6-amino nicotinamide loaded nano material for targeting disulfide death as well as preparation method and application of 6-amino nicotinamide loaded nano material in ovarian cancer

Abstract

The invention provides a 6-amino nicotinamide loaded nano material for targeting disulfide death, a preparation method of the 6-amino nicotinamide loaded nano material and application of the 6-amino nicotinamide loaded nano material in ovarian cancer, and belongs to the technical field of biological medicine and molecular biology. Specifically, experiments prove that the disulfide death induced by the 6-amino nicotinamide (6-AN) can efficiently inhibit the growth of the SLC7A11 high-expression ovarian cancer cells. Therefore, the folic acid modified liposome drug delivery system (FA-L at AI) is constructed for the first time for targeted delivery and controllable release of 6-AN on the basis of good biological safety and high drug loading rate of the liposome and by utilizing the characteristic of folic acid targeting cancer cells, so that the ovarian cancer cells are specifically mediated to disulfide death, and efficient treatment of ovarian cancer is realized; therefore, the method has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomedicine and molecular biology, and particularly relates to a 6-aminonicotinamide-loaded nanomaterial targeting ferroptosis, a preparation method thereof, and an application thereof in ovarian cancer. Background Art

[0002] The information disclosed in the background art of the present invention is only intended to increase the understanding of the overall background of the present invention, and does not necessarily be regarded as an admission or imply in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Ovarian cancer (OC) is prevalent and has a mortality rate as high as 70%, being the leading cause of death from gynecological cancers globally. Early diagnosis of OC can significantly increase 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 methods for OC are surgery combined with cisplatin or paclitaxel chemotherapy. Although it can relieve the symptoms in the short term, the five-year survival rate is only about 30-40%, and drug resistance is extremely likely to occur. Immunotherapy, although having few side effects, has a patient response rate of only 8-15%. Therefore, it is urgent to develop new methods for precise and effective treatment of ovarian cancer.

[0004] Cell death is a physiological process that maintains biological development and internal environmental homeostasis, and targeting cell death-related pathways to kill cancer cells is an effective means of cancer treatment. Solute carrier family 7 member 11 (SLC7A11; also known as xCT) can mediate the uptake of cystine and promote glutathione synthesis. Studies have shown that glucose deficiency can significantly promote the high expression of SLC7A11 (SLC7A11 high ) cell death. SLC7A11 high cells, when lacking glucose, will cause the depletion of reduced nicotinamide adenine dinucleotide phosphate (NADPH), hinder cystine decomposition, promote the increase of intracellular disulfide molecules, induce abnormal disulfide bonding between actin cytoskeleton proteins, and ultimately lead to the collapse of the actin network and rapid cell death. This new type of cell death is named ferroptosis, opening a new window for the effective treatment of SLC7A11 high cancer. SLC7A11 is highly expressed in patients with ovarian cancer. At the same time, analysis of the 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 the use of ferroptosis for the treatment of ovarian cancer has not been reported. Summary of the Invention

[0005] Aiming at the deficiencies in the prior art, the purpose of the present invention is to provide a 6-aminonicotinamide-loaded nanomaterial targeting ferroptosis, its preparation method and application in ovarian cancer. Specifically, the present invention proves through experiments that ferroptosis induced by 6-aminonicotinamide (6-AN) can efficiently inhibit the growth of SLC7A11 high ovarian cancer cells. Therefore, based on the good biosafety and high drug loading rate of liposomes, and taking advantage of the property of folic acid targeting cancer cells, the present invention constructs for the first time a folic acid-modified liposome drug delivery system (FA-L@AI) for the targeted delivery and controlled release of 6-AN, thereby specifically mediating ferroptosis of ovarian cancer cells and achieving efficient treatment of ovarian cancer. Based on the above research results, the present invention is completed.

[0006] In order to achieve the above technical objectives, the technical solutions provided by the present invention are as follows:

[0007] In the first aspect of the present invention, there is provided a 6-aminonicotinamide-loaded nanomaterial targeting ferroptosis, and the nanomaterial is specifically a liposome modified with folic acid and 6-aminonicotinamide; the liposome is a pH-sensitive liposome.

[0008] Furthermore, the liposome is also modified with a fluorescent indicator, so as to realize in-situ imaging of cancer tissues. In a specific embodiment of the present invention, the fluorescent indicator is indocyanine green (ICG).

[0009] In the second aspect of the present invention, there is provided a preparation method of the above-mentioned 6-aminonicotinamide-loaded nanomaterial targeting ferroptosis, and the preparation method includes preparing the above-mentioned nanomaterial in one step by the thin film hydration method.

[0010] In the third aspect of the present invention, there is provided an application of the above-mentioned 6-aminonicotinamide-loaded nanomaterial targeting ferroptosis in the preparation of anti-tumor drugs.

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

[0012] In the fourth aspect of the present invention, there is provided an anti-tumor drug, and the active ingredient of the anti-tumor drug contains the above-mentioned 6-aminonicotinamide-loaded nanomaterial targeting ferroptosis.

[0013] According to the present invention, when the product is a drug, the drug also includes at least one pharmaceutically inactive ingredient.

[0014] In the fifth aspect of the present invention, there is provided a method for treating tumors, and the method includes administering a therapeutically effective dose of the above-mentioned 6-aminonicotinamide-loaded nanomaterial targeting ferroptosis or drug to a subject.

[0015] Beneficial technical effects of the above one or more technical solutions:

[0016] Through bioinformatics analysis, the above technical solution found that SLC7A11 was significantly highly expressed in OC, and further proved the glucose dependence of SLC7A11 high ovarian cancer cells, and clarified that 6-AN-induced disulfidptosis could effectively inhibit SLC7A11 high the growth of ovarian cancer cells.

[0017] To enhance the therapeutic effect of 6-AN, the above technical solution prepared a novel FA-modified liposome nanodrug FA-L@AI through a simple one-step self-assembly. This strategy improved tumor uptake while fully retaining the efficacy of 6-AN.

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

[0019] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0020] Figure 1 For SLC7A11 in the embodiments of the present invention high 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 the SLC7A11 high-expression group and the 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 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) Under reducing and non-reducing conditions, SLC7A11 cultured in glucose-free medium for 0, 3, 6 hours highWestern blot analysis of actin cytoskeleton proteins in ovarian cancer cells. (TCEP = 2.5 mM, Trion = 50 μM, Fer-1, Z-VAD, Nec-1 = 20 μM). -Glu, 1640 medium without glucose; NC, 1640 medium with glucose. Values are expressed as mean ± standard deviation, n = 3.

[0021] Figure 2 In this example of the present invention, 6-AN induces SLC7A11 high Disulfidptosis of ovarian cancer cells. a) Correlation between SLC7A11, G6PD expression and OS in HGSOC patients (based on TCGA data). b) SLC7A11 high Dependence 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) Cystine levels increase in SLC7A11 high Ovarian cancer cells after 6-AN treatment. g-h) Western blot analysis of actin cytoskeleton proteins in OVCAR3 and TOV-21G cells cultured in 6-AN medium with or without TCEP under reducing and non-reducing conditions. i) F-actin fluorescence images of SLC7A11 high Ovarian cancer cells. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (red: rhodamine-phalloidin, blue: DAPI). (TCEP = 2.5 mM, Trion = 50 μM, 6-AN, Fer-1, Z-VAD, Nec-1 = 20 μM). Values are expressed as mean ± standard deviation, n = 3.

[0022] Figure 3 Synthesis and characterization of FA-L@AI in this example of the present invention. 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) Ultraviolet-visible 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 specified days. Values are expressed as mean ± standard deviation, n = 3.

[0023] Figure 4In vitro cytotoxicity of FA-L@AI in the embodiments of the present invention. a) Fluorescence images of ovarian cancer cells and normal cells after treatment with L@AI and FA-L@AI. The cell nuclei were stained with bisbenzimide (Hochest) (red: ICG in L@AI or FA-L@AI, blue: Hochest). b) Quantitative fluorescence intensity analysis of cells treated with L@AI and FA-L@AI. c) Cell viability of ovarian cancer cells and normal cells treated with 26.74 μM 6-AN (equivalent to 0.2 mg / mL FA-L@AI), 0.2 mg / mL L@AI, and 0.2 mg / mL FA-L@AI for 12 hours. The values are expressed as mean ± standard deviation, n = 3.

[0024] Figure 5 Specific induction of SLC7A11 by FA-L@AI in the embodiments of the present invention high Disulfide death of ovarian cancer cells. a) TCEP inhibits FA-L@AI-induced death of OVCAR3 and TOV-21G cells. b) In the presence of FA-L@AI, NADPH high decreases in ovarian cancer cells. c) The nanodrug increases the cystine level high in ovarian cancer cells. d-e) Reducing and non-reducing Western blot analysis of actin cytoskeleton proteins in OVCAR3 and TOV-21G cells cultured in FA-L@AI medium with or without TCEP. NPs: FA-L@AI. f) Fluorescence images of SLC7A11 high ovarian cancer cells stained with rhodamine-phalloidin. The cell nuclei were stained with DAPI. (FA-L@AI = 0.2 mg / mL, TCEP = 2.5 mM). The values are expressed as mean ± standard deviation, n = 3.

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

[0026] Figure 7Anti-tumor effect of FA-L@AI on ovarian cancer-bearing mice. a) Schematic diagram of the treatment protocol. b) Tumor volumes measured at different time points after different drug treatments. c) Tumor weights after different treatments. d) Relative body weight changes during the 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 presented as mean ± standard deviation, n = 5. Detailed implementation manners

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

[0028] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, 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 be further described in conjunction with specific examples below. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by reagent companies; the reagents, consumables, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0030] In a typical specific implementation manner of the present invention, a 6-aminonicotinamide-loaded nanomaterial targeting dual ferroptosis is provided, and the nanomaterial is specifically a liposome modified with folic acid and 6-aminonicotinamide; the liposome is a pH-sensitive liposome.

[0031] In another specific implementation manner of the present invention, the liposome is further modified with a fluorescent indicator, so as to realize in-situ imaging of cancer tissues. In a specific implementation manner of the present invention, the fluorescent indicator is indocyanine green (ICG).

[0032] In the present invention, the pH-sensitive liposome is a liposome that can change its structure in response to changes in pH value. By using specific chemical groups for modification, in a low-pH environment (such as tumor tissues), the membrane structure of the liposome changes, thereby promoting the release of the above-mentioned drugs.

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

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

[0035] Specifically, the present invention uses folic acid (FA) to enhance tumor uptake ability, indocyanine green (ICG) as a fluorescence indicator, and incorporates a PEOz acid-sensitive group to construct a novel multifunctional liposome material (FA-L@AI) loaded with 6-AN and ICG. This material can accumulate at the ovarian cancer site through the enhanced permeability and retention (EPR) effect, promote the binding of FA to the overexpressed folic acid receptor α (FRα) 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 activity of G6PD, thereby inhibiting the production of NADPH, increasing intracellular disulfides, and inducing cells to undergo disulfide death; ICG achieves in-situ imaging of cancer tissues under near-infrared light excitation. In vitro studies have confirmed that 6-AN has the function of inducing disulfide death in OVCAR3 and TOV-21G. The present invention has also successfully constructed a folic acid-modified liposome drug delivery system with a simple structure and good stability. The 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 invention, a method for preparing the above-mentioned 6-aminonicotinamide-loaded nanomaterial targeting disulfide death is provided. The preparation method includes one-step preparation of the above-mentioned nanomaterial by the thin-film hydration method.

[0037] Specifically, the preparation method includes: dissolving the liposome film material in an organic solvent, adding 6-aminonicotinamide thereto, removing methanol, adding PBS buffer thereto, heating and hydrating, and performing ultrasonic treatment, and then obtaining the product after filtration, extrusion, and centrifugation.

[0038] Among them, 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 invention, in the preparation method, the above-mentioned fluorescent indicator is further added to the solution in which the liposome membrane material is dissolved. Further, 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 carried out by water bath heating. The specific conditions are: heating at 50-80 °C for 10-60 minutes, preferably heating at 60 °C for 30 minutes.

[0044] The ultrasonic treatment can be carried out under the condition of 20-60Khz for 10-60 minutes, preferably under the condition of 40Khz for 30 minutes.

[0045] The filtration is specifically carried out by filtering with 0.45μm and 0.22μm membranes in sequence;

[0046] The extrusion can be carried out by a liposome extruder. Specifically, it is passed through a 100nm filter membrane for 10-20 times, preferably 15 times;

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

[0048] In another specific embodiment of the present invention, the above-mentioned 6-aminonicotinamide-loaded nanomaterial targeting dual ferroptosis is provided for use in the preparation of an anti-tumor drug.

[0049] It should be noted that tumors are used in the present invention as is known to those skilled in the art, and include benign tumors and / or malignant tumors. Benign tumors are defined as the overgrowth of cells that cannot form aggressive and metastatic tumors in the body. Conversely, malignant tumors are defined as cells with multiple cellular and biochemical abnormalities that can form a systemic disease (such as forming tumor metastases in distant organs).

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

[0051] In another specific embodiment of the present invention, an anti-tumor drug is provided, and the active ingredient of the anti-tumor drug comprises the above-mentioned 6-aminonicotinamide-loaded nanomaterial targeting disulfidptosis.

[0052] It should be noted that in addition to playing an anti-tumor (OC) role by relying on disulfidptosis, the drug of the present invention can also be used as a fluorescent indicator to detect tumor tissues, and thus can be used for the detection, monitoring and prognosis evaluation of tumors; at the same time, it can also realize the evaluation of the efficacy of the drug, etc., which will not be specifically limited here.

[0053] According to the present invention, when the product is a drug, the drug further comprises at least one pharmaceutically inactive ingredient.

[0054] According to the present invention, the drug may further comprise at least one other pharmaceutically inactive ingredient.

[0055] The pharmaceutically inactive ingredient may be a carrier, excipient, diluent, etc. commonly used in pharmacy. Moreover, according to the usual methods, it can be made into dosage forms such as powders, granules, suspensions, emulsions, syrups, sprays, etc. for oral administration, external use, suppositories and sterile injection solutions for use.

[0056] The non-drug active ingredients such as the carrier, excipient and diluent that can be included are well-known in the art, and those of ordinary skill in the art can determine that they meet clinical standards.

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

[0058] In another specific embodiment of the present invention, the drug of the present invention can be administered into the body by known means. For example, it can be delivered systemically via intravenous injection or locally injected (such as intratumoral injection) into the tissue of interest. Such administration can be carried out via a single dose or multiple doses. Those skilled in the art understand that the actual dose to be administered in the present invention can vary to a large extent depending on various factors such as target cells, biological type or its tissue, the general condition of the subject to be treated, the administration route, the administration method, and so on.

[0059] In yet another specific embodiment of the present invention, the subjects to which the drug is administered can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, chimpanzees, etc.

[0060] In yet another specific embodiment of the present invention, a method for treating tumors is provided, the method comprising administering to a subject a therapeutically effective dose of the above-mentioned 6-aminonicotinamide-loaded nanomaterial or drug targeting disulfidptosis.

[0061] The "subject" refers to an animal that has been the subject of treatment, observation or experiment, preferably a mammal, and most preferably a human. The "therapeutically effective amount" refers to the amount of an active compound or agent, including the compounds of the present invention, that can cause a biological or medical response in a tissue system, animal or human that is sought by a researcher, veterinarian, doctor or other medical personnel, which includes alleviating or partially alleviating the symptoms of the disease, syndrome, disorder or condition being treated. It must be recognized that the optimal dosage and interval of administration of the active ingredient of the present invention are determined by its nature and external conditions such as the form, route and site of administration and the particular mammal being treated, and this optimal dosage can be determined by conventional techniques. It must also be recognized that the optimal course of treatment, i.e., the daily dosage of the compound over a specified period of time, can be determined by methods well known in the art.

[0062] The present invention will be further explained and illustrated by the following examples, but this does not constitute a limitation on the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The test methods without specific conditions noted in the following examples are generally carried out under conventional conditions.

[0063] Examples

[0064] 1. Experimental methods

[0065] Cells and animals: The human ovarian cancer cell lines A2780, OVCAR3, TOV-21G and the normal cell line IOSE-29 were donated 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, the 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 carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health of the United States and were approved by the Animal Ethics Committee of Shandong University of Traditional Chinese Medicine (Jinan, China). The ethical approval number is SDUTCM20240102001. The mice were 2000mm3 The tumor volume was the human endpoint. In this study, the maximum tumor volume observed did not exceed this limit.

[0067] Western blot assay: The Western blot assay was performed as previously described. To analyze the expression of SLC7A11, A2780, OVCAR3, TOV-21G, and IOSE-29 cells were seeded in 12-well plates at a density of 1.5×10 5 cells / well. After overnight incubation, the cells were washed and lysed with 150 μL of RIPA buffer at 4 °C for 15 minutes. Cell debris was discarded by centrifugation at 12,000 rpm for 15 minutes at 4 °C, and the protein concentration was determined using the BCA protein assay. Then, 20 μL of 5×SDS was added to 80 μL of the sample, and the samples containing equal amounts of protein were separated by electrophoresis on a 12% polyacrylamide SDS gel, transferred to a nitrocellulose membrane, and blocked with 10% skim milk. The membrane was incubated with the SLC7A11 antibody (1:5000, abcam) overnight at 4 °C. After that, the anti-rabbit IgG secondary antibody (1:4000, ZSGB-Bio) was added and incubated for another 1 hour. Finally, the membrane was visualized by an 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 cytoskeletal 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 1.5×10 5 cells / well. After 12 hours, the medium was replaced with glucose-free RPMI-1640 medium and cultured for 3 or 6 hours, 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 and cultured for another 12 hours. Subsequently, the cells were lysed with 150 μL of RIPA buffer at 4 °C for 15 minutes. Protein samples were obtained by centrifugation. Each protein sample was divided into two equal parts. To one part (60 μL), 15 μL of non-reducing 5×SDS (Biosharp) was added, and the other part was used for reducing analysis. All samples were heated at 100 °C for 10 minutes. The Western blot procedure was performed as described, using the actin antibody (1:5000, abcam).

[0069] Preparation of FA-L@AI, FA-L@A and L@AI: FA-L@AI, FA-L@A and L@AI were synthesized using the one-step method reported previously. When preparing FA-L@AI, 10 mg of lecithin, 2.5 mg of cholesterol, 2 mg of DSPE-PEOz2000, and 3.5 mg of DSPE-PEG2000-FA were added to 18 mL of methanol. After dissolving by ultrasound (40 Khz), 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 sonicated (40 Khz) for 30 minutes, and the mixture was filtered sequentially through 0.45 μm and 0.22 μm membranes, and then extruded using a small liposome extruder (100 nm membrane, 15 times). Centrifugation was performed twice at 3500 rpm for 30 minutes using a 50KD ultrafiltration tube to obtain a product with a final concentration of 5.0 mg / mL.

[0070] To prepare FA-L@A, the materials included lecithin, cholesterol, DSPE-PEOz2000, DSPE-PEG2000-FA and 6-AN. To prepare L@AI, the materials included lecithin, cholesterol, DSPE-PEOz2000, DSPE-PEG2000-methyl, 6-AN and ICG. The synthesis procedure was the same as described above.

[0071] Rhodamine-phalloidin staining: Cells were seeded at a density of 1×10 4 cells / well on 12-well plate round cell slides (diameter 20 mm) and cultured for 12 hours. Then, the cells were treated with 20 μM 6-AN or 0.2 mg / mL FA-L@AI. After 12 hours, the medium was discarded and the cells were fixed with 4% formaldehyde for 10 minutes. After fixation, the cells were permeabilized with 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, the cells were washed twice and treated with DAPI for 10 minutes. After washing with PBS, images were taken using a Zeiss LSM 880 NLO microscope. (Red: λ ex = 543 nm, Blue: λ ex = 405 nm)

[0072] Cell uptake: Cells were seeded at a density of 1×10 4Cells were seeded at a density of ex cells per dish in 15-mm glass-bottom dishes and cultured for 12 h. The control group was left untreated to grow naturally. Other cells were incubated in medium containing 0.2 mg / mL L@AI or 0.2 mg / mL FA-L@AI for 1 h. Then, the original medium was removed, and 10 μM Hochest was added and incubated for another 10 min. Before imaging, all the medium in the dish was discarded, 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 = 643 nm, blue: λ

[0073] Detection of NADPH and cystine levels: Intracellular NADPH levels were measured according to a previously published method. Briefly, cells were seeded at a density of 1×10 6 cells per well in 6-well plates overnight and treated with different drugs (6-AN = 20 μM, FA-L@AI = 0.2 mg / mL) for 12 h. Then, the cells were lysed in 200 μL of extraction buffer and gently pipetted to facilitate cell lysis. Subsequent steps were carried out according to the instructions.

[0074] For the detection of intracellular cystine, cells were seeded at a density of 1×10 6 cells per well in 6-well plates and cultured for 12 h. Then, medium containing 20 μM 6-AN or 0.2 mg / mL FA-L@AI was added. After 12 h, the cells were washed with PBS and lysed in 200 μL of RIPA buffer at 4 °C for 15 min. Samples were collected by centrifugation at 12,000 rpm for 15 min at 4 °C. Subsequently, ELISA assays were performed according to the operation 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 via intravenous injection every other day. FA-L@AI and FA-L@A were administered at a concentration of 2 mg / mL (100 μL), and an equal amount of free 6-AN was injected at a dose of 267.4 μM (100 μL). The tumor volume and body weight of the mice were recorded before each drug injection. The formula for calculating the tumor volume was as follows: Tumor volume (V) = L×W 2 / 2, where L and W represent the length and width of the tumor, respectively. On day 20, all the mice were sacrificed and the tumors were dissected. After weighing, some tumors from each group were fixed in 4% formaldehyde for 3 days. After conventional dehydration and paraffin embedding, the above tumors were stained with H&E and Ki67. Other fresh tumor parts 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-treated samples and experimental samples. No data points of any animals 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, through TCGA database analysis, it was found that SLC7A11 was significantly highly expressed in ovarian cancer tissues ( Figure 1 a). Meanwhile, SLC7A11 high significantly prolonged the survival of ovarian cancer patients ( Figure 1 b). By scoring genes related to disulfide death, it was predicted that SLC7A11 high elevated the disulfide death activity of ovarian cancer patients, and the survival of patients with high disulfide death activity was prolonged ( Figure 1 c). The above results indicate 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 the effective treatment of ovarian cancer.

[0080] Subsequently, it was tested whether glucose deprivation could induce disulfide death in ovarian cancer cells. Through WB experiments, it was first confirmed that SLC7A11 protein was highly expressed in ovarian cancer cell lines OVCAR3 and TOV-21G ( Figure 1 d). Using the CCK8 assay, the sensitivity of SLC7A11 high ovarian cancer cell lines and normal cells to glucose was studied. The experimental results showed that the survival rates of OVCAR3 and TOV-21G decreased with the prolongation of glucose-free time, while the survival rate of A2780 cells decreased slowly ( Figure 1 e), suggesting that SLC7A11 high ovarian cancer cell lines have a high sensitivity to glucose. Further research found that this glucose deprivation-induced cell death could be significantly inhibited by the disulfide bond-specific reducing agent TCEP. Under the same conditions, the addition of other death inhibitors Fer-1, Trion, Z-VAD, and Nec-1 had no effect ( Figure 1f - g). More importantly, Western blot experiments showed that after glucose deprivation, there was a significant formation of disulfide bonds in Actin protein within OVCAR3 and TOV - 21G cells ( Figure 1 h - i). All of the above results demonstrated that glucose deprivation could induce disulfidptosis in OVCAR3 and TOV - 21G cells.

[0081] 2.2 6 - AN induces SLC7A11 high Disulfidptosis in ovarian cancer cells

[0082] To study the function of 6 - AN in inducing disulfidptosis in ovarian cancer cells. TCGA data showed that the survival time of patients in the SLC7A11 high G6PD low group in HGSOC was significantly longer than that of patients in the SLC7A11 low G6PD high group, suggesting that inhibiting G6PD might be a viable treatment strategy for SLC7A11 high OC ( Figure 2 a). Therefore, the focus was placed on the G6PD inhibitor 6 - AN and its potential to induce disulfidptosis in SLC7A11 high ovarian cancer cells. Experimental results showed that the survival rates of OVCAR3 and TOV - 21G cells decreased significantly with the increase in the concentration of 6 - AN treatment, while the viability of A2780 cells decreased to 60%, indicating that the death of SLC7A11 high ovarian cancer cells was highly dependent on 6 - AN ( Figure 2 b). Similar to glucose deprivation, the cell death induced by 6 - AN could be effectively rescued by TCEP, but not by Fer - 1, Trion, Z - VAD, and Nec - 1. This indicated that 6 - AN triggered the accumulation of intracellular disulfides ( Figure 2 c - d). Considering that inhibiting NADPH could prevent cystine from being reduced to cysteine and increase intracellular disulfides, the NADPH levels in SLC7A11 high ovarian cancer cells treated with 6 - AN were detected. The results showed that after 6 - AN treatment, the intracellular NADPH levels decreased significantly ( Figure 2 e). All of these data indicated that 6 - AN inhibited NADPH, blocked cystine breakdown, led to a large accumulation of intracellular disulfides, and ultimately induced disulfidptosis in SLC7A11 high ovarian cancer cells.

[0083] To prove the above conclusion, indicators related to disulfidptosis were detected. First, an ELISA kit was used to detect the cystine content in cells under different treatments. As Figure 2As shown in Fig. 6-AN group, the intracellular cystine concentration was significantly higher than that of the normal group, suggesting the accumulation of cystine in cells. The WB results showed that after treatment with 6-AN, a large number of disulfide bonds were formed in the Actin protein and could be effectively reduced by TCEP( Figure 2 g-h). Subsequently, the fluorescence confocal imaging technique was used to observe the morphology of F-actin. After treatment with 6-AN, obvious aggregation of F-actin appeared in cells( Figure 2 i), further indicating the formation of a large number of disulfide bonds in the Actin protein. The above results showed that 6-AN could inhibit the production of intracellular NADPH, resulting in the obstruction of cystine metabolism, the accumulation of disulfides, and the induction of cell 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 was simple and clear, and the specific synthesis steps are detailed in the method. Transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) images showed that FA-L@AI was spherical particles with a particle size of about 100 nm( Figure 3 b-c). Dynamic light scattering (DLS) showed that the hydrated particle size was about 116 nm and the polydispersity index (PDI) was about 0.21, indicating that the particle size distribution of the material was uniform( 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 map confirmed the effective incorporation of ICG into FA-L@AI( Figure 3 e). The absorption spectrum of FA-L@AI matched the absorption spectra 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 indicated 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 nanodrug was placed at 4°C, and samples were taken at the time points shown in the figure to measure its particle size and PDI. The results showed that the particle size of the material basically did not change with the increase of days, and the PDI was also relatively stable. At the same time, the particle size of the material in different dispersants also basically did not change with time, indicating that the material had good physical stability( Figure 3 g-h). In summary, FA-L@AI had good stability, which was a necessary prerequisite for its further application.

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

[0088] To investigate the characteristics of high uptake of nano-drugs by 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. As Figure 4 shown in a, after staining with L@AI, the fluorescence in the red channel in IOSE-29, OVCAR3, and TOV-21G cells was basically the same. However, in OVCAR3 and TOV-21G cells treated with FA-L@AI, the red fluorescence was significantly stronger than that in IOSE-29 cells, and the fluorescence output value was approximately 3 times that of normal cells ( Figure 4 b), indicating that the new cancer cell material has a high absorption rate. All the above results show that due to the high expression of FRα on the cancer cell membrane, FA-L@AI can be specifically and highly absorbed by cancer cells.

[0089] Subsequently, the toxicity of FA-L@AI to cells was tested. The CCK8 results showed that the survival rate of cancer cells was significantly dependent on the concentration of the nano-drug. At 0.2 mg / mL, obvious death of cancer cells occurred, and the survival rate of normal cells decreased to 65% ( Figure 4 c). Although FA-L@AI showed certain toxicity to IOSE-29 cells, the cytotoxicity was significantly lower than that to cancer cells. After calculation, the IC50 value of FA-L@AI for IOSE-29 cells was 0.38 mg / mL, and that for cancer cells was approximately 0.068 mg / mL, further suggesting that when the nano-drug dose was 0.2 mg / mL, it had less impact on normal cells but obvious toxicity to cancer cells. To prove that the toxicity of 6-AN was not affected after being loaded into liposomes, normal cells and cancer cells were treated with the same concentration of free 6-AN, FA-L@AI, and FA-L@A. The results showed that the drug had obvious killing effect on cancer cells but was basically non-toxic to normal cells, and the lethal effects of FA-L@AI and FA-L@A were the same as those of 6-AN, suggesting that FA-modified liposome carriers and ICG did not affect the toxicity of 6-AN. All the above data indicate that the nano-drug retains the efficacy of 6-AN and can specifically kill cancer cells.

[0090] 2.5 FA-L@AI specifically induces disulfide death of SLC7A11 high in ovarian cancer cells

[0091] Since FA-L@AI has the toxicity of 6-AN, it is speculated that the nano-drug kills cancer cells through disulfide death. As Figure 5As shown in Figure a, the cell death induced by FA-L@AI was effectively reversed by TCEP and was not affected by Fer-1, Trion, Z-VAD, or Nec-1, suggesting that FA-L@AI caused the accumulation of disulfides in ovarian cancer cells. The NADPH kit was used to detect the cells treated with the nanomedicine, and it was found that the level of NADPH was significantly decreased ( Figure 5 b). The above results indicate that FA-L@AI can inhibit the synthesis of NADPH in cells and induce the accumulation of disulfides in cells. Subsequently, the indicators related to disulfide death were detected. First, the ELISA kit was used to detect the cystine content in cells under different treatments. As Figure 5 shown in Figure c, the cystine concentration in the cells of the FA-L@AI group was significantly higher than that in the normal group, suggesting the accumulation of cystine in the cells. The WB results showed that after treatment with FA-L@AI, a large number of disulfide bonds were formed in the Actin protein and could be effectively reduced by TCEP ( Figure 5 d-e). Subsequently, the fluorescence confocal imaging technique was used to observe the morphology of F-actin. After treatment with FA-L@AI, obvious aggregation of F-actin appeared in the cells ( Figure 5 f), further indicating the formation of a large number of disulfide bonds in the Actin protein. The above results show that the lethal mechanism of FA-L@AI is the same as that of 6-AN, that is, by inhibiting the production of NADPH in cells, leading to the obstruction of cystine metabolism, the accumulation of disulfides, and the induction of cell disulfide death.

[0092] 2.6 FA-L@AI targets ovarian cancer tissues in vivo

[0093] To further evaluate the therapeutic potential of the nanomedicine, the uptake ability of the tumor to FA-L@AI in a tumor-bearing mouse model was studied. The OVCAR3 cancer cells were subcutaneously injected into BALB / c nude mice to create a tumor-bearing model. After intravenous injection of FA-L@AI, imaging was performed on a small animal in vivo imaging system. Notably, strong fluorescence was detected only at the tumor site, reaching a peak 4 h after administration and lasting up to 12 h, indicating that FA-L@AI aggregated in the tumor and had a long residence time ( Figure 6 a). Imaging of the major organs and tumor tissues showed bright fluorescence in the tumor tissue, while normal tissues (except the liver and kidneys) showed dim fluorescence. This finding indicates that due to the EPR effect and the high expression of FRα on the cancer cell membrane, FA-L@AI preferentially accumulates in the tumor region ( 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 kidneys, mainly due to the influence of the reticuloendothelial system. Taken together, these results confirm that FA-L@AI effectively aggregates and visualizes the tumor location, laying a foundation for the 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 the nanomedicine via the tail vein, respectively. Blood samples were collected 12 h later to detect relevant serum indices. The contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (CRE), and creatine kinase (CK) in the FA-L@AI group showed no significant changes compared with those in the PBS group, suggesting normal liver, kidney, and heart functions( Figure 6 d). To further evaluate the safety of FA-L@AI, the major organs of these 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 indicate that FA-L@AI has good biosafety.

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

[0096] Since FA-L@AI can highly accumulate in tumor tissues, whether it can exert a therapeutic effect on tumors remains to be explored. 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 injected with the corresponding drugs via the tail vein every other day. The tumor volume was measured continuously for 20 days to evaluate the therapeutic effect. The treatment regimens are shown in Figure 7 a. Compared with the PBS group, the tumor size in the free 6-AN group decreased, while the tumors in the FA-L@A and FA-L@AI groups were significantly inhibited. Notably, compared with the free 6-AN group, the FA-L@AI group showed significant growth inhibition, indicating that FA-L@AI has a potent antitumor effect and its toxicity is not affected by ICG loading( Figure 7 b). Studies have shown that FA can bind to FRα on tumor cells, and liposomes can accumulate at the tumor site through the enhanced permeability and retention (EPR) effect. Therefore, compared with free drugs, FA-L@AI has a better antitumor effect. Meanwhile, the change trend of tumor weight was consistent with the volume measurement( Figure 7 c. In addition, there was no significant change in the body weight of the mice in the FA-L@AI group, indicating that FA-L@AI has no systemic toxicity( Figure 7 d. In short, it was fully verified that the strategy using FA-L@AI not only effectively improved the absorption of ovarian cancer tissues but also significantly inhibited tumor growth through disulfidptosis.

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

[0098] In summary, FA-L@AI mainly has three major advantages: (1) highly absorbed by FA-modified ovarian cancer cells; (2) specifically mediating SLC7A11 high disulfidptosis of ovarian cancer cells, showing a strong cell damage ability; (3) selectively accumulating at the tumor site due to FA modification and the EPR effect, and showing superior anti-tumor efficacy, significantly inhibiting the growth of SLC7A11 high ovarian tumors with minimal systemic toxicity. In addition, the related therapeutic mechanism of disulfidptosis triggered by FA-L@AI was further elucidated by mRNA transcriptome analysis, which may provide an alternative to traditional therapies for OC treatment.

[0099] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A 6-aminonicotinamide-loaded nanomaterial targeting ferroptosis, characterized in that, The nanomaterial is specifically a liposome modified with folic acid and 6-aminonicotinamide; the liposome is a pH-sensitive liposome.

2. The nanomaterial according to claim 1, wherein The liposome is further modified with a fluorescent indicator, and further, the fluorescent indicator is indocyanine green ICG.

3. The nanomaterial according to claim 1, characterized in that, The pH-sensitive liposome contains a PEOz acid-sensitive group, further DSPE-PEOz2000.

4. The nanomaterial according to any one of claims 1 to 3, characterized in that, The nanomaterial is a spherical particle with a particle size of ~100 nm.

5. The preparation method of the 6-aminonicotinamide-loaded nanomaterial targeting disulfidptosis according to any one of claims 1-4, characterized in that, The preparation method includes one-step preparation of the nanomaterial according to any one of claims 1-4 by the thin film hydration method.

6. The preparation method according to claim 5, characterized in that, The preparation method includes: dissolving the liposome film material in an organic solvent, adding 6-aminonicotinamide thereto, removing methanol, adding PBS buffer thereto, heating and hydrating, and performing ultrasonic treatment, and then obtaining the product after filtration, extrusion, and centrifugation.

7. The preparation method according to claim 6, characterized in that, The liposome film 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; In the preparation method, a fluorescent indicator is further added to the solution in which the liposome film material is dissolved; further, the fluorescent indicator is indocyanine green; The mass ratio of the lecithin to 6-aminonicotinamide is 5-15:1-5; The mass ratio of the lecithin to indocyanine green is 5-15:1-5.

8. The preparation method according to claim 6, characterized in that, The heating and hydration can be carried out by a water bath heating method, and the specific conditions are: heating at 50-80 °C for 10-60 minutes; The ultrasonic treatment is carried out at 20-60 Khz for 10-60 minutes; The filtration is specifically carried out by filtering with 0.45 μm and 0.22 μm membranes in sequence; The extrusion is carried out through a 100 nm filter membrane for 10-20 times; The centrifugation is carried out by using a 30-80 KD ultrafiltration tube at 1000-5000 rpm for 10-60 minutes; further, centrifugation is carried out 1-3 times.

9. Use of the 6-aminonicotinamide-loaded nanomaterial targeting disulfidptosis according to any one of claims 1-4 in the preparation of an anti-tumor drug; Further, the tumor is an ovarian tumor; more specifically, it is SLC7A11 high Ovarian tumor.

10. An anti-tumor drug, characterized in that, The active ingredient of the anti-tumor drug contains the 6-aminonicotinamide-loaded nanomaterial targeting disulfidptosis according to any one of claims 1-4.

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