Application of gold nanocluster for enhancing breast cancer endoplasmic reticulum stress death

Abstract

The invention discloses application of a gold nanocluster for enhancing breast cancer endoplasmic reticulum stress death, and relates to the field of triple negative breast cancer treatment. A proper gold salt solution and natural peptide / protein are fully stirred according to the explored temperature and pH conditions, at the moment, high-valence Au ions in the gold salt are reduced, and the luminescent characteristic is shown. As a docetaxel enhancer, the nanocluster disclosed by the invention can effectively promote endoplasmic reticulum stress death of breast cancer cells and inhibit tumor cell activity; due to good biological safety and stability, effective treatment of breast cancer can be realized. The compound can be used for treating various breast cancers represented by triple-negative breast cancers.

Description

Technical Field

[0001] The present invention relates to the field of cell stress and triple-negative breast cancer treatment, and specifically to a preparation method of a nanoscale gold cluster compound that promotes tumor cell death through endoplasmic reticulum stress and its related characterization, as well as verifying the therapeutic effect of GA on triple-negative breast cancer cells through in vitro tumor cell biosafety tests, mechanism of action tests, and in vivo mouse model tests. Background Art

[0002] Cancer is one of the major diseases facing mankind, and breast cancer is one of the most common cancers in women. According to the latest data from the International Agency for Research on Cancer (IARC) survey in 2018, the incidence of breast cancer in women worldwide is 24.2%, ranking first among female cancers, of which 52.9% occur in developing countries. The incidence of breast cancer in women in my country has been increasing year by year. Although the overall survival of breast cancer is better than that of other cancers, the 5-year survival rate of advanced triple-negative breast cancer is only 11%. Negative breast cancer refers to breast cancer in which the results of immunohistochemical examination of cancer tissue are negative for estrogen receptor (ER), progesterone receptor (PR) and proto-oncogene Her-2. This type of breast cancer accounts for 10.0% to 20.8% of all breast cancer pathological types, has special biological behavior and clinical pathological characteristics, and has a worse prognosis than other types. Paclitaxel and its derivative docetaxel (docetaxel) can trigger mitotic arrest and endoplasmic reticulum stress, thereby promoting cell death, and are widely used to treat breast cancer, ovarian cancer, lung cancer and other types of tumors. However, with the widespread use of paclitaxel drugs, cancer cells have gradually developed resistance to them, so there is an urgent need for a drug to promote the efficacy of paclitaxel drugs.

[0003] Literature shows that docetaxel (DTX) is an M-phase cycle-specific drug that can promote the polymerization of cell microfilaments into microtubules and inhibit the depolymerization of microtubules, thereby destroying the microtubule network structure and causing cell apoptosis, and it can antagonize the effects of Bcl-2 and Bcl-xL. Docetaxel mainly acts through three pathways of endoplasmic reticulum stress: PERK pathway, ATF6 pathway and IRE1 pathway to ultimately cause cell apoptosis. The endoplasmic reticulum is an important organelle in cells for synthesizing proteins, lipids, etc., and is also related to functions such as maintaining intracellular calcium balance. When cells face environmental stress, such as oxidative stress, the endoplasmic reticulum will be damaged, resulting in abnormal protein folding and inducing "endoplasmic reticulum stress". A series of downstream reactions are triggered, which may eventually lead to cell death. However, as a first-line chemotherapy drug, docetaxel can cause certain damage to the human body, such as neutropenia, leukopenia, hematopoietic cell reduction, fatigue, anemia, neurotoxicity and hepatosplenic toxicity. Therefore, long-term or excessive use will cause great damage to the body. Therefore, an auxiliary drug is needed to enhance its efficacy in order to reduce the drug dosage.

[0004] As a precious metal, gold has the characteristics of high chemical stability, easy synthesis and biological inertness, and is becoming increasingly popular in the field of biology. Gold nanoclusters are a hot topic. Gold nanoclusters are widely used in tumor treatment, imaging diagnosis, biosensors and molecular biology. In addition, it is also used in medical imaging, medical testing and other fields. This is mainly because the surface of gold nanoclusters is easy to modify, and they have good solubility and biocompatibility; secondly, the size and shape of gold nanoclusters can be controlled at the nanoscale to achieve different functional applications; in addition, the surface optical properties of gold nanoclusters are excellent, and microscopic imaging can be achieved through surface plasmon resonance, surface Raman scattering and other technologies.

[0005] Gold nanoclusters have many advantages. The nanoclusters formed by the reaction of Au and polypeptides are small in size, can penetrate most barriers in the human body and enter cells, have good stability, clear molecular structure, good biocompatibility, are easy to modify and are non-toxic to the human body.

[0006] At present, the treatment of triple-negative breast cancer is relatively complicated, because its ER, PR and Her-2 are all negative, so progesterone therapy, estrogen therapy, endocrine therapy and targeted therapy cannot be used. At present, the main treatment method for triple-negative breast cancer is chemotherapy, and docetaxel is a commonly used chemotherapy drug. . However, the long-term use of docetaxel and chemotherapy can cause damage to the human body, and the concentration of docetaxel in the human body cannot exceed 0.74mg / ml. Therefore, it also has certain limitations. If the above problems can be solved by using the characteristic advantages of gold nanoclusters themselves, the effect of docetaxel can be enhanced, the therapeutic effect can be improved, the side effects of treatment can be reduced, and the economic cost of treatment can be reduced. The present invention provides a newly synthesized nano-sized gold cluster, by establishing an endoplasmic reticulum stress death model of breast cancer cells in vitro, verifying whether gold nanoclusters can enhance the effect of docetaxel, and exploring the mechanism of action of gold clusters, and finally verifying its effect in simulated in vivo and real animal bodies through 3D culture and animal experiments. Summary of the invention

[0007] The invention relates to the preparation and application of gold and a thiol-containing natural peptide, and the application of the gold and thiol-containing natural peptide to the preparation of drugs in the field of breast cancer treatment, especially drugs in the field of endoplasmic reticulum stress and triple-negative breast cancer treatment.

[0008] The gold nanoclusters formed by the gold and the thiol-containing natural peptide of the present invention are used for preparing drugs in the field of breast cancer treatment, especially drugs in the field of endoplasmic reticulum stress and triple-negative breast cancer treatment, and are especially used as a reinforcing adjuvant of docetaxel to enhance the drug application of docetaxel in the treatment of breast cancer.

[0009] A drug for treating breast cancer, characterized by comprising docetaxel and gold nanoclusters formed by gold, an enhancer of docetaxel, and a thiol-containing natural peptide. The drug for treating breast cancer is a drug in the field of endoplasmic reticulum stress and triple-negative breast cancer.

[0010] The specific synthesis of the gold nanoclusters formed by the thiol-containing natural peptide is: reacting the gold salt solution with the thiol-containing natural peptide at a specific pH and temperature for a period of time, and this process will reduce the gold ions from the high valence state to the low valence state. The composition of the synthesized gold nanoclusters can be determined qualitatively and quantitatively by mass spectrometry analysis, ultraviolet analysis, etc. The present invention is non-toxic and harmless to cells and organisms, but can enhance the killing of docetaxel on breast cancer cells, specifically by enhancing the endoplasmic reticulum stress of tumor cells, thereby causing them to be inhibited and eventually die, thereby achieving the purpose of enhancing drug efficacy and reducing drug dosage.

[0011] The preparation method of the reaction of gold and natural glutathione containing thiol to form nanoclusters is as follows: first, the gold salt solution and glutathione are mixed evenly, and then a reducing agent is added, and the reaction is stirred for a certain time under certain temperature and pH conditions. Gold reacts with thiol groups to finally generate Au. 29 SG 27 Complex.

[0012] The gold salt solution is a trivalent gold inorganic salt ion solution such as chloroauric acid, and the trivalent gold ions are reduced to monovalent gold ions or gold atoms during the synthesis process.

[0013] The reducing agent is sodium borohydride (NaBH4), sodium hydroxide (NaOH), vitamin C (vitamin C) or trisodium citrate dihydrate (Trisodium citrate dihydrate), etc., and is preferably selected according to actual synthesis.

[0014] The concentration of the thiol-containing natural peptide in the mixed solution is 0.1 μM-10M, and the concentration of the gold salt solution is 0.1 μM-10M. Generally, the best effect is achieved when the ratio of the gold salt solution to the polypeptide is 1:2, which is optimized according to the actual synthesis.

[0015] When the gold salt solution reacts with the polypeptide, the pH is generally controlled at a weakly alkaline to neutral level, and the temperature is controlled at about 50-70° C. The optimum is determined based on the actual synthesis conditions.

[0016] When the solution is mixed, start the stirring reaction in a water bath to avoid light. During the reaction, the color of the solution gradually changes from colorless to bright golden yellow, and the reaction time is about 6-10 hours. After the reaction is completed, the synthesized product needs to be ultrafiltered and purified, and the purified product can be used. The synthesized GA nanoclusters have good stability and microscale, and the hydrated particle size is generally 1.9nm and above nanometer level, such as 1.9-3nm.

[0017] The application of the complex synthesized by gold and binding peptide is used to promote endoplasmic reticulum stress of cancer cells, which has an adjuvant therapeutic effect on breast cancer. Compared with docetaxel alone, docetaxel and GA can significantly promote the efficacy of docetaxel when they act together, achieving better therapeutic effects, and the dosage of GA can be appropriately increased to reduce the dosage of docetaxel, so as to ensure the efficacy while minimizing damage to the human body.

[0018] The beneficial results of the present invention are:

[0019] (1) The present invention uses a green one-step synthesis method to prepare gold clusters, which has simple preparation steps, low cost, and less reagent loss.

[0020] (2) Experiments on animal models have shown that the gold nanoclusters have little toxicity and side effects and are relatively safe.

[0021] (3) The present invention can integrate the targeted binding function of polypeptides and the small size and good biocompatibility of gold complexes into the same compound, realizing cell death caused by endoplasmic reticulum stress of cancer cells in vitro at the 2D and 3D levels, providing a different new idea for anticancer drugs.

[0022] (4) The present invention can act synergistically with docetaxel in the triple-negative breast cancer animal model, and has a more obvious promoting effect compared with docetaxel alone in the treatment of tumors caused by triple-negative breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1a The images obtained by direct observation method in Example 1 of the present invention are photographs under sunlight (a) and 365nm ultraviolet light (b);

[0024] Figure 1b is a fluorescence spectrum diagram of Example 1 of the present invention, where ex represents the excitation spectrum and em represents the emission spectrum;

[0025] Figure 1c This is a DLS hydrated particle size image of Example 1 of the present invention, including PDI and Zeta potential, and the results show that the particle size is about 1.91 nm;

[0026] Figure 1d This is a transmission electron microscope photograph of Example 1 of the present invention;

[0027] Figure 2a This is the toxicity test on 4T1 cells in Example 1 of the present invention;

[0028] Figure 2bThis is the confocal imaging of enhancing tumor killing ability by promoting docetaxel-induced 4T1 cell apoptosis in a 4T1 cell 2D culture system according to Example 1 of the present invention;

[0029] Figure 2c This is the toxicity test on MDA-MB-231 cells in Example 1 of the present invention;

[0030] Figure 2d This is the confocal imaging of enhancing tumor killing ability by promoting docetaxel-induced 4T1 cell apoptosis in the MDA-MB-231 cell 2D culture system in Example 1 of the present invention;

[0031] Figure 2e This is a 2D image of 4T1 cell apoptosis after adding GA and docetaxel to the 4T1 cell 2D culture system after pretreatment with apoptosis inhibitor z-VAD, ferroptosis inhibitor Fer-1 and necrosis inhibitor Nec-1 in Example 1 of the present invention;

[0032] Figure 2f Western blot image of Example 1 of the present invention promoting docetaxel-induced apoptosis of 4T1 cells

[0033] Figure 2g This is an image of clone formation in Example 1 of the present invention that promotes docetaxel-induced apoptosis of 4T1 cells;

[0034] Figure 3a This is an image of promoting the polymerization of microfilaments into microtubules according to Example 1 of the present invention;

[0035] Figure 3b This is an image of promoting cell cycle arrest of 4T1 cells at the G2 / M phase in Example 1 of the present invention;

[0036] Figure 3c This is a Western blot image of the effect of Example 1 of the present invention on the IRE1α signaling pathway of endoplasmic reticulum stress induced by docetaxel;

[0037] Figure 3d This is a Western blot image of the PERK signaling pathway that promotes docetaxel-induced endoplasmic reticulum stress in Example 1 of the present invention;

[0038] Figure 3e This is a qPCR data diagram of the signaling pathway that promotes docetaxel-induced endoplasmic reticulum stress in Example 1 of the present invention;

[0039] Figure 4a The microtubule changes caused by adding Example 1 of the present invention and docetaxel to 4T1 cells after pretreatment with the endoplasmic reticulum stress inhibitor TUDCA;

[0040] Figure 4bThis is a qPCR data diagram of the signaling pathway of endoplasmic reticulum stress induced by docetaxel in Example 1 of the present invention after pretreatment with pathway inhibitors IRE1αi and PERKi and endoplasmic reticulum stress inhibitor TUDCA;

[0041] Figure 4c 2D images of 4T1 cell apoptosis after pretreatment with pathway inhibitors IRE1αi and PERKi and endoplasmic reticulum stress inhibitor TUDCA in a 4T1 cell 2D culture system, and then after addition of Example 1 of the present invention and docetaxel;

[0042] Figure 5a This is the confocal imaging of Example 1 of the present invention for enhancing the function of the endoplasmic reticulum stress inducer in a 3D tumor cell culture system to enhance the tumor killing ability;

[0043] Figure 5b Confocal imaging of Example 1 of the present invention in a 3D tumor cell culture system in combination with an endoplasmic reticulum stress inducer after adding the pathway inhibitor PERKi;

[0044] Figure 6a This is a graph showing the gold content of various tissues in the in vivo animal experiment of Example 1 of the present invention;

[0045] Figure 6b This is the in vivo animal administration situation of Example 1 of the present invention;

[0046] Figure 6c It is a line graph of the tumor growth curve of the in vivo animal experiment of Example 1 of the present invention;

[0047] Figure 6d This is a histogram of tumor size in the in vivo animal experiment of Example 1 of the present invention;

[0048] Figure 6e This is a tumor photo of the in vivo animal experiment in Example 1 of the present invention;

[0049] Figure 6f This is a histogram of tumor mass in the in vivo animal experiment of Example 1 of the present invention;

[0050] Figure 6g The results of TUNEL immunofluorescence staining of tumor tissue in the in vivo animal model of Example 1 of the present invention;

[0051] Figure 6h The results of immunohistochemical staining of cleaved caspase-3 in tumors of in vivo animal experiments in Example 1 of the present invention;

[0052] Figure 6i The results of p-PERK immunofluorescence staining of tumors in the in vivo animal experiment of Example 1 of the present invention;

[0053] Figure 6j The results of TNF-α immunofluorescence staining of tumors in the in vivo animal experiment of Example 1 of the present invention;

[0054] Figure 6k The results of IL-6 immunofluorescence staining of tumors in the in vivo animal experiment of Example 1 of the present invention;

[0055] Figure 6l This is the HE staining result of the in vivo animal experiment in Example 1 of the present invention; DETAILED DESCRIPTION

[0056] Typical embodiments that embody the features and advantages of the present invention will be described in detail in the following description. It should be understood that the present invention can have various changes in different embodiments without departing from the scope of the present invention, and the descriptions and illustrations therein are essentially used for illustration rather than for limiting the present invention.

[0057] The present invention provides a novel method for preparing gold nanoclusters combined with gold and polypeptides and their application in the diagnosis of triple-negative breast cancer. The core of the method is to form a gold-sulfur bond by reacting gold with thiol groups in polypeptides, wherein the polypeptide provides thiol groups and the gold salt solution provides gold elements; under a certain temperature, pH and reducing agent, the salt solution and the polypeptide solution are mixed to undergo a reduction reaction, reducing the high-valent gold in the gold salt to low-valent gold ions or gold atoms.

[0058] Preferably, a weak base may be added to the mixed solution to provide a weak alkaline environment so that the reducing agent has reducing properties, thereby reducing gold ions.

[0059] The gold salt solution used in the present invention is a trivalent gold salt solution, such as chloroauric acid solution. During the preparation process, trivalent gold is reduced to gold atoms or monovalent gold ions.

[0060] The present invention provides a method for preparing a gold nanocluster compound for promoting tumor death of triple-negative breast cancer, comprising:

[0061] The gold salt solution is mixed with the thiol-containing polypeptide solution, stirred for 3-5 minutes, so that the gold salt and the polypeptide are fully contacted to form a mixed solution; a reducing agent is added to the above-mentioned mixed solution to cause the system to undergo a reduction reaction, the pH is adjusted, and the reaction is carried out under light-proof conditions. When the color of the solution changes from colorless to light yellow, stirring is continued for 12 hours after the color change occurs, and finally a complex of gold and peptide is obtained. The obtained complex is centrifuged at high speed for 30 minutes to remove large particles in the product, and then the obtained product is washed with ethanol 3-5 times. After washing, 0.25M NAOH is used to stir and dissolve and the pH is controlled at about 7. Finally, the product is purified with a 3kDA ultrafiltration tube (MWCO.3kDA) to remove unreacted substances. The purified product is then concentrated, and the volume and concentration multiple are recorded. The molar ratio of gold salt to polypeptide is 1:1-1:5, preferably 1.5:1, and the reducing agent used can be sodium borohydride (NaBH4), sodium hydroxide (NaOH), vitamin C (vitamin C), etc., preferably trisodium citrate. The gold salt concentration used is 0.01 μM-10M, and the thiol polypeptide concentration is 0.1 μM-10M.

[0062] The hydrated particle size of the complex prepared by the present invention is about 2-3 nm. Each Au-peptide complex has a precise molecular composition: (Au)x(SG)y, wherein 1≤x≤35, 1≤y≤30, and has photoluminescence properties.

[0063] The preparation method of the present invention prepares GA, uses natural glutathione containing thiol as a ligand, utilizes the high affinity of gold for thiol, and synthesizes Au-peptide adduct. The method is characterized by using a one-step reduction method, being simple and easy to prepare, and the obtained complex has high stability, and is particularly suitable for the treatment of triple-negative breast cancer.

[0064] In the present invention, a thiol-containing GSH peptide is selected as a ligand and directly adducted with an Au salt to form a thiol-Au adduct. In addition, when preparing such complexes, their biocompatibility, water solubility, chemical stability, size, and surface properties can be designed and modified to meet the needs of specific biomedical applications.

[0065] The novel gold complex molecular probe Au-peptide adduct of the present invention has the following advantages in treating triple-negative breast cancer: (1) small size, only 2-3 nm, can penetrate the membrane structure in most cells, thereby reducing the loss of drug efficacy; (2) non-toxic. Although docetaxel as a chemotherapy drug has many adverse effects on the human body, such as neutropenia, peripheral neurotoxicity, local skin redness and swelling, etc., the complex synthesized by the present invention is non-toxic and harmless to the human body; (3) the present invention can enhance the efficacy of docetaxel, can be used as an auxiliary agent, and can also reduce the dosage of docetaxel to reduce the side effects on the human body.

[0066] The application of the complex formed by the combination of gold and glutathione is used for the future clinical treatment of triple-negative breast cancer.

[0067] The GA complex of the present invention, its preparation method and its application are further described below in conjunction with specific examples. The gold salt used in the examples is trivalent chloroauric acid. The fluorescence characteristics of the synthesized GA are characterized by direct observation and fluorescence spectrometer. The feasibility of the metal nanoclusters for preventing and treating breast cancer is verified by a series of cytotoxic experiments around breast cancer cells in vivo and therapeutic experiments on animal models in vitro.

[0068] Example 1. Preparation and characterization of GA complex

[0069] 1) At a certain temperature (preferably 70°C in this example), 9.2 mg of GSH peptide (γ-Glu-Cys-Gly) (first and last amide bonds cyclized) was dissolved in 10 mL of ultrapure water, and a stirring bar was added and stirred on a magnetic stirrer;

[0070] 2) Add 80 mL of chloroauric acid solution (25 mM) dropwise into the above solution;

[0071] 3) Then add 200uL of trisodium citrate solution into the system;

[0072] 4) Immediately add 1000uL of NaOH (1mol / L) solution dropwise to adjust the pH of the solution to near neutral, and then continue stirring for 12h;

[0073] The stirring bar was sucked out with a magnetic rod to obtain gold clusters emitting red fluorescence. The product was ultrafiltered using an ultrafiltration tube with a molecular weight cutoff of 3000 to remove free metal ions and unreacted peptides. The GA image obtained by direct observation is shown in Figure 1. Figure 1a As shown, GA appears bright yellow under sunlight ( Figure 1a 1), and it shows orange-red fluorescence under 365nm ultraviolet light ( Figure 1a 2). The fluorescence spectrum of GA is shown in Figure 1b As shown, the black line is the optimal excitation spectrum of GA, and the red line is the optimal emission spectrum, that is, the excitation peak of GA is at 460nm, and the emission peak is at 603nm.

[0074] The particle size of the nano-gold cluster GA was measured to be about 1.9 nm by dynamic light scattering (DLS). Figure 1c ). High-resolution transmission electron microscopy showed that the particle size of GA in the aqueous phase was uniform.

[0075] The relevant tests in the following examples all adopt the GA complex obtained in Example 1.

[0076] Example 2: GA sensitizes TNBC cells to docetaxel-induced apoptosis

[0077] Figure 2a The 24-hour toxicity test of GA at different concentrations on the mouse triple-negative breast cancer cell line, 4T1 cells, shows that even at high concentrations, GA has no obvious toxicity to 4T1 cells, indicating that GA alone has no killing effect on 4T1 cells.

[0078] Figure 2b The GA of the present invention promotes the killing effect of docetaxel on 4T1 cells under a 2D model. The well-grown 4T1 cells were inoculated in a 96-well plate, and after they were attached to the wall, docetaxel (50 μM) was used to induce endoplasmic reticulum stress, and the present invention (GA 100 μM) was added at the same time. After 24 hours, Calcein AM (2.5 μM) and PI / propidium iodide (2.5 μg / ml) were used to perform live / dead double staining of cells in each well for 30 minutes, followed by observation and statistical analysis by laser confocal imaging. The results show that GA significantly enhances the killing effect of docetaxel in the 2D model of mouse 4T1 cells.

[0079] Figure 2c The 24-hour toxicity test of GA at different concentrations on the human triple-negative breast cancer cell line, MDA-MB-231 cells, shows that even at high concentrations, GA has no obvious toxicity to MDA-MB-231 cells, indicating that GA alone has no killing effect on MDA-MB-231 cells.

[0080] Figure 2d The GA of the present invention promotes the killing effect of docetaxel on MDA-MB-231 cells under the 2D model. The well-grown MDA-MB-231 cells are inoculated in a 96-well plate, and after they are attached to the wall, DTX (50 μM) is used to induce endoplasmic reticulum stress, and the present invention (GA 100 μM) is added at the same time. After 24 hours, Calcein AM (2.5 μM) and PI / propidium iodide (2.5 μg / ml) are used to perform live / dead double staining for 30 min on each well cell, and then laser confocal imaging is used and statistical analysis is performed. The results show that the GA of the present invention can improve the effect of DTX in the MDA-MB-231 cell 2D model, strengthen the endoplasmic reticulum stress of tumor cells and kill.

[0081] Figure 2eThis is the way in which GA of the present invention promotes DTX to kill 4T1 cells under a 2D model. 4T1 cells were inoculated in a 96-well plate, and after they adhered to the wall, apoptosis inhibitor z-VAD (10 μM), ferroptosis inhibitor Fer-1 (2 μM) and necrosis inhibitor Nec-1 (10 μM) were first added for pretreatment for 1 hour, followed by the addition of DTX (50 μM) and the present invention (GA 100 μM). After 24 hours, Calcein AM (2.5 μM) and PI / propidium iodide (2.5 μg / ml) were used to perform live / dead double staining of cells in each well for 30 minutes, followed by laser confocal imaging and statistical analysis. The results showed that the apoptosis inhibitor Z-VAD completely inhibited cell death, while necrosis and ferroptosis inhibitors had no effect, suggesting that cell death induced by the combined treatment of GA and DTX was apoptosis.

[0082] Figure 2f The GA of the present invention promotes the protein expression of DTX for apoptosis of 4T1 cells. The well-grown 4T1 cells were inoculated in a 12-well plate, and after they were attached to the wall, DTX (50 μM) was used to induce endoplasmic reticulum stress, and the present invention (GA 100 μM) was added at the same time. After 1 hour, the cells were digested, collected and protein was extracted, and the expression of apoptotic protein Cleavedcaspase-3 was detected by Western blot. Consistent with the results obtained before, compared with the single DTX treatment, the addition of GA significantly increased the expression of Cleaved caspase-3, that is, increased the apoptosis of 4T1.

[0083] Figure 2g This is an experiment of cloning formation of GA in the present invention to promote the killing function of DTX (50 μM) on 4T1 cells. After treating cells with DTX, GA (100 μM) and GSH (100 μM) for 4 hours, 1500 cells were taken from each group for culture until obvious clones were formed. The results showed that the number of cell clones formed by the combined action of GA and DTX was significantly reduced, indicating that on the basis of DTX treatment to reduce 4T1 cells, the addition of GA further significantly reduced the cell survival rate, while GSH had no such effect, indicating that GA directly increased the apoptosis rate of 4T1 cells by DTX.

[0084] Example 3: GA promotes microtubule dysregulation and endoplasmic reticulum stress in TNBC cells

[0085] Literature has shown that docetaxel-induced apoptosis is achieved by increasing microtubule polymerization and stimulating endoplasmic reticulum stress. Endoplasmic reticulum stress mainly includes three pathways, so we conducted experiments to verify it and found that in 4T1 cells, docetaxel mainly induces endoplasmic reticulum stress through the PERK pathway, leading to cell apoptosis.

[0086] Figure 3aThe effect of GA of the present invention on intracellular tubulin. Well-grown 4T1 cells were inoculated in a 12-well plate, and after they were attached to the wall, DTX (50 μM) was used to induce endoplasmic reticulum stress, and the present invention (GA 100 μM) was added at the same time. After 24 hours of treatment, immunofluorescence staining was performed to observe the changes in tubulin α-tubulin. It was found that α-tubulin increased significantly after adding GA of the present invention, indicating that GA enhanced the tubulin stabilization effect of DTX.

[0087] Figure 3b The effect of GA of the present invention on the cell cycle. Well-grown 4T1 cells were inoculated in a 12-well plate, and after they adhered to the wall, DTX (50 μM) was used to induce endoplasmic reticulum stress, and the present invention (GA 100 μM) was added at the same time. After 24 hours of treatment, the cells were digested and collected, and stained with PBS containing 0.2% saponin, 10 μg / ml RNase and 50 μg / ml propidium iodide (PI). Subsequently, flow cycle detection was performed on it, and it was found that DTX can promote cell arrest in the G2 / M phase, and GA can enhance the efficacy of DTX.

[0088] Figure 3c Well-growing 4T1 cells were inoculated in a 12-well plate, and after they adhered to the wall, DTX (50 μM) was used to induce endoplasmic reticulum stress, and the present invention (GA 100 μM) was added at the same time. After 1 hour, the cells were digested and collected to extract proteins, and the expression of IRE pathway proteins was detected by Western blot, and it was found that DTX and GA had no effect on the IRE pathway.

[0089] Figure 3d Well-growing 4T1 cells were inoculated in a 12-well plate, and after they adhered to the wall, DTX (50 μM) was used to induce endoplasmic reticulum stress, and the present invention (GA 100 μM) was added at the same time. After 1 hour, the cells were digested and collected to extract proteins, and the expression of a series of proteins in the PERK pathway was detected by Western blot. It was found that DTX promoted the expression of proteins in the PERK pathway, and this was even more severe after the addition of GA.

[0090] Figure 3e Well-growing 4T1 cells were inoculated in a 12-well plate, and after they adhered to the wall, DTX (50 μM) was used to induce endoplasmic reticulum stress, and the present invention (GA 100 μM) was added at the same time. After 12 hours, the RNA of the cells was extracted, and the qPCR experiment was performed to detect the gene expression of the PERK pathway. It was found that DTX promoted the gene expression of the PERK pathway, and the addition of GA strengthened this situation.

[0091] Example 4: Sensitization of GA to endoplasmic reticulum stress

[0092] Figure 4a Well-growing 4T1 cells were inoculated in a 12-well plate, and after they adhered to the wall, the endoplasmic reticulum stress inhibitor TUDCA (1 μM) was added for pretreatment for 1 h, and then DTX (50 μM) was added to induce endoplasmic reticulum stress, and the present invention (GA 100 μM) was added at the same time. After treatment for 24 hours, immunofluorescence staining was performed to observe the changes in microtubule protein α-tubulin. It was found that α-tubulin increased significantly after adding GA of the present invention, but after adding TUDCA, the increase in microtubule protein caused by GA was inhibited, indicating that GA did increase the expression of α-tubulin through endoplasmic reticulum stress.

[0093] Figure 4b Well-growing 4T1 cells were inoculated in a 12-well plate. After they adhered to the wall, IRE pathway inhibitor IRE1α (1 μM), PERK pathway inhibitor PERKi (1 μM) and endoplasmic reticulum stress inhibitor TUDCA (1 μM) were added for pretreatment for 1 hour, followed by the addition of DTX (50 μM) to induce endoplasmic reticulum stress, and the present invention (GA 100 μM) was added at the same time. After 12 hours, the RNA of the cells was extracted, and the qPCR experiment was performed to detect the gene expression of the PERK pathway. It was found that the enhancement of the expression of several PERK pathway genes caused by GA did not change after the addition of IRE1αi, but was significantly inhibited after the addition of PERKi, and there was also an inhibitory effect after the addition of TUDCA. This shows that GA enhances endoplasmic reticulum stress through the PERK pathway.

[0094] Figure 4c The GA of the present invention promotes the killing effect of docetaxel on 4T1 cells under a 2D model. The well-grown 4T1 cells were inoculated in a 96-well plate. After they adhered to the wall, IRE pathway inhibitor IRE1αi (1 μM), PERK pathway inhibitor PERKi (1 μM) and endoplasmic reticulum stress inhibitor TUDCA (1 μM) were first added for pretreatment for 1 hour, and then docetaxel (50 μM) was added to induce endoplasmic reticulum stress, and the present invention (GA 100 μM) was added at the same time. After 24 hours, Calcein AM (2.5 μM) and PI / propidium iodide (2.5 μg / ml) were used to perform live / dead double staining of cells in each well for 30 minutes, and then observed and statistically analyzed by laser confocal imaging. It was found that the apoptosis enhancement effect brought about by GA was significantly inhibited after the addition of PERKi and TUDCA, but the addition of IRE1αi had no effect.

[0095] Example 5: 3D culture further verifies that gold nanocluster GA enhances tumor killing ability by promoting docetaxel function

[0096] Compared with traditional monolayer cell culture, 3D tumor spheroids have become an indispensable in vitro model for cancer research because they reproduce the structure and physiology of solid tumors.

[0097] Figure 5a The 3D spheres of mouse breast cancer 4T1 cells were counted with 1500 cells, and the cells were cultured in a low-adsorption U-shaped 96-well plate. The cells interacted with each other to cause the formation of spontaneous tumor spheres. The well-grown 3D spheres were induced to endoplasmic reticulum stress using DTX (50 μM), and GA (100 μM) of the present invention was added at the same time. After 24 hours, Calcein AM (2.5 μM) and PI / propidium iodide (2.5 μg / ml) were used to perform live / dead double staining of the 3D tumor spheres for 30 minutes, and then laser confocal imaging was used for statistical analysis. The results showed that GA of the present invention can also play a role in improving the tumor killing effect of DTX in the mouse 4T1 cell 3D tumor model.

[0098] Figure 5b The 3D spheres of mouse breast cancer 4T1 cells were counted with 1500 cells, and the cells were cultured in a low-adsorption U-shaped 96-well plate. The cells interacted with each other to cause the formation of spontaneous tumor spheres. The well-grown 3D spheres were first pretreated with the PERK inhibitor PERKi (1 μM) for 1 hour, and then DTX (50 μM) was used to induce endoplasmic reticulum stress, and GA (100 μM) of the present invention was added at the same time. After 24 hours, Calcein AM (2.5 μM) and PI / propidium iodide (2.5 μg / ml) were used to perform live / dead double staining on the 3D tumor spheres for 30 minutes, and then laser confocal imaging was used for statistical analysis. The results showed that GA of the present invention improves the tumor killing effect of DTX through the PERK pathway.

[0099] Example 6: 3D culture further verifies that gold nanocluster GA enhances tumor killing ability by promoting docetaxel function

[0100] Based on the good effect of GA on enhancing the tumor killing effect of endoplasmic reticulum stress inducer in 3D tumor spheroids in vitro, we further studied its therapeutic effect on orthotopic breast cancer model. 6 4T1 cells were subcutaneously implanted in the mammary glands of mice and divided into three groups (Saline, DTX, and DTX+GA) for treatment. DTX was given every 3 days, and GA was given every 1 day by intraperitoneal injection. The growth of the tumor inoculation site was observed daily, and the tumor volume was recorded with a vernier caliper. 肿瘤 (mm 3 )=0.5xlengthxwidth 2 .

[0101] Figure 6aAfter the treatment, ICP-MS was used to detect the gold content in the main organs and tumors of mice. The results showed that gold was enriched in the tumor, indicating that GA had indeed entered the tumor and resided there; most of the gold was in the kidneys, indicating that GA has good metabolism and can be excreted from the kidneys.

[0102] Figure 6b For the treatment dosing regimen, DTX was administered every 3 days at a concentration of 15 mg / kg, and GA was administered every 1 day at a concentration of 10 mg / kg. Both were administered by intraperitoneal injection.

[0103] Figure 6c-6f They are respectively a tumor growth curve, a tumor volume histogram, a tumor picture and a tumor mass histogram. It can be seen that the combination of GA (10 mg / kg) and DTX (15 mg / kg) of the present invention makes the tumor growth the slowest. In the animal model of breast cancer, the combined use of GA and DTX significantly enhanced the tumor-killing effect, and significantly delayed the progression of breast cancer compared with the use of DTX alone.

[0104] Figure 6g-6k The results of immunofluorescence and immunohistochemistry of tumor sections are shown. Figure 6g Immunofluorescence staining for apoptosis. Figure 6h This is the immunohistochemical staining of apoptosis. Combining the two pictures, it can be seen that GA significantly increased the apoptosis of tumor cells caused by DTX. Figure 6i Immunofluorescence staining of p-PERK. The results showed that the addition of GA on the basis of DTX significantly enhanced the activation of the PERK pathway. Figure 6j and Figure 6k The results of immunofluorescence staining of TNF-α and IL-6 were presented, indicating that the addition of GA significantly upregulated the expression of these two inflammatory factors. Figure 6g-6k , indicating that GA has a significant therapeutic effect.

[0105] Figure 6l The HE staining results of the main organs of mice, including heart, liver, spleen, lung and kidney, show that GA has no obvious toxicity to the main organs, reflecting good biosafety and biocompatibility.

Claims

1. An application of gold nanoclusters formed by gold and thiol-containing natural peptides for preparing drugs in the field of breast cancer treatment.

2. The use according to claim 1, for preparing drugs for the treatment of endoplasmic reticulum stress and triple-negative breast cancer.

3. The use according to claim 1 or 2, as a docetaxel enhancing adjuvant to enhance docetaxel in the treatment of breast cancer.

4. The use according to claim 1 or 2, wherein docetaxel and gold nanoclusters are used simultaneously to prepare corresponding drugs.

5. The use according to any one of claims 1 to 4, characterized in that: The gold nanoclusters are nanoclusters (Au)x(SG)y formed by the reaction of gold and natural glutathione containing thiol groups, wherein 1≤x≤35, 1≤y≤30; preferably Au 29 SG 27 ; The hydrated particle size is generally at the nanometer level of 1.9nm and above.

6. A drug for treating breast cancer, characterized in that: Includes gold nanoclusters formed by docetaxel and docetaxel enhancer gold and thiol-containing natural peptides.

7. The drug according to claim 5, wherein the drug for treating breast cancer is a drug in the field of endoplasmic reticulum stress and triple-negative breast cancer.

8. The drug according to claim 6 or 7, characterized in that The gold nanoclusters are nanoclusters (Au)x(SG)y formed by the reaction of gold and natural glutathione containing thiol groups, wherein 1≤x≤35, 1≤y≤30; preferably Au 29 SG 27 ; The hydrated particle size is generally at the nanometer level of 1.9 nm or above.

9. The method for preparing the gold nanoclusters according to any one of claims 1 to 8, characterized in that: The gold salt solution and the thiol-containing polypeptide solution are mixed and stirred for 3-5 minutes to allow the gold salt and the polypeptide to fully contact to form a mixed solution; a reducing agent is added to the above mixed solution to cause a reduction reaction in the system, the pH is adjusted, and the reaction is carried out under light-proof conditions. When the color of the solution changes from colorless to light yellow, stirring is continued for 12 hours after the color change occurs, and finally a complex of gold and peptide is obtained. The obtained complex is centrifuged at high speed for 30 minutes to remove large particles in the product, and then the obtained product is washed with ethanol for 3 to 5 times. After washing, 0.25M NAOH is used to stir and dissolve and the pH is controlled at about 7; finally, the product is purified with a 3kDA ultrafiltration tube (MWCO.3kDA) to remove unreacted substances. The molar ratio of gold salt to polypeptide is 1:1-1:5, preferably 1.5:

1. The reducing agent used is sodium borohydride (NaBH4), sodium hydroxide (NaOH), vitamin C (vitamin C), trisodium citrate, etc. The gold salt concentration used is 0.01μM-10M, and the thiol polypeptide concentration is 0.1μM-10M.

Images