Gold nanoclusters selectively binding to g-quadruplexes, and preparation method and application thereof

Abstract

The application discloses gold nanoclusters selectively combined with G-quadruplexes, a preparation method and application thereof, and the preparation method comprises the following steps: synthesizing gold nanoclusters by means of a hydrothermal reaction of reduced glutathione, N, N, N-trimethyl-(11-mercapto undecyl) ammonium chloride and chloroauric acid, wherein the preparation method is simple, the prepared gold nanoclusters are water-soluble gold nanoclusters capable of being specifically combined with G-quadruplexes, can easily enter into cell nuclei to be combined with G-quadruplex DNA, and exhibit an anti-tumor effect.

Description

Technical Field

[0001] This invention belongs to the field of gold nanocluster synthesis and application technology, specifically relating to a gold nanocluster that selectively binds G-quadruplexes, its preparation method, and its application. Background Technology

[0002] G-quadruplexes are secondary DNA structures consisting of four nucleotide chains linked by Hoogsteen hydrogen bonds within or between guanine-rich nucleic acid molecules. They typically require a specific cation (such as potassium ion) at the center of the structure for further stability. Studies have shown that G-quadruplex motifs are widely present in the genomes of plants, animals, bacteria, and viruses. These motifs are not randomly distributed across the genome but are found in important functional regions of chromosomes, such as telomere ends, gene promoter regions, and intron regions. The G-quadruplex structures formed by these motifs participate in a series of important life processes, including telomere maintenance, DNA replication, gene transcription / translation, and maintenance of genome stability. Furthermore, G-quadruplexes are also present in non-coding regions of RNA (including the 5′ and 3′ untranslated regions) to regulate the expression of some genes. Recent research indicates that tumor cells have higher levels of G-quadruplex formation compared to normal cells, and the content of G-quadruplexes is also higher in solid tumors such as breast cancer, head and neck cancer, gastric cancer, and liver cancer than in normal tissues. Given that G-quadruplexes exist at high levels in tumor cells / tissues and participate in some important processes of tumorigenesis and development (such as maintaining telomere homeostasis and regulating proto-oncogene expression as a gene expression regulator), G-quadruplexes are a promising anti-tumor target that has been developed in recent years for the development of novel anti-tumor drugs.

[0003] To date, numerous small molecules, peptides, and antibodies (including nanobodies) have been developed for the identification of G-quadruplex motifs, in-situ detection of G-quadruplex structures, monitoring of intracellular G-quadruplex kinetics, and regulation of G-quadruplex biological functions. However, only two small molecules have yet to undergo clinical trials for the treatment of neuroendocrine / carcinoid tumors and breast / ovarian cancer. These two small molecules were forced to terminate their trials due to low bioavailability, significantly underestimating the potential of G-quadruplexes as a novel antitumor target for cancer therapy. Among molecules that bind to G-quadruplexes, small molecules typically possess planar aromatic conjugated systems with poor water solubility, resulting in limited in vivo efficacy. Peptides and nanobodies not only readily lose their biological activity but also struggle to cross cell membranes to bind to G-quadruplexes. Therefore, there is an urgent need to develop more types of molecules or materials that can bind to G-quadruplexes. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a method for preparing gold nanoclusters.

[0005] The present invention also proposes a gold nanocluster prepared by the above preparation method.

[0006] This invention also proposes an application of the above-mentioned gold nanoclusters.

[0007] The present invention also proposes an antitumor drug containing the above-mentioned gold nanoclusters.

[0008] According to one aspect of the present invention, a method for preparing gold nanoclusters is provided, the method comprising the following steps: synthesizing gold nanoclusters by hydrothermal reaction of reduced glutathione, N,N,N-trimethyl-(11-mercaptoundecyl)ammonium chloride and chloroauric acid.

[0009] In some embodiments of the present invention, the molar ratio of the reduced glutathione to the N,N,N-trimethyl-(11-mercaptoundecyl)ammonium chloride is 1:(0.5-2).

[0010] In some embodiments of the present invention, the molar ratio of the reduced glutathione to the N,N,N-trimethyl-(11-mercaptoundecyl)ammonium chloride is 1:1.

[0011] In some embodiments of the present invention, the molar ratio of the reduced glutathione to the chloroauric acid is (1-4):(1-2).

[0012] In some embodiments of the present invention, the molar ratio of the reduced glutathione to the chloroauric acid is 2:1.

[0013] In some embodiments of the present invention, the molar ratio of N,N,N-trimethyl-(11-mercaptoundecyl)ammonium chloride to chloroauric acid is (1-4):(1-2).

[0014] In some embodiments of the present invention, the molar ratio of N,N,N-trimethyl-(11-mercaptoundecyl)ammonium chloride to chloroauric acid is 2:1.

[0015] In some embodiments of the present invention, the hydrothermal reaction temperature is 65°C to 75°C.

[0016] In some embodiments of the present invention, the hydrothermal reaction time is 10-24 hours.

[0017] In some embodiments of the present invention, the hydrothermal reaction is performed using an oil bath or a water bath.

[0018] In some embodiments of the present invention, the preparation method further includes the step of placing the obtained gold nanoclusters in a dialysis bag with a size of 3000-3500 Daltons and dialyzing them 2-5 times.

[0019] In some embodiments of the present invention, the single dialysis time is not less than 4 hours; for the purpose of fully removing unreacted ligands and chloroauric acid.

[0020] In some embodiments of the present invention, the preparation method further includes a step of concentrating the gold nanoclusters obtained by dialysis.

[0021] In some embodiments of the present invention, the concentration method is centrifugation, and the concentration conditions are as follows: the dialyzed gold nanocluster solution is transferred to an ultrafiltration tube with a molecular weight cutoff of 3,000 to 10,000 Daltons, and centrifuged at room temperature for 30 to 60 minutes at a speed of 6,000 to 8,000 rpm.

[0022] According to a second aspect of the present invention, gold nanoclusters prepared by the above method are provided.

[0023] In some embodiments of the present invention, the maximum excitation wavelength of the gold nanoclusters is located in the range of 330 to 335 nanometers, and the maximum emission wavelength is located in the range of 590 to 605 nanometers.

[0024] In some embodiments of the present invention, the gold nanoclusters have a particle size range of 0.9 to 1.3 nanometers.

[0025] In some embodiments of the present invention, the average particle size of the gold nanoclusters is 1.1 nanometers.

[0026] In some embodiments of the present invention, the gold nanoclusters are water-soluble gold nanoclusters.

[0027] In some embodiments of the present invention, the gold nanoclusters are potassium-resistant gold nanoclusters.

[0028] In some embodiments of the present invention, the gold nanoclusters can selectively bind G-quadruplex DNA and / or G-quadruplex RNA.

[0029] In some embodiments of the present invention, the sequence of the G-quadruplex DNA is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0030] In some embodiments of the present invention, the sequence of the G-quadruplex RNA is shown in SEQ ID NO:4.

[0031] According to a third aspect of the present invention, the application of the above-mentioned gold nanoclusters is proposed, wherein the application is in the preparation of antitumor drugs.

[0032] In some embodiments of the present invention, the tumor includes at least one of breast cancer, prostate cancer, liver cancer, non-small cell lung cancer, brain cancer, ovarian cancer, uterine cancer, stomach cancer, skin cancer, leukemia, head and neck cancer, colon cancer, bladder cancer, and rectal cancer.

[0033] In some embodiments of the present invention, the antitumor drug may further include chemotherapeutic drugs, photosensitizers, or photothermal agents.

[0034] In some embodiments of the present invention, the chemotherapeutic agent includes at least one of cyclophosphamide, cisplatin, podophyllotoxin, camptothecin, paclitaxel, doxorubicin, epirubicin, and 5-fluorouracil.

[0035] In some embodiments of the present invention, the application is in the preparation of C-Myc gene expression inhibitors.

[0036] In some embodiments of the present invention, the application is used in the detection of G-quadruplex structures.

[0037] According to a third aspect of the present invention, an antitumor drug is provided, the antitumor drug comprising the above-mentioned gold nanoclusters.

[0038] In some embodiments of the present invention, the antitumor drug may further include chemotherapeutic drugs, photosensitizers, or photothermal agents.

[0039] In some embodiments of the present invention, the chemotherapeutic agent includes at least one of cyclophosphamide, cisplatin, podophyllotoxin, camptothecin, paclitaxel, doxorubicin, epirubicin, and 5-fluorouracil.

[0040] In some embodiments of the present invention, the antitumor drug further includes pharmaceutically acceptable excipients.

[0041] According to some embodiments of the present invention, at least the following beneficial effects are achieved: The present invention synthesizes gold nanoclusters in one step by using reduced glutathione as a reducing agent and N,N,N-trimethyl-(11-mercaptoundecyl)ammonium chloride as a functional ligand, in a hydrothermal reaction with chloroauric acid. The preparation method is simple, and the prepared gold nanoclusters are water-soluble and can selectively bind to G-quadruplex DNA and G-quadruplex RNA. The gold nanoclusters can also easily enter the cell nucleus and bind to G-quadruplexes, exhibiting anti-tumor effects. Attached Figure Description

[0042] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0043] Figure 1The figures shown are characterization results of the gold nanoclusters prepared in Example 1 of Experimental Example 1 of this invention. In the figure, A is the transmission electron microscopy imaging result of the gold nanoclusters; B is the dynamic light scattering measurement result of the gold nanoclusters; C is the excitation and emission fluorescence spectrum of the gold nanoclusters; and D is the fluorescence imaging result of the gold nanoclusters in potassium chloride solutions of different concentrations.

[0044] Figure 2 The graph shows the interaction analysis results between the gold nanoclusters and G-quadruplexes prepared in Example 1 of Experimental Example 1 of this invention; wherein, A is the non-denaturing polyacrylamide gel electrophoresis result of the complexes of three different types of nucleic acids and gold nanoclusters; B is the result of biomembrane interference measurement of gold nanoclusters and three different types of nucleic acids; C is the curve of circular dichroism absorption of G-quadruplexes and G-quadruplex / gold nanocluster complexes as a function of temperature; D is the curve of emission fluorescence of gold nanoclusters as a function of G-quadruplex concentration; 0 indicates no gold nanoclusters were added;

[0045] Figure 3 The graph shows the results of the biological effect analysis of the gold nanoclusters prepared in Example 1 of Experiment 1 of this invention. A shows the change in the mass of gold entering the nucleus of human breast cancer cells with the concentration of gold nanoclusters; B shows the immunofluorescence results of the gold nanoclusters stabilizing the G-quadruplex structure in human breast cancer cells; C shows the immunoblotting results of the gold nanoclusters downregulating the expression level of C-Myc in human breast cancer cells; tubulin was used as an internal control; D shows the curve of the tumor volume formed by human breast cancer cells treated with gold nanoclusters in nude mice over time; 0 indicates no gold nanoclusters were added.

[0046] Figure 4 The above are non-deformable polyacrylamide gel electrophoresis results of the gold nanoclusters and G tetrachain mixtures prepared in Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4 of this invention. In the figure, the gold nanoclusters modified with 11-mercaptoundecanoic acid, 11-mercaptododecylphosphonic acid, 11-mercaptohexadecylsulfonic acid, and 4,6-diamino-2-mercaptopyrimidine are respectively comparative gold nanocluster 1, comparative gold nanocluster 2, comparative gold nanocluster 3, and comparative gold nanocluster 4. 0 indicates no gold nanoclusters were added.

[0047] Figure 5 The diagram shows the interaction analysis results between the gold nanoclusters prepared in Example 1 of Experimental Example 3 of this invention and different motif G quadruple strands. In this diagram, A is the affinity measurement result between the gold nanoclusters and two G quadruple strand DNAs (SEQ ID NO:2 and SEQ ID NO:3); B is the biomembrane interference measurement result between the gold nanoclusters and different concentrations of G quadruple strand RNA (SEQ ID NO:4). Detailed Implementation

[0048] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0049] Example 1

[0050] This embodiment provides a gold nanocluster, and the specific preparation process is as follows:

[0051] (1) Transfer a certain volume of freshly prepared reduced glutathione solution and N,N,N-trimethyl-(11-mercaptoundecyl)ammonium chloride solution into a round-bottom glass flask or flat-bottom glass bottle containing deionized water. The molar ratio between reduced glutathione and N,N,N-trimethyl-(11-mercaptoundecyl)ammonium chloride is 1:1. Stir on a magnetic stirrer for 10 minutes to fully mix the two ligands.

[0052] (2) Chloroauric acid solution was slowly added dropwise to the mixture of the two ligands. The molar ratio between reduced glutathione and chloroauric acid was 2:1, and the molar ratio between N,N,N-trimethyl-(11-mercaptoundecyl)ammonium chloride and chloroauric acid was 2:1. The mixture was placed in an oil bath at 70°C for 14 hours under stirring at 1000 rpm to obtain the reaction solution.

[0053] (3) After the temperature of the above reaction solution drops to room temperature, place the reaction solution in a dialysis bag with a size of 3500 Daltons and dialyze it 4 times (each time for no less than 4 hours) to remove unreacted ligands and chloroauric acid.

[0054] (4) The dialyzed gold nanoclusters were transferred to an ultrafiltration tube with a molecular weight cutoff of 10,000 Daltons and centrifuged at 6,500 rpm for 30 minutes at room temperature to obtain a gold nanocluster concentrate. The gold nanocluster concentrate did not settle after being placed at room temperature for 6 months.

[0055] Comparative Example 1

[0056] This comparative example provides a comparative gold nanocluster 1. The specific preparation process of gold nanocluster 1 is as follows: 2 mL of 11-mercaptoundecanoic acid solution (concentration 5 mmol / L) and 250 μL of chloroauric acid solution (concentration 20 mmol / L) are mixed, followed by the addition of 50 μL of sodium hydroxide solution (concentration 1 mol / L). Anhydrous ethanol is then added to the mixture to achieve a volume ratio of 20% ethanol. Subsequently, while continuously stirring, 50 μL of sodium borohydride solution (sodium borohydride dissolved in 0.2 mol / L sodium hydroxide, concentration 100 mmol / L) is added dropwise to the above mixture, and stirring is continued for 3 hours. After the reaction is completed, the gold nanocluster is purified using an ultrafiltration tube with a molecular weight cutoff of 3000 Daltons and deionized water.

[0057] Comparative Example 2

[0058] This comparative example provides a comparative gold nanocluster 2. The specific preparation process of gold nanocluster 2 is as follows: 2 mL of 11-mercaptododecylphosphonic acid solution (concentration 5 mmol / L) and 250 μL of chloroauric acid solution (concentration 20 mmol / L) are mixed, followed by the addition of 50 μL of sodium hydroxide solution (concentration 1 mol / L). Anhydrous ethanol is then added to the mixture to bring the volume ratio of ethanol in the mixture to 10%. Subsequently, while continuously stirring, 50 μL of sodium borohydride solution (sodium borohydride dissolved in 0.2 mol / L sodium hydroxide, concentration 100 mmol / L) is added dropwise to the above mixture, and stirring is continued for 3 hours. After the reaction is completed, the gold nanoclusters are purified using an ultrafiltration tube with a molecular weight cutoff of 3000 Daltons and deionized water.

[0059] Comparative Example 3

[0060] This comparative example provides a comparative gold nanocluster 3. The specific preparation process of gold nanocluster 3 is as follows: 2 mL of 11-mercaptohexadecylsulfonic acid solution (concentration 5 mmol / L) and 250 μL of chloroauric acid solution (concentration 20 mmol / L) are mixed, followed by the addition of 15 μL of sodium hydroxide solution (concentration 1 mol / L). Anhydrous ethanol is then added to the mixture to achieve a volume ratio of 20% ethanol. Subsequently, under continuous stirring, 50 μL of sodium borohydride solution (sodium borohydride dissolved in 0.2 mol / L sodium hydroxide, concentration 100 mmol / L) is added dropwise to the above mixture, and stirring is continued for 3 hours. After the reaction is completed, the gold nanoclusters are purified using an ultrafiltration tube with a molecular weight cutoff of 3000 Daltons and deionized water.

[0061] Comparative Example 4

[0062] This comparative example provides a comparative gold nanocluster 4. The specific preparation process of gold nanocluster 4 is as follows: A mixture of 4,6-diamino-2-mercaptopyrimidine (14 mg, dissolved in 10 mL of methanol, with 200 μL of glacial acetic acid and 40 mg of Tween 80 added) and chloroauric acid (41 mg, dissolved in 20 mL of methanol) is stirred on ice water for 10 minutes. Then, sodium borohydride solution (12 mg, dissolved in 5 mL of methanol) is added dropwise, and the mixture is stirred on ice water for 1 hour. After the reaction is complete, methanol is removed by rotary evaporation, an appropriate amount of deionized water is added, and the gold nanocluster solution is dialyzed in deionized water for 48 hours. Finally, it is filtered through a 0.22 μm filter membrane for sterilization to obtain gold nanocluster 4.

[0063] Experimental Example 1

[0064] This experimental example tested the properties of the gold nanoclusters prepared in Example 1. Among them:

[0065] 1. Characterization of gold nanoclusters

[0066] The gold nanoclusters prepared in Example 1 were characterized using transmission electron microscopy and dynamic light scattering.

[0067] The results are as follows Figure 1 As shown, from Figure 1 As shown in Figure A, the gold nanoclusters are uniform in size, with an average particle size of 1.1 nanometers; from Figure 1 As shown in Figure B, the dynamic light scattering measurement results indicate that the prepared gold nanoclusters are positively charged.

[0068] 2. Fluorescence property detection

[0069] The gold nanoclusters prepared in Example 1 were subjected to fluorescence spectroscopy analysis. Specifically, 200 μL of concentrated gold nanoclusters solution was pipetted into a micro-quartz cuvette, and then the excitation and emission spectra of the gold nanoclusters were scanned using a Hitachi F-4600 fluorescence spectrophotometer.

[0070] The results are as follows Figure 1 As shown, from Figure 1 As can be seen from Figure C, the synthesized gold nanoclusters exhibit fluorescence properties, with a maximum excitation wavelength of 332 nm and a maximum emission wavelength of 602 nm.

[0071] 3. Potassium ion tolerance test of gold nanoclusters

[0072] Different concentrations of potassium chloride (0, 5, 10, 20, 40, 50, and 100 mmol / L) were added to a 200 mg / L solution of gold nanoclusters, and the fluorescence properties of the gold nanoclusters were measured.

[0073] The results are as follows Figure 1 As shown, from Figure 1As can be seen from the D-plot, the fluorescence intensity of the gold nanoclusters remains unchanged even in a potassium chloride solution with a concentration of 100 mmol / L, indicating that the prepared gold nanoclusters are resistant to potassium ions.

[0074] 4. Interaction analysis between gold nanoclusters and G-quadruplexes

[0075] Detection method: 5 μL of gold nanoclusters prepared in Example 1 at different concentrations (10, 20, 40, 80, and 100 mg / L, respectively) were incubated with an equal volume of 2 μmol / L G-quadruplex DNA (to promote folding into a G-quadruplex structure, the G-quadruplex DNA was dissolved in a potassium chloride solution containing 100 mmol / L before reacting with the gold nanoclusters, then heated to 95°C for 10 minutes and slowly cooled for annealing), double-stranded DNA, and single-stranded DNA at room temperature for 10 minutes to obtain a mixture; a sample without added gold nanoclusters served as a control. The sequence of the G-quadruplex DNA is 5′-TGGGGAGGGTGGGGAGGGTGGGGAAGG-3′ (SEQ ID NO:1).

[0076] The resulting mixture was separated by 10% non-denaturing polyacrylamide gel electrophoresis (100V constant voltage electrophoresis for 45 minutes), and the separated products were analyzed by SYBR. TM Gold nucleic acid gel staining for 5 minutes was followed by washing the gel with deionized water. The gel was then placed in a gel imaging system for automatic exposure imaging. In the biomembrane interferometry assay, the sensor was first moistened with 50 mmol / L potassium chloride solution, then immersed in concentrated gold nanoclusters for adsorption. Subsequently, the sensor with adsorbed gold nanoclusters was immersed in different concentrations (1200, 600, 300, 150, 75, 37.5 nmol / L) of G-quadruplex DNA solution for 100 seconds, with the dissociation process measured for 200 seconds. In the circular dichroism spectroscopy assay for dissolution temperature, 10 μmol / L of G-quadruplex DNA was mixed with an equal volume of deionized water or gold nanoclusters and incubated for 1 minute to obtain the desired solution. For the mixed solution, 200 μL of the mixture was pipetted into a sample cup, and the circular dichroism chromatogram of the mixture between 220 nm and 320 nm was measured using a circular dichroism chromatogram. To determine the dissolution temperature of the mixture, a circulating water bath was used to control the temperature, and the circular dichroism chromatograms were continuously measured in the range of 25 to 97 degrees Celsius. Finally, the value at 265 nm was used to plot the value and calculate the dissolution temperature. The emission fluorescence intensity of the gold nanoclusters and the gold nanocluster / G-quadruplex mixture at 602 nm was measured using a fluorescence spectrometer. Specifically, 200 μL of the test solution was pipetted into a micro-volume quartz cuvette, and the emission fluorescence intensity was measured using a Hitachi F-4600 fluorescence spectrophotometer to determine the interaction between the gold nanoclusters and the G-quadruplex DNA.

[0077] The results are as followsFigure 2 As shown, from Figure 2 Figure A shows that the mixture of gold nanoclusters and G-quadruplex DNA was separated by non-denaturing polyacrylamide gel electrophoresis. As the concentration of gold nanoclusters gradually increased, less and less G-quadruplex DNA entered the gel and was absorbed by SYBR. TM Staining with Gold nucleic acid gel dye indicates an interaction between gold nanoclusters and G-quadruplex DNA. Conversely, gold nanoclusters do not affect the entry of double-stranded DNA (sequence: 5'-TAAAAAAAATAAAAAAAATAAAAAAGG-3' / 3'-ATTTTTTTTATTTTTTTTATTTTTTCC-5') or single-stranded DNA (sequence: 5'-TAAAAAAAATAAAAAAAATAAAAAAGG-3') into the gel and their absorption by SYBR. TM Staining with gold dye. From Figure 2 As shown in Figure B, biomembrane interferometry reveals an extremely high affinity between gold nanoclusters and G-quadruplex DNA (dissociation constant less than 1 picomolar per liter). From... Figure 2 As shown in Figure C, circular dichroism spectroscopy revealed that gold nanoclusters increase the melting temperature of G-quadruplex DNA. From... Figure 2 As shown in Figure D, fluorescence assays confirm that G-quadruplex DNA enhances the fluorescence intensity of gold nanoclusters, and these results confirm the interaction between gold nanoclusters and G-quadruplex DNA.

[0078] 4. Biological effects analysis of G-quadruplex-bound gold nanoclusters

[0079] To further test whether gold nanoclusters bind to intracellular G-quadruplexes and exert corresponding biological effects, different concentrations of gold nanoclusters were added to human breast cancer cells, followed by multiple biological assays.

[0080] (1) Determination of gold nanoclusters entering the nucleus of human breast cancer cells

[0081] 1) Human breast cancer cells were cultured in cell culture dishes with a diameter of 10 cm. When the cell density reached approximately 80%, a certain volume of the concentrated gold nanoclusters prepared in Example 1 was added to the culture medium, resulting in final gold nanocluster concentrations of 2.5, 5, 10, 20, and 40 mg / L. The group without added gold nanoclusters served as a control. The cells were then placed in a carbon dioxide incubator (37°C with 5% carbon dioxide purging) and cultured for another 24 hours. After incubation, the culture medium was discarded, and the cells were washed with phosphate-buffered saline.

[0082] 2) The nuclei of human breast cancer cells were isolated from the washed cells using a kit (purchased from Shanghai Sangon Biotech Co., Ltd.), and the mass of gold elements entering the nucleus was determined by inductively coupled plasma mass spectrometry.

[0083] The results are as follows Figure 3 As shown, from Figure 3 As shown in Figures A and B, gold nanoclusters can not only enter the nucleus of human breast cancer cells ( Figure 3 As shown in A), it can also stabilize the intracellular G-quadruplex structure. Treatment with gold nanoclusters significantly increases the level of intracellular G-quadruplex formation. Figure 3 (as shown in B).

[0084] (2) Detection of proto-oncogene C-Myc expression

[0085] The promoter region of the proto-oncogene C-Myc can form a G-quadruplex structure, which negatively regulates the transcription of C-Myc. Therefore, this experiment tested whether gold nanoclusters affect the expression of the C-Myc gene. The specific method was as follows: cell lysates were separated using sodium dodecyl sulfate polyacrylamide gel (separating gel concentration of 12%), and the separated products were transferred to a nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% skim milk powder for 1 hour. Then, the expression level of C-Myc protein in the control group and the treatment group was detected using a C-Myc-specific antibody and a secondary antibody modified with horseradish peroxidase that recognizes the C-Myc-specific antibody.

[0086] Immunoblotting results as follows Figure 3 As shown in Figure C, it can be seen that gold nanoclusters significantly downregulate the expression level of the C-Myc gene in cells, but do not affect the expression level of Max, the direct binding protein of C-Myc, indicating that the downregulation of C-Myc expression by gold nanoclusters is specific.

[0087] (3) Detection of the anticancer ability of gold nanoclusters

[0088] 1) Human breast cancer cells were cultured in 10 cm diameter cell culture dishes. When the cell density reached approximately 80%, a certain volume of concentrated gold nanoclusters was added to the culture medium to achieve final concentrations of 2.5, 5, 10, 20, and 40 mg / L. The group without added gold nanoclusters served as a control. The cells were then cultured in a CO2 incubator for 24 hours. After incubation, the culture medium was discarded, and the cells were washed with phosphate-buffered saline (PFS). Then, 1 mL of 0.25% trypsin was added for 3 minutes. Once the cells detached from the culture dish, 4 mL of DMEM medium containing fetal bovine serum was added. The cells were pipetted to achieve a single-cell state, then centrifuged at 1000 rpm for 5 minutes to settle the cells. The cells were then resuspended in 1 mL of PFS.

[0089] 2) Using a syringe, inject 1 ml of the cell suspension prepared in step 1) subcutaneously into nude mice. Each group contains 5 nude mice. The nude mice inoculated with human breast cancer cells were normally housed in the animal room for 15 days. Every two days, the major and minor axes of the tumor site were measured using calipers, with the major and minor axes perpendicular to each other. The formula was used: Tumor volume (millimeters in cubic meters) = 0.5 × major axis × minor axis. 2 To calculate tumor volume.

[0090] The results are as follows Figure 3 As shown, from Figure 3 As shown in Figure D, treatment with gold nanoclusters reduces the ability of human breast cancer cells to form tumors in nude mice, indicating that gold nanoclusters have anti-tumor effects.

[0091] Experimental Example 2

[0092] This experiment compares the binding of gold nanoclusters prepared in Example 1 and Comparative Examples 1-4 to G-quadruplex DNA.

[0093] Detection method: 5 μL of gold nanoclusters prepared in Examples 1 (10 and 100 mg / L) and Comparative Examples 1-4 (10 and 100 mg / L) at different concentrations were incubated with an equal volume of 2 μmol / L G-quadruplex DNA at room temperature for 10 minutes to obtain a mixture; a sample without added gold nanoclusters served as a control. The sequence of the G-quadruplex DNA was 5′-TGGGGAGGGTGGGGAGGGTGGGGAAGG-3′ (SEQ ID NO:1). The mixture was separated using a 10% non-denaturing polyacrylamide gel electrophoresis (100 V constant voltage electrophoresis for 45 minutes). The separated products were analyzed using SYBR Green gel electrophoresis. TM After staining with Gold nucleic acid gel dye for 5 minutes, the gel was washed with deionized water and then placed in a gel imaging system for imaging using automatic exposure mode.

[0094] The results are as follows Figure 4 As shown, from Figure 4 As can be seen, the gold nanoclusters prepared in Example 1 reduce the entry of G-quadruplex DNA into the gel and its absorption by SYBR. TM Gold nucleic acid gel dye staining was performed, but the gold nanoclusters prepared in Comparative Examples 1-4 did not, indicating that the interaction between the gold nanoclusters prepared in Example 1 and G-quadruplex DNA is specific.

[0095] Verification of the binding ability of gold nanoclusters with different motifs G-quadruplexes in Experiment 3, Example 1

[0096] The interactions between the gold nanoclusters prepared in Example 1 and two types of G-quadruplex DNA and one type of G-quadruplex RNA were determined using biomembrane interferometry. The sequences of the two types of G-quadruplex DNA were: 5'-AGGGGCGGGCGCGGGAGGAAGGGGGCGGGAGCGGGCTG-3' (SEQ ID NO:2) and 5'-CGGGCGGGAGCGCGGCGGGCGGGCGGCA-3' (SEQ ID NO:3), and the sequence of the G-quadruplex RNA was: 5'-GGGGGCCGUGGGGUGGGAGCUGGGG-3' (SEQ ID NO:4).

[0097] The results are as follows Figure 5 As shown, from Figure 5 As can be seen, gold nanoclusters can not only interact with these G quadruplex DNA and RNA, but also have a high affinity.

[0098] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. The application of gold nanoclusters in stabilizing intracellular G-quadruplex structures and increasing the level of intracellular G-quadruplex formation, characterized in that, The gold nanoclusters were prepared by the following method: reducing glutathione, N,N,N-trimethyl-(11-mercaptoundecyl)ammonium chloride and chloroauric acid were synthesized by hydrothermal reaction; the cells were human breast cancer cells.

2. The application according to claim 1, characterized in that, The molar ratio of the reduced glutathione to the N,N,N-trimethyl-(11-mercaptoundecyl)ammonium chloride is 1:(0.5~2); and / or, the molar ratio of the reduced glutathione to the chloroauric acid is (1~4):(1~2); and / or, the molar ratio of the N,N,N-trimethyl-(11-mercaptoundecyl)ammonium chloride to the chloroauric acid is (1~4):(1~2).

3. The application according to claim 1, characterized in that, The hydrothermal reaction is carried out at a temperature of 65℃~75℃ for 10-24 hours.

4. The application according to claim 1, characterized in that, The preparation method also includes the step of placing the prepared gold nanoclusters in a dialysis bag with a size of 3000-3500 Daltons and dialyzing them 2-5 times.

5. The application according to claim 4, characterized in that, The preparation method further includes a step of centrifuging and concentrating the dialyzed gold nanoclusters in an ultrafiltration tube with a size of 3000-10000 Daltons.

6. The application according to claim 1, characterized in that, The gold nanoclusters can selectively bind G-quadruplex DNA and / or G-quadruplex RNA.

7. The application according to claim 1, characterized in that, The gold nanoclusters have a particle size range of 0.9 to 1.3 nanometers; and / or, the gold nanoclusters are water-soluble gold nanoclusters; and / or, the maximum excitation wavelength of the gold nanoclusters is located in the range of 330 to 335 nanometers, and the maximum emission wavelength is located in the range of 590 to 605 nanometers.

8. Applications of gold nanoclusters in any of the following (1) to (2): (1) Preparation of antitumor drugs; (2) Preparation of C-Myc gene expression inhibitor; The gold nanoclusters were prepared by the following method: reducing glutathione, N,N,N-trimethyl-(11-mercaptoundecyl)ammonium chloride and chloroauric acid were synthesized by hydrothermal reaction. The tumor is breast cancer.

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