FAGNP-4-Au) activate the 1RE1-XBP1 splicing branch of UPR:

[0151] After A549 cells were digested and counted, they were then processed at a rate of 1×10⁻⁶. 5 Two mL of cell suspension was seeded into 6-well plates at a density of cells / mL and cultured at 37 °C and 5% CO2 for 24 h. Then, a dispersion of gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au) was added to a final concentration of 50 μg / mL, and the plates were cultured for another 24 h. After treatment, the culture medium was discarded and the plates were washed twice with pre-cooled phosphate-buffered saline (PBS). Total RNA was extracted by adding lysis buffer. The spliced ​​region of X-box binding protein 1 (XBP1) was amplified by RT-PCR and agarose gel electrophoresis was performed to distinguish between unspliced ​​XBP1u and spliced ​​XBP1s bands. The ratio of XBP1s / (XBP1s+XBP1u) was calculated using imaging software. Each sample was tested three times, and the experiment was biologically replicated and independently repeated at least three times.

[0152] The results are as follows Figure 9 As shown, compared with the negative control, the gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au) can significantly increase the XBP1 splicing ratio and are correlated with the upregulation of GRP78, suggesting that the gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au) can not only induce endoplasmic reticulum stress, but also induce the specific activation of the IRE1-XBP1 branch.

[0153] (3) Aromatic amine derivative-modified gold nanoparticles (FAGNP-1-Au~FAGNP-4-Au) activate the ATF6 branch of UPR:

[0154] 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 cultured at 37 °C and 5% CO2 for 24 h. Then, a dispersion of gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au) was added to a final concentration of 50 μg / mL, and the plates were cultured for another 24 h. After treatment, the culture medium was discarded and the plates were washed twice with pre-cooled phosphate-buffered saline (PBS). The total protein was extracted by adding lysis buffer, and Western blot was used to detect the full-length ATF6 protein and the lysed N-ATF6 (activated fragment). The N-ATF6 signal was quantified by grayscale. Each sample was tested three times, and the experiment was biologically replicated and independently repeated at least three times.

[0155] The results are as follows Figure 10 As shown, compared with the negative control, gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au) can significantly increase N-ATF6 signal.

[0156] Example 5

[0157] Quantitative experiments on protein steady-state disintegration induced by aromatic amine derivative-modified gold nanoparticles (FAGNP-1-Au~FAGNP-4-Au):

[0158] 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 cultured at 37 °C and 5% CO2 for 24 h. Then, a dispersion of gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au) was added to a final concentration of 50 μg / mL, and the plates were cultured for another 24 h. After treatment, the culture medium was discarded and the plates were washed twice with pre-cooled phosphate-buffered saline (PBS). The total protein was extracted by adding lysis buffer, and the levels of total ubiquitinated protein (Ub) and ubiquitin-binding protein p62 (p62 / SQSTM1) were detected by Western blot. The bands were quantified by grayscale. Each sample was tested three times, and the experiment was biologically replicated and independently repeated at least three times.

[0159] The results are as follows Figure 11As shown, aromatic amine derivative-modified gold nanoparticles (FAGNP-1-Au~FAGNP-4-Au) can induce significant accumulation of ubiquitinated proteins, enhanced protein aggregates, and accompanied by decreased proteasome activity or abnormal proteasome load within a window dose that does not cause significant acute death. This indicates that aromatic amine derivative-modified gold nanoparticles (FAGNP-1-Au~FAGNP-4-Au) can induce quantifiable protein homeostasis disintegration effects at the cellular level, which is corroborated by UPR activation.

[0160] Example 6

[0161] Cisplatin sensitization assay: After digesting and counting A549 cells, the cells were subjected to a reaction at a rate of 5 × 10⁻⁶. 3 100 μL of cell suspension was seeded into 96-well plates at a density of cells / well and cultured at 37 °C and 5% CO2 for 24 h for cell adhesion. Then, a dispersion of gold nanoparticles modified with aromatic amine derivatives (FAGNP-1-Au~FAGNP-4-Au) was added to a final concentration of 50 μg / mL for 6 h of pretreatment. Cisplatin working solution was then added to make the final concentrations of cisplatin 0, 1, 2, 4 and 8 μmol / L, and the plates were cultured for another 24 h.

[0162] Cell viability assay and sensitization evaluation: After treatment, cell viability in each group was determined using the CCK-8 assay, and the results were normalized to 1 for the untreated negative control group. The inhibition rate at different cisplatin concentrations was calculated, and the dose-response difference between the "cisplatin alone group" and the "gold nanoparticle + cisplatin combination group" was compared. The cisplatin half-maximal inhibitory concentration (IC50) was used as the crisplatin inhibitory concentration. 50 The reduction in the rate of decrease or the decrease in survival rate at the same cisplatin dose was used as a sensitization indicator; at least three replicates were set up for each sample and at least three independent replicate experiments were performed.

[0163] Results analysis: The results showed that pretreatment with 50 μg / mL of aromatic amine derivative-modified gold nanoparticles (FAGNP-1-Au~FAGNP-4-Au) significantly enhanced the inhibitory response of A549 cells to cisplatin, manifested as a further decrease in cell viability at the same cisplatin dose and a higher cisplatin IC50 value. 50 The sensitization intensity showed a decreasing trend; the sensitization amplitudes of samples modified with different surface aromatic amine ligands varied, suggesting that the differences in surface π electron density and polarizability determined by the ligand structure can further regulate endoplasmic reticulum stress intensity and protein homeostasis load, thereby affecting the cytotoxic threshold of cisplatin. These results demonstrate that aromatic amine derivative-modified gold nanoparticles (FAGNP-1-Au~FAGNP-4-Au) can be used in combination with cisplatin as endoplasmic reticulum stress inducers to improve the sensitivity of non-small cell lung cancer cells to cisplatin and enhance the efficacy of chemotherapy.

[0164] 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 gold nanoparticle modified with an aromatic amine derivative, characterized in that, It involves coating the surface of gold nanoparticles with aromatic amine derivative ligands, wherein the gold nanoparticles and the aromatic amine derivative ligands are connected by gold-sulfur bonds; The structural formula of the aromatic amine 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) or (Ⅴ): Formula (Ⅳ); Formula (V).

2. The method for preparing gold nanoparticles modified with aromatic amine derivatives according to claim 1, characterized in that, Includes the following steps: Aromatic amine 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 aqueous solution was added, the reaction was stirred, and the solid was collected to obtain gold nanoparticles modified with aromatic amine derivatives.

3. The preparation method according to claim 2, characterized in that, The preparation method of the aromatic amine derivative ligand includes the following steps: (1) Compound 1 was reacted with N-hydroxysuccinimide to synthesize compound 2; (2) Compound 2 reacts with 1,4-p-phenylenediamine to synthesize compound 3; (3) Compound 3 reacts with compounds 4, 5 and 6 to synthesize aromatic amine 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, 2, and 3, R1 is selected from one of formulas (II) or (III): Formula (II); Formula (Ⅲ); In compound 5, R2 is selected from one of formulas (Ⅳ) or (Ⅴ): Formula (Ⅳ); Formula (V).

4. The preparation method according to claim 2, characterized in that, The organic solvent is selected from N,N-dimethylformamide; The concentration of the aromatic amine derivative ligand in the organic solvent is 2.5~4 mmol / L; The gold salt is selected from chloroauric acid; The molar ratio of the aromatic amine derivative ligand to the gold salt is (0.8~1.2):(0.8~1.2). The molar ratio of the gold salt to sodium borohydride is 1:(2.5~4). The concentration of the sodium borohydride aqueous solution is 16~26 mmol / L; The concentration of sodium citrate in organic solvents is 2.5~4 mmol / L.

5. The use of the gold nanoparticles modified with aromatic amine derivatives as described in claim 1, or the gold nanoparticles modified with aromatic amine derivatives prepared by any one of claims 2 to 4, in combination with chemotherapeutic drugs in the preparation of drugs for non-small cell lung cancer.

6. The application as described in claim 5, characterized in that, The chemotherapy drug is cisplatin or a pharmaceutically acceptable salt thereof; The mass ratio of the aromatic amine derivative-modified gold nanoparticles to the chemotherapy drug is (0.5~20):

1.

7. A pharmaceutical composition for treating non-small cell lung cancer, characterized in that, The invention includes gold nanoparticles modified with aromatic amine derivatives as described in claim 1, or gold nanoparticles modified with aromatic amine derivatives prepared by the preparation method described in any one of claims 2 to 4, and chemotherapeutic drugs.

8. The pharmaceutical composition according to claim 7, characterized in that, The chemotherapy drug is cisplatin or a pharmaceutically acceptable salt thereof; The mass ratio of the aromatic amine derivative-modified gold nanoparticles to the chemotherapy drug is (0.5~20):1.

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