Preparation and application of nano material for targeting and enhancing nervous tumor ferroptosis

Abstract

The invention relates to preparation and application of a nano material for targeting and enhancing nervous tumor ferroptosis, and belongs to the field of medicines. The nano-scale polypeptide-gold complex cluster NG formed by gold and sulfydryl-containing targeting polypeptide is characterized in that NG molecules form AuxPy, x is equal to 1-35, y is equal to 1-30, and P is corresponding nervous system tumor targeting polypeptide, preferably Au17P7. The preparation method comprises the following steps: mixing a gold salt solution with nervous system tumor targeting polypeptide to form a mixed solution, and adding a reducing agent under certain temperature and pH conditions, so that Au interacts with a sulfydryl phase of the polypeptide to form the complex cluster NG. The compound is used for preparing a targeted drug for improving the tumor treatment effect of the ferroptosis inducer, and has a specific synergistic treatment effect on nervous system tumors when being used together with the ferroptosis inducer.

Description

Technical Field

[0001] The present invention relates to the fields of ferroptosis regulation and treatment of nervous system tumors, and can be summarized as a preparation method of a nanoscale gold cluster compound with the ability to target nervous system tumors and specifically enhance the therapeutic effect of ferroptosis inducers in the treatment of nervous system tumors, and in vitro and in vivo experiments verify that such complexes can enhance the therapeutic effect of ferroptosis inducers on nervous system tumors by regulating the intracellular iron ion concentration. Background Art

[0002] Adult nervous system tumors, especially gliomas, have a high incidence, high malignancy, complex treatment, are prone to recurrence and have a poor prognosis. The 5-year survival rate is only about 3%-5%, which is a type of disease that poses a great threat to human health. In recent years, the incidence of such tumors in China has shown a significant upward trend. Therefore, the effective treatment of nervous system malignancies with high mortality such as gliomas has always been an important indicator for promoting the health of national residents. The traditional treatment methods for nervous system tumors are surgical resection, supplemented by radiotherapy and chemotherapy after surgery. However, most patients do not have a significant remission of the disease, and the median survival time is only about 15 months, and it is easy to relapse. The reasons are as follows. First, different from other organs, the surgical resection of brain tumors cannot arbitrarily expand the resection range, resulting in the inability to completely remove the in-situ cancer by surgical methods. Moreover, the side effects of subsequent radiotherapy and chemotherapy are relatively large, greatly affecting the quality of life of patients. Second, the anti-tumor effect of the clinically standardized chemotherapy drug temozolomide is poor, and it cannot effectively enter the tumor site and accumulate to play a role. Moreover, due to the existence of the blood-brain barrier (BBB), most drugs or reagents cannot penetrate into the tumor for killing. The effective treatment of tumors largely depends on the effective killing of residual tumor cells and the targeting specificity of drugs. Therefore, it is urgent to explore more effective new therapies.

[0003] Ferroptosis is a newly discovered non-apoptotic cell death mode driven by reactive oxygen species (ROS) and membrane lipid peroxidation reaction. It is significantly different from necrosis, apoptosis, autophagy, etc. in terms of cell morphology, biochemistry, etc. In terms of mechanism, a decrease in the level of glutathione peroxidase 4 (GPX4) will cause lipid oxides to not be metabolized by the glutathione reduction reaction, and then divalent iron ions oxidize lipids in a manner similar to the Fenton reaction to generate a large amount of ROS, promoting cell ferroptosis. Recently, studies have found that in nervous system tumors, ferroptosis inducers can significantly induce ferroptosis of tumor cells and relieve the edema around the lesion, so it is considered a potential new therapy for the treatment of nervous system tumors.

[0004] The complex formed by the polypeptide and Au has a clear molecular structure, good biocompatibility, unique physical and chemical properties, such as stable fluorescence and enzyme-like catalytic properties, etc., as well as the ability to cross the blood-brain barrier. Thanks to these unique physical and chemical properties and the designability of the polypeptide, this type of material has great potential in biomedicine. In particular, due to the designability, targeting property and small side effects of the polypeptide, it also shows good application prospects in the field of tumor research. The polypeptide can be used as a specific ligand for receptor binding or substrate recognition to achieve the targeted delivery of polypeptide nanoclusters. In addition, it is found that Au has the function of increasing the expression of heme oxygenase 1 (HO-1). HO-1 mainly catalyzes the catabolism of heme into ferrous ions, which may greatly improve the function of ferroptosis inducers to promote the death of tumor cells. Therefore, it has great application prospects for improving the current treatment status of nervous system tumors. Based on the designability and targeting property of the polypeptide in the polypeptide nanoclusters, polypeptide nanoclusters that can target nervous system tumors can be designed to increase their targeting efficiency to tumors while retaining the property of gold elements to promote ferroptosis, and more effectively inhibit tumors.

[0005] An effective new therapy for nervous system tumors is to be able to kill tumors while specifically targeting tumors and reducing side effects on other normal brain cells. The latest research shows that the α7 subunit of the nicotinic acetylcholine receptor (α7nAChR) is highly expressed in classical nervous system cells. A 5-amino acid fragment sequence, -GCLRV-, shows a strong interaction with α7nAChR and has the potential to develop into a new targeting sequence. This provides data support for us to use the α7nAChR-targeting peptide as the "polypeptide shell" of the polypeptide nanoclusters. The present invention provides a newly synthesized polypeptide nanocluster targeting α7nAChR, named NG. It synergistically enhances the effect with ferroptosis inducers, enabling the ferroptosis inducers to more effectively and specifically kill nervous system tumors, while greatly reducing side effects. NG increases the content of ferrous ions in tumor cells, thereby significantly enhancing the anti-tumor killing effect of ferroptosis inducers in the presence of ferroptosis inducers, providing new ideas and theoretical basis for the application of nanodrugs in the treatment of the nervous system, and also providing new potential means for more effectively treating nervous system tumors clinically. Summary of the Invention

[0006] The present invention relates to the preparation and application of a nanoscale polypeptide-gold cluster formed by gold and a thiol-containing targeting polypeptide. The present invention can be used to promote the tumor-killing function of ferroptosis inducers. At the same time, compared with ordinary gold nanocompounds, it can increase the targeting efficiency to the lesion site and has no toxic side effects on normal cells.

[0007] The technical solution adopted by the present invention is as follows:

[0008] A preparation method and application of a nanoscale gold cluster compound targeting nervous system tumors, comprising the following steps:

[0009] (1) First, synthesize a polypeptide nanogold cluster NG with the ability to target nervous system tumors;

[0010] (2) Detect the synergistic killing effect and molecular mechanism of this gold cluster and ferroptosis inducer on nervous system tumor cells;

[0011] (3) Verify that this gold cluster and ferroptosis inducer can enhance the tumor killing effect of the ferroptosis inducer in a nervous system tumor model.

[0012] A preparation method of a nanoscale polypeptide-gold complex cluster NG, characterized in that it is a preparation method for forming a complex by the reaction of gold with the thiol group of a nervous system tumor-targeting polypeptide, comprising the following steps: Mix a gold salt solution with a nervous system tumor-targeting polypeptide, i.e., a corresponding polypeptide containing a thiol group, to form a mixed solution. Under certain temperature and pH conditions, add a reducing agent to cause a reduction reaction in the mixed solution system. Au forms the complex cluster NG by interacting with the thiol group of the polypeptide.

[0013] The gold salt solution is such that the gold compound is a trivalent inorganic salt compound, such as chloroauric acid. The trivalent gold is reduced to gold atoms or monovalent gold.

[0014] The nervous system tumor-targeting polypeptide is a polypeptide sequence targeting the α7nAChR protein, and further is: H2N-GCLRVKKKYCC-COOH (which can be prepared by conventional methods).

[0015] The reducing agent is sodium borohydride (NaBH4), sodium hydroxide (NaOH), vitamin C, or trisodium citrate dihydrate, etc., and is preferably selected according to actual synthesis.

[0016] In the mixed solution, the concentration of the nervous system tumor-targeting polypeptide is 0.1 μM - 10 M, and the gold salt concentration is 0.01 M - 10 M. Further, the molar ratio of the polypeptide to the gold salt is 1:1 - 2:1, and is preferably selected according to actual synthesis. Further, the resulting NG molecular composition is AuxPy, where x = 1 - 35, y = 1 - 30, and P is the corresponding nervous system tumor-targeting polypeptide, preferably Au 17 P 7 .

[0017] The reduction reaction of the mixed solution occurs at a reaction temperature of 20 - 75 °C, and the pH condition is weakly basic or neutral, which can be adjusted and preferably selected according to needs.

[0018] The mixed solution undergoes a reduction reaction, and is stirred in the dark at 20-75 °C for 5-15 hours. The color of the solution changes from light yellow, and the complex is obtained by purification methods such as ultrafiltration and dialysis. The hydrodynamic diameter of this complex is generally above 2 nm.

[0019] The application of the complex synthesized from gold and the binding peptide is used to prepare a targeted drug for improving the therapeutic effect of ferroptosis inducers on tumors (preferably, the molar concentration ratio of NG to ferroptosis inducer is 25-50:1, such as 50-100 μM:2 μM), and it has a synergistic therapeutic effect on nervous system tumors when used together with ferroptosis inducers.

[0020] The application of the complex of gold and the nervous system tumor-targeting binding peptide is used to prepare a drug for enhancing the effect of ferroptosis inducers on treating nervous system tumors. The nervous system tumors include but are not limited to gliomas such as neuroblastoma, astrocytoma, ependymoma, and medulloblastoma, meningioma, schwannoma, neurofibroma, and metastatic tumors of other tumors to the nervous system.

[0021] The beneficial effects of the present invention are:

[0022] (1) The polypeptide sequence adopted in the present invention naturally integrates its specific recognition sequence and mineralization sequence, without the need for other chemical modifications or linkages. The preparation steps are simple, the cost is low, and the required amount of reagents is small.

[0023] (2) The gold clusters synthesized with the polypeptide with good targeting ability specifically bind to specific membrane proteins on the surface of nervous system tumor cells, solving the problem that current nanomaterials and drugs lack effective specific treatment means for brain tumors.

[0024] (3) When administered alone, the present invention has little cytotoxicity to normal body cells and high safety.

[0025] (4) Compared with existing chemotherapy drugs, the present invention can integrate the targeting binding function of the polypeptide, the small size of the gold complex, and good biological activity on the same compound, realizing the enhancement of tumor killing ability by promoting the function of ferroptosis inducers in in vitro 2D and 3D tumor cell culture systems.

[0026] (5) In the animal model of nervous system tumors, the present invention can act synergistically with ferroptosis inducers. Compared with ferroptosis inducers alone, it can significantly delay the onset process of nervous system tumors. Description of the Drawings

[0027] Figure 1a It is an image obtained by the direct observation method in Example 1 of the present invention, and photos under sunlight irradiation (1) and 365 nm ultraviolet irradiation (2);

[0028] Figure 1b This is the fluorescence spectrum diagram of Example 1 of the present invention, where ex represents the excitation spectrum and em represents the emission spectrum;

[0029] Figure 1c This is the particle size statistical diagram of Example 1 of the present invention. The particle size of the present invention is about 2.3 nm;

[0030] Figure 1d This is the ultraviolet absorption spectrum diagram of Example 1 of the present invention. The ultraviolet absorptions of the GCLRV polypeptide and the formed gold nanoclusters are measured at 274 nm, 283 nm, and 243 nm respectively;

[0031] Figure 1e This is the matrix-assisted laser desorption / ionization time-of-flight mass spectrum (MALDI-TOF) of Example 1 of the present invention;

[0032] Figure 1f This is the transmission electron microscope photograph of Example 1 of the present invention;

[0033] Figure 2a This is the Western Blot diagram showing the high expression of α7nAChR in classical nervous system tumor cells;

[0034] Figure 2b This is the ICP-MS gold element quantitative analysis of the targeting of classical nervous system tumor cells glioblastoma GL261 and human neuroblastoma cells SH-SY5Y in Example 1 of the present invention;

[0035] Figure 2c This is the ICP-MS gold element quantitative analysis of the targeting blockade experiment on glioblastoma GL261 in Example 1 of the invention.

[0036] Figure 3a This is the toxicity test result diagram of glioblastoma GL261 cells and human neuroblastoma cells SH-SY5Y in Example 1 of the present invention;

[0037] Figure 3b This is the toxicity test result diagram of bone marrow cells (left) and brain cells (right) in Example 1 of the present invention;

[0038] Figure 4a This is the cck8 test result diagram after 24 h when Example 1 of the present invention enhances the death of GL261 cells induced by ferroptosis inducer in a non-target peptide-dependent form;

[0039] Figure 4b This is the cck8 test result diagram after 24 h when the ferroptosis inhibitor Fer-1 is added on the basis of enhancing the tumor killing effect of ferroptosis inducer in Example 1 of the present invention;

[0040] Figure 4cFlow cytometry diagram and statistical chart of PI staining for enhancing the tumor killing effect of ferroptosis inducer in Example 1 of the present invention;

[0041] Figure 4d Colony formation experiment for promoting the function of ferroptosis inducer in GL261 in Example 1 of the present invention;

[0042] Figure 4e Confocal imaging and statistical chart for detecting the degree of lipid peroxidation in cells treated differently after BODIPY C11 staining after enhancing the tumor killing effect of ferroptosis inducer in Example 1 of the present invention;

[0043] Figure 5a Confocal imaging for enhancing the tumor killing ability by promoting the function of ferroptosis inducer (RSL3) in the 3D culture system of GL261 cells in Example 1 of the present invention;

[0044] Figure 5b Confocal imaging for enhancing the tumor killing ability by promoting the function of ferroptosis inducer (Erastin) in the 3D culture system of GL261 cells in Example 1 of the present invention;

[0045] Figure 5c Confocal imaging for enhancing the tumor killing ability by promoting the function of ferroptosis inducer (Erastin) in the 3D culture system of SH-SY5Y cells in Example 1 of the present invention;

[0046] Figure 5d Confocal imaging for enhancing the tumor killing ability by promoting the function of ferroptosis inducer (RSL3) in the 3D culture system of U251 cells in Example 1 of the present invention;

[0047] Figure 6a Confocal images of the effect of Example 1 of the present invention on the concentration of ferrous ions in tumor cells;

[0048] Figure 6b Results of real-time quantitative PCR detection of the effect of Example 1 of the present invention on genes related to ferrous ion metabolism and genes related to ferrous ion carriers downstream thereof;

[0049] Figure 6c Confocal imaging for promoting ferritin autophagy especially through the increased transport of ferritin to lysosomes in Example 1 of the present invention.

[0050] Figure 6d Confocal images for enhancing the concentration of ferrous ions especially through the HO-1 pathway in Example 1 of the present invention;

[0051] Figure 6e Confocal imaging for enhancing the function of ferroptosis inducer through the HO-1 pathway to strengthen the tumor killing ability in the 3D tumor cell culture system in Example 1 of the present invention;

[0052] Figure 7a This is the line graph of the tumor growth curve for the in - vivo animal experiment of Example 1 of the present invention;

[0053] Figure 7b This is the bar graph of the tumor size for the in - vivo animal experiment of Example 1 of the present invention;

[0054] Figure 7c This is the bar graph of the tumor mass for the in - vivo animal experiment of Example 1 of the present invention;

[0055] Figure 7d This is the line graph of the body weight of mice during the entire cycle of the in - vivo animal experiment of Example 1 of the present invention; Detailed implementation manners

[0056] The present invention will be described in detail below in combination with the drawings and embodiments, so as to facilitate those skilled in the art to understand and implement the present invention, and further recognize the advantages of the present invention.

[0057] Unless otherwise defined in the specification of the present invention, all technical terms herein are used according to the conventional definitions commonly used and understood by those of ordinary skill in the art. The experimental methods described in the following embodiments are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0058] The present invention provides a preparation method of a novel gold - polypeptide - conjugated gold nanocluster and its targeted therapeutic application for nervous system tumors. Essentially, it is the reaction of gold with the sulfhydryl groups in the binding peptide to form gold - sulfur bonds, where the polypeptide provides sulfhydryl groups and the salt solution provides gold elements; at a certain temperature, pH and in the presence of a reducing agent, the salt solution and the polypeptide solution are mixed to undergo a reduction reaction, reducing the high - valence gold in the gold salt to gold atoms or low - valence gold ions.

[0059] Preferably, a weak base can be added to the mixture to provide a weakly alkaline environment, enabling the reducing agent to have reducibility and thus reducing gold ions.

[0060] The gold salt solution selected 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.

[0061] The present invention provides a preparation method of a gold nanocluster compound for targeted treatment of nervous system tumors to promote tumor ferroptosis, including:

[0062] Mix the gold salt solution with the targeting polypeptide solution and stir for 3 - 5 min to allow the gold salt and the polypeptide to come into full contact, forming a mixed solution; add a reducing agent to the above - mentioned mixed solution to cause a reduction reaction in the system, adjust the pH, react in the dark, and continue to stir for 12 hours after the color of the solution changes from light yellow, finally obtaining a complex of gold and peptide.

[0063] Further, the above complex can be centrifuged at high speed for 30 min to remove large particles, the product is washed once with ethanol, then the obtained precipitate is washed three times with 75% ethanol, and finally the precipitate is added with an appropriate amount of 0.25 M NaOH and stirred to dissolve, adjusting the pH = 7. Finally, an ultrafiltration tube (MWCO: 3 kDa) is used to purify the product to remove free peptides and unreacted substances. Finally, the purified product is concentrated and the volume and concentration multiple are recorded. The molar ratio of the gold salt to the thiol - containing polypeptide is 1:1 - 2:1, preferably 1.5:1. The reducing agent used can be sodium borohydride (NaBH 4 ), sodium hydroxide (NaOH), vitamin C, etc., preferably sodium citrate. The concentration of the gold salt used can be 0.01 μM - 10 M, such as a 25 mM chloroauric acid solution, which is mixed and reacted with a thiol - containing peptide with a concentration of 0.1 μM - 10 M according to the optimal molar ratio (such as 1:1).

[0064] The hydrated particle size of the complex prepared by the preparation method of the present invention is about 2.3 nm. Each Au - peptide complex cluster NG has an exact molecular composition AuxPy, where x = 1 - 35 and y = 1 - 30, and has a photoluminescence characteristic.

[0065] The preparation method of the NG complex of the present invention uses a targeting polypeptide containing a thiol group for nervous system tumors as a ligand, and utilizes the affinity of gold for thiol groups to synthesize an Au - peptide adduct. The one - step reduction synthesis method is simple, and the obtained complex has high stability, especially suitable for targeting nervous system tumors.

[0066] In the present invention, a targeting polypeptide containing a thiol group is selected as a ligand to directly adduct with the Au salt to form a thiol - Au adduct. In addition, when preparing this type of complex, properties such as its biocompatibility, water - solubility, chemical stability, size, and surface properties can all be adapted to the requirements of a specific biomedical application through independent design and modification.

[0067] The novel gold complex molecular probe Au - peptide adduct of the present invention has many advantages for the treatment of nervous system tumors: (1) Due to its ultra - small size, it can make up for the shortcoming that currently available nervous system tumor - treating drugs on the market cannot enter the blood - brain barrier; (2) It has good biosafety and will not affect the normal physiological activities of nerve cells even at a relatively high concentration; (3) Compared with existing chemotherapeutic drugs, it can combine the targeting function of polypeptides, increase the targeting efficiency of nervous system tumor sites, and reduce the side effects on other normal brain cells.

[0068] The application of the complex formed by the binding of gold and the targeting peptide is used for the preparation of drugs for treating nervous system tumors. The nervous system tumors include but are not limited to gliomas such as neuroblastoma, astrocytoma, ependymoma, and medulloblastoma, meningioma, schwannoma, neurofibroma, and metastatic tumors of other tumors to the nervous system.

[0069] Next, the NG complex of the present invention, its preparation method and application will be further described in combination with specific examples. Among them, the gold salt used in the examples is trivalent chloroauric acid. The fluorescence characteristics of the synthesized NG were characterized by the direct observation method and a fluorescence spectrometer. The feasibility of the gold nanoclusters in promoting ferroptosis and treating nervous system tumors was verified through a series of in - vitro cytotoxicity experiments around nervous system tumor cells and in - vivo animal tumor model treatment experiments.

[0070] Example 1: Preparation and Characterization of the NG Complex

[0071] 1) Dissolve 5 mg of the thiol - containing H2N - GCLRVKKKYCC - COOH polypeptide (a polypeptide targeting the α7nAChR protein) in 3.844 mL of ultrapure water to prepare a 1.0 mM polypeptide solution, place it in a clean and dry material reaction bottle, add a magnetic stirrer, and stir it at 600 rpm / min in a 37 °C water bath for 3 min;

[0072] 2) Prepare a 25 mM HAuCl4 solution from the HAuCl4 standard product, take 154 μL of the HAuCl4 solution and drop it into the polypeptide solution in (a), and continue to stir it at 600 rpm / min in a 37 °C water bath for 3 min to obtain a polypeptide - HAuCl4 mixture;

[0073] 3) Dissolve NaOH in ultrapure water to prepare a 0.5 M NaOH solution, take 384 μL of the 0.5 M NaOH solution and drop it into the mixture in step (2), and continue to stir the formed solution at 600 rpm / min in the dark at 37 °C for 12 h, and ultra - filter to obtain gold nanoclusters.

[0074] The magnetic stir bar was removed by a magnetic rod, and gold clusters emitting red fluorescence could be obtained. An ultrafiltration tube with a molecular weight cut-off of 3000 was used to remove free metal ions and unreacted polypeptides. The NG images obtained by the direct observation method are as Figure 1a shown. NG appears orange under daylight lamp irradiation ( Figure 1a 1 in Figure 1a ), and shows red fluorescence under 365 nm hand-held UV lamp irradiation ( Figure 1b 2 in

[0075] ). The fluorescence spectrum of NG is as Figure 1c shown. The black line is the best excitation spectrum of NG, and the red line is the best emission spectrum. It can be read from the figure that the excitation peak is at 504 nm and the emission peak is at 664 nm. Figure 1d ). The particle size of the gold nanoclusters was measured to be about 2.3 nm by dynamic light scattering (DLS) ( 17 7 ). And the ultraviolet absorptions of the polypeptide and the formed NG nanogold clusters were measured to be at 274 nm, 283 nm, and 243 nm respectively by ultraviolet-visible (UV-VIS) spectroscopy ( Figure 1e ). The exact composition of NG was obtained as Au Figure 1f by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). It can be seen from the transmission scanning electron micrograph that NG is of uniform size and evenly distributed in the aqueous phase (

[0076] ).

[0077] The relevant tests in the following examples were all carried out using the NG complex obtained in Example 1 of this embodiment.

[0078] Figure 2a To detect the expression level of α7nAChR protein in classical nervous system tumor cells, the results are shown in the figure. The classical nervous system tumor cells SH-SY5Y, U87, U251, and GL261 all highly express the α7nAChR protein, indicating that the targeting polypeptide of the present invention is accurately selected.

[0079] Figure 2b For ICP-MS gold element quantitative analysis, NG (50 μM) was co-incubated with 2x10 5 non-nervous system tumor cells (lymphocytes or bone marrow cells) or nervous system tumor cells for 2 hours. After collecting the cells, ICP-MS was used for quantitative analysis of intracellular gold elements. The results are shown in the figure. Under the same conditions, the uptake of NG by nervous system tumor cells (GL261, SH-SY5Y) is significantly stronger than that of other cells, indicating that NG has a targeting effect on nervous system tumor cells.

[0080] ​Figure 2c This is a column chart of ICP-MS gold element quantitative analysis for the blocking experiment of the co-incubation of NG of the present invention with its targeting polypeptide in GL261 cells. Under the same conditions, the cells were first incubated with the targeting polypeptide (50 μM) for 2 hours to block the α7nAChR receptors on the cell membrane. If NG (50 μM) needs to enter the cells by recognizing α7nAChR, the uptake of NG by the cells should be significantly reduced after the incubation with the targeting polypeptide. Indeed, after adding the targeting polypeptide, the uptake of NG by the cells was significantly decreased, and this experiment further demonstrated the targeting ability of NG of the present invention to nervous system tumor cells.

[0081] Example 3. Safety analysis of NG

[0082] Figure 3a This shows the 24-hour toxicity detection of NG of the present invention at different concentrations on GL261 and SH-SY5Y cells. It can be seen that even at a relatively high concentration, NG has no obvious toxicity to them. Therefore, NG alone has no killing effect on nervous system tumor cells.

[0083] Figure 3b This shows the 24-hour toxicity detection of NG of the present invention at different concentrations on bone marrow cells (left) and brain resident cells (right). It can be seen that even at a relatively high concentration, NG has no obvious toxicity to them, indicating the biosafety of the cluster NG.

[0084] Example 4. NG enhances the tumor killing effect of ferroptosis inducer at the 2D cell level

[0085] Figure 4a In GL261 cells, the ferroptosis inducer RSL3 (2 μM) was used to induce ferroptosis, and 50 or 100 μM NG nanogold clusters were added simultaneously. After 24 hours, the cell viability was detected using CCK-8. It was found that NG could significantly enhance the tumor killing ability by promoting the function of the ferroptosis inducer. However, the naked peptide of NG - GCLRV - could not promote ferroptosis, indicating that this ability of NG of the present invention does not depend on the polypeptide but mainly on the gold element.

[0086] Figure 4b Based on the promotion of the function of the ferroptosis inducer (RSL3, 2 μM) by NG (50 μM) of the present invention, the ferroptosis inhibitor Fer-1 (1 μM) was added. After 24 hours, it was found that the survival rate of tumor cells increased, corroborating that the cooperation between NG of the present invention and the ferroptosis inducer promotes ferroptosis rather than other secondary forms of cell death.

[0087] Figure 4cThis is the PI flow cytometry diagram of the present invention's NG (50 μM) enhancing the killing of glioma GL261 cells by the ferroptosis inducer (RSL3, 2 μM). After treating the cells with different reagents for 24 hours, the cells were collected for PI staining, and then flow cytometry was performed. The results were similar to Figure 4a -b. Compared with the control group, NG could significantly enhance the tumor cell killing ability of the ferroptosis inducer.

[0088] Figure 4d This is the colony formation experiment of the present invention's NG (50 μM) promoting the killing function of the ferroptosis inducer (RSL3, 2 μM) on nervous system tumor cells. After treating the cells with different reagents for 4 hours, 2000 cells were taken from each group for culture until obvious colony formation. The results showed that the number of cell colonies formed by the combined action of NG and the ferroptosis inducer RSL3 decreased significantly, indicating that on the basis of the ferroptosis inducer reducing the number of surviving tumor cells, the addition of NG further significantly reduced the cell survival rate; and the reversal of this result by Fer-1 (1 μM) indicated that NG directly enhanced the ferroptosis of RSL3 on tumor cells.

[0089] Figure 4e To induce ferroptosis in GL261 cells using the ferroptosis inducer RSL3 (2 μM), 50 or 100 μM NG nanoclusters were added simultaneously. After 24 hours, the cells were stained with BODIPY C11 (5 μM) for 30 min, and then imaged using a laser confocal microscope. The present invention's NG (50 μM) further significantly enhanced the lipid peroxidation level mediated by the ferroptosis inducer (RSL3, 2 μM). The physiological process of ferroptosis is accompanied by lipid peroxidation in cells. BODIPY C11 staining can change the color of lipid peroxidized cells from red to green. It can be seen from the figure that the addition of the present invention's NG resulted in the highest degree of intracellular lipid oxidation, while the ferroptosis inducer Fer-1 (1 μM) could inhibit the lipid peroxidation promoted by NG.

[0090] Example 5: 3D culture further verified that gold nanoclusters NG enhanced the tumor killing ability by promoting the function of ferroptosis inducers

[0091] Compared with traditional monolayer cell culture, 3D tumor spheroids have become an essential in vitro model for cancer research because they reproduce the structure and physiology of solid tumors.

[0092] Figure 5aIt is a 3D sphere of GL261 cells, a nervous system tumor in mice. 1500 cells were counted, and the cells were cultured using a low-attachment U-shaped 96-well plate. The interaction between cells caused the formation of spontaneous tumor spheres. The well-grown 3D spheres were induced to undergo ferroptosis using the ferroptosis inducer RSL3 (2 μM), and the present invention (NG 50 μM) was added simultaneously. After 24 hours, the 3D tumor spheres were double-stained for live / dead for 30 min using Calcein AM (2.5 μM) and PI / propidium iodide (2.5 μg / ml), and then laser confocal imaging was performed and statistical analysis was carried out. The results showed that NG of the present invention could also play a role in enhancing the tumor-killing effect of the ferroptosis inducer in the 3D tumor model of mouse GL261 cells.

[0093] Figure 5b It is a 3D sphere of GL261 cells, a nervous system tumor in mice. 1500 cells were counted, and the cells were cultured using a low-attachment U-shaped 96-well plate. The interaction between cells caused the formation of spontaneous tumor spheres. The well-grown 3D spheres were induced to undergo ferroptosis using the ferroptosis inducer Erastin (5 μM), and the present invention (NG 50 μM) was added simultaneously. After 24 hours, the 3D tumor spheres were double-stained for live / dead for 30 min using Calcein AM (2.5 μM) and PI / propidium iodide (2.5 μg / ml), and then laser confocal imaging was performed and statistical analysis was carried out. The results showed that NG of the present invention could also play a role in enhancing the tumor-killing effect of the ferroptosis inducer in the 3D tumor model of mouse GL261 cells.

[0094] Figure 5c It is a 3D sphere of SH-SY5Y cells, a nervous system tumor in humans. 1500 cells were counted, and the cells were cultured using a low-attachment U-shaped 96-well plate. The interaction between cells caused the formation of spontaneous tumor spheres. The well-grown 3D spheres were induced to undergo ferroptosis using the ferroptosis inducer Erastin (5 μM), and the present invention (NG 50 μM) was added simultaneously. After 24 hours, the 3D tumor spheres were double-stained for live / dead for 30 min using Calcein AM (2.5 μM) and PI / propidium iodide (2.5 μg / ml), and then laser confocal imaging was performed and statistical analysis was carried out. The results showed that NG of the present invention could also play a role in enhancing the tumor-killing effect of the ferroptosis inducer in the 3D tumor model of SH-SY5Y cells.

[0095] Figure 5dIt is a 3D sphere of U251 cells of human nervous system tumors. 1500 cells were counted, and the cells were cultured using a low-attachment U-bottom 96-well plate. Cell-cell interaction caused the formation of spontaneous tumor spheres. The well-grown 3D spheres were induced to undergo ferroptosis using the ferroptosis inducer RSL3 (2 μM), and the present invention (NG 50 μM) was added simultaneously. After 24 hours, the 3D tumor spheres were subjected to live / dead double staining with Calcein AM (2.5 μM) and PI / propidium iodide (2.5 μg / ml) for 30 min, and then laser confocal imaging was performed and statistical analysis was carried out. The results showed that NG of the present invention could also play a role in enhancing the tumor-killing effect of ferroptosis inducers in the 3D tumor model of SH-SY5Y cells.

[0096] Example 6, Gold nanoclusters NG enhance the Fe 2+ ion concentration in tumor cells by promoting HO-1, thereby enhancing the function of ferroptosis inducers

[0097] Figure 6a It is that NG (50 μM) of the present invention can further promote the level of ferrous ion concentration in cells induced by the ferroptosis inducer RSL3 (2 μM) after treating GL261 cells for 24 hours. The ferrous ion probe used was FerroOrange (1 μM).

[0098] Figure 6b It is that NG (50 μM) of the present invention can further increase the expression levels of key genes in the Nrf2-HO-1-Fth1 / Ftl pathway, which may be the reason for the increase in ferrous ion concentration. Under the stimulation of the ferroptosis inducer (RSL3, 2 μM) and NG, the genes Nrf2, HO-1, Fth1, and Ftl expressed by GL261 cells were all significantly upregulated.

[0099] Figure 6c It is that when NG (50 μM) of the present invention acts together with the ferroptosis inducer (RSL3, 2 μM), the co-localization of ferritin and lysosomes in cells increases, indicating that the transport of ferritin to lysosomes is significantly increased, promoting ferritin autophagy, and thus inducing ferroptosis. The lysosome probe used was LAMP1 (1 μM), and the ferritin antibody was ferritin (diluted 1:100).

[0100] Figure 6d It is that NG (50 μM) of the present invention lost the effect of increasing ferrous ion concentration in the presence of the HO-1 inhibitor Znpp (5 μM). The ferrous ion probe used was FerroOrange (1 μM).

[0101] Figure 6eIn the present invention, NG (50 μM) lost its ability to enhance the killing effect of the ferroptosis inducer RSL3 (2 μM) on 3D tumor spheroids in the presence of the HO-1 inhibitor Znpp (5 μM).

[0102] Example 7: Gold nanoclusters NG exert a therapeutic effect on nervous system tumor diseases in an in vivo tumor model

[0103] Based on the good ability of NG to enhance the tumor-killing effect of ferroptosis inducers on 3D tumor spheroids in vitro, we further investigated its therapeutic effect on a subcutaneous nervous system tumor model. 2x10^ 6 GL261 cells were subcutaneously inoculated at the back of the neck of the brain and divided into 3 groups (Saline, RSL3, RSL3+NG) for treatment. The drug was administered once every other day by intraperitoneal injection. The growth of the tumor inoculation site was observed daily, the body weight of the mice was weighed, and the tumor volume was recorded using a vernier caliper. V 肿瘤 (mm 3 ) = 0.5 x length x width 2 .

[0104] Figures 7a - 7c The tumor growth curve, tumor volume histogram, and tumor mass histogram are shown respectively. It can be seen that in the present invention, NG (10 mg / kg) combined with the ferroptosis inducer RSL3 (2.5 mg / kg) made the tumor grow the slowest, and could exert an enhanced tumor-killing effect of the ferroptosis inducer in the animal model of nervous system tumors. Compared with the ferroptosis inducer alone, it significantly delayed the onset process of nervous system tumors such as gliomas.

[0105] Figure 7d The body weight of the mice is shown. It can be seen that the body weight of the mice did not decrease daily, indicating that NG of the present invention has good biosafety.

Claims

1. A nanoscale polypeptide-gold complex cluster NG formed by gold and a thiol-containing targeting polypeptide, characterized in that, The NG molecule forms AuxPy, where x = 1 - 35, y = 1 - 30, and P is the corresponding nervous system tumor-targeting polypeptide, preferably Au 17 P 7 。 2. A nanoscale polypeptide-gold complex cluster NG formed by gold and a thiol-containing targeting polypeptide according to claim 1, characterized in that, P is GCLRVKKKYCC.

3. A method for preparing the nanoscale polypeptide-gold complex cluster NG formed by gold and a thiol-containing targeting polypeptide according to claim 1 or 2, characterized in that, The preparation method of the complex formed by the reaction of gold and the thiol group of the nervous system tumor targeting polypeptide includes the following steps: mixing a gold salt solution with the nervous system tumor targeting polypeptide, namely glutathione containing a thiol group, to form a mixed solution. Under certain temperature and pH conditions, a reducing agent is added to cause a reduction reaction in the mixed solution system. Au forms the complex cluster NG by interacting with the thiol group of the polypeptide.

4. According to the method described in claim 3, characterized in that, The gold salt solution is a trivalent inorganic salt compound of a gold compound, such as chloroauric acid; wherein the trivalent gold is reduced to gold atoms and / or monovalent gold.

5. According to the method described in claim 3, characterized in that, The nervous system tumor targeting polypeptide is a polypeptide sequence targeting the α7nAChR protein, and further is: H2N-GCLRVKKKYCC-COOH.

6. According to the method described in claim 3, characterized in that, The reducing agent is sodium borohydride (NaBH4), sodium hydroxide (NaOH), vitamin C (vitamin C), or trisodium citrate (Trisodium citrate dihydrate), etc., and is preferably selected according to actual synthesis. The concentration of the nervous system tumor targeting polypeptide in the mixed solution is 0.1 μM - 10 M, and the gold salt concentration is 0.01 M - 10 M; further, the molar ratio of the polypeptide to the gold salt is 1:1 - 2:1, and is preferably selected according to actual synthesis.

7. According to the method described in claim 3, characterized in that, The reduction reaction occurs in the mixed solution, the reaction temperature is 20 - 75 °C, and the pH condition is weakly alkaline or neutral, which can be adjusted and optimized as needed; The reduction reaction occurs in the mixed solution, stirring in the dark at 20 - 75 °C for 5 - 15 hours, the color of the solution changes from light yellow, and the complex is purified by methods such as ultrafiltration and dialysis; the hydrodynamic diameter of this complex is above 2 nm.

8. The application of the nanoscale polypeptide-gold complex cluster NG formed by gold and a thiol-containing targeting polypeptide according to claim 1 or 2, which is used to prepare a targeted drug for improving the therapeutic effect of ferroptosis inducers, and has a synergistic therapeutic effect specifically on nervous system tumors when used together with ferroptosis inducers.

9. According to the application described in claim 8, the molar concentration ratio of NG to the ferroptosis inducer is 25 - 50:1, such as 50 - 100 μM:2 μM.

10. The application of the complex of gold and the nervous system tumor-targeting binding peptide according to claim 8 or 9, wherein the nervous system tumors include, but are not limited to, gliomas such as astrocytoma, oligodendroglioma, ependymoma, and medulloblastoma, meningioma, schwannoma, neurofibroma, and metastatic tumors of other tumors to the nervous system, etc.

Images