6-aminonicotinamide-loaded nanomaterial targeting bithiophene death and its preparation method and application in ovarian cancer

Abstract

The application provides a 6-aminonicotinamide nano material targeting double sulfur death and a preparation method and application in ovarian cancer thereof, and belongs to the technical field of biological medicine and molecular biology. Specifically, the application proves by experiments that double sulfur death induced by 6-aminonicotinamide (6-AN) can efficiently inhibit the growth of SLC7A11 high-expression ovarian cancer cells. Therefore, based on the good biological safety and high drug loading rate of liposomes and the characteristics of folate targeting cancer cells, the application first constructs a folate-modified liposome drug delivery system (FA-L@AI) for targeted delivery and controlled release of 6-AN, thereby specifically mediating double sulfur death of ovarian cancer cells, realizing efficient treatment of ovarian cancer, and thus having good practical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine and molecular biology, and particularly relates to a 6-aminonicotinamide-loaded nanomaterial targeting disulfide death and a preparation method thereof and application thereof in ovarian cancer. BACKGROUND

[0002] The information disclosed in the background of the present application is only intended to increase the understanding of the overall background of the present application and should not necessarily be regarded as acknowledging or implying in any form that this information constitutes the prior art known to those skilled in the art.

[0003] Ovarian cancer (OC) is prevalent and has a high mortality rate of up to 70%, and is the leading cause of gynecological cancer death worldwide. Early diagnosis of OC can significantly improve the five-year survival rate of patients to 80%, but 75% of cancer patients are already in the advanced stage when they seek medical treatment. So far, the main treatment for OC is surgery combined with cisplatin or paclitaxel chemotherapy, which can alleviate the symptoms in the short term, but the five-year survival rate is only about 30-40%, and drug resistance is easily produced. Immunotherapy has a small side effect, but the patient response rate is only 8-15%. Therefore, it is urgent to develop a new method for precise and effective treatment of ovarian cancer.

[0004] Cell death is a physiological process for maintaining biological development and internal environment homeostasis, and killing cancer cells by targeting cell death-related pathways is an effective means of cancer treatment. Solute Carrier Family 7, Member 11 (SLC7A11; also known as xCT) can mediate cystine uptake and promote glutathione synthesis. Studies have shown that glucose deficiency can significantly promote cell death of SLC7A11 high ) cells with high expression of SLC7A11 high When cells are in glucose deficiency, it will trigger the depletion of reduced triphosphopyridine nucleotide (NADPH), hinder the decomposition of cystine, promote the increase of disulfide molecules in cells, induce abnormal disulfide bonding between actin cytoskeleton proteins, and ultimately lead to the collapse of actin network and rapid cell death. This new cell death mode is named disulfide death, which is a new target for cancer treatment. high Effective treatment of cancer opens a new window. SLC7A11 is highly expressed in ovarian cancer patients, and analysis of The Cancer Genome Atlas (TCGA) database shows that the expression of SLC7A11 in ovarian cancer tissues is significantly higher than that in normal tissues, but disulfide death has not been reported for the treatment of ovarian cancer. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application aims to provide a 6-aminonicotinamide-loaded nanomaterial targeting bithiophenic death and a preparation method and application thereof in ovarian cancer. high The growth of ovarian cancer cells. Therefore, based on the good biological safety and high drug loading rate of liposomes, and the characteristics of folate targeting cancer cells, the present application first constructs a folate-modified liposome drug delivery system (FA-L@AI) for targeted delivery and controlled release of 6-AN, thereby specifically mediating bithiophenic death of ovarian cancer cells and achieving efficient treatment of ovarian cancer. Based on the above research results, the present application is completed.

[0006] To achieve the above technical purposes, the technical solutions of the present application are as follows:

[0007] In a first aspect of the present application, a 6-aminonicotinamide-loaded nanomaterial targeting bithiophenic death is provided, which is specifically a liposome modified with folate and 6-aminonicotinamide; the liposome is a pH-sensitive liposome.

[0008] Further, the liposome is also modified with a fluorescent indicator, so that in situ imaging of cancer tissues can be achieved. In one specific embodiment of the present application, the fluorescent indicator is indocyanine green (ICG).

[0009] In a second aspect of the present application, a preparation method of the above-mentioned 6-aminonicotinamide-loaded nanomaterial targeting bithiophenic death is provided, which comprises one-step preparation of the above-mentioned nanomaterial by using a thin film hydration method.

[0010] In a third aspect of the present application, the above-mentioned 6-aminonicotinamide-loaded nanomaterial targeting bithiophenic death is provided for use in the preparation of an antitumor drug.

[0011] The tumor is an ovarian tumor, and further can be SLC7A11 high ovarian tumor.

[0012] In a fourth aspect of the present application, an antitumor drug is provided, wherein the active ingredient of the antitumor drug comprises the above-mentioned 6-aminonicotinamide-loaded nanomaterial targeting bithiophenic death.

[0013] According to the present application, when the product is a drug, the drug further comprises at least one non-active ingredient of the drug.

[0014] In a fifth aspect of the present application, a method for treating tumors is provided, which comprises administering a therapeutically effective dose of the above-mentioned 6-aminonicotinamide-loaded nanomaterial targeting bithiophenic death or a drug to a subject.

[0015] The beneficial technical effects of the one or more technical solutions above are:

[0016] The technical solutions above found that SLC7A11 is significantly highly expressed in OC through bioinformatics analysis, and further proved that SLC7A11 high The glucose dependence of ovarian cancer cells, and elucidated that 6-AN induced disulfide death can effectively inhibit SLC7A11 high The growth of ovarian cancer cells.

[0017] In order to enhance the treatment effect of 6-AN, the technical solutions above prepared a novel FA modified liposome nanomedicine FA-L@AI through a simple one-step self-assembly, which improved the tumor uptake while fully retaining the efficacy of 6-AN.

[0018] The technical solutions above are the first to use 6-AN induced SLC7A11 high disulfide death of ovarian cancer cells to achieve specific treatment of OC, providing a promising treatment method for OC and providing strong evidence for promoting disulfide death targeted therapy, and thus having good practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0019] The drawings accompanying the specification of the present application serve to provide further understanding of the present application, and the illustrative embodiments of the present application and their descriptions serve to explain the present application, and do not constitute an improper limitation on the present application.

[0020] Figure 1 SLC7A11 high The glucose dependence of ovarian cancer cells. a) Differential expression of SLC7A11 between high-grade serous ovarian cancer (HGSOC) tissues and normal tissues (based on TCGA-GTEx data). b) Correlation between SLC7A11 expression and overall survival (OS) in HGSOC patients (based on GSE32062 data). c) Comparison of disulfide death enrichment scores between SLC7A11 high expression group and SLC7A11 low expression group in HGSOC patients (based on GSE32062 data). d) Expression of SLC7A11 in ovarian cancer cells and normal cells. e) SLC7A11 high The significant sensitivity of ovarian cancer cells to glucose deprivation. f-g) TCEP inhibits glucose deprivation-induced cell death in OVCAR3 and TOV-21G cells. h-i) SLC7A11 highWestern blot analysis of actin cytoskeleton proteins in ovarian cancer cells. (TCEP = 2.5 mM, Trion = 50 mM, Fer-1, Z-VAD, Nec-1 = 20 mM). -Glu, 1640 medium without glucose; NC, 1640 medium with glucose. Values are expressed as mean ± SD, n = 3.

[0021] Figure 2 SLC7A11 in 6-AN-induced ovarian cancer cells high Bisulfide death in ovarian cancer cells. a) Correlation of SLC7A11, G6PD expression with OS in HGSOC patients (based on TCGA data). b) SLC7A11 high Dependency of ovarian cancer cells on 6-AN. c-d) TCEP inhibits 6-AN-induced SLC7A11 high Death of ovarian cancer cells. e) 6-AN deprives SLC7A11 high NADPH in ovarian cancer cells. f) SLC7A11 high Increased cysteine levels in ovarian cancer cells. g-h) Western blot analysis of actin cytoskeleton proteins in OVCAR3 and TOV-21G cells cultured with 6-AN medium with or without TCEP under reducing and non-reducing conditions. i) SLC7A11 high F-actin fluorescence images of ovarian cancer cells. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (red: rhodamine- phalloidin, blue: DAPI). (TCEP = 2.5 mM, Trion = 50 mM, 6-AN, Fer-1, Z-VAD, Nec-1 = 20 mM). Values are expressed as mean ± SD, n = 3.

[0022] Figure 3 Synthesis and characterization of FA-L@AI for the present application. a) Overall structure of FA-L@AI. b-c) Transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) images of FA-L@AI. d) Hydrodynamic size of FA-L@AI. e) Elemental distribution of FA-L@AI. f) UV-Vis spectra of 6-AN (black line), ICG (red line), and FA-L@AI (blue line). g-h) Hydrodynamic size and polydispersity index (PDI) of FA-L@AI at the indicated days. Values are expressed as mean ± SD, n = 3.

[0023] Figure 4a) Fluorescence images of ovarian cancer cells and normal cells after treatment with L@AI, FA-L@AI. Nuclei were stained with bisbenzimide (Hochest) (red: ICG in L@AI or FA-L@AI, blue: Hochest). b) Quantitative fluorescence intensity analysis of cells after treatment with L@AI and FA-L@AI. c) Cell viability of ovarian cancer cells and normal cells treated with 26.74 mM 6-AN (equivalent to 0.2 mg / mL FA-L@AI), 0.2 mg / mL L@AI, 0.2 mg / mL FA-L@AI for 12 h. Values are expressed as mean ± SD, n = 3.

[0024] Figure 5 FA-L@AI specifically induced SLC7A11 high Bisulfide death of ovarian cancer cells. a) TCEP inhibited FA-L@AI-induced cell death in OVCAR3, TOV-21G cells. b) SLC7A11 high NADPH reduction in ovarian cancer cells. c) Nanodrugs increased SLC7A11 high Cysteine levels in ovarian cancer cells. d-e) Reduction and non-reduction Western blot analysis of actin cytoskeleton proteins in OVCAR3 and TOV-21G cells cultured with FA-L@AI with or without TCEP. NPs: FA-L@AI. f) SLC7A11 high Fluorescence images of ovarian cancer cells. Nuclei were stained with DAPI. (FA-L@AI = 0.2 mg / mL, TCEP = 2.5 mM). Values are expressed as mean ± SD, n = 3.

[0025] Figure 6 FA-L@AI specifically targeted ovarian cancer tissues in the present embodiments. a) In vivo imaging of tumor sites in ovarian cancer tumor-bearing mice at different times after injection of FA-L@AI. b) Imaging of major organs (heart, liver, spleen, lung, and kidney) and tumor tissues of tumor-bearing mice taken 12 h after injection of FA-L@AI (2 mg / mL, 100 pL). c) Quantitative analysis of fluorescence intensity of major organs and tumors (n = 5). d) Serum biochemical indicators of normal nude mice treated with PBS or FA-L@AI for 12 h (n = 3). e) H&E staining of major organs of normal nude mice treated with PBS, FA-L@AI for 12 h.

[0026] Figure 7This study investigated the antitumor effect of FA-L@AI on ovarian cancer-bearing mice. a) Schematic diagram of the treatment regimen. b) Tumor volume measured at different time points after different drug treatments. c) Tumor weight after different treatments. d) Relative body weight changes during treatment. e) H&E and Ki67 staining of tumor tissues after different treatments. f) Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs) between the FA-L@AI and PBS groups. g) Gene set enrichment analysis (GSEA) of NADH-related biological processes. Data are expressed as mean ± standard deviation, n = 5. Detailed Implementation

[0027] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0029] The present invention will now be further illustrated with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Unless otherwise specified, experimental conditions not explicitly stated in the examples are generally performed under conventional conditions or as recommended by the reagent company. Unless otherwise specified, all reagents and consumables used in the following examples are commercially available.

[0030] In a typical embodiment of the present invention, a 6-aminonicotinamide-loaded nanomaterial targeting disulfide death is provided, wherein the nanomaterial is specifically a liposome modified with folic acid and 6-aminonicotinamide; the liposome is a pH-sensitive liposome.

[0031] In another specific embodiment of the present invention, the liposomes are further modified with a fluorescent indicator, thereby enabling in situ imaging of cancerous tissue. In one specific embodiment of the present invention, the fluorescent indicator is indocyanine green (ICG).

[0032] In this invention, the pH-sensitive liposome is a liposome that can change its structure in response to changes in pH value. By modifying it with specific chemical groups, the membrane structure of the liposome changes in a low pH environment (such as tumor tissue), thereby promoting the release of the aforementioned drug.

[0033] In the present application, the chemical group can be a PEOz acid-sensitive group, and specifically can be DSPE-PEOz2000, i.e., 1,2-distearoylphosphatidylethanolamine-poly(2-ethyl-2-oxazoline), which has good biocompatibility and biodegradability, and at the same time, due to its low antigenicity, can reduce the immune response that can be triggered in the drug delivery process.

[0034] In the present application, the nanomaterial is a spherical particle with a particle size of about 100 nm and has good stability.

[0035] Specifically, the present application uses folic acid (FA) to enhance the tumor absorption capacity, indocyanine green (ICG) as a fluorescent indicator, and incorporates a PEOz acid-sensitive group to construct a new type of folic acid-modified multifunctional liposome material (FA-L@AI) loaded with 6-AN and ICG. The material can accumulate at the site of ovarian cancer through the enhanced permeability and retention (EPR) effect, promote the binding of FA to the folic acid receptor alpha (FRa) overexpressed on the surface of ovarian cancer cells, mediate the phagocytosis of liposome drugs by cancer cells, and promote the hydrolysis of the material in lysosomes, thereby releasing 6-AN and ICG. 6-AN inhibits the production of NADPH by inhibiting G6PD activity, thereby increasing the amount of intracellular disulfide and inducing cells to undergo disulfide death; ICG realizes in situ imaging of cancer tissues under near-infrared light excitation. In vitro studies have confirmed that 6-AN has the function of inducing OVCAR3 and TOV-21G disulfide death. The present application also successfully constructs a folic acid-modified liposome drug delivery system with simple structure and good stability, and experimental results show that FA-L@AI can specifically target ovarian cancer cells and cause cells to undergo disulfide death. More importantly, FA-L@AI can effectively inhibit the growth of ovarian cancer through the disulfide death mechanism, and the effect is significantly higher than that of free 6-AN.

[0036] In another specific embodiment of the present application, a preparation method of the above-mentioned 6-aminonicotinamide-loaded nanomaterial for targeted disulfide death is provided, and the preparation method comprises one-step preparation of the above-mentioned nanomaterial by using a thin film hydration method.

[0037] Specifically, the preparation method comprises the following steps: dissolving a liposome membrane material in an organic solvent, then adding 6-aminonicotinamide thereto, removing methanol, then adding a PBS buffer solution thereto and performing heating hydration and ultrasonic treatment, and obtaining the nanomaterial after filtration, extrusion and centrifugation.

[0038] The liposome membrane material can be lecithin, cholesterol, DSPE-PEOz2000 and DSPE-PEG2000-FA; the mass ratio of the four is 5-15:1-5:1-5:2-8, preferably 10:2.5:2:3.5.

[0039] The organic solvent is methanol.

[0040] In another specific embodiment of the present application, the preparation method further comprises adding the above-mentioned fluorescent indicator to the solution in which the liposome membrane material is dissolved, and more preferably, the fluorescent indicator is indocyanine green.

[0041] The mass ratio of the lecithin to 6-aminonicotinamide is 5-15:1-5, preferably 10:3;

[0042] The mass ratio of the lecithin to indocyanine green is 5-15:1-5, preferably 10:3.

[0043] The heating and hydration can be performed by water bath heating, and the specific conditions are as follows: heating at 50-80℃ for 10-60 minutes, preferably heating at 60℃ for 30 minutes.

[0044] The ultrasonic treatment can be performed at 20-60Khz for 10-60 minutes, preferably at 40Khz for 30 minutes.

[0045] The filtration is specifically performed by using 0.45μm and 0.22μm membranes in sequence;

[0046] The extrusion can be performed by using a liposome extruder, and specifically, the extrusion is performed through a 100nm filter membrane for 10-20 times, preferably 15 times.

[0047] The centrifugation is specifically performed by using a 30-80KD (preferably 50KD) ultrafiltration tube at 1000-5000rpm (preferably 3500rpm) for 10-60 minutes (preferably 30 minutes), and the centrifugation can be performed 1-3 times.

[0048] In another specific embodiment of the present application, the above-mentioned 6-aminonicotinamide-loaded nanomaterial targeting bissulfide death is used for preparing an antitumor drug.

[0049] It should be noted that the tumor is used as known by those skilled in the art, which includes benign tumor and / or malignant tumor. The benign tumor is defined as excessive proliferation of cells that cannot form invasive and metastatic tumors in vivo. On the contrary, the malignant tumor is defined as cells with multiple cell abnormalities and biochemical abnormalities that can form systemic diseases (e.g. tumor metastasis in distant organs).

[0050] The tumor is an ovarian tumor, which can be further SLC7A11 high ovarian tumor.

[0051] In another embodiment of the present application, an anti-tumor drug is provided, wherein the active ingredient of the anti-tumor drug comprises the above-mentioned 6-aminonicotinamide-loaded nanomaterial targeting disulfide death.

[0052] It should be noted that, in addition to relying on disulfide death to exert the anti-tumor (OC) effect, the drug of the present application can also achieve the detection of tumor tissues based on the fluorescent indicator, and thus can be used for the detection, monitoring and prognosis evaluation of tumors; at the same time, the drug efficacy evaluation can also be achieved, which is not limited herein.

[0053] According to the present application, when the product is a drug, the drug further comprises at least one non-drug active ingredient.

[0054] According to the present application, the drug can further comprise at least one other non-drug active ingredient.

[0055] The non-drug active ingredient can be a carrier, excipient and diluent commonly used in pharmacy, etc. Moreover, according to the usual method, it can be made into a dosage form of powder, granule, suspension, emulsion, syrup, spray, etc. for oral administration, external use, suppository and sterile injection solution.

[0056] The non-drug active ingredient that can be contained, such as carrier, excipient and diluent, is well known in the art, and a person skilled in the art can determine that it meets the clinical standards.

[0057] In another embodiment of the present application, the carrier, excipient and diluent include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, etc.

[0058] In another embodiment of the present application, the drug of the present application can be administered into the body by known means. For example, it can be delivered into the tissue of interest by intravenous systemic delivery or local injection (such as intratumoral injection). Such administration can be performed via single dose or multiple doses. A person skilled in the art understands that the actual dose to be administered in the present application can vary to a great extent depending on various factors, such as target cells, biological types or tissues thereof, general condition of the subject to be treated, administration route, administration mode, etc.

[0059] In another embodiment of the present application, the subject to which the drug is administered can be a human and a non-human mammal, such as a mouse, a rat, a guinea pig, a rabbit, a dog, a monkey, a chimpanzee, and the like.

[0060] In another embodiment of the present application, a method for treating a tumor is provided, which comprises administering to a subject a therapeutically effective amount of the above-mentioned 6-aminonicotinamide-targeted nanomaterial or drug for bithiophosphine-induced death.

[0061] The subject refers to an animal, preferably a mammal, and most preferably a human, who has been the object of treatment, observation or experiment. The "therapeutically effective amount" refers to the amount of an active compound or pharmaceutical agent, including the compounds of the present application, which elicits the biological or medical response that is being sought in a tissue system, animal or human by a researcher, veterinarian, medical doctor or other medical person, including alleviation or partial alleviation of the symptoms of the disease, syndrome, condition, or disorder being treated. It must be appreciated that the optimal dosage and spacing of administration of the active ingredients of the present application are determined by their properties, and external conditions such as the form, route and site of administration, and the specific mammal being treated, and this optimal dosage can be determined using conventional techniques. It must also be appreciated that the optimal course of treatment, i.e. the daily dosage of the compound within a given period of time, can be determined using methods known in the art.

[0062] The present application is further explained by the following examples, which do not limit the present application. It should be understood that these examples are intended to illustrate the present application and not to limit the scope of the present application. In the following examples, the test methods are described for specific conditions, and generally follow conventional conditions.

[0063] Examples

[0064] 1. Experimental methods

[0065] Cells and animals: Human ovarian cancer cell lines A2780, OVCAR3, TOV-21G and normal cell line IOSE-29 were gifted by Shandong University (Jinan, China). All cells were cultured in RPMI-1640 medium (VivaCell, China) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. In the glucose deprivation experiment, cells were cultured in glucose-free RPMI-1640 medium (Solarbio) supplemented with 10% FBS.

[0066] Female BALB / c nude mice (4 weeks old) were purchased from Huafukang Biotechnology (Beijing, China). All experimental procedures involving mice were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of Shandong University of Chinese Medicine (Jinan, China). The ethical approval number was SDUTCM20240102001. Mice were housed in a pathogen-free facility under a 12-hour light / dark cycle with free access to food and water.3 Tumor volume was the human endpoint. In this study, the maximum tumor volume observed did not exceed this limit.

[0067] Western blotting experiments: Western blotting experiments were performed following the previously described method. To analyze the expression of SLC7A11, A2780, OVCAR3, TOV-21G and IOSE-29 cells were seeded in 12-well plates at a density of 1.5 x 10 5 cells / well. After overnight culture, cells were washed and lysed with 150 μL RIPA buffer at 4 °C for 15 min. Cell debris was discarded by centrifugation at 12000 rpm for 15 min at 4 °C, and protein concentration was determined using BCA protein assay. Then, 20 μL 5x SDS was added to 80 μL sample, and the above samples containing equal amounts of protein were electrophoretically separated on 12% polyacrylamide SDS gels, followed by transfer to nitrocellulose membranes and blocking with 10% skim milk. The membranes were incubated with SLC7A11 antibody (1:5000, abeam) at 4 °C overnight. Thereafter, anti-rabbit IgG secondary antibody (1:4000, ZSGB-Bio) was added for another 1 h incubation. Finally, the membranes were visualized by automatic chemiluminescence imaging analysis system (Tanon 5200). In addition, GAPDH was selected as an equal loading control.

[0068] To observe the disulfide bonds in actin cytoskeleton proteins induced by glucose starvation, 6-AN or FA-L@AI, OVCAR3 and TOV-21G cells were seeded in 12-well plates at a density of 1.5 x 10 5 cells / well. After 12 h, the medium was replaced with glucose-free RPMI-1640 medium for 3 or 6 h, or the medium was discarded and medium containing different drugs (6-AN = 20 μM, FA-L@AI = 0.2 mg / mL, TCEP = 2.5 mM) was added for another 12 h incubation. Subsequently, cells were lysed with 150 μL RIPA buffer at 4 °C for 15 min. Protein samples were obtained by centrifugation. Each protein sample was divided into two equal parts, 15 μL non-reducing 5x SDS (Biosharp) was added to one part (60 μL), and the other part was used for reduction analysis. All samples were heated at 100 °C for 10 min. The Western blotting procedure was performed as described, using actin antibody (1:5000, abeam).

[0069] Preparation of FA-L@AI, FA-L@A and L@AI: FA-L@AI, FA-L@A and L@AI were synthesized using a one-step method previously reported. When preparing FA-L@AI, 10 mg of egg phospholipid, 2.5 mg of cholesterol, 2 mg of DSPE-PEOz2000, 3.5 mg of DSPE-PEG2000-FA were added to 18 mL of methanol. After dissolving by ultrasonication (40Khz), 3.0 mg of 6-AN and 3.0 mg of ICG were added to the above solution. Methanol was removed by rotary evaporation, then 3.6 mL of PBS (pH = 7.4) was added and heated at 60°C for 30 minutes. The above liquid was ultrasonicated (40Khz) for 30 minutes, the mixture was filtered with 0.45 μm, 0.22 μm membrane in turn, then extruded with a small liposome extruder (100 nm filter membrane, 15 times). The product with a final concentration of 5.0 mg / mL was obtained by centrifugation twice at 3500 rpm for 30 minutes using a 50KD ultrafiltration tube.

[0070] Preparation of FA-L@A, materials include egg phospholipid, cholesterol, DSPE-PEOz2000, DSPE-PEG2000-FA and 6-AN. Preparation of L@AI, materials include egg phospholipid, cholesterol, DSPE-PEOz2000, DSPE-PEG2000-methyl, 6-AN and ICG. The synthesis procedure is the same as described above.

[0071] Rhodamine-phalloidin staining: Cells were seeded on 12-well plate round coverslips (20 mm in diameter) at a density of 1 x 10 4 cells / well for 12 hours. Then, cells were treated with 20 μM 6-AN or 0.2 mg / mL FA-L@AI. After 12 hours, the culture medium was discarded and cells were fixed with 4% formaldehyde for 10 minutes. After fixation, cells were permeabilized with a permeabilization buffer (0.5% Triton X-100) for 5 minutes. After washing with PBS, rhodamine-phalloidin (1:1000, containing 1% BSA) was added for staining. After 30 minutes, cells were washed twice and treated with DAPI for 10 minutes. After washing with PBS, images were taken by a Zeiss LSM 880 NLO microscope. (Red: λ ex = 543 nm, Blue: λ ex = 405 nm)

[0072] Cell uptake: Cells were seeded on 12-well plate round coverslips (20 mm in diameter) at a density of 1 x 10 4Cells were seeded at a density of 10 cells / dish in 15 mm glass-bottom culture dishes and cultured for 12 hours. The control group was left untreated and allowed to grow naturally. Other cells were incubated for 1 hour in medium containing 0.2 mg / mL L@AI or 0.2 mg / mL FA-L@AI. The original medium was then removed, and 10 μM Hochest was added for a further 10 minutes of incubation. Before imaging, all medium was discarded from the culture dishes, and the cells were washed three times with PBS to remove excess dye. Fluorescence images were obtained using an LSM880+Fast Airyscan microscope. (Red: λ) ex =643nm, blue: λ ex =405nm)

[0073] NADPH and Cystine Level Detection: Intracellular NADPH levels were measured according to previously published methods. Briefly, cells were loaded with 1 × 10⁻⁶ cells / cells. 6 Cells were seeded at a density of 10 cells / well overnight in 6-well plates and treated for 12 hours with different drugs (6-AN = 20 μM, FA-L@AI = 0.2 mg / mL). Cells were then lysed in 200 μL of extraction buffer and gently pipetted to promote lysis. Subsequent steps were performed according to the manufacturer's instructions.

[0074] For the detection of intracellular cystine, cells were sampled at a concentration of 1×10⁻⁶. 6 Cells were seeded at a density of 10 cells / well in 6-well plates and cultured for 12 hours. Then, medium containing 20 μM 6-AN or 0.2 mg / mL FA-L@AI was added. After 12 hours, cells were washed with PBS and lysed at 4°C with 200 μL RIPA buffer for 15 minutes. Samples were collected by centrifugation at 12,000 rpm for 15 minutes at 4°C. Subsequently, ELISA assays were performed according to the instruction manual.

[0075] In vivo antitumor experiment: Two weeks after tumor growth, tumor-bearing mice were randomly divided into four groups (n=5). Different drugs were administered intravenously every other day. FA-L@AI and FA-L@A were administered at a concentration of 2 mg / mL (100 μL), and an equal volume of free 6-AN was injected at a dose of 267.4 μM (100 μL). Tumor volume and body weight were recorded before each injection. The tumor volume was calculated using the following formula: Tumor volume (V) = L × W 2 / 2, where L and W represent the length and width of the tumor, respectively. On day 20, all mice were sacrificed and the tumors were dissected. After weighing, a portion of the tumors from each group was fixed in 4% formaldehyde for 3 days. After routine dehydration and paraffin embedding, the tumors were stained with H&E and Ki67. Other fresh tumor fractions from the FA-L@AI and PBS groups were used for mRNA transcriptome sequencing.

[0076] Statistics and reproducibility: Data plotting and statistical analysis were performed using GraphPad Prism 9.5.1 and Origin 2018. Each experiment was repeated at least three times, and quantitative results were expressed as mean ± standard deviation. Student’s t-test was used to compare control and experimental samples. No data points were excluded from the analysis. By Student’s t-test, *** P < 0.001, ** P < 0.01, * P < 0.05. ns, indicates not significant (P > 0.05).

[0077] 2. Results and discussion

[0078] 2.1 SLC7A11 high Glucose dependence of ovarian cancer cells

[0079] To demonstrate the high expression of SLC7A11 in ovarian cancer tissues, first by TCGA database analysis, it was found that SLC7A11 was significantly higher expressed in ovarian cancer tissues than in normal tissues Figure 1 a). At the same time, SLC7A11 high The survival of ovarian cancer patients was significantly prolonged Figure 1 b). By scoring the genes related to disulfide death, it was predicted that SLC7A11 high The disulfide death activity of ovarian cancer patients was increased, and the survival of patients with high activity of disulfide death was prolonged Figure 1 c). The above results show that the level of SLC7A11 in ovarian cancer tissues is higher than that in normal tissues, and disulfide death is expected to provide a new strategy for effective treatment of ovarian cancer.

[0080] Subsequently, whether sugar deprivation can induce disulfide death in ovarian cancer cells was tested. First, WB experiments confirmed that SLC7A11 protein was highly expressed in ovarian cancer cell lines OVCAR3 and TOV-21G Figure 1 d). Using CCK8 experiments, the sensitivity of SLC7A11 high ovarian cancer cell lines and normal cells to sugar was studied. The experimental results showed that the survival rate of OVCAR3 and TOV-21G decreased with the prolongation of sugar deprivation time, while the survival rate of A2780 cells decreased slowly Figure 1 e), suggesting that SLC7A11 high ovarian cancer cell lines have higher sensitivity to sugar. Further studies found that this sugar deprivation-induced cell death can be significantly inhibited by the disulfide bond-specific reducing agent TCEP, and under the same conditions, the addition of other death inhibitors Fer-1, Trion, Z-VAD, Nec-1 had no effect Figure 1f-g). More importantly, WB experiments showed that after sugar deprivation, Actin protein in OVCAR3, TOV-21G cells had obvious disulfide bond formation Figure 1 h-i). All the above results proved that sugar deprivation could induce disulfide death in OVCAR3, TOV-21G.

[0081] 2.2 6-AN induced SLC7A11 high Disulfide death of ovarian cancer cells

[0082] The function of 6-AN in inducing disulfide death of ovarian cancer cells was studied. TCGA data showed that SLC7A11 high G6PD low The survival rate of patients in the SLC7A11 low G6PD high group was significantly longer than that in the SLC7A11 high OC, suggesting that inhibiting G6PD might be a viable treatment strategy for SLC7A11 Figure 2 a). Therefore, the focus was on G6PD inhibitor 6-AN, and its potential to induce disulfide death in SLC7A11 high ovarian cancer cells was studied. The experimental results showed that the survival rate of OVCAR3, TOV-21G cells decreased significantly with increasing 6-AN treatment concentration, while the activity of A2780 cells decreased to 60%, indicating that SLC7A11 high ovarian cancer cell death is highly dependent on 6-AN( Figure 2 b). Similar to glucose deprivation, 6-AN-induced cell death can be effectively rescued by TCEP, but not by Fer-1, Trion, Z-VAD and Nec-1. This indicates that 6-AN triggers the accumulation of intracellular disulfides( Figure 2 c-d). Considering that inhibiting NADPH can prevent cystine from being reduced to cysteine, increasing intracellular disulfides, the NADPH level in 6-AN-treated SLC7A11 high ovarian cancer cells was detected. The results showed that after 6-AN treatment, the intracellular NADPH level decreased significantly( Figure 2 e). All these data suggest that 6-AN inhibits NADPH, hinders cystine decomposition, leads to a large accumulation of intracellular disulfides, and ultimately induces SLC7A11 high disulfide death of ovarian cancer cells.

[0083] To prove the above conclusion, the disulfide death-related indicators were detected. First, the intracellular cystine content under different treatments was detected by ELISA kit. As Figure 2f). As shown in Fig. 6A, the intracellular cystine concentration in 6-AN group was significantly higher than that in the normal group, suggesting that cystine accumulated in the cells. WB results showed that a large amount of disulfide bonds were generated in Actin protein after 6-AN treatment, and could be effectively reduced by TCEP Figure 2 g-h). Subsequently, the morphology of F-actin was observed using fluorescence confocal imaging technology, and F-actin in the cells after 6-AN treatment showed obvious aggregation Figure 2 i), further indicating that a large amount of disulfide bonds were generated in Actin protein. The above results show that 6-AN can inhibit the production of NADPH in cells, leading to blocked cystine metabolism, accumulation of disulfides, and induction of cellular disulfide death.

[0084] 2.3 Synthesis and characterization of FA-L@AI

[0085] To improve the therapeutic effect of 6-AN, a liposome system FA-L@AI was designed and synthesized Figure 3 a). The construction of FA-L@AI is simple and clear, and the specific synthesis steps are described in the method. Transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) images show that FA-L@AI is spherical particles, and the particle size is about 100 nm Figure 3 b-c). Dynamic light scattering (DLS) shows that the hydrated particle size is about 116 nm, and the polydispersity index (PDI) is about 0.21, indicating that the material has uniform particle size distribution Figure 3 d). To further verify the successful assembly of FA-L@AI, energy dispersive spectrometer (EDS) analysis was performed. The detection of sulfur (S) in the elemental spectrum confirms the effective incorporation of ICG into FA-L@AI Figure 3 e). The absorption spectrum of FA-L@AI matches that of free 6-AN and free ICG, verifying the effective loading of the two compounds Figure 3 f). According to the formula, the loading and encapsulation efficiency of 6-AN were calculated to be 14% and 2.3%, respectively. For ICG, these values reached 58% and 9.7%, respectively. These findings indicate the successful preparation of FA-L@AI.

[0086] Good stability is a prerequisite for the use of nanomaterials, therefore, the optical and physical properties of FA-L@AI were studied. The nanoparticle was placed at 4°C, and the particle size and PDI were measured at the time nodes shown in the figure. The results show that the particle size of the material does not change with the increase of days, and the PDI is also stable. At the same time, the particle size of the material in different dispersants also does not change with time, indicating that the material has good physical stability Figure 3 g-h). In summary, FA-L@AI has good stability, which is a necessary prerequisite for its further application.

[0087] 2.4. In vitro cytotoxicity of FA-L@AI

[0088] To investigate the characteristics of highly absorbable nanomedicines in cancer cells, FA-L@AI and L@AI were co-cultured with IOSE-29, OVCAR3, and TOV-21G cells, respectively, and fluorescence imaging was performed using a fluorescence confocal microscope. Figure 4 As shown in Figure a, after L@AI staining, the red channel fluorescence in IOSE-29, OVCAR3, and TOV-21G cells was basically the same. However, the red fluorescence in OVCAR3 and TOV-21G cells treated with FA-L@AI was significantly stronger than that in IOSE-29 cells, with a fluorescence output value approximately three times that of normal cells. Figure 4 b) indicates that the new material for cancer cells has a high absorption rate. All the above results suggest that the high expression of FRα on the cancer cell membrane mediates the specific high absorption of FA-L@AI by cancer cells.

[0089] Subsequently, the cytotoxicity of FA-L@AI was tested. CCK8 results showed that cancer cell survival was significantly dependent on the concentration of the nanomedicine; at 0.2 mg / mL, cancer cells showed significant death, and the survival rate of normal cells decreased to 65%. Figure 4 c). Although FA-L@AI showed some cytotoxicity to IOSE-29 cells, its cytotoxicity was significantly lower than that of cancer cells. Calculations showed that the IC50 value of FA-L@AI for IOSE-29 cells was 0.38 mg / mL, while for cancer cells it was approximately 0.068 mg / mL, further suggesting that a nanomedicine dose of 0.2 mg / mL had little effect on normal cells but produced significant toxicity to cancer cells. To demonstrate that the toxicity of 6-AN was not affected after being loaded onto liposomes, normal cells and cancer cells were treated with the same concentrations of free 6-AN, FA-L@AI, and FA-L@A. The results showed that the drug had significant killing power against cancer cells, while being essentially non-toxic to normal cells. Furthermore, the lethal effects of FA-L@AI and FA-L@A were the same as those of 6-AN, suggesting that FA modification of the liposome carrier and ICG did not affect the toxicity of 6-AN. All of the above data indicate that the nanomedicine retained the efficacy of 6-AN and could specifically kill cancer cells.

[0090] 2.5 FA-L@AI-specific induction of SLC7A11 high Disulfide death in ovarian cancer cells

[0091] Because FA-L@AI exhibits 6-AN toxicity, it is speculated that the nanomedicine kills cancer cells through disulfide death. Figure 5As shown in Fig. 2a, the cell death induced by FA-L@AI was effectively reversed by TCEP, and was not affected by Fer-1, Trion, Z-VAD, Nec-1, suggesting that FA-L@AI caused disulfide accumulation in ovarian cancer cells. Using the NADPH kit to detect the cells treated with nanodrugs, it was found that the NADPH level was significantly reduced Figure 5 b). The above results show that FA-L@AI can inhibit the synthesis of NADPH in cells, inducing disulfide accumulation in cells. Subsequently, the disulfide death-related indicators were detected. First, the cysteine content in cells under different treatments was detected by ELISA kit. As shown in Fig. 2c, the cysteine concentration in the FA-L@AI group was significantly higher than that in the normal group, indicating that cysteine accumulated in the cells. WB results showed that after FA-L@AI treatment, a large number of disulfide bonds were generated in Actin protein, and could be effectively reduced by TCEP Figure 5 Figure 5 d-e). Subsequently, the morphology of F-actin was observed by fluorescence confocal imaging technology, and F-actin in the cells after FA-L@AI treatment showed obvious aggregation Figure 5 f), further indicating that a large number of disulfide bonds were generated in Actin protein. The above results show that the death mechanism of FA-L@AI is the same as that of 6-AN, that is, by inhibiting the production of NADPH in cells, causing cysteine metabolism to be blocked, disulfide accumulation, and triggering cell disulfide death.

[0092] 2.6 FA-L@AI in vivo targeting ovarian cancer tissue

[0093] To further evaluate the therapeutic potential of the nanodrug, the uptake ability of the tumor to FA-L@AI in tumor-bearing mouse models was studied. OVCAR3 cancer cells were injected subcutaneously into BALB / c nude mice to create tumor-bearing models. After intravenous injection of FA-L@AI, imaging was performed on a small animal live imaging system. Notably, strong fluorescence was detected only at the tumor site, reaching a peak at 4 h after administration and lasting up to 12 h, indicating that FA-L@AI accumulated in the tumor and had a long residence time Figure 6 a). Imaging of major organs and tumor tissue showed bright fluorescence in tumor tissue, while normal tissues (except liver and kidney) showed dim fluorescence. This finding indicates that FA-L@AI preferentially accumulates in the tumor area due to the EPR effect and the high expression of FRa on the membrane of cancer cells Figure 6 b). In addition, it was found that the fluorescence intensity in the tumor tissue was 10 times that in the heart, spleen and lung Figure 6 c). At the same time, bright fluorescence was also observed in the liver and kidney, which was mainly due to the influence of the reticuloendothelial system. In summary, these results confirm that FA-L@AI effectively accumulates and visualizes the tumor site, laying the foundation for efficient treatment of OC.​

[0094] Subsequently, the toxicity of FA-L@AI was evaluated. BALB / c nude mice were randomly divided into two groups, and injected with PBS and nanodrugs, respectively. Blood samples were taken 12 h later, and serum-related indicators were detected. The alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN), creatinine (CRE), and creatine kinase (CK) contents of the FA-L@AI group showed no significant changes compared with the PBS group, indicating that the liver, kidney, and heart functions were normal Figure 6 d) To further evaluate the safety of FA-L@AI, the main organs of the two groups of mice were collected and subjected to hematoxylin-eosin (H&E) staining. No organ damage was found in the FA-L@AI group compared with the PBS group Figure 6 e) The above results show that FA-L@AI has good biological safety.

[0095] 2.7 Anti-tumor effect of FA-L@AI on ovarian cancer-bearing mice

[0096] Since FA-L@AI can be highly accumulated in tumor tissues, it is necessary to explore whether it can exert a therapeutic effect on tumors. Therefore, the anti-ovarian cancer effect of FA-L@AI was further tested in the aforementioned tumor-bearing mouse model. The tumor-bearing mice were randomly divided into four groups, and the corresponding drugs were injected intravenously every other day for 20 consecutive days to measure the tumor volume and evaluate the therapeutic effect. The treatment regimen is shown in Figure 7 a. Compared with the PBS group, the tumor size of the free 6-AN group was reduced, while the tumors of the FA-L@A and FA-L@AI groups were significantly inhibited. Notably, the FA-L@AI group showed significant growth inhibition compared with the free 6-AN group, indicating that FA-L@AI has a strong anti-tumor effect and does not affect its toxicity due to ICG loading Figure 7 b). Studies have shown that FA can bind to FRa on tumor cells, and liposomes can accumulate at the tumor site through the EPR effect. Therefore, FA-L@AI has a better anti-tumor effect compared with free drugs. Meanwhile, the change trend of tumor weight is consistent with the volume measurement Figure 7 c). In addition, the body weight of the mice in the FA-L@AI group did not change significantly, indicating that FA-L@AI has no systemic toxicity Figure 7 d). In short, the strategy of using FA-L@AI not only effectively improves the absorption of ovarian cancer tissues, but also significantly inhibits the growth of tumors through bithiophene death.

[0097] To further demonstrate the anti-tumor efficacy of FA-L@AI in vivo, H&E and Ki67 staining of tumor tissues were performed Figure 7e) No cell damage was observed in the free 6-AN group compared to the PBS group. In contrast, tumor cells in the FA-L@A and FA-L@AI groups exhibited obvious cell destruction, indicating that FA-L@AI has a very high therapeutic effect in vivo. The expression of Ki67 in the FA-L@A and FA-L@AI groups was lower than in the other groups, indicating that they have a strong ability to inhibit tumor proliferation. All results indicate that FA-L@AI has a stronger anti-tumor ability than free 6-AN. In addition, tumor tissues were collected from the PBS and FA-L@AI groups for mRNA sequencing. Gene ontology analysis enriched biological processes related to actin cytoskeleton proteins and glucose metabolism, which means that the nanodrug can cause intracellular changes in actin cytoskeleton dynamics by affecting glucose metabolism Figure 7 f-g) These results further strengthen the fact that FA-L@AI triggers bithiophenol death in cancer cells, thereby effectively inhibiting tumor growth in vivo.

[0098] In summary, FA-L@AI has three major advantages: (1) ovarian cancer cells highly absorb FA modification; (2) specifically mediates SLC7A11 high bithiophenol death in ovarian cancer cells, showing strong cell damage ability; (3) due to FA modification and EPR effect, selectively accumulates at the tumor site, and shows superior anti-tumor efficacy, significantly inhibiting SLC7A11 high expression in ovarian tumors, and has minimal systemic toxicity. In addition, mRNA transcriptome analysis further elucidates the relevant therapeutic mechanisms of bithiophenol death triggered by FA-L@AI, which may provide an alternative to traditional therapy for OC treatment.

[0099] The above examples are only for illustrating the technical concepts and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and to implement it, and cannot limit the protection scope of the present application. Any equivalent changes or modifications made in accordance with the spirit and essence of the present application shall be covered within the protection scope of the present application.

Claims

1. Application of 6-aminonicotinamide-loaded nanomaterials targeting bithiophene death in the preparation of anti-tumor drugs; the tumor is SLC7A11 high ovarian tumor; The targeting bithiophene death 6-aminonicotinamide nanomaterial is a liposome modified with folic acid and 6-aminonicotinamide; the liposome is a pH-sensitive liposome.

2. Use according to claim 1, wherein The liposome is further modified with a fluorescent indicator.

3. Use according to claim 2, wherein the compound is ###0002### The fluorescent indicator is indocyanine green ICG.

4. The use according to claim 1, wherein The pH-sensitive liposome contains PEOz acid-sensitive groups.

5. The use according to claim 4, wherein the compound is ###0002### The pH-sensitive liposome contains DSPE-PEOz2000.

6. Use according to any one of claims 1 to 5, characterized in that, The nanomaterial is a spherical particle with a particle size of ~100 nm.

7. Use according to any one of claims 1 to 5, wherein The preparation method of the targeting bithiophene death 6-aminonicotinamide nanomaterial comprises one-step preparation of the nanomaterial by using a thin film hydration method.

8. Use according to claim 7, wherein the compound is ###0002### The preparation method comprises the following steps: dissolving a liposome membrane material in an organic solvent, then adding 6-aminonicotinamide thereto, removing the organic solvent, then adding a PBS buffer thereto and performing heating hydration and ultrasonic treatment, and then filtering, extruding and centrifuging to obtain the nanomaterial.

9. Use according to claim 8, wherein the compound is ###0002### The liposome membrane material is lecithin, cholesterol, DSPE-PEOz2000 and DSPE-PEG2000-FA; the mass ratio of the four is 5-15:1-5:1-5:2-8; The organic solvent is methanol; The preparation method further comprises adding a fluorescent indicator to the solution in which the liposome membrane material is dissolved; The mass ratio of the lecithin to the 6-aminonicotinamide is 5-15:1-5; The mass ratio of the lecithin to the fluorescent indicator is 5-15:1-5.

10. Use according to claim 9, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The fluorescent indicator is indocyanine green.

11. Use according to claim 8, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The heating hydration is performed by using a water bath heating method, and the specific conditions are as follows: heating at 50-80 ℃ for 10-60 minutes; The ultrasonic treatment is performed under the condition of 20-60 Khz for 10-60 minutes; The filtering is specifically performed by using 0.45 μm and 0.22 μm membranes in sequence; The extrusion is performed through a 100 nm filter membrane for 10-20 times; The centrifugation is performed by using a 30-80 KD ultrafiltration tube at 1000-5000 rpm for 10-60 minutes.

12. The use according to claim 11, wherein the compound is ###0002### The centrifugation is performed 1-3 times.

Images